THE
BIOLOGICAL BULLETIN
PUBLISHED BY
THE MARINE BIOLOGICAL LABORATORY
Editorial Board
GARY N. CALKINS, Columbia University E. E. JUST, Howard University
E. G. CONKLIN, Princeton University FRANK R. LlLLIE, University of Chicago
1LS: 5t™VPf nCeKt0nTTUniVerSity CARL R' MOORE, University of Chicago
SELIG HECHT, Columbia University ~ _ T.-
LEIGH HOADLEY, Harvard University GEORGE T. MOORE, Missouri Botanical Garden
L. IRVING, Swarthmore College T. H. MORGAN, California Institute of Technology
M. H. JACOBS, University of Pennsylvania G. H. PARKER, Harvard University
H. S. JENNINGS, Johns Hopkins University F. SCHRADER, Columbia University
ALFRED C. REDFIELD, Harvard University
Managing Editor
VOLUME LXXVII
AUGUST TO DECEMBER, 1939
Printed and Issued by
LANCASTER PRESS, Inc.
PRINCE &. LEMON STS.
LANCASTER, PA.
11
THE BIOLOGICAL BULLETIN is issued six times a year. Single
numbers, $1.75. Subscription per volume (3 numbers), $4.50.
Subscriptions and other matter should be addressed to the
Biological Bulletin, Prince and Lemon Streets, Lancaster, Pa.
Agent for Great Britain : Wheldon & Wesley, Limited, 2, 3 and
4 Arthur Street, New Oxford Street, London, W.C. 2.
Communications relative to manuscripts should be sent to the
Managing Editor, Marine Biological Laboratory, Woods Hole,
Mass., between June 1 and October 1 and to the Biological Labo-
ratories, Divinity Avenue, Cambridge, Mass., during the remainder
of the year.
Entered October 10, 1902, at Lancaster, Pa., as second-class matter under
Act of Congress of July 16, 1894.
LANCASTER PRESS, INC., LANCASTER, PA.
CONTENTS
No. 1. A.UGUST, 1939
PAGE
FORTY-FIRS^ ^ x ^INE BIOLOGICAL LABORATORY. 1
irematodes of Woods Hole. II. The life
o "' .. •£ » "••
- 01 Stephanostomum tenue (Linton) 65
IIARVEY, ETHEL BROWNE
An Hermaphrodite Arbacia 74
ROOSEN-RUNGE, EDWARD C.
Karyokinesis during Cleavage of the Zebra fish Brachydanio
rerio 79
MATTHEWS, SAMUEL A.
The Effects of Light and Temperature on the Male Sexual
Cycle in Fundulus 92
BURGER, J. WENDELL
Some Experiments on the Relation of the External Environ-
ment to the Spermatogenetic Cycle of Fundulus heteroclitus
(L.) 96
BROWN, F. A., JR., AND ONA CUNNINGHAM
Influence of the Sinusgland of Crustaceans on Normal Via-
bility and Ecdysis 104
MACGINITIE, G. E.
The Method of Feeding of Chaetopterus 115
WELSH, JOHN H.
The Action of Eye-stalk Extracts on Retinal Pigment Migra-
tion in the Crayfish, Cambarus bartoni 119
CROZIER, W. J., AND ERNST WOLF
The Flicker-response Contour for the Crayfish. II. Retinal
pigment and the theory of the asymmetry of the curve 126
LAWSON, CHESTER A.
The Significance of Germaria in Differentiation of Ovarioles
in Female Aphids 135
51145
111
11
THE BIOLOGICAL BULLETIN is issued six times a year. Single
numbers, $1.75. Subscription per volume (3 numbers), $4.50.
Subscriptions and other matter shou) v° addressed to the
Biological Bulletin, Prince and Lemon Lancaster, Pa.
Agent for Great Britain: Wheldon & \v A. 2, 3 and
4 Arthur Street, New Oxford Street, London,
Communications relative to manuscripts should ,^
Managing Editor, Marine Biological Laboratory, Woo.
Mass., between June 1 and October 1 and to the Biological i^.
ratories, Divinity Avenue, Cambridge, Mass., during the remaindei
of the year.
Entered October 10, 1902, at Lancaster, Pa., as second-class matter under
Act of Congress of July 16, 1894.
LANCASTER PRESS, INC., LANCASTER, PA.
CONTENTS
No. 1. AUGUST, 1939
PAGE
FORTY- FIRST REPORT OF THE MARINE BIOLOGICAL LABORATORY. 1
MARTIN, W. E.
Studies on the Trematodes of Woods Hole. II. The life
cycle of Stephanostomum tenue (Linton) 65
HARVEY, ETHEL BROWNE
An Hermaphrodite Arbacia 74
ROOSEN-RUNGE, EDWARD C.
Karyokinesis during Cleavage of the Zebra fish Brachydanio
rerio 79
MATTHEWS, SAMUEL A.
The Effects of Light and Temperature on the Male Sexual
Cycle in Fundulus 92
BURGER, J. WENDELL
Some Experiments on the Relation of the External Environ-
ment to the Spermatogenetic Cycle of Fundulus heteroclitus
(L.) 96
BROWN, F. A., JR., AND ONA CUNNINGHAM
Influence of the Sinusgland of Crustaceans on Normal Via-
bility and Ecdysis 104
MACGINITIE, G. E.
The Method of Feeding of Chaetopterus 115
WELSH, JOHN H.
The Action of Eye-stalk Extracts on Retinal Pigment Migra-
tion in the Crayfish, Cambarus bartoni 119
CROZIER, W. J., AND ERNST WOLF
The Flicker-response Contour for the Crayfish. II. Retinal
pigment and the theory of the asymmetry of the curve 126
LAWSON, CHESTER A.
The Significance of Germaria in Differentiation of Ovarioles
in Female Aphids 135
51145
111
iv CONTENTS
No. 2. OCTOBER, 1939
PAGE
SOUTHWICK, WALTER E.
Activity-preventing and Egg-Sea-Water Neutralizing Sub-
stances from Spermatozoa of Echinometra subangularis .... 147
SOUTHWICK, WALTER E.
The "Agglutination" Phenomenon with Spermatozoa of
Chiton tuberculatus 157
KANDA, SAKYO
The Luminescence of a Nemertean, Emplectonema kandai,
Kato 166
FAWCETT, DON WAYNE
Absence of the Epithelial Hypophysis in a Fetal Dogfish
Associated with Abnormalities of the Head and of Pigmenta-
tion 174
GOODRICH, H. B., AND PRISCILLA L. ANDERSON
Variations of Color Pattern in Hybrids of the Goldfish,
Carassius auratus 184
GOODRICH, H. B., AND J. P. TRINKAUS
The Differential Effect of Radiations on Mendelian Pheno-
types of the Goldfish, Carassius auratus 192
JOHNSON, W. H., AND J. E. G. RAYMONT
The Reactions of the Planktonic Copepod, Centropages
typicus, to Light and Gravity 200
ROSE, S. MERYL
Embryonic Induction in the Ascidia 216
PORTER, K. R.
Androgenetic Development of the Egg of Rana pipiens 233
BUTCHER, EARL O.
The Illumination of the Eye Necessary for Different Melano-
phoric Responses of Fundulus heteroclitus 258
BRAGG, ARTHUR N.
Observations upon Amphibian Deutoplasm and its Relation
to Embryonic and Early Larval Development 268
VON BRAND, THEODOR, NORRIS W. RAKESTRAW AND CHARLES E.
RENN
Further Experiments on the Decomposition and Regeneration
of Nitrogenous Organic Matter in Sea Water 285
PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS PRESENTED AT
THE MARINE BIOLOGICAL LABORATORY, SUMMER OF 1939. 297
CONTENTS v
No. 3. DECEMBER, 1939
PAGE
KlTCHING, J. A.
The Effects of a Lack of Oxygen and of Low Oxygen Tensions
on Paramecium 339
RAYMONT, J. E. G.
Dark Adaptation and Reversal of Phototropic Sign in
Dineutes 354
BlSSONNETTE, THOMAS HUME AND ALBERT GEORGE CSECH
Modified Sexual Photoperiodicity in Cotton-tail Rabbits . . . 364
LlTTLEFORD, ROBERT A.
The Life Cycle of Dactylometra quinquecirrha, L. Agassiz in
the Chesapeake Bay 368
BROWN, MORDEN G.
The Blocking of Excystment Reactions of Colpoda duo-
denaria by Absence of Oxygen 382
MAST, S. O.
The Relation between Kind of Food, Growth, and Structure
in Amoeba 391
ANGERER, C. A.
The Effect of Electric Current on the Relative Viscosity of
Sea-Urchin Egg Protoplasm 399
BEADLE, G. W., E. L. TATUM AND C. W. CLANCY
Development of Eye Colors in Drosophila: Production of v+
Hormone by Fat Bodies 407
TATUM, E. L., AND G. W. BEADLE
Effect of Diet on Eye-Color Development in Drosophila
melanogaster 415
RUSSELL, ALICE
Pigment Inheritance in the Fundulus-Scomber Hybrid 423
CHILD, GEORGE
The Effect of Increasing Time of Development at Constant
Temperature on the Wing Size of Vestigial of Drosophila
melanogaster 432
MACGINITIE, G. E.
The Method of Feeding of Tunicates 443
DEWEY, VIRGINIA C.
Test Secretion in Two Species of Folliculina 448
INDEX FOR VOLUME 77 . 457
Vol. LXXVII, No. 1 August, 1939
THE
BIOLOGICAL BULLETIN
PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY
THE MARINE BIOLOGICAL LABORATORY
FORTY-FIRST REPORT, FOR THE YEAR 1938 —
FIFTY-FIRST YEAR
I. TRUSTEES AND EXECUTIVE COMMITTEE (AS OF AUGUST 9,
1938) 1
STANDING COMMITTEES 3
II. ACT OF INCORPORATION 3
III. BY-LAWS OF THE CORPORATION 4
IV. REPORT OF THE TREASURER 5
V. REPORT OF THE LIBRARIAN 10
VI. REPORT OF THE DIRECTOR 11
Statement 11
Addenda :
1. Report of the Committee on Policy 15
2. The Staff, 1938 27
3. Investigators and Students, 1938 30
4. Tabular View of Attendance 41
5. Subscribing and Cooperating Institutions, 1938 .... 41
6. Evening Lectures, 1938 42
7. Shorter Scientific Papers, 1938 43
8. General Scientific Meeting, 1938 45
9. Members of the Corporation 50
I. TRUSTEES
EX OFFICIO
FRANK R. LILLIE, President of the Corporation, The University of Chicago.
CHARLES PACKARD, Associate Director, Columbia University.
LAWRASON RIGGS, JR., Treasurer, 120 Broadway, New York City.
PHILIP H. ARMSTRONG, Clerk of the Corporation, Syracuse University and
Medical College.
EMERITUS
H. C. BUMPUS, Brown University.
E. G. CONKLIN, Princeton University.
C. R. CRANE, New York City.
R. A. HARPER, Columbia University.
H. S. JENNINGS, Johns Hopkins University.
M. M. METCALF, Waban, Mass.
T. H. MORGAN, California Institute of Technology.
I MARINE BIOLOGICAL LABORATORY
G. H. PARKER, Harvard University.
W. B. SCOTT, Princeton University.
E. B. WILSON, Columbia University.
TO SERVE UNTIL 1942
E. R. CLARK, University of Pennsylvania.
OTTO C. GLASER, Amherst College.
Ross G. HARRISON, Yale University.
E. N. HARVEY, Princeton University.
M. H. JACOBS, University of Pennsylvania.
F. P. KNOWLTON, Syracuse University.
FRANZ SCHRADER, Columbia University.
B. H. WILLIER, University of Rochester.
TO SERVE UNTIL 1941
W. R. AMBERSON, University of Tennessee.
W. C. CURTIS, University of Missouri.
H. B. GOODRICH, Wesleyan University.
I. F. LEWIS, University of Virginia.
R. S. LILLIE, The University of Chicago.
A. C. REDFIELD, Harvard University.
C. C. SPEIDEL, University of Virginia.
D. H. TENNENT, Bryn Mawr College.
TO SERVE UNTIL 1940
H. B. BIGELOW, Harvard University.
R. CHAMBERS, Washington Square College, New York University.
W. E. GARREY, Vanderbilt University Medical School.
CASWELL GRAVE, Washington University.
S. O. MAST, Johns Hopkins University.
A. P. MATHEWS, University of Cincinnati.
C. E. McCLUNG, University of Pennsylvania.
C. R. STOCKARD, Cornell University Medical College.
TO SERVE UNTIL 1939
W. C. ALLEE, The University of Chicago.
GARY N. CALKINS, Columbia University.
B. M. DUGGAR, University of Wisconsin.
L. V. HEILBRUNN, University of Pennsylvania.
L. IRVING, University of Toronto.
W. J. V. OSTERHOUT, Member of the Rockefeller Institute for Medical Re-
search.
A. H. STURTEVANT, California Institute of Technology.
LORANDE L. WOODRUFF, Yale University.
EXECUTIVE COMMITTEE OF THE BOARD OF TRUSTEES
FRANK R. LILLIE, Ex. Off. Chairman.
CHARLES PACKARD, Ex. Off.
LAWRASON RIGGS, JR., Ex. Off.
CASWELL GRAVE, to serve until 1939.
C. E. MCCLUNG, to serve until 1939.
ACT OF INCORPORATION
LAURENCE IRVING, to serve until 1940.
S. O. MAST, to serve until 1940.
THE LIBRARY COMMITTEE
E. G. CON KLIN, Chairman.
WILLIAM R. AMBERSON.
C. O. ISELIN, II.
C. C. SPEIDEL.
A. H. STURTEVANT.
WILLIAM R. TAYLOR.
THE APPARATUS COMMITTEE
L. V. HEILBRUNN, Chairman.
W. R. AMBERSON.
D. J. EDWARDS.
W. E. CARREY.
E. N. HARVEY.
L. IRVING.
M. H. JACOBS.
B. LUCKE.
THE SUPPLY DEPARTMENT COMMITTEE
LAURENCE IRVING, Chairman.
T. H. BlSSONNETTE.
H. B. GOODRICH.
A. C. REDFIELD.
C. C. SPEIDEL.
THE EVENING LECTURE COMMITTEE
B. H. WILLIER, Chairman.
M. H. JACOBS.
CHARLES PACKARD.
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 Sedg-
wick 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 scien-
tific study and investigation, and a school for instruction in biology and
natural history, and have complied with the provisions of the statutes of this
Commonwealth in such case made and provided, as appears from the cer-
tificate 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 Massachusetts, do hereby certify that said A. Hyatt, W. S. Stevens,
W. T. Sedgwick, E. G. Gardiner, "S. Minns, C. S. Minot, S. Wells, W.
4 MARINE BIOLOGICAL LABORATORY
G. Farlow, A. D. Phillips, and B. H. Van Vleck, their associates and suc-
cessors, are legally organized and established as, and are hereby made, an
existing Corporation, under the name of the MARINE BIOLOGICAL
LABORATORY, 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 annual meeting of the members shall be held on the second
Tuesday in August, at the Laboratory, in Woods Hole, Mass., at 11.30 A.M.,
daylight saving time, in each year, and at such meeting the members shall
choose by ballot a Treasurer and a Clerk to serve one year, and eight Trustees
to serve four years. There shall be thirty-two Trustees thus chosen divided
into four classes, each to serve four years, and in addition there shall be two
groups of Trustees as follows: (a) Trustees ex officio, who shall be the
President of the Corporation, the Director of the Laboratory, the Associate
Director, the Treasurer and the Clerk; (&) 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 Cor-
poration and he shall become eligible for election as Trustee Emeritus for
life. The Trustees ex officio and Emeritus shall have all 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.
II. Special meetings of the members may be called by the Trustees to
be held in Boston or in Woods Hole at such time and place as may be
designated.
III. Inasmuch as the time and place of the Annual Meeting of Members
is 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 said 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.
IV. Twenty-five members shall constitute a quorum at any meeting.
V. The Trustees shall have the control and management of the affairs
of the Corporation; they shall present a report of its condition at every
annual meeting; they shall elect one of their number President of the Cor-
poration who shall also be 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
REPORT OF THE TREASURER
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. They shall from time to time elect members to the Corporation
upon such terms and conditions as they may think best.
VI. Meetings of the Trustees shall be called by the President, or by
any two Trustees, and the Secretary shall give notice thereof by written
or printed notice sent to each Trustee by mail, postpaid. Seven Trustees
shall constitute a quorum for the transaction of business. The Board of
Trustees shall have 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.
VII. The accounts of the Treasurer shall be audited annually by a
certified public accountant.
VIII. 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 de-
termined by the affirmative vote of two-thirds of the Board of Trustees.
IX. These By-laws may be altered at any meeting of the Trustees, pro-
vided that the notice of such meeting shall state that an alteration of the
By-laws will be acted upon.
X. Any member in good standing may vote at any meeting, either in
person or by proxy duly executed.
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 1938.
The accounts have been audited by Messrs. Seamans, Stetson and
Tuttle, certified public accountants. A copy of their report is on file
at the Laboratory and is open to inspection by members of the Cor-
poration.
At the end of the year 1938, the book value of the Endowment
Funds in the hands of the Central Hanover Bank and Trust Company
as Trustee, was
General Fund, Securities (market $862,409.23) $ 916,855.70
9,235.71
858.45
173,918.24
20,102.88
319.46
Real Estate
Cash, principal
Library Fund, Securities (market $162,008.68)
Real Estate
Cash
$1,121,290.44
- •
ash.
6 MARINE BIOLOGICAL LABORATORY
The income collected from these Funds was as follows :
General Endowment $36,382.94
Library 6,665.66
$43,048.60
The income in arrears on these Funds at the end of the year was :
Arrears General Fund $13,518.86
Arrears Library Fund 3,450.00
$16,968.86
Arrears at the end of the year 1937 $12,755.86
showing an increase of $ 4,213.00
The dividends from the General Biological Supply House totalled
$14,224.00.
Retirement Fund: A total of $4,060 was paid in pensions of which
$197.20 was advanced from current funds. The Fund at the end of the
year consisted of mortgages and real estate at the book value of
$17,462.08.
Plant Assets: The land (exclusive of Gansett and Devil's Lane),
buildings, equipment and library represent an investment of
$1,789,884.74
less reserve for depreciation 517,178.00
or a net of $1,272,706.74
The hurricane water damage to the inventory and equipment and
plant amounted to $30,399.02
of which $2,387.97 was charged to Plant Fund and $28,011.05 to Cur-
rent Surplus. Early this year The Carnegie Corporation of New York
most generously contributed $20,000 toward the repair of the hurricane
damage.
Income and Expenses: Income including a donation of stock valued
at $7,250 exceeded expense, including $24,481.56 depreciation, by
$11,432.64.
There was expended from current funds for plant account a net of
$15,083.21 and in addition $6,500 in reduction of mortgage and note
indebtedness.
At the end of the year the Laboratory owed $5,500 on mortgages
and $7,000 on notes all for property purchased in earlier years. It had
accounts and. notes receivable of $12,305.69 and $8,531.29 in cash and
bank accounts in its current funds.
REPORT OF THE TREASURER 7
A gift of 200 shares of Crane Company stock was received from
Dr. Frank R. Lillie, to which he has since added 300 shares.
Following is the balance sheet, the condensed statement of income
and outgo, and the surplus account all as set out by the accountants :
EXHIBIT A
MARINE BIOLOGICAL LABORATORY BALANCE SHEET,
DECEMBER 31, 1938
Assets
Endowment Assets and Equities :
Securities and Cash in Hands of Central Hanover
Bank and Trust Company, New York, Trustee
—Schedules I-a and I-b $1,121,290.44
Securities and Cash— Minor Funds— Schedule II .. 8,742.81 $1,130,033.25
Plant Assets :
Land— Schedule IV $ 110,884.58
Buildings— Schedule IV 1,239,161.81
Equipment— Schedule IV 165,567.34
Library— Schedule IV 274,271.01 $1,789,884.74
Less Reserve for Depreciation 517,178.00
$1,272,706.74
Cash in Dormitory Building Fund 223.24
Cash in Reserve Fund 24.65 $1,272,954.63
Current Assets :
Cash $ 8,531.29
Accounts and Notes — Receivable 12,305.69
Inventories :
Supply Department $ 37,672.27
Biological Bulletin 9,762.64 47,434.91
Investments :
Devil's Lane Property $ 44,398.34
Gansett Property 5,822.49
Stock in General Biological Supply
House, Inc 12,700.00
Other Investment Stocks 7,250.00
Securities and Real Estate — Re-
tirement Fund List — Sched-
ule V, viz.,
Retirement Fund Por-
tion 17,264.88
Current Account Portion . 197.20 87,632.91
Prepaid Insurance 3,193.90
Items in Suspense (Net) 693.98 $ 159,792.68
$2,562,780.56
8
MARINE BIOLOGICAL LABORATORY
Liabilities
Endowment Funds :
Endowment Funds— Schedule III $1,120,581.61
Reserve for Amortization of Bond
Premiums 708.83 $1,121,290.44
Minor Funds — Schedule III
8,742.81 $1,130,033.25
Plant Liabilities and Funds :
Mortgage— Payable, Howes Property $ 5,500.00
Notes— Payable a/c Bar Neck Property Purchase . 7,000.00
Donations and Gifts— Schedule III 1,038,402.61
Other Investments in Plant from Gifts and Cur-
rent Funds 222,052.02 $1,272,954.63
Current Liabilities and Surplus :
Accounts — Payable
Reserve for Additional Repairs and Replacements on
account of Hurricane Water — Damage
Current Surplus — Exhibit C
EXHIBIT B
4,077.37
17,518.12
138,197.19
159,792.68
$2,562,780.56
MARINE BIOLOGICAL LABORATORY INCOME AND EXPENSE,
YEAR ENDED DECEMBER 31, 1938
Total
Net
Expense Income Expense Income
Income :
General Endowment Fund $ 36,382.94 $ 36,382.94
Library Fund 6,665.66 6,665.66
Donations 7,250.00 7,250.00
Instruction 8,356.14 9,960.00 1,603.86
Research 4,215.07 16,312.50 12,097.43
Evening Lectures 58.56 58.56
Biological Bulletin and Membership
Dues 9,691.33 10,362.75 671.42
Supply Department— Schedule VI . 40,814.01 38,134.93 2,679.08
Mess— Schedule VII 25,899.67 25,759.83 139.84
Dormitories— Schedule VIII 22,609.70 12,973.34 9,636.36
( Interest and Depreciation
charged to above 3 Departments
—See Schedules VI, VII, and
VIII) 23,731.15 23,731.15
Dividends, General Biological Sup-
ply House, Inc 14,224.00 14,224.00
Rents :
Bar Neck Property 3,568.46 3,568.46
Bay Shore Property 206.57 91.75 114.82
Howes Property 196.64 480.00 283.36
Janitor House 23.19 360.00 336.81
Newman Cottage 81.43 250.00 168.57
Danchakoff Cottage 324.30 750.00 425.70
REPORT OF THE TREASURER
Sale of Library Duplicates 390.73 390.73
Apparatus Rental 991.30 991.30
Interest on Notes— Receivable 150.00 150.00
Sundry Income 38.54 38.54
Maintenance of Plant :
Buildings and Grounds 22,482.48 22,482.48
Chemical and Special Apparatus
Expense 14,121.00 14,121.00
Library Expense 7,576.77 7,576.77
Truck Expense 1,249.70 1,249.70
Workmen's Compensation
Insurance 507.51 507.51
Sundry Expense 19.50 19.50
General Expenses :
Administration Expense 12,199.94 12,199.94
Endowment Fund Trustee and
Safe-keeping 1,001.95 1,001.95
Interest on Notes and Mortgage
-Payable 829.83 829.83
Bad Debts 448.39 448.39
Reserve for Depreciation 24,481.56 24,481.56
$173,664.09 $185,096.73 $ 97,547.29 $108,979.93
Excess of income over Expense
carried to Current Surplus-
Exhibit C 11,432.64 11,432.64
$185,096.73 $108,979.93
EXHIBIT C
MARINE BIOLOGICAL LABORATORY, CURRENT SURPLUS ACCOUNT,
YEAR ENDED DECEMBER 31, 1938
Balance, January 1, 1938 $153,266.82
Add:
Excess of Income over Expense for Year as shown in
Exhibit B $11,432.64
Reserve for Depreciation Charged to Plant Funds 24,481.56 35,914.20
$189,181.02
Deduct :
Payments from Current Funds during Year for Plant
Assets as shown in Schedule IV,
Buildings $ 939.80
Equipment 4,818.61
Library 9,499.80
$15,258.21
Less Received for Plant Assets Disposed of 175.00
$15,083.21
Payment on Plant Mortgage and Note— Payable $ 4,500.00
Pensions Paid $4,060.00
10 MARINE BIOLOGICAL LABORATORY
Expenses on Account of Retirement Fund
Securities 36.79
$4,096.79
Less Retirement Fund Income and Gain
from Security Sale 707.22 3,389.57
Hurricane Water Damage (except portion Charged
to Plant Funds) 28,011.05 50,983.83
Balance, December 31, 1938— Exhibit A $138,197.19
Respectfully submitted,
LAWRASON RIGGS, JR.,
Treasurer.
V. THE REPORT OF THE LIBRARIAN
A report of the expenditures from the $18,800, appropriated to the
Library in 1938, follows: books, $351.67; current serials, $5,319.86;
binding, $1,171.38 ($45.00 of this on insurance); express, $181.48;
supplies, $1,070.07 (includes $37.53 for new boxes to ship books to the
bindery; $707.73 for new catalogue cases); salaries, $7,150.00; back
sets, $1,795.33; total, $17,039.79.
For various reasons such as lack of space in the Library and the
difficulty of securing the present lacks except in Germany, where prices
are high, it seemed best to allow the $1,760.21 available for back sets
besides $390.73 for the Library sale of duplicates, to revert to the
General Fund of the Laboratory. Also a correction of the printed 1937
report must be made here. An order for the back set of " Flora "
placed in Germany failed to come through and the order was finally
cancelled by the Librarian, allowing another sum of $2,430.50 to drop
from the Library expenditures.
The usual appropriation to the Library of $600.00 by the Woods
Hole Oceanographic Institution was expended to the amount of $591.98
and separately accounted.
This year the Library lists but 1,306 current serials of which 426
are subscriptions, 385 (11 new) purchases of the Marine Biological
Laboratory, 41 (1 new) of the Woods Hole Oceanographic Institution;
666 are exchanges, 596 (4 new) with the BIOLOGICAL BULLETIN and
70 (1 new) with the Woods Hole Oceanographic Institution publica-
tions; and 207 come as gifts to the former and 7 as gifts to the latter.
The record shows 47 books purchased, 41 by the Marine Biological
Laboratory and 6 by the Woods Hole Oceanographic Institution, 19
presented by the authors and 41 from publishers; while a contribution
from Dr. Alfred Meyer enabled the Library to purchase a new " Ameri-
REPORT OF THE DIRECTOR
can Medical Directory"; and Dr. Douglas M. Whitaker presented a
copy of Beaumont's " Experiments and Observations on the Gastric
Juice and the Physiology of Digestion." Completed back sets of serials
number 36; as purchases of the Marine Biological Laboratory, 20, of
the Woods Hole Oceanographic Institution, 2 ; while purchases partially
completing back sets number 15 for the former and 1 for the latter;
through exchange of duplicates, 11 completed back sets for the former,
and 1 for the latter ; besides many additions to still incomplete back sets ;
and 2 sets for the Marine Biological Laboratory completed by gifts, with
4 partially completed. Reprint additions number 6,905 : current for
1937, 1,897; current for 1938, 894, and of date previous to 1937, 4,114;
about 200 of the latter kindly presented by Dr. M. A. Bigelow and 70 by
Mrs. H. H. Donaldson. A summary of the current holdings of the
Library proper is therefore 44,897 bound volumes and 108,927 reprints.
VI. THE REPORT OF THE DIRECTOR
To THE TRUSTEES OF THE MARINE BIOLOGICAL LABORATORY :
Gentlemen: I beg to present herewith a report of the fifty-first
session of the Marine Biological Laboratory for the year 1938.
1. Attendance. The number of investigators and their assistants
present during the summer of 1938 was somewhat less than in 1937, but
it taxed the facilities of the Laboratory to the utmost. Attendance has
risen steadily, with minor fluctuations, since 1933 ; now the number
present is greater than the optimum which can be cared for under
existing conditions. We are rapidly approaching the time when selec-
tion among the applicants for research space must be made, a situation
referred to in the report of the Committee on Future Policy in the
following words : " It will be necessary to adopt more definite policies
concerning the admission of investigators than in the past. These should
not, however, be of too binding a character, but rather a definition of
principles within which the Director will have free scope for the exercise
of his best judgment." The definition of these principles deserves the
most careful consideration.
2. The Library. The continued growth of the Library is a source
of satisfaction to the investigator, but it presents a serious problem to
the Librarian who must find a place for new volumes and reprints.
Each year's increment of bound volumes requires a space about equal
to one complete stack. Since the present stacks are already practically
filled, it will presently be necessary to use rooms now employed for
cataloguing or other Library purposes. This will disturb the present
orderly arrangement of serials and will at best provide only temporary
relief. An addition to the Library is urgently needed.
c
(LI »v
12 MARINE BIOLOGICAL LABORATORY
3. The Board of Trustees. At the meeting of the Corporation held
Tuesday, August 9, 1938, Dr. H. S. Jennings, Trustee since 1905, was
elected Trustee Emeritus. To fill his place in the Class of 1942, Dr.
M. H. Jacobs, the retiring Director, was chosen. At the same meeting,
Dr. P. H. Armstrong was elected Clerk of the Corporation in place of
Dr. Charles Packard who resigned when appointed Assistant Director.
The Board has suffered heavy losses by death. Mr. Charles R.
Crane, Trustee from 1901 and President of the Board from 1902 to
1925, " the best friend the Laboratory ever had " ; Dr. Edmund B.
Wilson, Trustee continuously from 1890, whose contributions from
this Laboratory were instrumental in establishing its scientific eminence ;
Dr. Charles R. Stockard, Trustee from 1920, whose counsels, vigor-
ously expressed, were always highly valued ; and Dr. J. Playf air Mc-
Murrich, Trustee from 1892 to 1900, active in the early days of this
Laboratory.
4. The Hurricane and Flood. We may be profoundly thankful that
in the storm of September 21, 1938 no one connected with the Laboratory
lost his life. Some were rescued from desperate situations, and many
suffered heavy material loss. Damage to Laboratory property was due
almost entirely to water which poured into the basement of the Brick
Building, into the Supply Department and the Dormitory, washed away
most of the foundations of the Club House, and carried the Bathhouse
far inland. The old laboratory buildings and the Mess were above the
flood level.
During the height of the storm our staff worked heroically to protect
the buildings and equipment. Mr. Larkin organized a bucket brigade
and saved the apparatus in the Pump House ; Mr. Mclnnis and his crew
protected the motor boats ; others barricaded doors in the Brick Build-
ing against the rising waters, but to no avail for the flood broke through
the windows in the sub-basement of the Library; Mr. MacNaught
opened the Apartment House to those who had been driven from their
homes.
The greatest loss occurred in the Brick Building where the water,
four feet deep, submerged microscopes and electrical apparatus, overran
the storage battery, the switchboard and motors, and reduced the chemi-
cal and storage rooms to utter confusion.
The work of repair began at once. Dr. Pond and his assistants
examined all the apparatus which had been wet with salt water, recon-
ditioned much of it in our workship, and sent some to the manufacturers
for servicing. To restore the Chemical Room required many weeks of
hard work. Mr. Mclnnis and his men quickly reduced the confusion
in the Supply Department where the damage was not great, and were
REPORT OF THE DIRECTOR 13
able, within a few days, to resume regular business. Under the direc-
tion of Mr. Larkin, the storage battery was cleaned and recharged, and
the various motors were dried and set in place. None were lost, but
some needed repairs. The switchboard was damaged but has been re-
conditioned. Mrs. Montgomery saved many of the more important
duplicate reprints which had been water-soaked. Fortunately the regu-
lar reprint collection and the bound volumes were never in danger. The
bathhouse, after being put back on new foundations, was damaged by a
second storm. By order of the Executive Committee it was removed
entirely.
These very extensive repairs to the buildings and the equipment have
been made almost entirely by our permanent staff who have given un-
sparingly of their time and energy. To them the Laboratory owes a
debt of gratitude.
In the Treasurer's Report the loss due to the storm is set at $30,400.
This sum includes all of the various items which were lost. Inasmuch
as many of these were of little actual value, and need not be replaced,
the actual cost of restoring the damage will undoubtedly be less than
$25,000. Since the Laboratory carried no insurance against this type
of loss, the financial burden thus imposed upon us was serious. But we
are fortunate in our friends. The Carnegie Corporation of New York,
a benefactor of former years, has presented to the Laboratory the sum
of $20,000 to be used for purposes of restoration. We are sincerely
grateful for this generous and timely gift.
5. Research in Botany. For some years it has been apparent that
the number of investigators at the Laboratory carrying on research in
Botany has declined. This situation is due in part to the fact that some
of the members of the Research Staff have been unable to attend the
summer session, and in part to the lack of facilities for pursuing re-
search in the dynamic phases of Botany. Following the resignation of
Drs. Ivy M. Lewis, C. E. Allen and W. J. Robbins from the staff after
many years of active service, Dr. E. W. Sinnott, of Columbia University,
and Dr. D. R. Goddard, of the University of Rochester, were appointed.
The lack of facilities for research has been stressed by many botanists
who have expressed the opinion that more laboratory space is needed,
that a suitable plot of ground for raising plants should be provided, and
that a greenhouse is an essential part of an active botanical laboratory.
These requirements should be met at the earliest opportunity.
6. Gifts. The .sum of $20,000 given by the Carnegie Corporation of
New York, to be used for the purpose of restoring the damage done by
the flood, has already been mentioned. The Marine Biological Labora-
tory also gratefully acknowledges gifts amounting to $17,775 presented
by Dr. F. R. Lillie.
14 MARINE BIOLOGICAL LABORATORY
7. The Committee on Future Policy. At the meeting of August 11,
1937, the Board of Trustees authorized the President to appoint a com-
mittee to formulate a statement concerning the policies and future of the
Marine Biological Laboratory. The members of this Committee are :
E. G. Conklin, Chairman, G. N. Calkins, W. C. Curtis, H. B. Goodrich,
M. H. Jacobs, T. H. Morgan, G. H. Parker, A. C. Redfield and C. R.
Stockard. After many discussions during the summers of 1937 and
1938 a report was drawn up by Dr. Lillie. This was studied and
amended by the Committee and is now presented on p. 15 of this Annual
Report.
8. Lectures and Scientific Meetings. During the summer of 1938
there were ten regular evening lectures and seven seminars at which
shorter papers were discussed. In addition to these there were several
informal exhibitions of motion pictures of scientific interest and a
number of discussion groups. At the final scientific meetings, held
August 30 and August 31, numerous investigators reported the results
of their work during the current summer. In addition, many demon-
strations were on display, both at the Laboratory and at the Fish Com-
mission.
One of the regular seminar evenings was devoted to an informal
celebration of the fiftieth anniversary of the founding of the Laboratory.
Dr. Conklin reviewed the history of the early days, and Dr. Lillie spoke
of those who have contributed to the scientific and material welfare of
the institution. At the close of the meeting he presented to the Labora-
tory, in behalf of the Trustees, a portrait of Mr. Crane. It was a great
source of satisfaction that Mr. Crane could be present to receive greet-
ings from his many friends.
As in previous years, the Laboratory was host to the Genetics So-
ciety of America, which held its meetings on August 31 and Sep-
tember 1.
There are appended as parts of the report :
1. The Report of the Committee on Policies and Future of the Marine
Biological Laboratory.
2. The Staff, 1938.
3. Investigators and Students, 1938.
4. A Tabular View of Attendance, 1934-38.
5. Subscribing and Cooperating Institutions, 1938.
6. Evening Lectures, 1938.
7. Shorter Scientific Papers, 1938.
8. General Scientific Meeting, 1938.
9. Members of the Corporation, 1938.
Respectfully submitted,
CHARLES PACKARD,
Associate Director.
REPORT OF THE DIRECTOR 15
1. REPORT OF THE COMMITTEE APPOINTED ON RE-
QUEST OF THE BOARD OF TRUSTEES, AUGUST 10,
1937, TO FORMULATE A STATEMENT CONCERNING
THE POLICIES AND FUTURE OF THE MARINE BIO-
LOGICAL LABORATORY
I. INTRODUCTION
By way of introduction, it is important to remind ourselves of the
aims of the founders of the Marine Biological Laboratory. For this
purpose a series of quotations follows. It is not the intention to present
a history in any detail because it will be found that the original state-
ments of policies and aims have been carefully observed during the
entire history of the Laboratory for the fifty years of its existence. As
the first director early remarked, " These policies should be the germ
of an indefinite future development " ; and this has been the case.
In the First Annual Report of the Marine Biological Laboratory for
the year 1888, the Trustees made the following statements :
"Foundation. — The Marine Biological Laboratory is an outgrowth of
a sea-side laboratory maintained at Annisquam, Mass., from 1880 to
1886, by the Women's Education Association of Boston, in cooperation
with the Boston Society of Natural History. In 1886, efforts were
made by the Association to place the Laboratory on an independent and
broader foundation. A circular letter was addressed to many of the
leading biologists of the country, reciting what had been already done
at Annisquam, and asking for cooperation and counsel. The replies
received were most encouraging, testifying to a general and hearty
approval of the enterprise, and promising cooperation and support."
(P. 7.)
" At the first meeting held by this committee, its members showed by
votes that it was their desire to found a laboratory that should give
opportunity for original research as well as for instruction, and soon
after appointed the following
TRUSTEES
Prof. William G. Farlow, Prof. Charles S. Minot,
Miss Florence M. Gushing, Miss Susan Minns.
Prof. Alpheus Hyatt, Prof. William T. Sedgwick,
Mr. Samuel Wells." (P. 8.)
The first announcement issued in 1888 contained the following
statements :
' The Trustees of the Marine Biological Laboratory earnestly desire
to enlist your co-operation in the support of a sea-side laboratory for
instruction and investigation in Biology."
" It is the desire of the Trustees that the enterprise shall enlist the
active support of the universities and colleges of the country. To pre-
16 MARINE BIOLOGICAL LABORATORY
vent its becoming a simply local undertaking, they wish to see all who
aid in its support by subscribing to investigators' tables share with the
other members of the Corporation in the annual election of Trustees.
The Trustees will, therefore, invite each institution which holds an
investigator's table to name five persons for members of the Corporation
during the term of subscription."
Dr. Whitman commented on these statements in the Eighth Annual
Report, for the year 1895 as follows :
" Here we see sketched the elemental basis of our germ-organization
— mainly potentialities of a theoretical nature, but ' instinct with spirit.'
The aim was a permanent biological station ; the function was to be
instruction and investigation ; the formative principle relied upon was
co-operation." (P. 19.)
Whitman himself was the most influential person in determining the
policies and aims of the new laboratory. In his first annual report as
Director in 1888 he stated his personal viewpoint as follows :
" The new Laboratory at Woods Hole is nothing more, and, I trust,
nothing less, than a first step towards the establishment of an ideal
biological station, organized on a basis broad enough to represent all
important features of the several types of laboratories hitherto known
in Europe and America. It should be provided eventually with means
for sending men to different points of the coast to undertake the investi-
gation of subjects of special interest, thus adding to the advantages of
a fixed station those of an itinerant laboratory.
" The research department should furnish just the elements required
for the organization of a thoroughly efficient department of instruction.
Other things being equal, the investigator is always the best instructor.
The highest grade of instruction in any science can only be furnished by
one who is thoroughly imbued with the scientific spirit, and who is
actually engaged in original work. Hence the propriety — and, I may
say, the necessity — of linking the function of instruction with that of
investigation. The advantages of so doing are not by any means con-
fined to one side. Teaching is beneficial to the investigator, and the
highest powers of acquisition are never reached where the faculty of
imparting is neglected. Teaching is an art twice blest; it blesseth him
that gives and him that takes. To limit the work of the Laboratory
to teaching would be a most serious mistake; and to exclude teaching
would shut out the possibilities of the highest development. The com-
bination of the two functions in mutually stimulating relations is a
feature of the Laboratory to be strongly commended." (Pp. 16-17.)
In his lecture on " Specialization and Organization " (Biological Lec-
tures, 1890) he remarked :
" Among the ways of bringing together our scattered forces into some-
thing like organic union, the most important, and the most urgent at
REPORT OF THE DIRECTOR 17
this moment, is that of a national marine biological station. Such an
establishment, with a strong endowment, is unquestionably the great
desideratum of American biology. There is no other means that would
bring together so large a number of the leading naturalists of the coun-
try, and at the same time place them in such intimate helpful relations
to one another. The larger the number of specialists working together,
the more completely is the organized whole represented, and the greater
and the more numerous the mutual advantages." (P. 24.)
In 1893 he wrote in his lecture on " Work and Aims of the Marine
Biological Laboratory" (Biological Lectures, 1893):
" To those who by word and example have encouraged cooperation,
this record will certainly be gratifying; and perhaps it will be accepted
by all as an assurance that good-will and united effort have not been
fruitless. For six years the Marine Biological Laboratory has stood
for the first and the only cooperative organization in the interest of
Marine Biology in America." (P. 236.)
The same year he remarked in his article " A Marine Observatory the
Prime Need of American Biologists " (Atlantic Monthly, June, 1893,
pp. 808-815):
'The Marine Biological Laboratory attaches itself to no single insti-
tution, but holds itself rigidly to the impartial function of serving all
on the same terms. It depends not upon one faculty for its staff of
instructors, but seeks the best men it can find among the higher in-
stitutions of the land. The board of trustees is a growing body, every
year adding to its number, until it now comprises a very large proportion
of the leading biologists of America. The whole policy is national in
spirit and scope. The laboratory exists in the interest of biology at
large, and not to nurse the prestige of any university or the pride of
individual pretension." (P. 811.)
" Representative character, devotion to biology at large, independent
government, — such are the essential elements of a strong and progressive
organization." (P. 812.)
Again in 1898 he returned to the theme in an article " Some of the
Functions and Features of a Biological Station " (Science, N.S., Vol. 7,
No. 159, January 14, 1898, pp. 11-12) :
" It now remains to briefly sketch the general character and to emphasize
some of the leading features to be represented in a biological station.
' The first requisite is capacity for growth in all directions con-
sistent with the symmetrical development of biology as a whole. The
second requisite is the union of the two functions, research and instruc-
tion, in such relations as will best hold the work and the workers in the
natural coordination essential to scientific progress and to individual
development. It is on this basis that I would construct the ideal and
test every practical issue.
18 MARINE BIOLOGICAL LABORATORY
" A scheme that excludes all limitations except such as nature pre-
scribes is just broad enough to take in the science, and that does not
strike me as at all extravagant or even as exceeding by a hair's breadth
the essentials. Whoever feels it an advantage to be fettered by self-
imposed limitations will part company with us here. If any one is
troubled with the question: Of what use is an ideal too large to be
realized ? I will answer at once. It is the merit of this ideal that it can
be realized just as every sound ideal can be realized, only by gradual
growth. An ideal that could be realized all at once would exclude
growth and leave nothing to be done but to work on in grooves. That
is precisely the danger we are seeking to avoid.
" The two fundamental requisites which I have just defined scarcely
need any amplification. Their implications, however, are far-reaching,
and I may, therefore, point out a little more explicitly what is involved.
I have made use of the term ' biological station ' in preference to those
in more common use, for the reason that my ideal rejects every artificial
limitation that might check growth or force a one-sided development.
I have in mind, then, not a station devoted exclusively to zoology, or
exclusively to botany, or exclusively to physiology ; not a station limited
to the study of marine plants and animals ; not a lacustral station deal-
ing only with land and fresh-water faunas and floras ; not a station
limited to experimental work, but a genuine biological station, embrac-
ing all these important divisions, absolutely free of every artificial
restriction.
"Now, that is a scheme than can grow just as fast as biology
grows, and I am of the opinion that nothing short of it could ever
adequately represent a national center of instruction and research in
biology. Vast as the scheme is, at least in its possibilities, it is a true
germ, all the principal parts of which could be realized in respectable
beginnings in a very few years and at no enormous expense. With
scarcely anything beyond our hands to work with, we have already
succeeded in getting zoology and botany well started at Woods Hole,
and physiology is ready to follow."
II. FUTURE PLANS AND POLICIES
A. The Problem of Expansion vs. Consolidation
Since the erection of the " New Laboratory " in 1923, there has been
a steady growth in the attendance of investigators, subject to some
recession during the depression, but reaching a peak in 1937 which
strained our accommodations to the limit during the greater part of the
session. The question is therefore forced upon our attention whether
we should limit arbitrarily the number of investigators as we have long
since done in the case of students in classes. The only alternative would
be to increase our accommodations. Decision of this point would affect
various policies, and it should therefore receive first consideration,
REPORT OF THE DIRECTOR 19
The Committee have given careful attention to the question of ex-
pansion and have reached the unanimous conclusion that it would be wise
at this time to consolidate and develop our present plant and organization,
and to postpone the question of expansion, or of new construction except
as noted below under Library and under Instruction.
The main reasons for this opinion are two : first, that the problems
of housing and adequate care of a considerably larger number of persons
would be difficult in the restricted community in which we find ourselves,
and second, the need of prudence which rests upon economic uncertain-
ties. It is by no means certain that we may not have to face another
period of depression before many years, and this should not find us
over-expanded. Each of these considerations can, of course, be de-
veloped in detail.
B. The Principle of Cooperation
Whitman spoke of cooperation as the " formative principle " of the
Laboratory. It is illustrated in the national scope of the Laboratory
and in its fundamental organization and government. The principles
involved in nation-wide institutional representation and cooperation, and
in comprehensive membership of the Corporation, are so rooted in our
practices and have proved so fruitful as to require only emphasis.
C. Organization and Government
The inter-relations of Trustees and Corporation as given in the
By-laws have operated harmoniously and effectively for a long time.
Rules concerning nomination and election of Trustees and members
of the Corporation by the respective bodies have been formulated as
follows :
1. By the Corporation: — August 11, 1931.
1) After considering various methods by which those engaged in in-
struction might be represented upon the Board of Trustees, it is
believed that the following action by the Corporation will be the best
means of insuring such representation :
1 The Corporation affirms its position that instruction in course
work is a fundamental part of the work of the Laboratory and
should be adequately represented upon the Board of Trustees."
2) ' That the Committee of the Corporation for nomination of
Trustees consist of five members, of whom not less than two shall
be non-Trustee members and not less than two shall be Trustee mem-
bers of the Corporation."
20 MARINE BIOLOGICAL LABORATORY
3) " That on or about July first of each year, the Clerk shall send a
circular letter to each member of the Corporation giving the names
of the Nominating Committee and stating that this committee desires
suggestions regarding nomination."
4) " That the Nominating Committee shall post the list of nomina-
tions at least one week in advance of the annual meeting of the
Corporation."
(Memo: The same committee also makes nominations annually for
Treasurer and Clerk of the Corporation.)
2. By the Trustees :— August 10, 1937.
" Proposals for membership in the Corporation shall be made to the
Nominating Committee on or before the first Tuesday of August
upon a regular form and endorsed by two members of the Corpora-
tion.
" With the recognition that rigid and completely standardized
requirements for membership in the Corporation of the Marine
Biological Laboratory are neither practicable nor desirable, it is
recommended that future members of the Corporation shall, in
general, be selected from among persons who, by engaging in active
research at the Marine Biological Laboratory during substantial
portions of at least two summers, shall have become acquainted with
the work, aims, and peculiar problems of the Laboratory, and who,
by papers published over a period of several years shall have demon-
strated a capacity for sustained scientific productiveness not less
than that required for full membership in such national societies as
the American Society of Zoologists, the Botanical Society of America,
and the American Physiological Society.
" It is further recommended that in doubtful or border-line cases
action on applications for membership shall be deferred until a time
when, in the opinion of the Nominating Committee then serving, the
status of the applicant has become entirely clear."
D. Administration
In the course of the years we have developed methods of adminis-
tration of the various service departments of the Laboratory that have
worked well. It should be the function of the Director and Assistant
Director to control the operation of such services.
Dr. Jacobs' greatly regretted resignation as Director raises very
directly the question of the higher administration. The first two Direc-
tors of the Laboratory served without salary, and the routine admin-
REPORT OF THE DIRECTOR 21
istration was performed by an Assistant Director on pay, at first part
time but later on full time. Then Dr. Jacobs performed the services
both of Director and Assistant Director on half time and half pay, and
the Business Manager became able with experience to take over many of
the duties formerly exercised by the Assistant Director. Though this
arrangement worked admirably for the period of its duration, experi-
ence showed that it is not reasonable to expect a man of the scientific
experience and reputation expected of the Director of this Laboratory
to endure indefinitely the limitations of scientific activity imposed by
such an arrangement. It seems probable that we cannot return to this
plan.
As soon as possible we should provide for a full-time resident
Director or Assistant Director. This would afford continuous super-
vision of the business of the Laboratory and in addition would permit
this officer to continue his research work under favorable conditions.
Such a resident scientist would attract other scientists during the portion
of the year when the Laboratory is little used and would thus help to
make it an all-year-round institution.
E. Research and Instruction
Research and instruction have been companion principles since the
foundation of the Laboratory as cited in the introduction to this report.
In the maintenance of research and instruction side by side throughout its
history, the Marine Biological Laboratory has been outstanding, if not
strictly unique. We have stood by the principle that it is the business
of the Laboratory to help to produce investigators as well as investiga-
tion ; and we believe that it can be shown that our courses of instruction
have contributed in an important way to this purpose, and, moreover,
that they have been an important factor in the improvement of biological
instruction and research throughout the country. Although there has
been some opinion among members of the Laboratory since the courses
ceased to be an important source of income that we would be better off
without courses, this opinion has never prevailed. We believe that our
problem is in the way of improvement, not elimination, of instruction.
The Laboratory has no program of its own in research, except as
defined in its name, and it therefore promotes no specific research proj-
ects as official undertakings. It operates entirely on the principle of
furnishing facilities to competent investigators, and to beginning in-
vestigators who are working under qualified direction. No biological
subjects are specifically excluded except such as are ruled out by lack of
facilities or suitable conditions, as in the case of pathogenic organisms
for example. This has been the rule from the foundation of the Labora-
22 MARINE BIOLOGICAL LABORATORY
tory, and the range of research has consequently steadily increased with
improvement of facilities. Changes of fashion have of course also
occurred, and are reflected in the annual reports.
The policy has been to interest the strongest biologists and promising
young investigators to bring their work to Woods Hole ; and the degree
of success of this policy has been the measure of success and influence
of the Laboratory. The future of the Laboratory depends upon the
continuance of this policy, and the elimination of conditions that tend
to restrict its operation, whether these are based on inadequacy of equip-
ment, administrative regulations, or community conditions. This is
the most important policy of the Laboratory, if one may be allowed to
rank essentials, for it ensures leadership and reputation. To supple-
ment this policy the attendance of as many promising young investiga-
tors as possible should be encouraged.
If the number of investigators admitted is to be definitely restricted,
and if the tendency towards an increase in numbers continues, it will
be necessary to adopt more definite policies concerning admission of in-
vestigators than in the past. These should not, however, be of too
binding a character, but rather a definition of principles within which the
Director will have free scope for the exercise of his best judgment.
The established fees for research accommodations should be con-
tinued, and paid by the institution represented as far as possible. When
this cannot be done it has been a frequent policy, more in the past than
at present, to waive fees for distinguished investigators. Such arrange-
ments have often been doubly blessed, in giving and in taking. The
cooperation by institutions in the expenses of investigation of their
representatives has been a strong stabilizing factor in the history of the
Laboratory in more ways than one. This plan has never been more
effective than at the present time, but it is important constantly to
cultivate it.
The Committee recommends the continuance of our historical policy
of maintaining courses of instruction. These should be contributory to
research, and based upon the advantages of marine material, so that they
are in no sense duplications of courses that may equally well be offered
by universities. Of such courses there are several kinds. As con-
tributory to research it is not meant that all necessarily lead directly to
research as a final preparatory step, but that they may sometimes fill
essential gaps in education for the kind of biological research intended by
the individual. Preference for admission to courses should be given to
students whose promise or declared intention indicates a professional
career in the field of biology. Such students should, and do, derive great
profit, not only from the actual instruction, but also from the scientific
contacts that they make at Woods Hole.
REPORT OF THE DIRECTOR 23
The Trustees should maintain control of courses to see that proper
content and principles of admission are preserved. The Executive
Committee has for some time held a conference with the heads of courses
each year with these purposes in mind.
Strict limitation of the numbers admitted to each course should be
observed in the future as in the past. It should also be a policy to
provide better and more stable laboratory accommodations.
F. Buildings, Equipmnt and Grounds
The first question is whether our holdings of real estate are adequate
for the future. This can be answered substantially in the affirmative.
We already have considerable undeveloped harbor frontage ; we now
own all the land on the block on which the original buildings of the
Laboratory stood ; in the block immediately north there is only one parcel
of land on Center Street not now in our possession ; and there is no
immediate reason for attempting to complete our ownership of the re-
mainder of the block. For residential purposes we still have unsold lots
in the Gansett tract, and no subdivision whatever has been made of the
100 acres in the Devil's Lane Tract.
The second question concerns the buildings. Here three main needs
present themselves.
In the first place, additional stack space for the library is needed.
At the present rate of growth the stacks will be fully occupied in very
few years. It is essential for the work of the Laboratory that this
growth should be continued. Additional space can be provided by a
wing to the east of the present library. It has been suggested that the
present reading room might be utilized for additional stack space and
the catalogue room be converted into a reading room with other neces-
sary readjustments ; other suggestions for temporary relief have also
been offered. But at most only a short postponement would be afforded
in such ways. The problem should be faced and estimates secured for
building additional stack space.
The second main need is to replace the present wooden buildings with
a fireproof building of solid construction. The work of the classes and
investigators in the wooden buildings is seriously hampered by vibration,
and the buildings do not lend themselves readily to modern installations.
These buildings range in age from forty to fifty years, and they con-
stitute a real fire hazard. This need should also receive the earnest
attention of the Trustees.
Additional space is also needed for various technical services neces-
sitated by the increasing complexity of important kinds of biological
research in recent years, and which are not adequately provided for at
24 MARINE BIOLOGICAL LABORATORY
present. Among these needs are those for space for autoclaves and
sterilizers, which must now be used in rooms occupied by investigators,
space for stills, which are now very disadvantageously housed in the
boiler room in the basement of the Brick Building, additional shop space,
particularly for use by investigators for relatively simple operations
which they can carry out themselves, additional space for housing small
animals, dehumidified and air-conditioned rooms, additional dark rooms,
etc. Doubtless most of these needs could be cared for on the lower
floors of the proposed addition for the Library. They ought, in any
event, not to be forgotten. Furthermore, since needs of this sort are
likely to increase in future years and are less predictable than the growth
of the library, ample reserve space should be provided for them.
Our waterfront should be improved by landscaping and other ways
so as to furnish a dignified frontage and water approach to the Labora-
tory. The George M. Gray Museum should have more adequate hous-
ing, and there are numerous other desirable small improvements that
should be undertaken as soon as possible.
It is becoming increasingly important that the Supply Department
be enabled to collect material for research from a wider area. To this
end there should be a larger motor boat, and it is highly desirable that
a resident naturalist be associated with the department who could study
ecological conditions from year to year with a view to establishing
sources of more abundant and more varied material for research. The
standing Committee in the Supply Department should be asked to
formulate the aims and policies of the Department.
G. Library
The Library Committee should be asked to formulate the aims and
policies of the library.
H. Apparatus
Similarly, the Apparatus Committee should be asked to formulate its
aims and policies.
/. Finances and Fiscal Policies
In 1932 the income from our endowment funds was $55,668, rep-
resenting a return of 5 per cent on book value. It is now approximately
$43,000, representing a return of 3.8 per cent on book value. The
decrease in yield has been due partly to the necessity of refunding opera-
tions at lower interest rates ; but the most drastic reductions in income
have been suffered on the mortgage participations, some of which have
been foreclosed, and others have had the interest rate much reduced.
REPORT OF THE DIRECTOR 25
The outstanding arrears of income amounted to $18,094 in 1935 but
were reduced to $12,775 at the end of 1937. For three years the
income has been supported by payment of arrears, a condition that
cannot continue indefinitely.
With a loss of annual income from endowment amounting to over
$12,000 there has been a considerable increase in attendance, which has
not been compensated for by increased fees for research space. The
cost of most apparatus and materials has recently risen appreciably and
is likely to rise still farther. It is also certain that the progress of
biological research will continually create new demands for special ap-
paratus and equipment which must be met if the Laboratory is to retain
its present position in scientific research. As a matter of fact, the budget
of the Laboratory has been kept balanced since the beginning of the de-
pression only by economies which have considerably handicapped the
work of many of our investigators. Furthermore, although necessary
upkeep has been maintained, certain desirable repairs to the buildings
and equipment have been postponed since the early years of the depres-
sion but cannot be deferred much longer. Among the more expensive
items that will require attention within the next few years are battery
replacement, a new heating system for the Brick Building, repairs of the
salt water system, painting and waterproofing of the brick buildings, etc.
Reserves should also be built up to cover further depreciation of the
buildings and equipment owned by the Laboratory, and to provide for
the retirement of the Howes mortgage and for future purchases of
property, etc. The problem of sewage disposal which may arise at any
time is also likely to involve very considerable expense.
It is clear that substantial increase of the endowment of the Labora-
tory is necessary if we are to aim to restore the income to its pre-
depression value, to provide adequately for the upkeep of the present
plant, for the establishment of necessary reserves, and to meet increas-
ing costs of operation.
As a partial offset to the loss of endowment income since 1931, the
dividends of the General Biological Supply House increased from $2,032
in 1931 to $12,700 in 1936. The income from fees of students and in-
vestigators cannot be increased much unless considerably higher rates
are established, which seems undesirable.
The Committee agrees that the most important fiscal policies to pur-
sue are first to increase endowment and second to establish cash reserves
for depreciation and contingencies. It is fair to point out in the latter
connection that cash reserves previously accumulated have been used for
purchase of real estate and that considerable sums have also gone each
year into capital improvements. The Committee recommends that
26 MARINE BIOLOGICAL LABORATORY
additional endowments secured be placed in the same trusts as the
present major endowment funds, or in another trust under the same
principles.
/. Community Arrangements and Responsibilities
As our community has grown and assumed a more settled character,
community needs have increased. The primitive needs of food and
lodging have from the beginning been recognized as an official respon-
sibility of the Laboratory ; the present arrangements for low-cost hous-
ing do not appear to be entirely adequate for a community of our size.
Their administration should be in the hands of the Business Manager
subject to control by the superior administrative officers. An advisory
committee is not recommended.
For those who desire to own their own homes, the Laboratory
possesses ample real estate in the Gansett and Devil's Lane tracts, sale-
able to members at reasonable rates and terms. The acquisition of these
tracts has aided to prevent unreasonable increase of price of village
properties.
These provisions should be regarded as terminating the direct and
exclusive official responsibility of the Laboratory for community pur-
poses. While the Laboratory should aid in securing recreational facili-
ties, the responsibility for operating them should be in the hands of the
community itself. This principle has operated well in the case of the
" M. B. L. Club " and the " M. B. L. Tennis Club." The Laboratory
has furnished land and buildings, and from time to time has made loans
for improvements, and it may yet appear desirable to provide an addition
to the building of the M. B. L. Club. But these organizations should
operate under their own membership and fees. With the acquisition of
the bathing beach the question arises whether the same principles could
not be made to operate there.
K. Summary of Principal Recommendations
1. That the Marine Biological Laboratory pursue a policy of consolida-
tion rather than expansion for the present.
2. That in pursuit of this policy steps be taken to provide the following
major improvements :
a. Secure additional funds for endowment.
/'. Provide additional stack room to accommodate approximately
100,000 additional volumes, together with study cubicles.
c. Replace present wooden laboratories by a building, or buildings,
of stable fireproof construction, providing an intermediate court
to set off the present main building.
REPORT OF THE DIRECTOR 27
d. In connection with the library construction provide adequate space
for expansion of various technical services as described in II F.
e. Make provision for the series of miscellaneous needs enumerated
in the body of the report.
3. Maintain the principles of cooperation (II B.), organization and gov-
ernment (II C.), administration (II D.), research and instruction,
(II E.), that have served so well in the past, as the basis for future
development.
4. Additional endowment funds as received should be placed, like the
present main endowment funds, in trust. Reserves for depreciation,
contingencies, improvements and retirement fund should be set up
out of income (II I.).
5. Responsibility for recreational facilities should be placed as far as
possible on voluntary organizations within our scientific community
(II J.).
2. THE STAFF, 1938
CHARLES PACKARD, Associate Director, Assistant Professor of Zoology,
Institute of Cancer Research, Columbia University.
ZOOLOGY
I. INVESTIGATION
GARY N. CALKINS, Professor of Protozoology, Columbia University.
E. G. CONKLIN, Professor of Zoology, Princeton University.
CASWELL GRAVE, Professor of Zoology, Washington University.
H. S. JENNINGS, Professor of Zoology, Johns Hopkins University.
FRANK R. LILLIE, Professor of Embryology Emeritus, The University of
Chicago.
C. E. McCujNG, Professor of Zoology, University of Pennsylvania.
S. O. MAST, Professor of Zoology, Johns Hopkins University.
T. H. MORGAN, Director of the Biological Laboratory, California Institute
of Technology.
G. H. PARKER, Professor of Zoology Emeritus, Harvard University.
E. B. WILSON, Professor of Zoology Emeritus, Columbia University.
LORANDE L. WOODRUFF, Professor of Protozoology, Yale University.
II. INSTRUCTION
T. H. BISSONNETTE, Professor of Biology, Trinity College.
P. S. CROWELL, JR., Instructor in Zoology, Miami University.
C. E. HADLEY, Associate Professor of Biology, New Jersey State Teachers
College at Montclair.
F. R. KILLE, Assistant Professor of Zoology, Swarthmore College.
A. M. LUCAS, Associate Professor of Zoology, Iowa State College.
S. A. MATTHEWS, Assistant Professor of Biology, Williams College.
A. J. WATERMAN, Assistant Professor of Biology, Williams College.
28 MARINE BIOLOGICAL LABORATORY
JUNIOR INSTRUCTORS
W. F. HAHNERT, Associate Professor of Zoology, Ohio Wesleyan Univer-
versity.
J. S. RANKIN, Teaching Fellow in Zoology, Amherst College.
PROTOZOOLOGY
I. INVESTIGATION
(See Zoology)
II. INSTRUCTION
GARY N. CALKINS, Professor of Protozoology, Columbia University.
G. W. KIDDER, Assistant Professor of Biology, Brown University.
ELIZABETH DRUMTRA HUGHES, Lecturer in Zoology, Barnard College.
EMBRYOLOGY
I. INVESTIGATION
(See Zoology)
II. INSTRUCTION
W. W. BALLARD, Assistant Professor of Biology and Anatomy, Dartmouth
College.
HUBERT B. GOODRICH, Professor of Biology, Wesleyan University.
VIKTOR HAMBURGER, Assistant Professor of Zoology, Washington Univer-
sity.
OSCAR SCHOTTE, Assistant Professor of Biology, Amherst College.
DOUGLAS M. WHITAKER, Professor of Zoology, Stanford University.
PHYSIOLOGY
I. INVESTIGATION
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.
M. H. JACOBS, Professor of General Physiology, University of Pennsylvania.
RALPH S. LILLIE, Professor of General Physiology, The University of
Chicago.
ALBERT P. MATHEWS, Professor of Biochemistry, University of Cincinnati.
II. INSTRUCTION
Teaching Staff
LAURENCE IRVING, Professor of Biology, Swarthmore College.
ROBERT CHAMBERS, Professor of Biology, New York University.
REPORT OF THE DIRECTOR 29
J. K. W. FERGUSON, Assistant Professor of Physiology, Ohio State Univer-
sity.
KENNETH C. FISHER, Assistant Professor of Experimental Biology, Uni-
versity of Toronto.
C. LADD PROSSER, Assistant Professor of Physiology, Clark University.
CARL F. SCHMIDT, Professor of Pharmacology, University of Pennsylvania.
F. J. M. SICHEL, Instructor in Physiology, University of Vermont, College
of Medicine.
BOTANY
I. INVESTIGATION
C. E. ALLEN, Professor of Botany, University of Wisconsin.
S. C. BROOKS, Professor of Zoology, University of California.
B. M. DUGGAR, Professor of Physiological and Economic Botany, University
of Wisconsin.
IVEY F. LEWIS, Professor of Biology, University of Virginia.
WM. J. ROBBINS, Professor of Botany, University of Missouri.
II. INSTRUCTION
WILLIAM RANDOLPH TAYLOR, Professor of Botany, University of Michigan.
FRANCIS DROUET, Research Fellow, Yale University.
B. F. D. RUNK, Research Fellow, University of Virginia.
GENERAL OFFICE
F. M. MACNAUGHT, Business Manager.
POLLY L. CROWELL, Assistant.
EDITH BILLINGS, Secretary.
RESEARCH SERVICE AND GENERAL MAINTENANCE
SAMUEL E. POND, Technical Mgr. LESTER F. Boss, Technician.
G. FAILLA, X-ray Physicist. J. D. GRAHAM, Glassblower.
T. E. LARKIN, Superintendent. J. T. SIMONTON, Assistant.
W. C. HEMENWAY, Carpenter. ELBERT P. LITTLE, X-ray.
LIBRARY
PRISCILLA B. MONTGOMERY (Mrs. Thomas H. Montgomery, Jr.), Librarian.
DEBORAH LAWRENCE, Secretary.
MARY A. ROHAN, S. MABELL THOMBS, Assistants.
SUPPLY DEPARTMENT
JAMES MC!NNIS, Manager. GEOFFREY LEHY, Collector.
MILTON B. GRAY, Collector. WALTER KAHLER, Collector.
A. M. HILTON, Collector. F. N. WHITMAN, Collector.
A. W. LEATHERS, Shipping Dept. RUTH S. CROWELL, Secretary.
GRACE HARMAN, Secretary.
30 MARINE BIOLOGICAL LABORATORY
THE GEORGE M. GRAY MUSEUM
GEORGE M. GRAY, Curator Emeritus.
3. INVESTIGATORS AND STUDENTS, 1938
Independent Investigators
ABRAMOWITZ, A. A., Research Assistant, Harvard University.
ADAMS, MARK H., Fellowship in Pneumonia Research, Rockefeller Institute for
Medical Research.
AMBERSON, WILLIAM R., Professor of Physiology, University of Maryland, School
of Medicine.
ANDERSON, R. L., Professor of Biology, Johnson C. Smith University.
ANDERSON, RUBERT S., Research Associate, Princeton University.
ANGERER, C. A., Instructor, University of Pennsylvania.
APPEL, FREDERICK W., Associate Professor of Biology, St. John's College.
ARMSTRONG, PHILIP B., Professor of Anatomy, University of Alabama, School of
Medicine.
BALLARD, WILLIAM W., Assistant Professor in Zoology and Anatomy, Dartmouth
College.
EARTH, LESTER G., Assistant Professor of Zoology, Columbia University.
BECK, LYLE V., Research Fellow, University of Pennsylvania, School of Medicine.
BEDICHEK, SARAH, Associate Professor of Biology, North Texas Agricultural
College.
BERNSTEIN, FELIX, Professor of Biometrics, New York University, College of
Medicine.
BERTALANFFY, LUDWIG VON, Privatdozent an der Universitat Wien, Wien, Germany.
BISSONNETTE, T. H., Professor and Head of Biology Department, Trinity College.
BLACK, LINDSAY MACLEOD, Assistant, Rockefeller Institute for Medical Research.
BOCHE, ROBERT D., Research Assistant, Department of Embryology, Carnegie
Institution of Washington.
BODIAN, DAVID, National Research Council Fellow in Medicine, University of
Michigan.
BOETTIGER, EDWARD G., Graduate Student, Harvard University.
BOERNSTEIN, WALTER, Honorary Research Fellow, Yale University, School of
Medicine.
BOTSFORD, E. FRANCES, Assistant Professor of Zoology, Connecticut College.
BOZLER, EMIL, Assistant Professor of Physiology, Ohio State University.
BRADLEY, HAROLD C., Professor of Physiological Chemistry, University of Wis-
consin.
BRAMBEL, CHARLES E., Instructor in Zoology, Johns Hopkins University.
BRONFENBRENNER, J. J., Professor of Bacteriology and Immunology, Washington
University, School of Medicine.
BUCK, JOHN B., Research Assistant, Department of Embryology, Carnegie Insti-
tution of Washington.
BUDINGTON, ROBERT A., Professor of Zoology, Oberlin College.
BURTON, ALAN C., Fellow in Medical Physics, Johnson Foundation, University
of Pennsylvania.
CABLE, RAYMOND M., Assistant Professor of Parasitology, Purdue University.
CALKINS, GARY N., Professor of Protozoology, Columbia University.
CAROTHERS, E. ELEANOR, Research Associate, University of Iowa.
CARPENTER, RUSSELL L., Assistant Professor of Anatomy, College of Physicians
and Surgeons, Columbia University.
CHAMBERS, ROBERT, Research Professor of Biology, Washington Square College,
New York University.
REPORT OF THE DIRECTOR 31
CHENEY, RALPH H., Chairman of Biology Department, Professor of Biology, Long
Island University.
CLAFF, C. LLOYD, 5 Van Beal Road, Randolph, Massachusetts.
CLARK, ELEANOR L., Department of Anatomy, University of Pennsylvania, School
of Medicine.
CLARK, ELIOT R., Professor of Anatomy, University of Pennsylvania, School of
Medicine.
CLAUDE, ALBERT, Associate, Rockefeller Institute for Medical Research.
CLOWES, G. H. A., Director of Research, Lilly Research Laboratories.
COLE, ELBERT C., Professor of Biology, Williams College.
COLE, KENNETH S., Associate Professor of Physiology, College of Physicians and
Surgeons, Columbia University.
COLWIN, ARTHUR L., Research Fellow, Osborn Zoological Laboratory, Yale
University.
COMMONER, BARRY, Graduate Student, Harvard University.
CONKLIN, EDWIN G., Professor Emeritus of Biology, Princeton University.
COOPER, KENNETH W., Lydig Fellow, Columbia University.
COPELAND, D. EUGENE, Assistant, Harvard University.
COPELAND, MANTON, Professor of Biology, Bowdoin College.
CORSON, SAMUEL A., Instructor, Cell Physiology, Division of General Education,
Washington Square College, New York University.
COSTELLO, DONALD P., Assistant Professor of Zoology, University of North
Carolina.
COWLES, RHEINART P., Professor of Zoology, Johns Hopkins University.
Cox, EDWARD H., Professor of Chemistry, Swarthmore College.
CROASDALE, HANNAH T., Technical Assistant in Zoology, Dartmouth College.
CROUSE, HELEN V., Research Assistant, Carnegie Institution of Washington.
CROWELL, PRINCE S., JR., Instructor in Zoology, Miami University.
CURTIS, W. C., Professor of Zoology, University of Missouri.
DENNY, MARTHA, Instructor, Connecticut College.
DILLER, IRENE COREY, Research Associate in Zoology, University of Pennsylvania.
DILLER, WILLIAM F., Assistant Professor of Zoology, Dartmouth College.
DROUET, FRANCIS, Theresa Seessel Research Fellow, Yale University.
DURYEE, WILLIAM R., Research Associate in Biology, Washington Square College,
New York University.
ELFTMAN, HERBERT, Assistant Professor of Zoology, Columbia University.
ELWYN, ADOLPH, Associate Professor of Neurology, College of Physicians and
Surgeons, Columbia University.
FAILLA, G., Physicist, Memorial Hospital, New York City.
FENNELL, RICHARD A., Instructor in Zoology, Michigan State College.
FERGUSON, J. K. W., Assistant Professor of Physiology, Ohio State University.
FISHER, KENNETH C., Assistant Professor of Experimental Biology, University of
Toronto.
FLORKIN, MARCEL, Professor of Biochemistry, University of Liege, Belgium.
FORBES, HENRY S., Associate in Neuropathology. Harvard Medical School.
FRIES, E. F., Assistant Professor, College of the City of New York.
FRISCH, JOHN A., Professor of Biology, Canisius College.
FRY, HENRY J., Visiting Investigator, Cornell University Medical College.
FURTH, JACOB, Assistant Professor of Pathology, Cornell University Medical
College.
CARREY, WALTER E., Professor of Physiology, Vanderbilt University, School of
Medicine.
GELDARD, FRANK A., Professor of Psychology, University of Virginia.
GILMAN, MR. LAUREN C., Laboratory Instructor in Biology, Johns Hopkins
University.
GLASER, OTTO, Professor of Biology, Amherst College.
MARINE BIOLOGICAL LABORATORY
GOODRICH, H. B., Professor of Biology, Wesleyan University.
GORDON-KONIGES, HELMUT, Fellow, Rockefeller Foundation.
GRABAR, PIERRE, Fellow, Rockefeller Foundation, Chef de Laboratoire a 1'Institut
Pasteur, Paris, France.
GRANT, RONALD, Lecturer in Physiology, McGill University.
GRAVE, CASWELL, Professor of Zoology, Washington University.
GRAY, PETER, Lecturer in Vertebrate Embryology, Edinburgh University.
GUTHRIE, MARY J., Associate Professor of Zoology, University of Missouri.
HADLEY, CHARLES E., Associate Professor of Biology, Montclair State Teachers'
College.
HAHNERT, WILLIAM F., Associate Professor of Zoology, Ohio Wesleyan Univer-
sity.
HAMBURGER, VIKTOR, Assistant Professor, Washington University.
HARRIS, DANIEL L., Instructor, University of Pennsylvania.
HARROLD, C. M., Graduate Assistant, New York University.
HARTMAN, FRANK A., Chairman and Professor of Physiology, Ohio State Uni-
versity.
HARVEY, ETHEL BROWNE, Investigator, Princeton University.
HARVEY, E. NEWTON, Professor of Physiology, Princeton University.
HEILBRUNN, L. V., Associate Professor of Zoology, University of Pennsylvania.
HENSHAW, PAUL S., Biophysicist, Memorial Hospital, New York City.
HETZER, H. O., Associate Animal Husbandman, United States Department of
Agriculture, Washington, D. C.
HICKSON, ANNA KELTCH, Research Chemist, Lilly Research Laboratories.
HIESTAND, WILLIAM A., Associate Professor of Physiology, Purdue University.
HILL, EDGAR S., Instructor in Biochemistry, Washington University.
HILL, SAMUEL E., Assistant in General Physiology, Rockefeller Institute for
Medical Research.
HODGE, CHARLES, 4th, Assistant Professor, Temple University.
HODGKIN, ALAN L., Demonstrator in Physiology, Cambridge, England.
HOPKINS, DWIGHT L., Research Assistant, Johns Hopkins University.
HOWE, H. E., Editor, Industrial and Engineering Chemistry, Washington, D. C.
HUGHES, ELIZABETH DRUMTRA, Lecturer in Zoology, Barnard College.
HUGHES, ROSCOE D., Assistant in Zoology, Columbia University.
HUNNINEN, ARNE V., Professor of Biology, Oklahoma City University.
HUNTER, GEORGE W., Ill, Assistant Professor of Biology, Wesleyan University.
HUNTER, LAURA N., Assistant Professor, Pennsylvania College for Women.
HUTCHINGS, Lois M., Teacher of Biology, Weequahic High School, Newark, N. J.
IRVING, LAURENCE, Professor of Biology, Swarthmore College.
JACOBS, M. H., Professor of General Physiology, University of Pennsylvania.
JEFFERS, KATHARINE R., Instructor in Zoology, Duke University.
JENKINS, GEORGE B., Professor of Anatomy, George Washington University.
JOHLIN, J. M., Associate Professor of Biochemistry, Vanderbilt University, School
of Medicine.
JONES, E. RUFFIN, JR., Associate Professor, College of William and Mary.
JONES, RUTH McCLUNG, Instructor in Botany and Zoology, Swarthmore College.
KARADY, STEPHEN, Assistant Professor, Internal Clinic of the Francis Joseph
University, Hungary.
KIDDER, GEORGE W., Assistant Professor of Biology, Brown University.
KIESE, MANFRED, Rockefeller Fellow, Assistant, Pharmacological Institute of the
University of Berlin.
KILLE, FRANK R., Assistant Professor of Zoology, Swarthmore College.
KINDRED, J. E., Associate Professor of Histology and Embryology, University
of Virginia.
KNOWLTON, FRANK P., Professor of Physiology, Syracuse University, College of
Medicine.
KOPAC, M. J., Research Associate, Washington Square College, New York
University.
REPORT OF THE DIRECTOR
KORR, IRVIN M., Instructor in Physiology, New York University, College of
Medicine.
KRAHL, M. E., Research Chemist, Lilly Research Laboratories.
KREEZER, GEORGE, Assistant Professor of Psychology, Cornell University.
KRIEG, WENDELL J. S., Instructor in Anatomy, New York University, College of
Medicine.
KUNITZ, MOSES, Associate, Rockefeller Institute for Medical Research.
LANCEFIELD, DONALD E., Associate Professor in Biology, Queens College.
LEVY, MILTON, Assistant Professor in Chemistry, New York University, College
of Medicine.
LIEBMANN, EMIL, Fisheries Service of the British Government in the Near East.
LILLIE, FRANK R., Professor of Embryology, Emeritus, The University of Chicago.
LILLIE, RALPH S., Professor of General Physiology, The University of Chicago.
LOEB, LEO, Professor Emeritus of Pathology, Washington University, School of
Medicine.
LUCAS, ALFRED M., Associate Professor of Zoology, Iowa State College.
LUCAS, MIRIAM SCOTT, Iowa State College.
LUDWIG, DANIEL, Associate Professor of Biology, New York University.
LYNN, W. GARDNER, Instructor, Johns Hopkins University.
McCANN, LEWIS P., Graduate Assistant, University of Maryland.
McCLUNG, C. E., Director Zoological Laboratory, University of Pennsylvania.
McCuRDY, MARY DERRICKSON, Graduate Student, Duke University.
McCuRDY, HAROLD G., Research Assistant, Duke University.
MACDOUGALL, MARY STUART, Head of Biology Department, Agnes Scott College.
McFARLAND, ELSIE LAITY, Instructor in Zoology, Wheaton College.
MACLENNAN, RONALD F., Associate Professor of Zoology, State College of
Washington.
MAGRUDER, SAMUEL R., Assistant in Anatomy, Cornell University Medical College.
MALOEUF, N. S. ROYSTON, Honorary Research Fellow, Yale University.
MARTIN, W. E., Assistant Professor of Zoology, DePauw University.
MAST, S. O., Professor of Zoology in Charge of General Physiology, Johns
Hopkins University.
MATHEWS, ALBERT P., Andrew Carnegie Professor of Biochemistry, University
of Cincinnati.
MATTHEWS, SAMUEL A., Assistant Professor of Biology, Williams College.
MAYOR, JAMES W., Professor of Biology, Union College.
MILLER, JAMES A., Instructor in Anatomy, University of Michigan.
MOLTER, JOHN A., Instructor, University of Notre Dame.
MORGAN, LILIAN V., Pasadena, California.
MORGAN, T. H., Professor of Biology, California Institute of Technology.
MORRILL, CHARLES V., Associate Professor of Anatomy, Cornell University
Medical College.
MULLER, H. J., Institute of Animal Genetics, University of Edinburgh.
NAVEZ, ALBERT E., Instructor in Biology, Milton Academy.
NEWTON, WILLIAM H., Reader in Physiology, Institute of Physiology, University
College, London, England.
NONIDEZ, JOSE F., Professor of Anatomy, Cornell University Medical College.
NORTHROP, JOHN H., Member, Rockefeller Institute for Medical Research.
OBRESHKOVE, VASIL, Professor of Biology, Bard College, Columbia University.
O'BRIEN, JOHN P., Johns Hopkins University.
OLSON, MAGNUS, Instructor in Zoology, University of Minnesota.
ORR, PAUL R., Assistant Professor, Brooklyn College.
OSTER, ROBERT H., Assistant Professor, University of Maryland, School of
Medicine.
OSTERHOUT, W. J. V., Member, Rockefeller Institute for Medical Research.
PACKARD, CHARLES, Assistant Professor of Zoology, Institute of Cancer Research,
Columbia University.
34 MARINE BIOLOGICAL LABORATORY
PARKER, G. H., Professor of Zoology Emeritus, Harvard University.
PARMENTER, CHARLES L., Associate Professor, University of Pennsylvania.
PARPART, ARTHUR K., Associate Professor, Princeton University.
PATRICK, RUTH, Associate Curator of Department of Microscopy, Academy of
the Natural Sciences of Philadelphia.
PIERCE, MADELENE, Vassar College.
PIERSON, BERNICE F., Graduate Student, Johns Hopkins University.
PIPKIN, C. A., University of Texas.
PLOUGH, HAROLD H., Professor of Biology, Amherst College.
POLLISTER, ARTHUR W., Assistant Professor of Zoology, Columbia University.
POND, SAMUEL E., Technical Manager, Marine Biological Laboratory.
PROSSER, C. LADD, Assistant Professor of Physiology, Clark University.
RABINOWITCH, E., Research Associate, University College, London, England.
RANKIN, JOHN S., JR., Teaching Fellow in Biology, Amherst College.
ROOT, RAYMOND W., Assistant Professor of Biology, College of the City of New
York.
Rous, PEYTON, Member in Pathology and Bacteriology, Rockefeller Institute for
Medical Research.
RUGH, ROBERTS, Instructor in Zoology, Hunter College.
RUNK, B. F. D., Research Fellow, University of Virginia.
RUSSELL, ALICE MARY, Instructor in Zoology, University of Pennsylvania.
SABIN, ALBERT B., Associate, Pathology and Bacteriology, Rockefeller Institute
for Medical Research.
SANDOW, ALEXANDER, Assistant Professor of Biology, Washington Square College,
New York University.
SASLOW, GEORGE, Instructor in Physiology, Harvard School of Public Health.
SAYLES, LEONARD P., Assistant Professor of Biology, College of the City of New
York.
SCHAEFFER, ASA A., Professor of Biology, Temple University.
SCHECHTER, VICTOR, Instructor, College of the City of New York.
SCHMIDT, CARL F., Professor of Pharmacology, University of Pennsylvania.
SCHMIDT, IDA GENTHER, Assistant Professor of Anatomy, University of Cincin-
nati, College of Medicine.
SCHMIDT, L. H., Research Fellow, Christ Hospital and University of Cincinnati,
College of Medicine.
SCHOTTE, OSCAR E., Associate Professor of Biology, Amherst College.
SCOTT, ALLAN C., Assistant Professor of Biology, Union College.
SCOTT, SISTER FLORENCE MARIE, Professor of Zoology, Seton Hill College.
SHAW, MYRTLE, Senior Bacteriologist, New York State Department of Health.
SICHEL, ELSA KEIL, Assistant Professor of Zoology, Rutgers University.
SICHEL, F. J. M., Instructor in Physiology, University of Vermont, College of
Medicine.
SLIFER, ELEANOR H., Assistant Professor, State University of Iowa.
SMITH, DIETRICH C., Associate Professor of Physiology, University of Maryland,
School of Medicine.
SMITH, JAY A., Instructor in Biology, Johns Hopkins University.
SMITH, MARSHALL E., Student, Johns Hopkins University, Medical School.
SOLBERG, ARCHIE N., Instructor in Biology, University of Toledo.
SOUTHWICK, MILDRED D., Ecologist, Department of Botany, Vassar College.
SPEIDEL, CARL C., Professor of Anatomy, University of Virginia, Medical School.
STANLEY, W. M., Associate Member, Rockefeller Institute for Medical Research.
STANNARD, J. NEWELL, Instructor in Physiology, University of Rochester, Medical
School.
STEINBACH, H. BURR, Assistant Professor of Zoology, Columbia University.
STEVEN, DAVID M., Magdalen College, Oxford, England.
STEINHARDT, JACINTO, Research Fellow, Rockefeller Foundation, Harvard Medi-
cal School.
REPORT OF THE DIRECTOR
STOCKARD, CHARLES R., Professor of Anatomy, Cornell University Medical College.
STOKEY, ALMA G., Professor of Botany, Mount Holyoke College.
TAYLOR, WM. RANDOLPH, Professor of Botany, University of Michigan.
THORNTON, CHARLES S., Assistant Professor of Biology, Kenyon College.
TOWN SEND, GRACE, Professor, Great Falls Normal College.
TROMBETTA, VIVIAN V., Assistant in Botany, Barnard College, Columbia University.
TURNER, C. L., Professor of Zoology, Northwestern University.
TURNER, JOHN P., Assistant Professor of Zoology, University of Minnesota.
UHLENHUTH, EDUARD, Professor of Anatomy, University of Maryland, School of
Medicine.
VANDEBROEK, GEORGES, Assistant in the Laboratory of Embryology and Histology,
Faculty of Medicine, University of Ghent, Belgium.
VICARI, EMELIA M., Associate in Anatomy, Cornell University Medical College.
VISSCHER, J. PAUL, Professor of Biology, Western Reserve University.
WALZL, EDWARD M., Instructor, Johns Hopkins University, School of Medicine.
WATERMAN, A. J., Assistant Professor of Biology, Williams College.
WEISS, PAUL, Associate Professor, The University of Chicago.
WENRICH, D. H., Professor of Zoology, University of Pennsylvania.
WHITAKER, D. M., Professor of Biology, Stanford University.
WHITE, MICHAEL J. D., Lecturer in Zoology, University College, London, England.
WHITE, THOMAS N., JR., Assistant Biophysicist, United States Public Health
Service, National Institute of Health.
WHITING, ANNA R., Guest Research Investigator, University of Pennsylvania.
WHITING, P. W., Associate Professor of Zoology, University of Pennsylvania.
WICHTERMAN, RALPH, Instructor, Temple University.
WIEMAN, H. L., Professor of Zoology, University of Cincinnati.
WIERSMA, CORNELIS A. G., Associate Professor of Physiology, California Institute
of Technology.
WILHELMI, RAYMOND W., Graduate Assistant, New York University.
WILLEY, CHARLES H., Assistant Professor of Biology, New York University.
WILLIER, BENJAMIN H., Chairman, Division of Biological Sciences, University
of Rochester.
WILSON, EDMUND B., Professor Emeritus in Residence, Columbia University.
WOLF, E. ALFRED, Associate Professor of Biology, University of Pittsburgh.
WOLF, OPAL M., Assistant Professor of Biology, Goucher College.
WOODRUFF, L. L., Professor of Protozoology, Yale University.
YANCEY, P. H., Chairman, Department of Biology, Spring Hill College.
YOUNG, ROGER A., Graduate Student, University of Pennsylvania.
Beginning Investigators
ALGIRE, GLENN H., Weaver Research Fellow in Anatomy, University of Maryland.
ARENA, JULIO F. DE LA, Auxiliary Professor of Biology, Universidad de la Habana.
BALLENTINE, ROBERT, Graduate Student, Princeton University.
BELCHER, JANE C., Graduate Assistant in Zoology, University of Missouri.
BELDA, WALTER H., Graduate Student in Zoology, Johns Hopkins University.
BISHOP, DAVID W., Instructor, University of Pennsylvania.
BLISS, ALFRED F., Laboratory Assistant, Department of Biophysics, Columbia
University.
BRILL, EDMUND R., Graduate Student in Biology, Harvard University.
CASTLE, RUTH M., Assistant in Zoology, Vassar College.
CHURNEY, LEON, Instructor in Zoology, University of Pennsylvania.
COOPER, RUTH SNYDER, Assistant in Zoology, Columbia University.
CORNMAN, IVOR, Teaching Fellow, Washington Square College, New York Uni-
versity.
CROWELL, VILLA BAILEY, Miami University.
36 MARINE BIOLOGICAL LABORATORY
DONNELLON, J. A., Graduate Student, University of Pennsylvania.
FERGUSON, FREDERICK P., Undergraduate Assistant, Wesleyan University.
FRANK, JOHN A., Medical Student, Yale University.
GLANCY, ETHEL, Teaching Fellow, Washington Square College, New York Uni-
versity.
GOLDIN, ABRAHAM, Graduate Student, Columbia University.
GRAVE, CASWELL, II, Assistant, Washington University.
GUTTMAN, RITA, Graduate Student in Physiology, College of Physicians and
Surgeons, Columbia University.
HALL, THOMAS S., Graduate Student, Yale University.
HIATT, EDWIN P., Research Fellow, University of Maryland, School of Medicine.
HINCHEY, M. CATHERINE, Graduate Student, University of Pennsylvania.
HOBSON, LAWRENCE B., Graduate Assistant in Zoology, University of Cincinnati.
HOLLINGSWORTH, JOSEPHINE, Graduate Student, University of Pennsylvania.
HUTCHINS, Louis W., Graduate Student, Yale University.
KRIETE, BERTRAND C., Graduate Assistant in Zoology, University of Cincinnati.
LAMBERT, BARBARA, Graduate Assistant in Physiology, Mount Holyoke College.
LEVENSON, ALFRED S., Graduate Student, University of Pittsburgh.
LIPMAN, HARRY J., Graduate Assistant, University of Pittsburgh.
LUDWIG, FRANCIS W., Graduate Student, University of Pennsylvania.
MAYO, MERCEDES, Assistant Professor of Biology, Universidad de la Habana.
MOORE, ANNA BETTY CLARK, Graduate Student, Columbia University.
MOORE, JOHN A., Assistant in Zoology, Columbia University.
MULLINS, LORIN J., Graduate Student, University of California.
RAMSEY, HELEN J., Purdue University.
RAY, D. T., Assistant Professor of Biology, Johnson C. Smith University.
ROSE, S. MERYL, Assistant in Zoology, Columbia University.
RYAN, FRANCIS J., Assistant in Zoology, Columbia University.
SCHENTHAL, JOSEPH E., Weaver Fellow in Anatomy, University of Maryland,
School of Medicine.
SCHOENBORN, HENRY W., Graduate Assistant, New York University.
SCHOEPFLE, G. M., Research Assistant, Princeton University.
SHAVER, JOHN R., Museum Assistant, University of Pennsylvania.
SILBER, ROBERT H., Assistant and Graduate Student, Washington University.
SINGER, MARCUS, Student Worker, University of Pittsburgh.
SMITH, AUDREY U., Assistant in Physiology, Vassar College.
STEWART, BROOKS, Graduate Student, University of Pennsylvania.
VON DACH, HERMAN, Assistant, Ohio State University.
WEINBERG, VICTOR, The University of Chicago.
WHITE, ELIZABETH C., Student, University of Pennsylvania.
WILBUR, KARL M., Harrison Fellow, University of Pennsylvania.
WISE, JOHN S., Medical Student, University of Pennsylvania, School of Medicine.
ZWILLING, EDGAR, Assistant, Columbia University.
Research Assistants
ALLEY, ARMINE, Research Assistant, McGill University.
ANDERSON, KATHERINE, Research Technician, Vanderbilt University.
ARMSTRONG, CHARLES W. J., Demonstrator in Biology, University of Toronto.
ARMSTRONG, LOUISE S., Research Assistant, University of Alabama.
AURINGER, JACK, Research Assistant, Columbia University.
BAKER, LINVILLE A., Lilly Research Laboratories.
BECK, NAOMI, Graduate Student, The University of Chicago.
BENDER, JOSEPH C., Research Assistant, Swarthmore College.
BERNSTEIN, MARIANNE, 325 E. 41st Street, New York City.
BERTALANFFY, MARIA M. VON, Universitat Wien, Wien, Germany.
REPORT OF THE DIRECTOR 37
BIEN, BETTINA H., Wheaton College.
BIRNBAUM, SANFORD M., University Scholar, University of Cincinnati.
BIRNBAUM, WILLIAM F., Research Assistant, New York University, College of
Medicine. )
BLACK, EDGAR C, Research Associate, Swarthmore College.
BOWEN, WILLIAM J., Bruce Fellow, Johns Hopkins University.
BROVVNELL, KATHARINE A., Research Assistant, Ohio State University.
BURNETT, JACK M., Graduate Student, Washington University.
CECIL, SAM, Assistant, Vanderbilt University, School of Medicine.
CHAMBERS, EDWARD L., Research Assistant, New York University.
COHEN, IRVING, Memorial Hospital, New York City.
COSTELLO, HELEN MILLER, University of North Carolina.
CRAWFORD, JOHN D., Milton Academy, Milton, Massachusetts.
CURTIS, HOWARD J., Associate in Physiology, College of Physicians and Surgeons,
Columbia University.
DIENES, PRISCILLA, 27 Walker Street, Cambridge, Massachusetts.
DOWDING, GRACE L., Research Technician, University of Maryland, School of
Medicine.
DOWNS, J. HUNTER, Undergraduate, Colgate University.
DUGAL, LOUIS-PAUL, Instructor in Biology, University of Montreal.
DUMM, MARY E., 13 Sampson Avenue, Madison, New Jersey.
DZIEMIAN, ARTHUR J., Graduate Student, Princeton University.
EVANS, HIRAM J., Assistant in Biology, Williams College.
FINK, HAROLD K., Student, Princeton University.
FINKEL, ASHER J., Research Assistant, The University of Chicago.
FOSTER, RICHARD, Milton Academy, Milton, Massachusetts.
Fox, ERNEST L., Research Assistant, Miami University.
FUNKHOUSER, ELIZABETH M. J., Swarthmore College.
GETTEMANZ, JOHN F., Laboratory Assistant, Rockefeller Institute for Medical
Research.
GRAND, C. G., Research Associate, Washington Square College, New York
University.
HAMDI, TURGUT N., University of Pennsylvania.
HATCH, CLEORA, Technician, Cornell University Medical College.
HORN, EDWARD C., Assistant, Trinity College.
HOWELL, CHARLES D., Professor of Biology, Elizabethtown College.
HUTCHENS, JOHN, Lilly Research Laboratories.
KEEFE, EUGENE L., Research Assistant, Washington University.
KEMP, EMILY J., Instructor in Physiology, University of Maryland, School of
Medicine.
LEVIN, Louis, Student, University of Cincinnati, College of Medicine.
LEWIS, LENA, Research Assistant in Physiology, Ohio State University.
LINSCHEID, MARTHA, Research Assistant, Western Reserve University.
LYON, RHEA C., Research Technician, University of Maryland, School of Medicine.
MCDONALD, MARGARET RITCHIE, Senior Technician, Rockefeller Institute for Medi-
cal Research.
MARTIN, MARY S., University of Rochester, School of Medicine.
MARTIN, ROSEMARY D. C., Assistant, University of Toronto.
MELLAND, AMICIA M., Research Worker, Carnegie Institution of Washington.
MILFORD, JOHN J., Student, New York University.
MUSSER, RUTH E., Student, Goucher College.
NAUMANN, RUDOLPH V., Fellow in Physiology, New York University, College of
Medicine.
NETSKY, MARTIN, Research Assistant, University of Pennsylvania.
NORRIS, CHARLES H., Graduate Student, Princeton University.
OSBORN, CLINTON M., Research Fellow, Harvard University.
38 MARINE BIOLOGICAL LABORATORY
PRATT, DAVID M., Student, Williams College.
RAWLES, MARY E., Research Assistant, University of Rochester.
SAFFORD, VIRGINIA, Assistant, Swarthmore College.
SALZBURG, FREDERICK P., Research Assistant, University of Minnesota.
SAWYER, ELIZABETH L., Associate Professor of Biology, Converse College.
SCROLL, SAMUEL M., Research Assistant, University of Toledo.
SELVERSTONE, BERTRAM, Student, Harvard Medical School.
SIMMONS, ERIC L., Research Assistant, Swarthmore College.
SISSON, WARREN R., JR., Assistant, Milton Academy.
SMITH, CARL C, Iglauer Fellow in Biochemistry, University of Cincinnati.
SNEIDER, ELIZABETH, Arnold Biological Fellow, Brown University.
SPENCER, JOSEPH M., Research Assistant, College of Physicians and Surgeons,
Columbia University.
STENGER, ALBERT H., Technician, New York University.
STOCKER, GAIL, Research Assistant, University of Pennsylvania.
STRICKLAND, J. C., Graduate Instructor, University of Richmond.
SUDDATH, E. E., Technician, Washington University.
TANERI, BEDIA, Graduate Student, New York University, College of Medicine.
THOMPSON, RAYMOND K., Research Assistant, University of Maryland.
TUM SUDEN, CAROLINE, Research Fellow in Physiology, Boston University, School
of Medicine.
WAGNER, CARROLL E., Research Technician, University of Maryland.
WIGHTMAN, JOHN C., Assistant in Biology, Brown University.
WILSON, JOHN WOODROVV, Graduate Assistant in Zoology, Duke University.
YOUNG, SAUL B., Technician, Rockefeller Institute for Medical Research.
Students
BOTANY
BADER, JOAN E., Montclair State Teachers College.
BIEN, BETTINA H., Student, Wheaton College.
BONNER, JOHN T., Student, Harvard University.
FENDER, FLORA S., Preparator, University of Pennsylvania.
GRAVES, E. IRENE, Senior Instructor in Biology, Bridgewater State Teachers Col-
lege.
HOFFMAN, ELIZABETH D., Mount Holyoke College.
MARKLE, JANE C., Smith College.
POSTEL, FRANCES H., Wellesley College.
RUTLEDGE, ALMA W., Graduate Student, Johns Hopkins University.
SCHALLEK, WILLIAM B., Harvard University.
SIEGEL, MARION T., New Jersey College for Women.
WARD, HENRY S., JR., Alabama Polytechnic Institute.
EMBRYOLOGY
ALLEY, ARMINE, Research Assistant, McGill University.
ARMSTRONG, FLORENCE H., Student, Dalhousie University.
BERRY, CLYDE, JR., Washington University.
BLANCHARD, JOSEPH, Student, Wesleyan University.
BOOKHOUT, CAZLYN G., Instructor in Zoology, Duke University.
BRUSH, HELEN V., Vassar College.
COLLIER, JANE G., Assistant, University of Missouri.
COPPOLA, ARMANDO R., Brothers College of Drew University.
DOBLER, MARIAN, Goucher College.
DRURY, HORACE F., Harvard University.
REPORT OF THE DIRECTOR 39
DUNHAM, DONALD W., Assistant in Zoology, Ohio State University.
EDDS, MAC VINCENT, JR., Amherst College.
FINK, HAROLD K., Graduate Student, Princeton University.
FINKEL, ASHER J., Research Assistant, The University of Chicago.
HARROLD, CHARLES M., JR., Graduate Assistant, New York University.
KLEIN, ETHEL L., University of Rochester.
KURTZ, ELIZABETH L., Wilson College.
LEWISOHN, MARJORIE G., University of Michigan.
MILNE, WALTER S., Graduate Assistant, University of Missouri.
PHILIPS, FREDERICK S., Graduate Assistant, University of Rochester.
ROGICK, MARY D., Professor of Biology, College of New Rochelle.
ROGOFF, WILLIAM M., Graduate Student, Yale University.
ROTHERMEL, JULIA E., Professor of Biology, Western College.
SODERWALL, ARNOLD L., Assistant in Zoology, University of Illinois.
SPANGLER, JULIET M., Wheaton College.
STABLEFORD, Louis T., Laboratory Assistant, Yale University.
STEVENS, FLORENCE F., New Jersey College for Women.
TAYLOR, HARRIETT E., Radcliffe College.
TERZIAN, ANNETTE V., Mount Holyoke College.
TOWLE, HARRIET N., Assistant in Zoology, Wellesley College.
WADDILL, SAMUEL F., Washington and Jefferson College.
WILLIAMS, JOHN L., Graduate Assistant, New York University.
WOODWARD, ARTHUR A., JR., Student, Oberlin College.
WORDEN, FREDERIC G., Student, Dartmouth College.
PHYSIOLOGY
ALBRINK, WILHELM S., Assistant in Biology, Yale University.
ALLEN, PAUL J., Graduate Assistant in Botany, University of Rochester.
ARMSTRONG, CHARLES W. J., Demonstrator in Biology, University of Toronto.
BECK, NAOMI E., The University of Chicago.
BLAIR, JOHN H., Graduate Assistant, Wesleyan University.
BLISS, ALFRED F., Columbia University.
BRISCOE, PRISCILLA M., Graduate Student, Ohio State University.
CASEY, MARGARET T., Graduate Assistant in Physiology, Mount Holyoke College.
CROWELL, HAMBLIN H., Graduate Assistant, Ohio State University.
CURTIS, HOWARD J., Fellow, Rockefeller Foundation.
GRAVE, CASWELL, II, Assistant, Washington University.
HENSON, MARGARET, Assistant in Physiology, Wellesley College.
LEVINE, HARRY PHILIP, Zoology Instructor, University of Vermont.
MARTIN, ROSEMARY D. C, Assistant, University of Toronto.
MOORE, IMOGENE, Instructor in Zoology, New Jersey College for Women.
MULLINS, LORIN J., University of California.
O'BRIEN, JOHN P., Johns Hopkins University.
OWENS, WILLIAM C., St. John's College.
SMITH, AUDREY U., Assistant in Physiology, Vassar College.
VON DACH, HERMAN, Assistant in Zoology, Ohio State University.
WIEGHARD, CHARLOTTE, 4544 Harris Avenue, St. Louis, Missouri.
WILSON, JOHN W., Graduate Assistant in Zoology, Duke University.
PROTOZOOLOGY
BEVEL, NELL H., Assistant in Zoology, Duke University.
BURBANCK, WILLIAM D., Graduate Assistant, The University of Chicago.
COLE, ROGER M., Undergraduate Assistant, Massachusetts State College.
EWING, WILLIAM H., Fellow in Biology, Washington and Jefferson College.
40 MARINE BIOLOGICAL LABORATORY
FINKELSTEIN, NATHANIEL, Johns Hopkins University.
HIERHOLZER, CAROLYN ANNE, Instructor in Biology, Adelphia College.
KORNBLUM, LUCILE, Student, Columbia University.
MAYO, MERCEDES, Assistant Professor of Biology, Universidad de la Habana.
WELLS, WAYNE W., Associate Professor of Science, Southern Oregon State
Normal.
WILKINSON, ELIZABETH J., Student, Columbia University.
INVERTEBRATE ZOOLOGY
ACOSTA, JOSEFINA, Goucher College.
ALEXANDER, ROBERT S., Graduate Assistant, Amherst College.
ARNSTEIN, MARGERY, Simmons College.
BELDA, WALTER H., Graduate Student, Johns Hopkins University.
BIGLER, FRANCES B., Western Reserve University.
BROWN, HENRY, Student, College of the City of New York.
COONEY, MARILYN R., Student, Smith College.
CRANE, TODD, Student, Wilson College.
DAVIS, JAMES O., Graduate Assistant in Zoology, University of Missouri.
DELISA, DOMINICK A., Student, Union College.
DERINGER, MARGARET K., Johns Hopkins University.
DOBBELAAR, MARK E., Teacher of Science, Oradell High School.
FAHL, HELEN, Student, Oberlin College.
FERGUSON, FREDERICK P., Undergraduate Assistant, Wesleyan University.
FLEMING, ROBERT S., Science Critic Teacher, East Carolina Teachers College.
FRASER, LEMUEL A., Student, American University.
GALE, SHIRLEY, Radcliffe College.
GRAVES, E. IRENE, Senior Instructor in Biology, Bridgewater State Teachers
College.
GRIFFITHS, RAYMOND B., Graduate Research Assistant, Princeton University.
HAINES, WILLIAM J., Wabash College.
HALL, LYDIA R., Graduate Assistant, Mount Holyoke College.
HAMANN, CECIL B., Assistant, Purdue University.
HARRIS, NELLIE R., Undergraduate Assistant, Montclair State Teachers College.
HOAGLAND, MARY, Swarthmore College.
JAEGER, LUCENA, Graduate Assistant in Zoology, University of Missouri.
JORDON, ELIZABETH L., Barnard College.
JOSEPH, SAMUEL, Student Laboratory Assistant, DePauw University.
KELLOGG, MARGARET P., Graduate Student, Cornell University.
KERRIGAN, SYLVIA, Graduate Assistant, University of Cincinnati.
LINSCHEID, ALFRED G., Western Reserve University.
LOVE, GENEVIEVE, Pennsylvania College for Women.
MCDONALD, BROWN, Laboratory Assistant, DePauw University.
MORRISON, PETER R., Swarthmore College.
NADLER, EVELYN R., Brooklyn College.
PIERSON, MARY E., Graduate Assistant, Mount Holyoke College.
REYER, RANDALL W., Cornell University.
ROLLASON, HERBERT D., JR., Middlebury College.
ROOT, CHARLOTTE C, Student, Mount Holyoke College.
RYAN, THOMAS L, Instructor, Boston College.
SACKETT, JOHN T., Graduate, University of Pennsylvania.
SANDERS, MARY ELIZABETH, Depauw University.
SCHAEFFER, BoBB, Graduate Student, Columbia University.
SCHNEIDER, MATHILDA E. C., University of Illinois.
SHEEHAN, ELEANOR L., Instructor, University of New Hampshire.
SMITH, RALPH I., Harvard University.
REPORT OF THE DIRECTOR
41
SNEDECOR, JAMES, Student, Iowa State College.
SPERRY, ROGER W., Oberlin College.
TABER, ELSIE, Instructor in Biology, Lander College.
TOWLE, HARRIET N., Assistant in Zoology, Wellesley College.
TROWBRIDGE, CAROLYN F., University of Iowa.
WARD, HELEN L., Assistant in Biology, Purdue University.
WELCH, D'ALTE A., Johns Hopkins University.
WELLS, LORNA A., Graduate Assistant, Oberlin College.
WILLIAMS, EDITH M., Student, Elmira College.
4. TABULAR VIEW OF ATTENDANCE
1934 1935 1936 1937 1938
INVESTIGATORS— Total 323 315 359 391 380
Independent 222 208 226 256 246
Under Instruction 49 56 76 74 53
Research Assistants 52 51 57 61 81
STUDENTS— Total 131 130 138 133 132
Zoology 54 55 55 57 54
Protozoology 11 16 17 16 10
Embryology 30 33 34 35 34
Physiology 23 20 22 16 22
Botany 13 6 10 9 12
TOTAL ATTENDANCE 454 445 497 524 512
Less Persons Registered as Both Students
and Investigators 15 16 24 13 16
439 429 473 511 496
INSTITUTIONS REPRESENTED — Total 131 143 158 165 151
By Investigators 98 111 120 134 125
By Students 75 70 77 79 67
SCHOOLS AND ACADEMIES REPRESENTED
By Investigators 1 2 3 4
By Students 5 3 3 2 1
FOREIGN INSTITUTIONS REPRESENTED
By Investigators 4 7 9 16 14
By Students 1 1 5 3
5. SUBSCRIBING AND COOPERATING INSTITUTIONS IN
1938
American University
Amherst College
Barnard College
Belgian American Education Founda-
tion, Inc.
Bowdoin College
Brothers College of Drew University
Brown University
Bryn Mawr College
Carnegie Institute of Washington
College of Physicians and Surgeons
College of William and Mary
Columbia University
Purdue University
Radcliffe College
Rockefeller Foundation
Rockefeller Institute for Medical Re-
search
Rutgers University
St. John's College
Smith College
Spring Hill College
State University of Iowa
Swarthmore College
Syracuse University
Temple University
42
MARINE BIOLOGICAL LABORATORY
Connecticut College
Cornell University Medical College
Dalhousie University
Dartmouth College
DePauw University
Duke University
Elmira College
General Education Board
Goucher College
Harvard University
Harvard University Medical School
Hunter College
Industrial & Engineering Chemistry, of
the American Chemical Society
Iowa State College
Johns Hopkins University
Kenyon College
Eli Lilly & Company
Long Island University
Massachusetts State College
Memorial Hospital, New York City
Mount Holyoke College
New York State Department of Health
New York University
New York University Medical School
Northwestern University
Oberlin College
Pennsylvania College for Women
Princeton University
Toledo University
Tufts College
Union College
University of Chicago
University of Cincinnati
University of Illinois
University of Maryland Medical School
University of Minnesota
University of Missouri
University of Notre Dame
University of Pennsylvania
University of Pittsburgh
University of Rochester
University of Rochester Medical School
University of Vermont
University of Virginia
Vanderbilt University Medical School
Vassar College
Wabash College
Washington University
Washington University Medical School
Wellesley College
Wesleyan University
Western Reserve University
Wheaton College
Williams College
Wilson College
Yale University
6. EVENING LECTURES, 1938
Tuesday, June 21
DR. E. H. MYERS "Life Cycle of Foraminifera."
Friday, July 1
DR. M. H. JACOBS " Blood and Zoological Classification."
Friday, July 8
DR. S. O. MAST " The Synthesis of Living Substance,
as Exemplified in Chilomonas
paramecium."
Friday, July 15
DR. G. H. PARKER " The Color Changes in Animals and
Neurohumoral Transmission."
Wednesday, July 20
DR. ROBERT CHAMBERS AND
DR. WILLIAM DURYEE " Micromanipulation Studies on Cells
and Nuclei."
Friday, July 22
DR. O. E. SCHOTTE " Induction of Embryonic Organs in
Regenerates and Neoplasms."
Friday, July 29
DR. EDUARD UHLENHUTH "A Quantitative Approach to the Se-
cretion Process of the Thyroid."
REPORT OF THE DIRECTOR 43
Friday, August 5
DR. ROBERT CHAMBERS " Structural Aspects of Cell Division."
Tuesday, August 9
DR. E. G. CONKLIN AND
DR. F. R. LILLIE " Informal Memorial of the Fiftieth
Anniversary of the Founding of
the Marine Biological Laboratory."
Friday, August 12
DR. L. G. EARTH " Studies of the Factors Influencing
Regeneration."
Friday, August 19
MR. COLUMBUS ISELIN " The Influence of Fluctuations in the
Major Ocean Currents on the Cli-
mate and the Fisheries."
Friday, August 26
DR. PETER GRAY " The Possibility of Affecting Develop-
mental Patterns by Electrical
Means."
Thursday, September 1 (Under the joint auspices of the Genetics Society of
America and the Marine Biological Laboratory)
DR. H. J. MULLER " The Remaking of Chromosomes."
7. SHORTER SCIENTIFIC PAPERS, 1938
Tuesday, July 5
DR. W. H. NEWTON " Endocrine Activity of the Placenta
in Mice."
DR. J. K. W. FERGUSON AND
DR. H. O. HATERIUS " Evidence for Hormonal Control of
Uterine Motility by the Hypoph-
ysis in the Rabbit."
DR. ROBERTS RUGH " Experimental Studies on the Genital
System of the Male Anuran."
Tuesday, July 12
DR. G. W. KlDDER AND
DR. C. A. STUART " The Role of Chromogenic Bacteria
in Ciliate Growth."
MR. J. A. SMITH " Some Effects of Temperature on the
Reproduction of Chilomonas para-
mecium."
DR. D. L. HOPKINS " Adjustment of the marine Amoeba,
Flabellula mira Schaeffer, to
changes in the Total Salt Concen-
tration of the Outside Medium."
MR. C. L. CLAFF " Phenomena of Excystment in Col-
poda cucullus."
Tuesday, July 19
DR. K. C. FISHER AND
MR. R. OHNELL " The Steady State Frequency of the
Embryonic Fish Heart at Differ-
ent Cyanide Concentrations."
44 MARINE BIOLOGICAL LABORATORY
DR. LENA A. LEWIS " Studies on the Refractory State Re-
sulting from the Repeated Injec-
tions of Adrenal Extract."
DR. EMIL BOZLER " Action Potentials of Visceral Smooth
Muscles."
DR. L. IRVING " Rhythmical Changes in Blood Flow
Through Muscles."
Tuesday, July 26
DR. B. H. WlLLIER AND
DR. MARY E. RAWLES " Skin Transplants between Embryos
of Different Breeds of Fowl."
DR. ARTHUR COLWIN " Induction by Cauterization in the
Amphibian Egg."
DR. VIKTOR HAMBURGER " The Innervation of Transplanted
Limbs in Chick Embryos."
DR. PAUL WEISS " The Effect of Mechanical Stress on
Cartilage Differentiated in Vitro."
Tuesday, August 2
DR. J. P. VISSCHER " Some Recent Studies on Barnacles."
DR. E. R. JONES, JR " Observations on some of the Lower
Turbellaria of the Eastern United
States."
DR. o'A. A. WELCH " Some Problems of Distribution and
Variation in the Hawaiian Tree
Snail Achatinella."
Tuesday, August 16
DR. D. P. COSTELLO " Studies on Fragments of Centrifuged
Nereis Eggs."
DR. VICTOR SCHECHTER " Calcium and Magnesium in Relation
to the Longevity of Egg Cells."
DR. J. B. BUCK AND
DR. R. D. BOCHE " Some Properties of Living Chromo-
somes."
DR. A. M. LUCAS " Some Cytological Studies on Virus-
Infected Cells."
DR. W. R. DURYEE "A Microdissection Study of Am-
phibian Chromosomes."
Tuesday, August 23
MR. KARL WILBUR " The Relation of the Magnesium Ion
to Ultraviolet Stimulation in the
Nereis Egg."
DR. E. ELEANOR CAROTHERS " Cytological Effects of X-Rays on
Grasshopper Embryos."
DR. J. FURTH " Quantitative Studies on the Effect
of X-Rays on Mammalian Cells,
and on the Mode of X-Ray Ac-
tion."
DR. P. S. HENSHAW " The Effect of X-Rays on Arbacia
punctulata Sperm."
DR. T. N. WHITE " Recovery of Arbacia Eggs from
High Intensity X-Ray Effects."
REPORT OF THE DIRECTOR 45
8. GENERAL SCIENTIFIC MEETING, 1938
Tuesday, August 30
Miss A. M. MELLAND " Isolation of Salivary Gland Nuclei."
MR. GLENN H. ALGIRE " Cytological Studies on the Living
Thyroid of the Salamander."
DR. RALPH H. CHENEY " Micro-Structural Changes in Muscle
Fibers after Caffeine."
DR. CARL C. SPEIDEL " Some Features of Contraction Nodes
and Retraction Clots as Observed
in Single Fibers of Cardiac and
Skeletal Muscle of Both Verte-
brates and Invertebrates."
DR. MICHAEL J. D. WHITE " The Heteropycnosis of Sex Chromo-
somes and its Interpretation in
Terms of Spiral Structure."
DR. JOHN P. TURNER " Mitochondria and other Inclusions in
the Ciliate Tillina canalifera."
DR. ROBERT CHAMBERS " Cytoplasmic Inclusions and Matrix
of the Arbacia Egg."
DR. M. J. KOPAC " The Devaux Effect at Oil-Proto-
plasm Interfaces."
DR. M. H. JACOBS AND
DR. A. K. PARPART " Further Studies on the Permeability
of the Erythrocyte to Ammonium
Salts."
MR. A. J. DziEMIAN AND
DR. A. K. PARPART " Permeability and the Lipoid Content
of the Erythrocyte."
MR. LOUIS-PAUL DUGAL AND
DR. LAURENCE IRVING " The Relation of the Shell to An-
aerobic Metabolism in Venus
mercenaria."
DR. ALEXANDER SANDOW AND
DR. KENNETH MORITZ " Tension Output of Muscles in Hypo-
tonic Solutions."
DR. DWIGHT L. HOPKINS " The Mechanism for the Control of
the Intake and the Output of
Water by the Vacuoles in the
Marine Amoeba, Flabellula mira
Schaeffer."
DR. N. S. R. MALOEUF "On the Kidney of the Crayfish and
the Uptake of Chlorid from Fresh
Water by this Animal."
DR. N. S. R. MALOEUF " The Osmo-regulative Function of the
Alimentary Tract of the Earth-
worm, and on the Uptake of Chlo-
rid from Fresh Water by this
Animal."
46 MARINE BIOLOGICAL LABORATORY
DR. ETHEL BROWNE HARVEY " Development of Half-Eggs of
Chaetopterus Obtained by Cen-
trifugal Force."
DR. PAUL S. HENSHAW " The Question of Whether the Delay
in Cleavage of Arbacia Eggs Pro-
duced with X-Rays is Caused by a
General Slowing of the Cleavage
Process or by a Block at Some
Particular Stage."
MR. E. L. CHAMBERS AND
DR. ROBERT CHAMBERS " The Resistance of Fertilized Arbacia
Eggs to Immersion in KC1 and
NaCl Solutions."
DR. ALBERT E. NAVEZ " Indolphenoloxidase in Arbacia Eggs
and the Nadi Reaction."
DR. K. C. COLE AND
DR. HOWARD J. CURTIS " Electric Impedance of Nerve Dur-
ing Activity."
DR. FRANK A. GELDARD " The Vibratory Response of the Skin
and its Relation to Pressure Sen-
sitivity."
DR. E. ALFRED WOLF " Reversal of Phototropic Reaction in
Daphnia by the Use of Photosensi-
tizing Dyes."
DR. CARL C. SPEIDEL " Motion Picture Showing Microscopic
Changes in Fibers of Cardiac and
Skeletal Muscle of Invertebrates
and Vertebrates during Contrac-
tion, Retraction, and Clotting."
DR. W. R. DURYEE "The Action of Direct Currents on
the Cell Nucleus."
DR. W. R. DURYEE " Hydration and Dehydration of Fol-
licle Cell Nuclei."
Wednesday, August 31
DR. HERBERT ELFTMAN "The Function of Muscles in Loco-
motion."
DR. WILLIAM J. BOWEN " The Effects of Copper and of Vana-
dium on the Frequency of Divi-
sion."
DR. SARAH BEDICHEK " Sex Balance in the Progeny of Trip-
loid Drosophila."
DR. EDUARD UHLENHUTH,
MR. JAMES U. THOMPSON AND
MR. JOSEPH E. SCHENTHAL " The Antihormone Problems in the
Salamander."
DR. ROBERTS RUGH "The Effect of the Sex-Stimulating
Factor of the Anterior Pituitary
Gland on the Testis of the Bull-
frog."
REPORT OF THE DIRECTOR 47
DR. J. PAUL VISSCHER " Studies on Barnacle Larvae."
DR. GRACE TOWNSEND ''The Spawning Reaction of Nereis
limbata with Emphasis Upon
Chemical Stimulation."
DR. GRACE TOWNSEND " Physiological Assays Concerning the
Nature of Fertilizin."
DR. ELBERT C. COLE "A Study of the Integument of the
Squid, During Staining with
Methylene Blue."
MR. CARL C. SMITH AND
MR. Louis LEVIN " The Use of the Clam Heart as a
Test Object for Acetylcholine."
DR. OSCAR W. RICHARDS AND
Miss KATHARINE J. HAWLEY " The Elimination of Molds."
DR. S. E. POND,
MR. E. P. LITTLE,
MR. A. M. SMITH, AND
MR. J. D. GRAHAM "A Comparative Study of Water
Aspirators."
PAPERS READ BY TITLE
DR. C. A. ANGERER " The Effect of Electric Current on
the Physical Consistency of Sea
Urchin Eggs."
MR. C. W. J. ARMSTRONG AND
DR. K. C. FISHER " The Effect of Sodium Azide on the
Frequency of the Embryonic
Fundulus Heart."
MR. ROBERT BALLANTINE " Reducing Activity of Fertilized and
Unfertilized Arbacia Eggs."
DR. LUDWIG VON BERTALANFFY ..." Studies on the Mechanism of Growth
in Planaria maculata."
MRS. RUTH SNYDER COOPER "Probable Absence of a Chromato-
phore Activator in Limulus poly-
phemus."
MR. C. G. GRAND " Intracellular pH Studies on the Ova
of Mactra solidissima."
DR. W. R. DURYEE " The Action of Fixatives on the Iso-
lated Cell Nucleus."
DR. ADOLPH ELWYN " The Melanophore-Expanding Ac-
tivity of the Ascidian Neural
Gland."
MR. RICHARD W. FOSTER,
MR. JOHN D. CRAWFORD AND
DR. ALBERT E. NAVEZ " Cardiac Rhythm in Pecten irra-
dians (Lamarck)."
DR. STEPHEN KARADY " The Alarm Reaction and Adaptation
Syndrome in Lower Vertebrates
(Fundulus majalis)."
48 MARINE BIOLOGICAL LABORATORY
DR. M. J. KOPAC " Micro-estimation of Protein Adsorp-
tion at Oil-Protoplasm Interfaces."
DR. M. J. KOPAC AND
DR. R. CHAMBERS " Effect of the Vitelline Membrane on
Coalescence of Arbacia Eggs with
Oil-drops."
DR. GEORGE SASLOW "The Osmotic Pressure of Gum
Acacia Solutions."
DR. A. A. SCHAEFFER " Differences Between Scottish and
American Amebas of the Species
Chaos diffluens Miiller."
DR. VICTOR SCHECHTER " Induction in Griffithsia."
DR. VICTOR SCHECHTER " Bacteria in Relation to Longevity of
Egg Cells."
DR. J. N. STANNARD " The Effect of Sodium Azide on the
Respiration of Frog Muscle."
DR. A. J. WATERMAN " Respiratory Stimulants and Gastru-
lation in Arbacia."
DR. RALPH WICHTERMAN "Does Transfer of Pronuclei ever
Occur in Conjugation of Para-
mecium caudatum ? "
DR. E. ALFRED WOLF AND
MR. A. S. LEVENSON " Studies in Calcification. IV. A
Contribution to the Problem of
Skeletal Calcification in the Tele-
ost, Fundulus heteroclitus."
DR. OPAL M. WOLF " Mitotic Activity of the Islands of
Langerhans and Parathyroids of
Rats Following Pituitary Extract
and Colchicine Injections."
DR. OPAL M. WOLF " Oviducts of Pituitary Stimulated
Females, Rana pipiens."
Miss R. A. YOUNG " The Effects of Roentgen Irradiatior
on Cleavage and Early Develop-
ment in the Annelid, Chaetopterm
pergamentaceus."
MR. E. ZWILLING " The Effect of Perisarc Removal on
Regeneration in Tubularia crocea."
DEMONSTRATIONS
Wednesday, August 31
DR. MICHAEL J. D. WHITE " The Spiral Structure of Animal
Chromosomes."
DR. P. S. HENSHAW " Cellular Abnormalities Produced by
X-Rays."
DR. K. S. COLE AND
H. J. CURTIS " Electrical Impedance Changes in the
Squid Giant Axon Following Ex-
citation."
REPORT OF THE DIRECTOR
49
DR. E. R. CLARK AND
MRS. ELEANOR LINTON CLARK ....a) "Marked Macrophages."
b) " Arterio-venous Anastomoses as
Observed in the Living Mammal."
MR. C. H. NORRIS " Method of Studying Elastic Tension
of Marine Eggs."
MR. G. H. ALGIRE " Apparatus for the Cytological Study
of the Thyroid in the Living Sala-
mander."
DR. J. P. TURNER " Mitochondria and Other Inclusions
in the Ciliate Tillina canalifera."
MR. A. S. LEVENSON " Microscopic Sections through Head
and Trunk Regions of Fundulus
heteroclitus, Prepared by the
Gomore Silver Nitrate Method for
the Study of Calcification."
DR. A. K. PARPART AND
MR. S. B. YOUNG " A Simple Glass Electrode System."
DR. S. E. POND AND
MR. E. P. LITTLE " Water Aspirator Tests and Compari-
sons."
DR. E. C. COLE a) " Methylene Blue Preparations of
the Chromatophores of the Squid."
b) "A Low Voltage Lamp for Gen-
eral Microscopic Use."
c) "Methyl Methacrylate as a Mount-
ing Medium for Macroscopic Prep-
arations."
DR. GRACE TOWNSEND " Spawning Reactions of Male Nereis
limbata in Response to Gluta-
thione."
MR. C. C. SMITH AND
MR. Louis LEVIN " The Use of the Clam Heart as a
Test Object for Acetylcholine."
DR. F. J. M. SICHEL AND
DR. S. E. POND " Multi Contact Rheotome."
DR. ROBERTS RUGH " Urogenital System of the Male Frog
Rana pipiens. Injected to Show
the Course of Spermatozoa from
the Seminiferous Tubules to the
Wolffian Ducts."
DR. P. S. GALTSOFF " Sex Reversal in Adult Oysters."
DR. P. S. GALTSOFF " Method of Measuring and Recording
the Rate of Flow of Water
Through the Gills of the Oyster."
DR. P. S. GALTSOFF AND
MR. GEORGE MISHTOWT " Respiration of the Oyster."
50 MARINE BIOLOGICAL LABORATORY
9. MEMBERS OF THE CORPORATION
1. LIFE MEMBERS
ALLIS, MR. E. P., JR., Palais Carnoles, Menton, France.
ANDREWS, MRS. GWENDOLEN FOULKE, Baltimore, Maryland.
BILLINGS, MR. R. C, 66 Franklin St., Boston, Massachusetts.
CONKLIN, PROF. EDWIN G., Princeton University, Princeton, New
Jersey.
CRANE, MR. C. R., New York City.
EVANS, MRS. GLENDOWER, 12 Otis Place, Boston, Massachusetts.
FOOT, Miss KATHERINE, Care of Morgan Harjes Cie, Paris, France.
GARDINER, MRS. E. G., Woods Hole, Massachusetts.
JACKSON, Miss M. C., 88 Marlboro St., Boston, Massachusetts.
JACKSON, MR. CHAS. C., 24 Congress St., Boston, Massachusetts.
KING, MR. CHAS. A.
LEE, MRS. FREDERIC S., 279 Madison Ave., New York City.
LEE, PROF. F. S., College of Physicians and Surgeons, New York City.
LEWIS, PROF. W. H., Johns Hopkins University, Baltimore, Maryland.
LOWELL, MR. A. L., 17 Quincy St., Cambridge, Mass.
McMuRRiCH, PROF. J. P., Toronto, Canada.
MEANS, DR. J. H., 15 Chestnut St., Boston, Mass.
MOORE, DR. GEORGE T., Missouri Botanical Gardens, St. Louis, Mo.
MORGAN, MR. J. PIERPONT, JR., Wall and Broad Sts., New York City.
MORGAN, PROF. T. H., Director of Biological Laboratory, California
Institute of Technology, Pasadena, California.
MORGAN, MRS. T. H., Pasadena, California.
MORRILL, DR. A. D., Hamilton College, Clinton, N. Y.
NOYES, Miss EVA J.
PORTER, DR. H. C., University of Pennsylvania, Philadelphia, Pennsyl-
vania.
SEARS, DR. HENRY F., 86 Beacon St., Boston, Massachusetts.
SHEDD, MR. E. A.
THORNDIKE, DR. EDWARD L., Teachers College, Columbia University,
New York City.
TREADWELL, PROF. A. L., Vassar College, Poughkeepsie, N. Y.
TRELEASE, PROF. WILLIAM, University of Illinois, Urbana, Illinois.
WALLACE, LOUISE B., 359 Lytton Avenue, Palo Alto, Calif.
WILSON, DR. E. B., Columbia University, New York City.
2. REGULAR MEMBERS, 1938
ABRAMOWITZ, DR. ALEXANDER A., Biological Laboratories, Harvard
University, Cambridge, Massachusetts.
REPORT OF THE DIRECTOR 51
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.
ALLEE, DR. W. C, The University of Chicago, Chicago, Illinois.
ALLYN, DR. HARRIET M., Mount Holyoke College, South Hadley,
Massachusetts.
AMBERSON, DR. WILLIAM R., Department of Physiology, University
of Maryland, School of Medicine, Lombard and Greene Streets,
Baltimore, Maryland.
ANDERSON, DR. E. G., California Institute of Technology, Pasadena,
California.
ANDERSON, DR. RUBERT S., Guyot Hall, Princeton University, Prince-
ton, New Jersey.
ARMSTRONG, DR. PHILIP B., 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., University of Pennsylvania, Philadelphia, Pennsyl-
vania.
BALDWIN, DR. F. M., University of Southern California, Los Angeles,
California.
BALL, DR. ERIC G., Johns Hopkins Medical School, Baltimore, Mary-
land.
BALLARD, DR. WILLIAM W., Dartmouth College, Hanover, New Hamp-
shire.
BARD, PROF. PHILIP, Johns Hopkins Medical School, Baltimore, Mary-
land.
BARRON, DR. E. S. GUZMAN, Department of Medicine, The Univer-
sity of Chicago, Chicago, Illinois.
EARTH, DR. L. G., Department of Zoology, Columbia University, New
York City.
BEADLE, DR. G. W., School of Biological Sciences, Stanford Univer-
sity, California.
BECKWITH, DR. CORA J., Vassar College, Poughkeepsie, New York.
BEHRE, DR. ELINOR H., Louisiana State University, Baton Rouge,
Louisiana.
BENNITT, DR. RUDOLF, University of Missouri, Columbia, Missouri.
BIGELOW, DR. H. B., Museum of Comparative Zoology, Cambridge,
Massachusetts.
BIGELOW, PROF. R. P., Massachusetts Institute of Technology, Cam-
bridge, Massachusetts.
52 MARINE BIOLOGICAL LABORATORY
BINFORD, PROF. RAYMOND, Guilford College, Guilford College, North
Carolina.
BISSONNETTE, DR. T. HUME, Trinity College, Hartford, Connecticut.
BLANCHARD, PROF. KENNETH C, Washington Square College, New
York University, New York City.
BODINE, DR. J. H., Department of Zoology, State University of Iowa,
Iowa City, Iowa.
BORING, DR. ALICE M., Yenching University, Peking, China.
BOZLER, DR. EMIL, Ohio State University, Columbus, Ohio.
BRADLEY, PROF. HAROLD C., University of Wisconsin, Madison, Wiscon-
sin.
BRIDGES, DR. CALVIN B., California Institute of Technology, Pasadena,
California.
BRONFENBRENNER, DR. JACQUES J., Department of Bacteriology, Wash-
ington University Medical School, St. Louis, Missouri.
BRONK, DR. D. W., University of Pennsylvania, Philadelphia, Pennsyl-
vania.
BROOKS, DR. S. C., University of California, Berkeley, California.
BROWN, DR. DUGALD E. S., New York University, College of Medicine,
New York City.
BUCKINGHAM, Miss EDITH N., Sudbury, Massachusetts.
BUDINGTON, PROF. R. A., Oberlin College, Oberlin, Ohio.
BULLINGTON, DR. W. E., Randolph-Macon College, Ashland, Virginia.
BUMPUS, PROF. H. C., Duxbury, Massachusetts.
BYRNES, DR. ESTHER E., 1803 North Camac Street, Philadelphia, Penn-
sylvania.
CALKINS, PROF. GARY N., Columbia University, New York City.
CALVERT, PROF. PHILIP P., University of Pennsylvania, Philadelphia,
Pennsylvania.
CANNAN, PROF. R. K., New York University College of Medicine, 477
First Avenue, New York City.
CARLSON, PROF. A. J., Department of Physiology, The University of
Chicago, Chicago, Illinois.
CAROTHERS, DR. E. ELEANOR, Department of Zoology, State University
of Iowa, Iowa City.
CARPENTER, DR. RUSSELL L., Tufts College, Tufts College, Massa-
chusetts.
CARROLL, PROF. MITCHELL, Franklin and Marshall College, Lancaster,
Pennsylvania.
CARVER, PROF. GAIL L., Mercer University, Macon, Georgia.
CATTELL, DR. MC!VEEN, Cornell University Medical College, 1300 York
Avenue, New York City.
REPORT OF THE DIRECTOR 53
CATTELL, PROF. J. McKEEN, Garrison-on-Hudson, New York.
CATTELL, MR. WARE, Garrison-on-Hudson, New York.
CHAMBERS, DR. ROBERT, Washington Square College, New York Uni-
versity, Washington Square, New York City.
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.
CLAFF, MR. C. LLOYD, Department of Biology, Brown University,
Providence, Rhode Island.
CLARK, PROF. E. R., University of Pennsylvania Medical School, Phila-
delphia, Pennsylvania.
CLARK, DR. LEONARD B., Union College, Schenectady, New York.
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, Wil-
liamstown, Massachusetts.
COLE, DR. KENNETH S., College of Physicians and Surgeons, Columbia
University, 630 W. 168th Street, New York City.
COLE, DR. LEON J., College of Agriculture, Madison, Wisconsin.
COLLETT, DR. MARY E., Western Reserve University, Cleveland, Ohio.
COLTON, PROF. H. S., Box 601, Flagstaff, Arizona.
COONFIELD, DR. B. R., Brooklyn College, 80 Willoughby Street, Brook-
lyn, New York.
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 Carolina, Chapel Hill, North Carolina.
COWDRY, DR. E. V., Washington University, St. Louis, Missouri.
CRAMPTON, PROF. H. E., Barnard College, Columbia University, New
York City.
CRANE, MRS. C. R., Woods Hole, Massachusetts.
CROWELL, DR. P. S., JR., Department of Zoology, Miami University,
Oxford, Ohio.
CURTIS, DR. MAYNIE R., Crocker Laboratory, Columbia University,
New York City.
CURTIS, PROF. W. C, University of Missouri, Columbia, Missouri.
DAN, DR. KATSUMA, Misaki Biological Station, Misaki, Japan.
54 MARINE BIOLOGICAL LABORATORY
DAVIS, DR. DONALD W., College of William and Mary, Williamsburg,
Virginia.
DAWSON, DR. A. B., Harvard University, Cambridge, Massachusetts.
DAWSON, DR. J. A., The College of the City of New York, New York
City.
DEDERER, DR. PAULINE H., Connecticut College, New London, Con-
necticut.
DILLER, DR. WILLIAM F., Dartmouth College, Hanover, New Hamp-
shire.
DODDS, PROF. G. S.. Medical School, University of West Virginia, Mor-
gantown, West Virginia.
DOLLEY, PROF. WILLIAM L., University of Buffalo, Buffalo, New York.
DONALDSON, DR. JOHN C., University of Pittsburgh, School of Medi-
cine, Pittsburgh, Pennsylvania.
DuBois, DR. EUGENE F., Cornell University Medical College, 1300 York
Avenue, New York City.
DUGGAR, DR. BENJAMIN M., University of Wisconsin, Madison, Wis-
consin.
DUNGAY, DR. NEIL S., Carleton College, Northfield, Minnesota.
DURYEE, DR. WILLIAM R., Department of Biology, Washington Square
College, New York University, New York City.
EDWARDS, DR. D. J., Cornell University Medical College, 1300 York
Avenue, New York City.
ELLIS, DR. F. W., Monson, Massachusetts.
FAURE-FREMIET, PROF. EMMANUEL, College de France, Paris, France.
FERGUSON, DR. JAMES K. W., Department of Physiology, Ohio State
University, Columbus, Ohio.
FISCHER, DR. ERNST, Department of Physiology, Medical College of
Virginia, Richmond, Virginia.
FISHER, DR. KENNETH C., Department of Biology, University of To-
ronto, Toronto, Canada.
FLEISHER, DR. MOYER S., School of Medicine, St. Louis University, St.
Louis, Missouri.
FORBES, DR. ALEXANDER, Harvard University Medical School, Boston,
Massachusetts.
FRY, DR. HENRY J., Cornell University Medical College, 1300 York
Avenue, New York City.
FURTH, DR. JACOB, Cornell University Medical College, 1300 York Ave-
nue, New York City.
GAGE, PROF. S. H., Cornell University, Ithaca, New York.
GALTSOFF, DR. PAUL S., 420 Cumberland Avenue, Somerset, Chevy
Chase, Maryland.
REPORT OF THE DIRECTOR 55
CARREY, PROF. W. E., Vanderbilt University Medical School, Nashville,
Tennessee.
GATES, PROF. R. RUGGLES, University of London, London, England.
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.
GOLDFORB, PROF. A. J., College of the City of New York, Convent Ave-
nue and 139th Street, New York City.
GOODRICH, PROF. H. B., Wesleyan University, Middletown, Connecticut.
GOTTSCHALL, DR. GERTRUDE Y., 230 Central Park West, New York
City.
GRAHAM, DR. J. Y., University of Alabama, University, Alabama.
GRAVE, PROF. B. H., DePauw University, Greencastle, Indiana.
GRAVE, PROF. CASWELL, Washington University, St. Louis, Missouri.
GRAY, PROF. IRVING E., Duke University, Durham, North Carolina.
GREGORY, DR. LOUISE H., Barnard College, Columbia University, New
York City.
GUTHRIE, DR. MARY J., University of Missouri, Columbia, Missouri.
GUYER, PROF. M. F., University of Wisconsin, Madison, Wisconsin.
HADLEY, DR. CHARLES E., Teachers College, Montclair, New Jersey.
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 Univer-
sity, St. Louis, Missouri.
HANCE, DR. ROBERT T., University of Pittsburgh, Pittsburgh, Pennsyl-
vania.
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 Uni-
versity, New York City.
HARPER, PROF. R. A., Columbia University, New York City.
HARRISON, PROF. Ross G., Yale University, New Haven, Connecticut.
HARTLINE, DR. H. KEFFER, University of Pennsylvania, Philadelphia,
Pennsylvania.
HARVEY, DR. ETHEL BROWNE, 48 Cleveland Lane, Princeton, New
Jersey.
HARVEY, DR. E. NEWTON, Guyot Hall, Princeton University, Princeton,
New Jersey.
HAYDEN, DR. MARGARET A., Wellesley College, Wellesley, Massachu-
setts.
56 MARINE BIOLOGICAL LABORATORY
HAYES, DR. FREDERICK R., Zoological Laboratory, Dalhousie University,
Halifax, Nova Scotia.
HAYWOOD, DR. CHARLOTTE, Mount Holyoke College, South Hadley,
Massachusetts.
HAZEN, DR. T. E., Barnard College, Columbia University, New York
City.
HECHT, DR. SELIG, Columbia University, New York City.
HEILBRUNN, DR. L. V., Department of Zoology, University of Penn-
sylvania, Philadelphia, Pennsylvania.
HENDEE, DR. ESTHER CRISSEY, Russell Sage College, Troy, New York.
HENSHAW, DR. PAUL S., Memorial Hospital, 2 West 106th Street, New
York City.
HESS, PROF. WALTER N., Hamilton College, Clinton, New York.
HIBBARD, DR. HOPE, Department of Zoology, Oberlin College, Oberlin,
Ohio.
HILL, DR. SAMUEL E., Department of Biology, Princeton University,
Princeton, New Jersey.
HISAW, DR. F. L., Harvard University, Cambridge, Massachusetts.
HOADLEY, DR. LEIGH, Harvard University, Cambridge, Massachusetts.
HOBER, DR. RUDOLF, University of Pennsylvania, Philadelphia, Penn-
sylvania.
HODGE, DR. CHARLES, IV., Temple University, Department of Zoology,
Philadelphia, Pennsylvania.
HOGUE, DR. MARY J., 503 N. High Street, West Chester, Pennsylvania.
HOLLAENDER, DR. ALEXANDER, c/o National Institute of Health, Labora-
tory of Ind. Hygiene, 25th and E Street, N.W., Washington, D. C.
HOOKER, PROF. DAVENPORT, University of Pittsburgh, School of Medi-
cine, Department of Anatomy, Pittsburgh, Pennsylvania.
HOPKINS, DR. DWIGHT L., Mundelein College, 6363 Sheridan Road,
Chicago. Illinois.
HOPKINS, DR. HOYT S., New York University, College of Dentistry,
New York City.
HOWE, DR. H. E., 2702 36th Street, N.W.. Washington, D. C.
HOWLAND, DR. RUTH B., Washington Square College, New York Uni-
versity, Washington Square East, New York City.
HOYT, DR. WILLIAM D., Washington and Lee University, Lexington,
Virginia.
HYMAN, DR. LIBBIE H., 85 West 166th Street, New York City.
IRVING, PROF. LAURENCE, Swarthmore College, Swarthmore, Pennsyl-
vania.
JACKSON, PROF. C. M., University of Minnesota, Minneapolis, Minne-
sota.
REPORT OF THE DIRECTOR 57
JACOBS, PROF. MERKEL H., School of Medicine, University of Pennsyl-
vania, Philadelphia, Pennsylvania.
JENKINS, DR. GEORGE B., George Washington University, 1335 M
Street, N.W., Washington, D. C.
JENNINGS, PROF. H. S., Johns Hopkins University, Baltimore, Mary-
land.
JEWETT, PROF. J. R., 44 Francis Avenue, Cambridge, Massachusetts.
JOHLIN, DR. J. M., Vanderbilt University Medical School, Nashville,
Tennessee.
JONES, DR. E. RUFFIN, JR., College of William and Mary, Norfolk,
Virginia.
JUST, PROF. E. E., Howard University, Washington, D. C.
KAUFMANN, PROF. B. P., Carnegie Institution, Cold Spring Harbor,
Long Island, New York.
KEEFE, REV. ANSELM M., St. Norbert College, West Depere, Wisconsin.
KEIL, PROF. ELSA M., Zoology Department, New Jersey College for
Women, New Brunswick, New Jersey.
KIDDER, DR. GEORGE W., Brown University, Providence, Rhode Island.
KILLE, DR. FRANK R., Swarthmore College, Swarthmore, Pennsylvania.
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.
KINGSBURY, PROF. B. F., Cornell University, Ithaca, New York.
KNOWER, PROF. H. McE., Woods Hole, Massachusetts.
KNOWLTON, PROF. F. P., Syracuse University, Syracuse, New York.
KOPAC, DR. M. J., Washington Square College, New York University,
New York City.
KORR, DR. I. M., Department of Physiology, Washington Square Col-
lege, New York University, New York City.
KRAHL, DR. M. E., Lilly Research Laboratories, Indianapolis, Indiana.
KRIEG, DR. WENDELL J. S., New York University, College of Medicine,
477 First Avenue, New York City.
LANCEFIELD, DR. D. E., Queens College, Flushing, New York.
LANGE, DR. MATHILDE M., Wheaton College, Norton, Massachusetts.
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.
LINTON, PROF. EDWIN, University of Pennsylvania, Philadelphia, Penn-
sylvania.
LOEB, PROF. LEO, Washington University Medical School, St. Louis,
Missouri.
58 MARINE BIOLOGICAL LABORATORY
LOWTHER, MRS. FLORENCE DEL., Barnard College, Columbia University,
New York City.
LUCAS, DR. ALFRED M., Zoological Laboratory, Iowa State College,
Ames, Iowa.
LUCAS, DR. MIRIAM SCOTT, Department of Zoology, Iowa State College,
Ames, Iowa.
LUCRE, PROF. BALDUIN, University of Pennsylvania, Philadelphia, Penn-
sylvania.
LUSCOMBE, MR. W. O., Woods Hole, Massachusetts.
LYNCH, DR. CLARA J., Rockefeller Institute, 66th Street and York Ave-
nue, New York City.
LYNCH, DR. RUTH STOCKING, Maryland State Teachers College, Tow-
son, Maryland.
MACCARDLE, DR. Ross C, School of Medicine, Duke University, Dur-
ham, North Carolina.
MACDOUGALL, DR. MARY S., Agnes Scott College, Decatur, Georgia.
MACLENNAN, DR. RONALD F., State College of Washington, Pullman,
Washington.
McCLUNG, PROF. C. E., University of Pennsylvania, Philadelphia, Penn-
sylvania.
MCGREGOR, DR. J. H., Columbia University, New York City.
MACKLIN, DR. CHARLES C., School of Medicine, University of Western
Ontario, London, Canada.
MAGRUDER, DR. SAMUEL R., Department of Anatomy, Tufts Medical
School, Boston, Massachusetts.
MALONE, PROF. E. F., College of Medicine, University of Cincinnati,
Department of Anatomy, Cincinnati, Ohio.
MANWELL, DR. REGINALD D., Syracuse University, Syracuse, New
York.
MARSLAND, DR. DOUGLAS A., Washington Square College, New York
University, New York City.
MARTIN, PROF. E. A., Department of Biology, Brooklyn College, 80
Willoughby Street, Brooklyn, New York.
MAST, PROF. S. O., Johns Hopkins University, Baltimore, Maryland.
MATHEWS, PROF. A. P., University of Cincinnati, Cincinnati, Ohio.
MATTHEWS, DR. SAMUEL A., Thompson Biological Laboratory, Wil-
liams College, Williamstown, Massachusetts.
MAYOR, PROF. JAMES W., Union College, Schenectady, New York.
MAZIA, DR. DANIEL, Department of Zoology, University of Missouri,
Columbia, Missouri.
MEDES, DR. GRACE, Lankenau Research Institute, Philadelphia, Penn-
sylvania.
REPORT OF THE DIRECTOR
MEIGS, DR. E. B., Dairy Division Experimental Station, Beltsville,
Maryland.
MEIGS, MRS. E. B., 1736 M Street, N.W., Washington, D. C.
METCALF, PROF. M. M., 51 Annawan Road, Waban, Massachusetts.
METZ, PROF. CHARLES W., Johns Hopkins University, Baltimore, Mary-
land.
MICHAELIS, DR. LEONOR, Rockefeller Institute, 66th Street and York
Avenue, New York City.
MILLER, DR. J. A., Department of Anatomy, University of Michigan,
Ann Arbor, Michigan.
MITCHELL, DR. PHILIP H., Brown University, Providence, Rhode Is-
land.
MOORE, DR. CARL R., The University of Chicago, Chicago, Illinois.
MOORE, PROF. J. PERCY, University of Pennsylvania, Philadelphia,
Pennsylvania.
MORGULIS, DR. SERGIUS, University of Nebraska, Omaha, Nebraska.
MORRILL, PROF. C. V., Cornell University Medical College, 1300 York
Avenue, New York City.
NAVEZ, DR. ALBERT E., Department of Biology, Milton Academy, Mil-
ton, Massachusetts.
NEAL, PROF. H. V., Tufts College, Tufts College, Massachusetts.
NELSEN, DR. OLIN E., Department of Zoology, University of Pennsyl-
vania, Philadelphia, Pennsylvania.
NEWMAN, PROF. H. H., The University of Chicago, Chicago, Illinois.
NICHOLS, DR. M. LOUISE, Rosemont, Pennsylvania.
NOBLE, DR. GLADWYN K., American Museum of Natural History, New
York City.
NONIDEZ, DR. JOSE F., Cornell University Medical College, 1300 York
Avenue, New York City.
NORTHROP, DR. JOHN H., The Rockefeller Institute, Princeton, New
Jersey.
OKKELBERG, DR. PETER, Department of Zoology, University of Michi-
gan, Ann Arbor, Michigan.
OSBURN, PROF. R. C., Ohio State University, Columbia, Ohio.
OSTERHOUT, MRS. W. J. V., Rockefeller Institute, 66th Street and York
Avenue, New York City.
OSTERHOUT, PROF. W. J. V., Rockefeller Institute, 66th Street and
York Avenue, New York City.
PACKARD, DR. CHARLES, Columbia University, Institute of Cancer Re-
search, 168th Street and Broadway, New York City.
PAGE, DR. IRVINE H., Lilly Laboratory Clinical Research, Indianapolis
City Hospital, Indianapolis, Indiana.
60 MARINE BIOLOGICAL LABORATORY
PAPPENHEIMER, DR. A. M., Columbia University, New York City.
PARKER, PROF. G. H., Harvard University, Cambridge, Massachusetts.
PARMENTER, DR. C. L., Department of Zoology, University of Penn-
sylvania, Philadelphia, 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.
PEARL, PROF. RAYMOND, Institute for Biological Research, 1901 East
Madison Street, Baltimore, Maryland.
PEEBLES, PROF. FLORENCE, Chapman College, Los Angeles, California.
PINNEY, DR. MARY E., Milwaukee-Downer College, Milwaukee, Wis-
consin.
PLOUGH, PROF. HAROLD H., Amherst College, Amherst, Massachusetts.
POLLISTER, DR. A. W., Columbia University, New York City.
POND, DR. SAMUEL E., Marine Biological Laboratory, Woods Hole,
Massachusetts.
PRATT, DR. FREDERICK H., Boston University, School of Medicine,
Boston, Massachusetts.
PROSSER, DR. C. LADD, Clark University, Worcester, Massachusetts.
RAFFEL, DR. DANIEL, Institute of Genetics, Academy of Sciences, Mos-
cow, U. S. S. R.
RAND, DR. HERBERT W., Harvard University, Cambridge, Massachu-
setts.
RANKIN, DR. JOHN S., Biology Department, Amherst College, Amherst,
Massachusetts.
REDFIELD, DR. ALFRED C., Harvard University, Cambridge, Massa-
chusetts.
REESE, PROF. ALBERT M., West Virginia University, Morgantown,
West Virginia.
DERENYI, DR. GEORGE S., Department of Anatomy, University of Penn-
sylvania, Philadelphia, Pennsylvania.
REZNIKOFF, DR. PAUL, Cornell University Medical College, 1300 York
Avenue, New York City.
RICE, PROF. EDWARD L., Ohio Wesleyan University, Delaware, Ohio.
RICHARDS, PROF. A., University of Oklahoma, Norman, Oklahoma.
RICHARDS, DR. O. W., Research Department, Spencer Lens Company, 19
Doat Street, Buffalo, New York.
RIGGS, LAWRASON, JR., 120 Broadway, New York City.
ROGERS, PROF. CHARLES G., Oberlin College, Oberlin, Ohio.
ROMER, DR. ALFRED S., Harvard University, Cambridge, Massachusetts.
REPORT OF THE DIRECTOR 61
ROOT, DR. R. W., Department of Biology, College of the City of New
York, Convent Avenue and 139th Street, New York City.
ROOT, DR. W. S., College of Physicians and Surgeons, Department of
Physiology, 630 West 168th Street, New York City.
RUGH, DR. ROBERTS, Department of Zoology, Hunter College, New
York City.
SASLOW, DR. GEORGE, Harvard School of Public Health, 55 Shattuck
Street, Boston, Massachusetts.
SAYLES, DR. LEONARD P., Department of Biology, College of the City of
New York, 139th Street and Convent Avenue, New York City.
SCHAEFFER, DR. ASA A., Biology Department, Temple University, Phila-
delphia, Pennsylvania.
SCHECHTER, DR. VICTOR, College of the City of New York, 139th Street
and Convent Avenue, New York City.
SCHMIDT, DR. L. H., Christ Hospital, Cincinnati, Ohio.
SCHRADER, DR. FRANZ, Department of Zoology, Columbia University,
New York City.
SCHRADER, DR. SALLY HUGHES, Department of Zoology, Columbia Uni-
versity, New York City.
SCHOTTE, DR. OSCAR E., Department of Biology, Amherst College, Am-
herst, Massachusetts.
SCHRAMM, PROF. J. R., University of Pennsylvania, Philadelphia, Penn-
sylvania.
SCOTT, DR. ALLAN C., Union College, Schenectady, New York.
SCOTT, DR. ERNEST L., Columbia University, New York City.
SCOTT, PROF. WILLIAM B., 7 Cleveland Lane, Princeton, New Jersey.
SEMPLE, MRS. R. BOWLING, 140 Columbia Heights, Brooklyn, New
York.
SEVERINGHAUS, DR. AURA E., Department of Anatomy, College of
Physicians and Surgeons, 630 W. 168th Street, New York City.
SHAPIRO, DR. HERBERT, Department of Biology, Clark University,
Worcester, Massachusetts.
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.
SIVICKIS, DR. P. B., Pasto Deze 130, Kaunas, Lithuania.
SLIFER, DR. ELEANOR H., Department of Zoology, State University of
Iowa, Iowa City, Iowa.
62 MARINE BIOLOGICAL LABORATORY
SMITH, DR. DIETRICH CONRAD, Department of Physiology, University
of Maryland, School of Medicine, Lombard and Greene Streets,
Baltimore, Maryland.
SNOW, DR. LAETITIA M., Wellesley College, Wellesley, Massachusetts.
SOLLMAN, DR. TORALD, Western Reserve University, Cleveland, Ohio.
SONNEBORN, DR. T. M., Johns Hopkins University, Baltimore, Mary-
land.
SPEIDEL, DR. CARL C., University of Virginia, University, Virginia.
SPENCER, DR. W. P., Department of Biology, College of Wooster,
Wooster, Ohio.
STABLER, DR. ROBERT M., Department of Zoology, University of Penn-
sylvania, Philadelphia. Pennsylvania.
STARK, DR. MARY B., New York Homeopathic Medical College and
Flower Hospital, New York City.
STEINBACH, DR. HENRY BURR, Columbia University, New York City.
STERN, DR. CURT, Department of Zoology, University of Rochester,
Rochester, New York.
STEWART, DR. DOROTHY R., Skidmore College, Saratoga Springs, New
York.
STOCK ARD, PROF. C. R., Cornell University Medical College, 1300 York
Avenue, New York City.
STOKEY, DR. ALMA G., Department of Botany, Mount Holyoke College,
South Hadley, Massachusetts.
STRONG, PROF. O. S., College of Physicians and Surgeons, Columbia
University, New York City.
STUNKARD, DR. HORACE W., New York University, University Heights,
New York City.
STURTEVANT, DR. ALFRED H., California Institute of Technology, Pasa-
dena, California.
SUMMERS, DR. FRANCIS MARION, Department of Biology, College of the
City of New York, New York City.
SUMWALT, DR. MARGARET, Department of Pharmacology, University of
Michigan, Ann Arbor, Michigan.
SWETT, DR. FRANCIS H., Duke University Medical School, Durham,
North Carolina.
TAFT, DR. CHARLES H., JR., University of Texas Medical School, Gal-
veston, Texas.
TASHIRO, DR. SHIRO, Medical College, University of Cincinnati, Cin-
cinnati, Ohio.
TAYLOR, DR. WILLIAM R., University of Michigan, Ann Arbor, Michi-
gan.
REPORT OF THE DIRECTOR 63
TENNENT, PROF. D. H., Bryn Mawr College, Bryn Mawr, Pennsylvania.
TsWiNKEL, DR. L. E., Department of Zoology, Smith College, North-
ampton, Massachusetts.
TURNER, DR. ABBY, Department of Physiology, Mount Holyoke College,
South Hadley, Massachusetts.
TURNER, PROF. C. L., Northwestern University, Evanston, Illinois.
TYLER, DR. ALBERT, California Institute of Technology, Pasadena, Cali-
fornia.
UHLENHUTH, DR. EDUARD. University of Maryland, School of Medi-
cine, Baltimore, Maryland.
UNGER. DR. W. BYERS. Dartmouth College, Hanover, New Hampshire.
VISSCHER, DR. J. PAUL, Western Reserve University. Cleveland, Ohio.
WAITE, PROF. F. C., Western Reserve University Medical School, Cleve-
land, Ohio.
WARD, PROF. HENRY B., University of Illinois. Urbana, Illinois.
WARREN, DR. HERBERT S., 1405 Greywall Lane, Overbrook Hills, Penn-
sylvania.
WATERMAN, DR. ALLYN J., Department of Biology, Williams College,
Williamstown, Massachusetts.
WEISS, DR. PAUL A., Department of Zoology, The University of Chi-
cago, Chicago, Illinois.
WENRICH, DR. D. H., University of Pennsylvania, Philadelphia, Penn-
sylvania.
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 Zoology, University of Rochester,
Rochester, New York.
WILSON, PROF. H. V., University of North Carolina, Chapel Hill, North
Carolina.
WILSON, DR. J. W., Brown University, Providence, Rhode Island.
WITSCHI, PROF. EMIL, Department of Zoology, State University of
Iowa, Iowa City, Iowa.
64 MARINE BIOLOGICAL LABORATORY
WOLF, DR. ERNST, Biological Laboratories, Harvard University, Cam-
bridge, Massachusetts.
WOODRUFF, PROF. L. L., Yale University, New Haven, Connecticut.
WOODWARD, DR. ALVALYN E., Zoology Dej irtment, University of
Michigan, Ann Arbor, Michigan.
YNTEMA, DR. C. L., Department of Anatomy, Cornell University Medi-
cal College, 1300 York Avenue, New York City.
YOUNG, DR. B. P., Cornell University, Ithaca, New York.
YOUNG, DR. D. B., 7128 Hampden Lane, Bethesda, Maryland.
ZELENY, DR. CHARLES, University of Illinois, Urbana, Illinois.
STUDIES ON THE TREMATODES OF WOODS HOLE
II. THE LIFE CYCLE OF STEPHANOSTOMUM TENUE (LINTON) l
W. E. MARTIN
(From DePauw University and the Marine Biological Laboratory,
Woods Hole, Massachusetts)
This paper deals with the results of a study of a member of the
trematode family Acanthocolpidae obtained during the summers of
1936 and 1938 at the Marine Biological Laboratory at Woods Hole,
Mass. No previous experimental work has been done on the life
cycles in this family, and consequently the systematic relationships
have been in question. This paper throws some light on these prob-
lems. The results obtained may be of some economic importance
because the adult members of this family are parasitic in marine fishes,
several of which are food fishes.
A synopsis of this work was given before the American Society of
Parasitologists at the 1938 meeting at Richmond, Virginia.
HISTORICAL
Some of the members of the family Acanthocolpidae were at first
assigned to the old pseudogenus, Distomum, and, due to the presence
of spines encircling the mouth, were thought to be related to the
echinostomes. Nicoll (1915) placed some of the acanthocolpids in the
family Allocreadiidae because of the similarity in the arrangement of
the reproductive organs in the two groups. Winfield (1929) criticizes
Nicoll's classification, stating, "The Stephanochasminae should be ex-
cluded (from the Allocreadiidae) because of the Y-shaped excretory
bladder, the circle of head spines, and the armed cirrus and vagina."
The family name, Acanthocolpidae, was created by Liihe in 1909 to
include trematodes whose principal diagnostic characters are: a well-
developed prepharynx and pharynx, a very short esophagus, a Y-
shaped excretory bladder, the ovary in front of the testes, the uterus
between the ovary and the ventral sucker, the cirrus and vagina
armed with spines, and the genital opening medially located anterior
to the ventral sucker. At present the following seven genera are
included in the family: Stephanostomum Looss 1899, Dihemistephanus
Looss 1901, Deropristis Odhner 1902, Acanthocolpus Odhner 1905,
1 This work was made possible through the use of the laboratory facilitirs
maintained by Purdue University at the Marine Biological Laboratory.
65
66 W. E. MARTIN
Acanthopsolus Liihe 1906, Tormopsolus Poche 1925, and Echinostepha-
nus Yamaguti 1934. Because of the presence of connections between
the excretory bladder and the ceca in the genus Echinostephanus,
Yamaguti separated it from the genus Stephanostomum. However,
McFarlane (1936) described such connections in Stephanostomum
casum (Linton) and indications of them in S. tristephanum. This
suggests that a more extensive and intensive study of this character
is needed.
Reports of observations pertaining to the life cycles of members of
this family have appeared from time to time. Lebour (1907) de-
scribed a cercaria that developed in rediae in the limpet, Patella vul-
gata, which she believed to be the larval form of some member of the
genus Stephanostomum. However, this cercaria lacked eyespots, had
a long esophagus and a small sac-shaped excretory bladder, all of
which were contrary to observations on the adult worms. The same
author (1910) described a cercaria from Buccinum undatum which she
thought was the larval form of Acanthopsolus lageniformis. This cer-
caria possessed eyespots and general characteristics which agreed with
the structures of the adult. No experimental work was done to test
the validity of her assumption. Some of the cercariae had tails while
the majority did not, which, in conjunction with the absence of large
glands in the body, was interpreted by Lebour to indicate that no
second intermediate host was required. This seems questionable since
Linton (1898), Stafford (1904), Liihe (1906), Nicoll and Small (1909),
Nicoll (1910), and others have found metacercariae of this family in
various species of fishes. Linton (1898) found cysts of Distomum
valdeinflatum attached to the peritoneum of Alutera schoepfi and
Menidia menidia notata. Stafford (1904) found the cysts of Stephano-
chasmus histrix on the fins of Pseudopleuronectes americanus. Liihe
(1906) found Stephanochasmus ceylonicus encysted in the subcutaneous
tissue of Narcine timlei taken off Dutch Bay, Ceylon. Lebour (1907)
reported Stephanochasmus metacercariae, probably 5. baccatus, under
the skin of the dab, witch, and long rough dab. Nicoll and Small
(1909) discovered the cysts of Stephanochasmus baccatus under the
skin of Pleuronectes limanda. They state, " It is not at all improbable
that the cercariae of 5. caducus, S. triglae, and 5. baccatus are all to
be found encysted in young pleuronectid fishes." Nicoll (1910) re-
ported finding cysts of S. baccatus in Drepanopsetta platessoides.
Yamaguti (1934) found cysts of Stephanochasmus sp. with 46 collar
spines in Lotella physis and Engraulis japonica, S. sp. with 36 collar
spines in Argentina kagoshimae, and 5. sp. with 54 collar spines in
Bothrocara zesta and Furcimarius sp. He also found Echinostephanus
LIFE CYCLE OF STEPHANOSTOMUM TENUE 67
sp. with 40 collar spines encysted in the flesh of Argentina kagoshimae.
The same author (1937) reported Stephanochasmus bicoronatus cysts
in the body cavity of Acanthogobius hasta and on the gills of Sciaena
sp. and Taenioides lacepedi; Echinostephanus hispidis cysts in the flesh
of Psendorhombus pentophthalmus and Neopercis sexfasciatus and Tor-
mopsolus larvae encysted near the gills of Leiognathus rivulata.
MATERIAL AND METHODS
The snail, Nassa obsoleta, which serves as the first intermediate
host, Menidia menidia notata the second intermediate host, and the
puffer, Spheroides maculatus, which serves as the experimental defini-
tive host, were all collected in the vicinity of Woods Hole. Naturally
infected snails were used as sources of cercariae. Some Menidia and
Spheroides were used for experimental feedings while others were re-
tained as controls.
Living material was used in the study of many of the cercarial
structures. Bouin's solution and a saturated aqueous solution of
mercuric chloride were used as fixatives. Mayer's paracarmine was
used to stain toto mounts, while sectioned material was stained with
Ehrlich's hematoxylin. Infected snails, isolated in finger bowls filled
with sea water, furnished a plentiful supply of cercariae for the experi-
mental infection of Menidia.
OBSERVATIONS AND DESCRIPTIONS
The life cycle of Stephanostomum tenue involves the production of
rediae and cercariae in the digestive gland of the marine snail, Nassa
obsoleta, the development of the metacercariae in cysts in the liver of
the small fish, Menidia menidia notata, and the maturation of the worm
in the intestine of the puffer, Spheroides maculatus.
All measurements listed in this paper are expressed in millimeters.
The Redia (Figs. 3 and 4)
Natural infections of this trematode were found in about A per
cent of the several thousand Nassa obsoleta under observation. Some
increase in the number of infected snails in the latter part of the sum-
mer was noted, which may be correlated with the migratory habits
of the hosts of the adult worms. The redia is an elongate, saccular
structure with a pharynx and short rhabdocoel gut. The length of
the gut, however, varies with age since it is nearly two-thirds the
length of the very young redia (Fig. 4). The young redia also exhibits
marked motility. The length of the redia varies from 0.14 to 0.66
with an average of about 0.5; the width varies from 0.03 to 0.14 with
68 W. E. MARTIN
an average of about 0.10. The pharynx varies from about 0.025 long
by 0.028 wide to 0.052 long by 0.029 wide. The number of germ balls
and cercariae per redia varies from 0 to 14 for the former and 0 to 5
for the latter. No ambulatory processes were present and no birth
pore was observed.
The Cercaria (Fig. 1)
The cercaria is of the ophthalmoxiphidio type with a simple tail.
In swimming the tail is lashed back and forth while the body is held
almost straight. In finger bowls of sea water the cercariae swim about
for a short time and then settle to the bottom to which they adhere
by the tips of their tails. No special glandular bodies were found in
the distal region of the tail that might account for this adhesive action.
This behavior may be of importance in the completion of the life cycle
since the cercariae may become attached to food particles and may be
eaten by fishes. The cercaria exhibits a positive response to light.
The cuticula of the body is spinous with larger spines on the an-
terior end. In addition to the spines there are seven or eight setae
projecting from each side of the body. These are irregularly spaced
along the entire body length. The oral sucker bears two rows (of
21 each) of alternating large spines about 0.005 long. These spines
are easily detached under even slight cover-glass pressure. The body
length varies with the degree of contraction from 0.145 to 0.38 with
an average of 0.24, while the body width varies from 0.045 to 0.086
with an average of 0.064. The tail averages about 0.183 long by
0.031 wide. The oral sucker averages about 0.031 long by 0.030 wide
while the ventral sucker averages about 0.033 long by 0.030 wide.
The ventral sucker bears two rows of small papillae with about 65
papillae in each row. Projecting anteriorly above the oral sucker
there is a simple spear-shaped stylet about 0.014 long. The mouth
EXPLANATION OF PLATE
All drawings were made with the aid of a camera lucida. Abbreviations used:
CG, cephalic gland; EB, excretory bladder; EG, esophageal gland; ES, eyespot;
G, genital anlage; GB, germ ball; GP, genital pore; /, intestine; 0, oral sucker; OS,
oral spines; OV, ovary; P, pharynx; PP, prepharynx; S, stylet; T, testes; V, vitellaria;
VG, vesicular gland; VS, ventral sucker.
FIG. 1. Ventral view of cercaria.
FIG. 2. Stylet of cercaria.
FIG. 3. Redia with germ balls and cercaria.
FIG. 4. Young redia showing elongate intestine.
FIG. 5. Metacercaria.
FIG. 6. Adult.
LIFE CYCLE OF STEPHANOSTOMUM TENIIE
J.
5
70 W. E. MARTIN
opens into a long narrow prepharynx approximately 0.038 long. The
pharynx is subglobular and measures about 0.012 in length and
width. The esophagus is extremely short. The rudimentary intes-
tine branches just anterior to the ventral sucker and the branches do
not extend beyond this organ. Two conspicuous eyespots are located,
one on each side of the body, near the oral sucker. Four cephalic
glands are located on each side of the body immediately lateral and
anterior to the ventral sucker. On each side of the body the ducts
from two glands pass anteriad median to the eyespot while the ducts
from the other two glands pass anteriad lateral to the eyespot. The
ducts of all four glands open to the exterior at the anterior end of the
body. Other glands include numerous vesicular glands along the wall
of the excretory bladder. The weakly Y-shaped excretory bladder
extends almost to the ventral sucker. In some specimens the anterior
wall of the bladder has a scalloped appearance. The main collecting
ducts arise from the anterior margin of the excretory bladder and pass
anteriad to the level of the eyespots where they bend on themselves
and pass posteriad to supply both sides of the body. The flame cells
are in seven groups of threes, with the first group given off just after
the main duct bends posteriorly at the eyespot level. The other
groups are given off at intervals along the side of the body.
The reproductive system is represented by a mass of deeply stain-
ing cells located just posterior to the ventral sucker and partially
surrounded by the anterior wall of the excretory bladder.
The Metacercaria (Fig. 5)
The cercariae are taken into the digestive tract of the second
intermediate host, Menidia menidia notata, where they work their
way through the intestinal wall and encyst in the liver or mesenteries.
No cercariae were observed to penetrate the bodies of the fishes
through the skin. The metacercaria increases to several times the
size of the cercaria. The 42 collar spines also increase in size until
they are approximately 0.050 long. The eyespots and the glandular
cells surrounding the excretory bladder undergo disintegration. There
is a marked increase in the size of the pharynx. The branches of the
intestine develop until they reach to near the posterior end of the
body. The metacercaria is held within a rather tough, loose encyst-
ment sac.
The Adult (Fig. 6)
Nearly mature adult worms were obtained by feeding pieces of
Menidia liver containing metacercariae to young puffers, Spheroides
maculatus. The puffers were examined about two weeks after the
LIFE CYCLE OF STEPHANOSTOMUM TENUE 71
initial feeding and the worms were recovered from the intestine.
Remnants of the eyespots were still present. The oral spines were
the same in number and approximately of the same size as in the
metacercaria. The relative proportions of the suckers and the pharynx
were about the same as in the metacercaria. Advances in develop-
ment over the conditions found in the metacercaria are: the differen-
tiated testes and ovary located in the posterior one-third of the body,
the reproductive tubes extending from these organs to the genital
pore located on the mid-ventral side of the body immediately anterior
to the ventral sucker, and the small clusters of vitelline cells extending
along the sides of the body from the posterior end of the pharynx to
near the posterior end of the body. Complete functional maturity of
the reproductive systems had not been attained since no eggs had
been produced.
The following measurements and description are based on but a
few worms so that the range of variation is probably less than would
be found with a larger number of individuals. Body length 1.9 to
2.2, width 0.5; oral sucker 0.13 long by 0.18 wide; ventral sucker 0.22
long by 0.25 wide; prepharynx from 0.19 to 0.31 in length by about
0.015 in width near the oral sucker to 0.031 at its widest point near
the pharynx; pharynx about 0.22 long by 0.16 to 0.18 in width; esoph-
agus 0.04 to 0.07 long; ovary about 0.057 long by 0.03 to 0.038 wide;
anterior testis 0.10 to 0.136 long by 0.04 to 0.07 wide, posterior testis
0.09 to 0.14 long by 0.04 to 0.07 wide.
Linton (1898) described Distomum tenue from the rectum of the
striped bass, Roccus lineatus, collected at Woods Hole. The descrip-
tion he gave, with measurements in millimeters, is as follows: oral
spines 0.051 long by 0.018 wide at base; esophagus 0.44 long by 0.34
wide (he undoubtedly has used the term esophagus for the pharynx) ;
vitellaria voluminous, peripheral in the posterior region; genital aper-
ture immediately in front of the ventral sucker; ova not numerous and
comparatively large, lying close behind the ventral sucker; ova length
0.088, width 0.044; body length 2.9, width 0.28; diameter of oral
sucker 0.26, of ventral sucker 0.38.
DISCUSSION AND CONCLUSIONS
Most descriptions of the adult members of this family show them
to have remnants of eyespots. This may indicate that the family
represents a fairly compact, closely related group. When the excre-
tory bladder is mentioned at all in the descriptions of species, it is
described as Y-shaped. However, in my study of living specimens of
Deropristis inflata, a simple tubular or sac-shaped bladder was found.
72 W. E. MARTIN
There is very little in the literature on the rest of the excretory system
although Pratt (1916) in his description of Stephanochasmus casum
showed that the main collecting tubes pass anteriad to near the level
of the eyespots without giving off secondary tubes.
The arrangement of the reproductive organs in the family Acan-
thocolpidae, as was pointed out by Nicoll (1915), is similar to the
arrangement of these organs in the family Allocreadiidae. There is
also some suggestion of similarity in the excretory systems of these
two groups. In addition, the members of both families are primarily
parasites of fishes. This suggests a rather close relationship between
the two families. However, the elucidation of the life cycles of other
genera is needed before a positive statement can be made.
The family Acanthocolpidae seems to be cosmopolitan in distri-
bution since some of its members have been found in European, Green-
land, North American, Japanese, and Ceylonese waters.
There has been some confusion in the literature concerning the
generic name Stephanostomum. This confusion resulted from Looss'
first (1899) naming the genus Stephanostomum and then changing it
to Stephanochasmus (1900) because of its similarity to the genus
Stephanostoma Danielson and Koren, a genus of Gephyrean worms.
SUMMARY
It was found that the life cycle of Stephanostomum, tenue involves
the development of rediae and cercariae in the marine snail, Nassa
obsoleta, the utilization of the small fish, Menidia menidia notata, as
the second intermediate host, and the development of the adult worm
in the intestine of the puffer, Spheroides maculatus. Although the
puffer may serve as the experimental definitive host, the striped bass,
Roccus lineatus, is probably a natural one.
About .4 per cent of the Nassa obsoleta observed were infected with
this parasite.
The excretory system of the cercaria is represented by the formula
The arrangement of the reproductive organs, some similarity in
the excretory systems, and the fact that fishes serve as hosts to the
adult worms suggest an affinity of the Acanthocolpidae to the family
Allocreadiidae.
LITERATURE CITED
LEBOUR, MARIE V., 1907. Fish trematodes of the Northumberland coast. North-
umberland Sea Fish. Rep. 23—67.
LEBOUR, MARIE V., 1910. Acanthopsolus lageniformis n. sp., a trematode in the
catfish. Northumberland Sea Fish. Comm. Rep. 1909-1910, pp. 29-35.
LINTON, E., 1898. Notes on trematode parasites of fishes. Proc. U. S. Nat. Mnx.,
20: 507-548.
LIFE CYCLE OF STEPHANOSTOMUM TENUE
Looss, A., 1899. Weitere Beitrage zur Kenntniss der Trematodenfauna Aegyptens,
zugleich Versuch einer natiirlichen Gliederungdes Genus Distomum Retzius.
Zool. Jahrb. Abt. Syst., 12: 521-784.
Looss, A., 1900. Nachtragliche Bemerkungen zu den Namen der von mir vor-
geschlagenen Distomidengattungen. Zool. Anz., 23: 601-608.
Looss, A., 1901. Ueber die Fasciolidengenera Stephanochasmus, Acanthochasmus
und einigeandere. Centralbl. Bakt. Parasit., 29: 595-606, 628-634, 654-661.
LUHE, MAX, 1906. Trematode parasites from the marine fishes of Ceylon. Ceylon
Pearl Oyster Fish, and Marine Biol., Pt. 5: 97-108.
LUHE, MAX, 1909. Parasitische Plattwiirmer 17. I. Trematodes. In A. Brauer's,
Die Siisswasserfauna Deutschlands.
MARTIN, W. E., 1938. The life cycle of Stephanostomum tenue (Linton), family
Acanthocolpidae. (Abstract.) Jour. ParasitoL, 24 (Supplement): 27.
McFARLANE, S. H., 1936. A study of the endoparasitic trematodes from marine
fishes of Departure Bay, B.C. Jour. Biol. Bd. Canada, 2: 335-347.
NICOLL, WM., 1910. On the entozoa of fishes from the Firth of Clyde. ParasitoL,
3: 322-359.
NICOLL, WM., 1915. A list of the trematode parasites of British marine fishes.
ParasitoL, 7: 339-378.
NICOLL, WM., AND WM. SMALL, 1909. Notes on larval trematodes. Ann. Mag. Nat.
Hist. (Ser. 8), 3: 237-246.
ODHNER, TH., 1902. Mittheilungen sur Kenntniss der Distomen. II. Drei neue
Distomen aus der Gallenblase von Nilfischen. Centralbl. Bakter. Orig. (4)
31: 152-162.
ODHNER, TH., 1905. Die Trematoden des arktischen Gebietes. Fauna Arctica,
(2) 4: 291-372.
POCHE, FRANZ, 1925. Das System der Platoderia. Arch. Naturg., 91: 1-458.
PRATT, H. S., 1916. The trematode genus Stephanochasmus Looss in the Gulf ol
Mexico. ParasitoL, 8: 229-238.
STAFFORD, J., 1904. Trematodes from Canadian fishes. Zool. Anz., 27: 481-495.
WINFIELD, G. F., 1929. Plesiocreadium typicum, a new trematode from Amia calva.
Jour. ParasitoL, 16: 81-87.
YAMAGUTI, S., 1934. Studies on the helminth fauna of Japan. Pt. 2. Jap. Jour.
Zool., 5: 249-541.
YAMAGUTI, S., 1937. Studies on the helminth fauna of Japan. Pt. 20. Larval
trematodes from marine fishes. Jap. Jour. Zool., (3) 7: 491-499.
AN HERMAPHRODITE ARBACIA
ETHEL BROWNE HARVEY
(From the Biological Laboratory, Princeton University, and the
Marine Biological Laboratory, Woods Hole, Mass.}
Among the many thousands of Arbacia punctulata opened in the
course of ten summers at Woods Hole, and many hundreds of Arbacia
pustulosa, Spliaer echinus granularis, Paracentrotus lividus and Par echi-
nus microtuberculatus opened during several springs at Naples, and
many hundreds of Strongylocentrotus drobachiensis, from Maine, I ob-
served last summer for the first time an hermaphrodite sea urchin, an
Arbacia punctulata, opened on July 4, 1938. One other case of her-
maphroditism in Arbacia punctulata has been described by Shapiro
(1935); it was found late in the season of 1934 at Woods Hole. His
animal had four testes and one ovary. It was fertile inter se, and all
the eggs formed fertilization membranes, but the cleavages were de-
layed and abnormal. Many blastulae were obtained and 30 per cent
gave rise to gastrulae; there was apparently no further development.
James Gray (1921) "described a Strongylocentrotus lividus in which three
of the gonads were completely female, another almost completely so
and the fifth contained both eggs and sperm which were fertile inter se;
development of the eggs is not described. Gadd (1907) described a
case of hermaphroditism in Strongylocentrotus drobachiensis at the
Mourmanschen Biological Station which had four female gonads and
one male, but he does not give any details. The above are the only
recorded cases of hermaphroditism in sea urchins, and it is indeed a
rare phenomenon.
The hermaphrodite Arbacia which I found last summer was quite
normal in external appearance and of average size. On removing the
ventral portion of the shell, as usual in preparing the eggs, the gonads
looked normal except that four were red ovaries and the fifth a white
testis with sperm oozing out. Photograph 1 is of the five gonads
immediately after removal to sea water. Microscopic observation of
the living gonads showed that none of them was entirely male or
female. The ovaries had a few tubules containing sperm and the
testis contained some ova in various stages of development; that is,
the gonads were really ovo-testes but predominantly female or male.
A portion of a gonad, living and unstained, is shown in Photograph 2;
the ovarian tubules are dark (from the red pigment) and the testis
tubules are light with scattered pigment spots; a few eggs have been
liberated and lie free in the space between the tubules. A stained
74
EXPLANATION OF PLATES
-•.* ^-t
• -•- JtJ* •••
•• »* vss&Fn
PLATE I
PHOTOGRAPH 1. Gonads of the hermaphrodite Arbacia, immediately after re-
moval from the shell; one testis (white) and four ovaries (black). Note the small
piece of testis (white) at edge of the lower right ovary.
PHOTOGRAPH 2. Part of a living gonad, showing testis tubules (white with
pigment spots) and ovarian tubules (black) containing eggs, as seen under the micro-
scope. A few eggs are seen free in the space between the tubules.
PHOTOGRAPH 3. Prepared stained section of a gonad predominantly female
containing eggs in various stages of maturity. One testis tubule is seen at lower
right.
PHOTOGRAPH 4. Prepared stained section of a gonad, predominantly mile,
containing mostly ripe sperm. One ovarian tubule is seen at lower right.
76 ETHEL BROWNE HARVEY
section of a predominantly female gonad is shown in Photograph 3;
all the tubules are filled with eggs in various stages of development
except the lower right which is mostly testis. Photograph 4 is a
section of the predominantly male gonad ; the tubules are all filled with
sperm except one at the lower right wrhich contains eggs. Photo-
graph 5 is a section of a predominantly female gonad showing greater
detail. Sections of normal ovaries and testes are exactly like these
except that there is no mixture. As far as I could tell, especially from
a study of the living gonads, the eggs and sperm in the hermaphrodite
gonad are separate in the small tubules, and do not lie together with-
out any partition. The eggs are not fertilized until they have been
liberated from the tubules into the sea water, probably because the
sperm are not motile until in sea water. As soon as the eggs have
poured out from the tubules into the sea water, they are immediately
fertilized by the sperm which have poured into the sea water and
become active. At any one time, therefore, the fertilized eggs are
found in various stages of development.
The eggs of the hermaphrodite are perfectly fertile with its own
sperm. Normal fertilization membranes are formed, first cleavage
takes place normally and at the normal time, and the later cleavages
also, and practically all the eggs develop. The only unusual phe-
nomenon was the occurrence of giant eggs. These were about 1 per
cent of the total and were all of the same size, 96 /u in diameter, giving
a volume of 463,000 yu3 whereas the normal egg has a diameter of 74 /u
and a volume of 212,000/u3; the giant eggs are approximately twice
the normal volume. The origin of the giant eggs is not known, but
they do not arise from fusion of ripe eggs since giant immature eggs
also occur. I have found similar giant eggs in other Arbacias but
very rarely, and I have also found in another Arbacia normal-sized
eggs with giant nuclei. These nuclei measured 25.6 /u in diameter
giving a volume of 8,785 /u3, whereas the normal nucleus measures
11.5 fj. in diameter, giving a volume of 796 /u3; the giant nuclei are thus
about eleven times the normal volume.
Eggs in late cleavages (3^ hours, and less, after fertilization at
PHOTOGRAPH 5. Prepared stained section of a gonad under higher magnifica-
tion to show greater detail. The gonad was predominantly female, but the portion
photographed predominantly male.
PHOTOGRAPH 6. Self-fertilized eggs 3| hours (21° C.) after opening the animal.
Most of the eggs are in late cleavage stages, but some are not so far advanced since
they have come from the tubules and been fertilized later than the others. Note
the giant eggs, also developing normally.
PHOTOGRAPHS 7-9. Normal development of self-fertilized eggs.
PHOTOGRAPH 7. Very early pluteus, self-fertilized, 31 hours old.
PHOTOGRAPH 8. Pluteus, self-fertilized, 35 hours.
PHOTOGRAPH 9. Pluteus, self-fertilized, 48 hours.
o
78 ETHEL BROWNE HARVEY
21° C.) are shown in Photograph 6, and one may observe here the
giant eggs. The eggs, including the giant ones, develop quite nor-
mally and become swimming blastulae at the normal time, 9 hours
after fertilization. The blastulae develop into perfectly normal plutei
(Photographs 7-9), and these were kept for nine days. The plutei
from the giant eggs were indistinguishable from the others which vary
greatly in size according to age.
The sperm were perfectly normal in fertilizing other eggs as well
as the hermaphrodite eggs (98 per cent), and the eggs from the her-
maphrodite could be fertilized perfectly well by sperm from another
sea urchin. This latter fact was ascertained by putting a small part
of an ovary into fresh water for about | minute to kill the sperm on
the outside; then the ovary was transferred to sea water. After an
hour, only 1 per cent of the eggs shed were fertilized (by a few sperm
liberated from the ovotestis after washing). But when the shed eggs
were transferred to sea water containing sperm from another animal,
98 per cent were fertilized. The fertilization was therefore made by
the sperm of the normal animal. These eggs developed quite nor-
mally. The hermaphrodite animal is therefore fertile with other males
and females as well as inter se.
I think this the first recorded case in which the eggs of an hermaph-
rodite sea urchin, self-fertilized, developed absolutely normally to
perfect plutei.
SUMMARY
1. A rare case of perfect functional hermaphroditism in the sea
urchin Arbacia punctulata is described. There were four red gonads
predominantly female and one white gonad predominantly male; there
were a few tubules of the opposite sex in all the gonads.
2. Fertilization occurred as soon as the sexual products were liber-
ated in sea water.
3. The development of the self-fertilized eggs was absolutely nor-
mal, in time and morphology, and normal plutei were raised, nine
days old.
4. There occurred about 1 per cent of giant eggs; these were twice
the normal volume, and they also developed normally.
5. Both the eggs and the sperm also functioned perfectly normally
with other normal males and females.
LITERATURE CITED
GADD, G., 1907. Ein Fall von Hermaphroditismus bei dem Strongylocentrotus
droebachiensis O. F. Mull. Zool. Anz., 31: 635.
GRAY, J., 1921. Note on true and apparent hermaphroditism in sea urchins. Proc.
Camb. Phil. Soc., 20: 481.
SHAPIRO, H., 1935. A case of functional hermaphroditism in the sea-urchin, Arbacia
punctulata, and an estimate of the sex-ratio. Am. Nat., 69: 286.
KARYOKINESIS DURING CLEAVAGE OF THE ZEBRA
FISH BRACHYDANIO RERIO
EDWARD C. ROOSEN-RUNGE
(From the Department of Biology, Brown University}
INTRODUCTION
The results presented in this paper have been obtained in the
course of a comprehensive study on the periodicity of cell division and
mitotic rate during development. A discussion of these results, con-
fined to observations on teleosts, is a necessary preliminary to the more
complete investigation, with history and literature, to be presented
later.
To obtain a definite picture of the role of cell division in develop-
ment, it is necessary to determine not only the number of mitoses which
occur at given times and at given places, but also the duration of a
single mitosis and the manner in which it proceeds in different stages
of development. In spite of many careful investigations on the rate
of mitosis, the duration of mitosis at different periods of development
has not been sufficiently determined.
The most successful method in investigating the role of cell division
in development has been that of Richards (1935) and others who tried
to determine mitotic activity by means of a mitotic index or the per-
centage number of dividing cells. However, it is not possible to tell
by this method whether mitoses occur at periodic cycles or are evenly
distributed, so that the counting at any time will actually furnish a
figure which approximates the average mitotic rate. The present
paper deals with the manner and duration of mitosis only during
cleavage.
The egg of the zebra fish (Brachydanio rerio] , recently described as
a favorable laboratory subject (Roosen-Runge, 1938), is especially
adapted to this study, because of its rapid development, its trans-
parency, and more particularly because the cell nuclei can be easily
observed in the living egg. Three lines of investigation will be de-
scribed in this paper, namely, (1) the morphology of the living and of
the fixed nuclei; (2) the duration of divisions and of mitotic phases;
and (3) their reaction to temperature changes.
MATERIALS AND METHODS
Information concerning the propagation and raising of the eggs of
the zebra fish may be found in an earlier paper (Roosen-Runge). For
79
80 EDWARD C. ROOSEN-RUNGE
observation of the living egg, a slide with a covering about 1 mm. thick
of a mixture of bee's wax and paraffine was used. A hole, the diameter
of the egg, was then cut through the layer of wax in order to let the
light come through, with a glass ring, 22 mm. wide and 9 mm. high,
added to prevent currents from moving the egg. The slide was im-
mersed in water in a large dish of about 150 cc. capacity, to insure an
abundant oxygen supply. The egg was then oriented in the hole and
all observation carried on, with the slide so immersed, by means of a
water-immersion lens (Zeiss, X 40), having sufficient depth of focus
to make visible the cells inside the cell membrane. Although the
use of an oil-immersion lens is also feasible, it is only useful to check
up on details which on the whole can be seen just as clearly with the
water-immersion lens.
The temperature was regulated with an ordinary desk lamp shining
from varying distances upon the observation dish. This simple device
proved sufficient to keep the temperature constant within the range of
half a degree Centigrade, since the amount of water in which the egg
was kept, being fairly large, made it possible to control the temperature
almost continuously during the period of development. Thus the eggs
continued to develop under the microscope without the least sign of
disturbance from the beginning of the second to the end of the tenth
cleavage, that is, for a period of about three hours.
Bouin's solution was used for fixation. The egg membrane and in
most cases the yolk were removed after fixation, for it is then quite
easy to tear off the membrane from the hardened egg and to remove
the brittle yolk. Dioxan or alcohol + benzol was used for dehydra-
tion, but the former is the simpler method and, therefore, to be pre-
ferred. All sections were stained in Heidenhain's haematoxylin and
cut 6 or 8 microns in thickness.
MORPHOLOGY OF THE NUCLEI
It is impossible to study the nucleus in the living egg before the
first cleavage since the delicate structure is then hidden by coarse
granules which are whirled up at the base of the cell by the streaming
of the protoplasm into the blastodisc. During the first cleavage the
streaming still continues, offering some difficulties to the observer.
Accurate observation of the nucleus becomes possible only when the
cytoplasm clears at the end of the first cleavage. The two nuclei
appear as ovals with a longitudinal axis of approximately 18 M- The
outlines are fine and smooth. Two or three, sometimes more, very
delicate curved lines divide the nucleus into several sections (Fig. 1).
The first signs of mitosis are the swelling of the nucleus and the
KARYOKINESIS DURING CLEAVAGE OF ZEBRA FISH EGG 81
appearance of irregularities in its oval shape when tiny indentations
can be seen at the poles which appear flattened so that the nucleus
assumes a barrel-like shape. Short rays which point toward the
center of the nucleus seem to radiate from the depths of the indenta-
tions. Very often the nucleus appears to be divided lengthwise into
halves by a fine channel which is filled with some substance a shade
darker than the nuclear sap.
In the living egg the appearance of the indentations marks the
beginning of a very rapid disintegration of the nuclear membrane.
The whole circumference appears strongly wrinkled and rapidly fades
out, together with the partition lines inside of the nucleus. In a short
time no traces of nuclear structures are left. By watching very closely,
one can for a moment fancy where the nucleus has been, because this
area appears somewhat lighter and free from the tiny granules which
are a part of the cytoplasm throughout the cell. Before nuclear
structures become visible again, the cell almost completes its division.
The changes in the cytoplasm and the shape of the cell during mitosis
have already been described (Roosen-Runge, 1938).
Sometime after the furrow has completely cut through, there ap-
pears in the center of each daughter cell a group of tiny dark granules.
These granules represent the chromosomes. They swell, become
lighter, and finally appear as little circles or vesicles with very distinct
outlines. The vesicles go on swelling rapidly until they come into
contact with each other, eventually forming one body with a common
but irregularly curved contour. The outlines of the individual vesicles
remain visible for a time, some of them fading out finally, while others
do not disappear until the breakdown of the nuclear membrane in the
next prophase.
Observations on the living nuclei confirm some of the results ob-
tained from sectioned material. The outstanding feature in the
karyokinesis of the teleost blastomeres is the formation of chromosomal
vesicles during the telophase. These chromosomal vesicles are quite
commonly found in early development and are supposed to persist
through the interkinetic phase into the prophase. This interpretation
has been made very probable by A. Richards (1917) and B. G. Smith
(1929) from the study of sections. It can be proved by the study of
living nuclei, in which some of the walls of the vesicles can actually be
seen to persist in the interphase nucleus. Some of the walls, however,
do not remain visible, but this seems to be due to their thinning out
and not to their complete disappearance, since the sections also show
some partitions, very dark and distinct, while others are delicate and
inconspicuous. In many instances the sectioned nuclei can be seen
EDWARD C. ROOSEN-RUNGE
divided into halves, inside of which the vesicles are visible. The halves
are separated by a gap, apparently filled with cytoplasm, which corre-
sponds to the observations on the living nuclei. The halves represent
the paternal and the maternal parts of the chromosome set, as first
described by Moenkhaus (1904) in teleost hybrids, and by many early
workers on other forms.
How the vesicles arise from the anaphase chromosomes and how the
chromosomes are formed from the vesicles in the prophase, cannot be
determined accurately from fixed material, nor do observations of the
living nuclei solve any of these problems. Richards (1917) concluded
that the vesicles are formed by a swelling of the chromosomes so that
finally the walls contain the chromatin material and enclose a space
"filled in from the fluid portion of protoplasm." Smith (1929), on
the other hand, studied the karyokinesis in Cryptobranchus eggs and
found that the vesicular membrane was of cytoplasmic origin, de-
veloped under the influence of the chromosome within. Pictures like
those of Smith certainly cannot be seen in sections of either Fundulus
or Brachydanio eggs. The study of living nuclei only confirms the
impression that the chromosomes actually swell during the telophase
and that the vesicular wall represents the surface of the chromosome
rather than a structure formed de novo from the cytoplasm. My own
material does not show some of the details as distinctly as they appear
(according to Richards) in Fundulus, although the formation of the
vesicles and their persistence through the interphase could be clearly
seen in the sections as well as in the living egg. Nevertheless, the
behavior of the chromosome material still remained puzzling. That
the reader may be better able to appreciate its actual appearance, I
have used photographs (see plate) rather than drawings, as Richards
and others have done. Attempts at drawing present possibly too
great a temptation to express a prejudiced interpretation not justified
by the actual material.
The prophase stage in the karyokinesis of the living blastomere has
already been described. The appearance of the nucleus as a whole cor-
responds very well with the observations of the sections. Because of
the rapidity with which the chromosomes reappear and arrange them-
selves, only a few figures in these phases wall be found in material fixed
at random. However, by closely watching the living nuclei and taking
into account the time necessary for sufficient penetration of the fixing
fluid to arrest the mitosis (about half a minute for Bouin's fluid), it is
possible to fix material in any desired stage. It can then be seen that
the individual chromosomes become clearly visible only immediately
before the breakdown of the nuclear membrane. They seem to begin
KARYOKINESIS DURING CLEAVAGE OF ZEBRA FISH EGG 83
6
PLATE I
EXPLANATION OF FIGURES
FIG. 1. One of the eight blastomeres of a zebra fish egg, living. Nucleus with
a few partition lines within, in the center. X 500.
FIGS. 2-7. Nuclei, fixed in Bouin's, Heidenhain'shaematoxylin, 6-8 yu. X 1100.
The different sizes of the nuclei are due to their belonging to different cleavage stages.
FIG. 2. Prophase in the beginning. Vesicles still visible. Indentations at the
poles.
FIG. 3. Advanced prophase, chromosomes appearing.
FIG. 4. Chromosomes forming metaphase plate. Outline of nucleus still visible.
FIG. 5. Nuclei from cells after the twelfth cleavage, showing typical spireme
formation in the prophase.
FIG. 6. Early telophase. Formation of chromosomal vesicles.
FIG. 7. Late telophase. Vesicles in contact. Paternal and maternal half of
chromosomes apparently separate.
84 EDWARD C. ROOSEN-RUNGE
the arrangement into a plate while still inside of the membrane (Fig. 4).
This fact has been confirmed by observations on the living eggs of
another teleost, Epiplaty chaperi, in which the chromosomes are some-
what more easily discernible in life. Directly after the breakdown of
the nuclear membrane the chromosomes can be seen arranged in a
metaphase plate but very soon afterwards they begin separating.
This observation shows that the disappearance of the membrane
actually occurs relatively late. Individual chromosomes inside the
separate vesicles, as pictured by Richards, could not be found in
sections of the zebra fish egg.
THE DURATION OF CELL DIVISION
The absolute duration of cell division varies tremendously in dif-
ferent animals, and in different cells of the same animal, despite the
fact that karyokinesis is supposed to occur essentially in the same way
in all of them. The duration of mitosis is characteristic for the dif-
ferent kinds of cells. It can only be measured accurately through the
direct observation of living material. The relative time of mitotic
phases has been estimated by using the percentage number of cells
active in the different stages, but in many cases, as will be pointed out
later, this method is very erroneous. It seems, therefore, that direct
observation is the safest method for determining the relative intervals
in cell division.
The most considerable error in measuring the duration of cell
division in life arises from the difficulty in finding any definite point of
departure. Neither the beginning of the prophase nor the last stage
of the telophase can be defined accurately, so that only a very few
events are established sharply enough to serve as marks by which
stages may be measured. In the blastomeres of the zebra fish the
swelling of the nucleus at the beginning of the prophase, the breakdown
of the nucleus, the appearance of the furrow, the completion of the
furrow, the first appearance of the chromosomes in the telophase, and
finally the completion of the rounded nucleus, furnish seven criteria
of very different value. The time of the formation of the furrow, which
means the division of the cytoplasm, can only be used indirectly for
the determination of karyokinetic stages, although it may serve to
subdivide the interval in which nuclear structures cannot be observed
at all. The moment when the nucleus seems completely rounded and
smoothly outlined is almost impossible to define, and its determination
involves a considerable error. The swelling of the nucleus in the early
prophase is also difficult to observe, but it is possible to determine its
approximate beginning somewhat better with the aid of a micrometer
KARYOKINESIS DURING CLEAVAGE OF ZEBRA FISH EGG 85
eye-piece, the scale of which will permit accurate observation of small
changes in size. The reappearance of the chromosomes as tiny gran-
ules in the telophase is an event more easily determined. Under
favorable conditions it is quite possible to watch the optically empty
central area of the cell and to see the chromosomes become visible.
I estimate the possible error under optimal conditions to be not more
than 30 seconds or 3 per cent of the whole time of cleavage. By far
the best mark, because of its rapidity of occurrence, is the breakdown
of the nucleus. The nuclear membrane not only disappears in from
15 to 30 seconds, but the onset of this event is foreshadowed by a series
of preparatory events, namely, the swelling of the nucleus and the
wrinkling of the membrane, which makes it possible to predict the
time of breakdown quite accurately. The error in determining the
precise time of this occurrence is certainly not greater than 15 seconds,
which is about 1.5 per cent of the whole time of cleavage. We have
thus found two marks which seem reliable, because their errors can be
estimated with considerable accuracy at only 1.5 to 3 per cent of the
entire duration of cleavage. All other marks certainly have a higher
error in determination, and if they are to be used for an estimate of
the duration of the mitotic phases, this uncertainty has to be kept
in mind.
The time for each cleavage from the first to the tenth is almost the
same in different eggs, provided that a constant temperature is main-
tained and the oxygen supply is sufficient. During the process of
cleavage the cell divisions follow each other without a typical resting
stage, therefore the cleavage time was measured from the breakdown
of the nucleus to the breakdown of the daughter nuclei. In Table I
the results are compared with those of Jordan and Eycleshymer (1894)
on amphibian blastomeres. The numbers concerning the zebra fish
egg are all averages of at least 10 eggs. It can be seen from Table I
that in every species the divisions show a characteristic duration. In
four of the six animals the divisions show a trend towards acceleration
before they finally begin to slow down. (The more complicated curve
for the Amblystoma egg cannot be discussed here.) The turning point
for this trend comes at different times. In the egg of the zebra fish
the acceleration is at its height during the fifth cleavage. It seems
significant that this is the last division when only one cell layer is
involved, for the sixth cleavage is horizontal and divides the blastoderm
into two layers. The sixth cleavage takes a slightly longer time than
the preceding division, and from then on the process of cleavage grad-
ually becomes slower and slower. Acceleration and retardation seem
to involve the whole mitotic process uniformly and not any of its
86
EDWARD C. ROOSEN-RUNGE
phases differentially. Only during the ninth and tenth cleavages has
a prolonged interkinetic phase been recorded, but as the error in deter-
mining this phase is even greater than for any other, no conclusion can
be drawn from observations made at these stages of development.
TABLE I
Duration of cleavage divisions in amphibian and teleost eggs.* The times enclosed
in brackets refer to individual cases and are not averages.
Temperature, ° C.
Ambly-
stoma
punctatum
Rana
palustris
Diemec-
tylus
viridescens
Bufo
variabilis
Epiplaly
chaperi
Brachy-
danio
rerio
18
18
18
18
24
25
Duration of Cleavage
Divisions
Fertilization to first
cleavage
10 hrs.?
4-5 hrs.
10 hrs.
4-5 hrs.
25 min.?
First to second
cleavage
1 hour
1 hour
2 hrs.
1 hour
2 hrs.
20 min.
Second to third
cleavage
50 min.
1 hour
15 min.
1 hour
1 hour
5 min.
1 hour
2 min.
44 min.
19| min.
Third to fourth
cleavage
55 min.
2 hrs.
15 min.
45 min.
1 hour
1 hour
(43 min.)
19 min.
Fourth to fifth
cleavage
1 hour
40 min.
1 hour
1 hour
(41 min.)
18 min.
Fifth to sixth
cleavage
40 min.
(1 hour
50 min.
(2 hrs.
(39 min.)
\1\ min.
Sixth to seventh
cleavage . ...
35 min.)
(1 hour
45 min.)
(2 hrs.
(39 min.)
18^ min.
Seventh to eighth
cleavage
25 min.)
(1 hour
45 min.)
(2 hrs.)
(40 min.)
19 min.
Eighth to ninth
cleavage
25 min.'
(1 hour
20 i min.
Ninth to tenth
cleavage
25 min.)
20 min.
* The data on amphibian eggs are taken from Jordan and Eycleshymer (1894).
In measuring the relative duration of the mitotic phases every
cleavage can, of course, be observed. Most observations, however,
were made during the sixth to ninth cleavages, since these stages had
to be studied also for the periodicity of divisions, which will be dis-
cussed later. The arbitrary definition of the stages is obviously
KARYOKINESIS DURING CLEAVAGE OF ZEBRA FISH EGG 87
a matter of terminology so long as the fundamental mechanism of
mitosis is not understood.
The prophase was denned as extending from the first swelling of the
nucleus until the break-up of the membrane. The time from the
break-up until the chromosomes reappeared was assumed to be the
duration of the metaphase plus the anaphase. As to the duration of
both of these phases, it can only be stated that the metaphase is much
shorter than the anaphase. This is true for two reasons, namely: (1)
Nuclei which were observed up to the breakdown of the membrane
and then immediately fixed always showed the chromosomes already
slightly apart, and (2) the very rapid passing of the metaphase as de-
TABLE II
Duration of mitosis and mitotic phases.
Material
Total
dura-
tion
Pro-
phase
Meta-
phase
Ana-
phase
Telo-
phase
Author
Protozoon :
Rhagostotna schilssleri
min-
utes
32.5
179
150
180
35
16
18
per
cent
18.5
22.5
19.5
20.0
18.5
16.5
per
cent
12.5
14.0
12.0
i
per
cent
18.5
3.5
40.0
19.5
per
cent
53.5
60.0
46.5
50.0
i
after
Darlington
after
Darlington
Jolly
Wassermann
(after Jolly)
Strangeways
Roosen-Runge
Protozoan :
Ruglypha sp
Erythrocytes, Triton
The same
Chorioidea, cartilage in
chicken, culture
i
80.0
i
26.0
33.5
Blastomeres, Brachydanio . . .
The same, interkinetic phase
counted as telophase
i
55.5
i
50.0
scribed can actually be seen in the egg of Epiplaty chaperi. From
these observations it must be concluded that the metaphase probably
takes not much longer than one minute, or about 5.5 per cent of the
total division time.
The telophase was measured from the appearance of the chromo-
somes until the nuclei were completely rounded, with only a few
partitions within. It was assumed that the reappearance of the
chromosomes in life actually indicated a break in the process of mitosis,
inasmuch as they become visible at the moment when they begin to
take up fluid and pass from more or less solid bodies into vesicles.
In Table II, some results on the relative duration of mitotic phases
in various animals have been compared. They were all obtained by
direct observation. Interesting data like those of Lewis and Lewis
EDWARD C. ROOSEN-RUNGE
(1917) have been omitted since they are given too inaccurately for the
present purpose. They seem, however, not to be in general disagree-
ment with the figures presented here. The significance of the data
compared lies in the fact that they agree surprisingly well, in spite of
the different kinds of material used by the different investigators as
well as the great disparity in the observations made with relation to the
total duration of mitosis and the definition of its phases.
The relative time for the prophase varies only from 18.5 to 22.5
per cent in cells as different as those of protozoa, chicken cartilage, and
fish blastomeres. The reported times of the metaphase vary also only
slightly. However, in both the anaphase and the telophase there is
considerable variation although it is smaller in the anaphase than in
the telophase.
TABLE III
The duration of cleavage divisions under different temperatures.*
The times are minutes.
Cleavage
2
3
4
5
6
7
8
9
10
23° C.
(21)
23|
(20)
(20)
(21)
24
20
19.5
24^
21
19
(20)
(19)
25
19.5
18.5
18.5
18
18.5
19
19.5
20
254
(18)
18
17
17
18
26
(17)
17.5
18
20
26|
(16)
* The times enclosed in brackets refer to individual cases and are not averages.
The telophase in Triton erythrocytes is reported to take 50 per cent
of the total time of mitosis, and in protozoa 53.5 and 60 per cent.
Lewis and Lewis state that the telophase "which can be more ac-
curately recorded than the other phases, shows a striking similarity in
all types of cells and much less variation." If we take their telophase
and reconstruction periods together as corresponding to the definition
of the telophase used here, we find that the telophase in cultures of
chicken mesenchyme and smooth muscles lasts about 50 per cent of
the whole time of division, while the telophase of the zebra fish blasto-
meres takes only about half of this relative time, that is, 26 per cent.
Even if the interkinetic phase, the delimitation of which is not at all
clear, is added quite arbitrarily to the telophase, there is not more
than 33.5 per cent of duration time accounted for. The certainty
with which this result is obtained leads to the conclusion that the rela-
tive shortness of the telophase is actually significant for the type of
karyokinesis we are dealing with, which involves the formation of
KARYOKINESIS DURING CLEAVAGE OF ZEBRA FISH EGG 89
chromosomal vesicles in the telophase, and has no actual "resting
phase."
The effect of different temperatures on the duration of cleavage is
shown in Table III. The results cover only a part of the large range
of temperature which the eggs can stand. The only conclusions,
therefore, which can be safely drawn are that the duration of cleavage
divisions is influenced by even slight changes in temperature, but that
the general trend of acceleration for the first six cleavages, and the
following retardation, are practically unchanged so that the duration
of mitosis may be said to be constant under constant conditions.
Many investigators, however, have found the duration of mitosis
varying up to several hundred per cent for the same kind of cells.
Observations of cells in tissue cultures in particular have yielded results
which were very inconsistent with respect to the total duration of
division. In all these cases the inconsistency can be attributed only
to varying conditions of nutrition, oxygen supply, and temperature.
A comparison of the results given in the literature and the observations
on the eggs of the zebra fish, show that under constant conditions the
duration of mitosis is constant and characteristic for the different
types of cells.
DISCUSSION
The process of cleavage is characterized by continuous and often
synchronous cell divisions, which frequently follow a definite pattern.
In general there is no morphological differentiation during cleavage,
but very often there is a segregation of different materials in different
cells. At the end of the cleavage period there is a break in the develop-
ment, the divisions cease to be continuous and synchronous, and the
period of cell migration and arrangement begins, often together with
the first histological differentiation. On the other hand, cleavage is
regarded as "but a continuation ... of that series of cell-divisions
which has been going on uninterruptedly, though with periodic pauses,
since the most remote antiquity. The divisions of the egg during
cleavage are in all essentials of the same type as those of adult cells;
such differences as may appear — e.g., the prominence of asters, the
frequent asymmetry of the amphiaster, and the consequent inequality
of cleavage — are of minor importance, though often interesting for
analyzing the mechanism of mitosis." (E. B. Wilson, 1928, page 981.)
The general conception is that cleavage divisions are dynamically
somewhat different from the divisions in the older animal, but that
their variation is not correlated with any essentially different mecha-
nism. There are, however, observations which point to a difference
in mechanism. Investigators of the chromosomal vesicles, which so
90 HOWARD C. ROOSEN-RUNGE
frequently occur in the telophase of cleavage divisions, have often
suspected that this particular feature of mitosis might be immediately
connected with the fact that cleavage divisions go on continuously and
almost without interphases.
The study of karyokinesis in zebra fish blastomeres reveals that the
formation of chromosomal vesicles is obviously in itself a process of
much shorter duration than the common type of telophase and, further-
more, that it represents a condition which permits of an almost im-
mediate start of the next division without a "resting stage" and with-
out a spireme formation in the prophase. No nucleoli are formed in
this type of mitosis. All these features are characteristic only for the
divisions during cleavage. About the time of the twelfth cleavage an
entirely different type of mitosis appears, which shows no chromosomal
vesicles in the telophase, but nucleoli and a very distinct spireme in
the prophase (Fig. 5). In my material no transitional forms have been
observed between these two types, though it is quite possible that a
more thorough investigation may reveal such transitions.
Chromosomal vesicles have been found in the eggs of very many
species and almost all classes of animals with the possible exception
of birds and mammals. (A review of the literature has been given by
Richards, 1917.) The suggestion seems obvious that the type of
mitosis which is characterized most strikingly by the formation of
chromosomal vesicles in the telophase, is due to some aspect of the
division mechanism that is peculiar to the cleavage divisions. We
have not yet arrived, however, at any definite conclusions concerning
the possibly different dynamics involved.
SUMMARY
The nuclei in the blastomeres of Brachydanio rerio can be observed
easily in life. They are visible in the prophase and telophase as well
as in the interkinetic phase. This discovery is used (1) to confirm
and consolidate the results obtained from sectioned material; (2) to fix
the blastomeres in any desired mitotic phase; and (3) to determine the
duration of mitosis and its phases.
The duration of mitosis and its phases under constant conditions,
particularly with respect to temperature, is found to be constant for
each cleavage. The time from the breakdown of 32 nuclei to the break-
down of 64 nuclei is 18 minutes at 25° C. This places the cleavage
divisions of the zebra fish among the most rapid ever observed. The
first six cleavages show a trend towards acceleration, the sixth being
the most rapid one. From then on the speed of the divisions slows
down. This trend is essentially undisturbed by changes in temperature.
KARYOKINESIS DURING CLEAVAGE OF ZEBRA FISH EGG 91
The nuclear divisions during cleavage are characterized (1) by the
formation of chromosomal vesicles in the telophase (some of these
vesicles can frequently be seen in life to persist through the interphase) ;
(2) by a comparatively short duration of this type of telophase; (3)
by a very short, if any, true interphase; (4) by the lack of nucleoli; and
(5) by the absence of a typical spireme formation in the prophase.
The very short duration of the telophase has been recorded for the
first time. The other observations have been found in the cleavage
divisions of a majority of the species examined. In the zebra fish egg
they continue until about the twelfth cleavage, when the form of
mitosis typical for the adult first appears. It is suggested that this
type of mitosis is probably associated with the rapid sequence of
divisions and is generally characteristic of cleavage mitoses. The
most characteristic feature of this type of karyokinesis is the formation
of the chromosomal vesicles, but the shortening of the interphase and
telophase, and the lack of spireme formation in the prophase are also
obvious.
I am indebted to Professor J. W. Wilson, Brown University, for his
very valuable advice and to Professor H. E. Walter for his assistance
in editing the paper.
LITERATURE CITED
DARLINGTON, C. D., 1937. Recent Advances in Cytology. Philadelphia.
JOLLY, J., 1904. Recherches experimentales sur la division indirecte des globules
rouges. Arch. Anat. Micros., 6: 455.
JORDAN, E. O., AND A. C. EYCLESHYMER, 1894. On the cleavage of amphibian ova.
Jour. Morph., 9: 407.
LEWIS, W. H., AND M. R. LEWIS, 1917. The duration of the various phases of mito-
sis in the mesenchyme cells of tissue cultures. Anat. Rec., 13: 359.
MOENKHAUS, W. J., 1904. The development of the hybrids between Fundulus
heteroclitus and Menidia notata with especial reference to the behavior of
the maternal and paternal chromatin. Am. Joiir. Anat., 3: 29.
RICHARDS, A., 1917. The history of the chromosomal vesicles in Fundulus and the
theory of genetic continuity of chromosomes. Biol. Bull., 32: 249.
RICHARDS, A., 1935. Analysis of early development of fish embryos by means of the
mitotic index. I. The use of the mitotic index. Am. Jour. Anat., 56: 355.
RoosEN-RuNGE, E. C., 1938. On the early development — bipolar differentiation
and cleavage — of the zebra fish, Brachydanio rerio. Biol. Bull., 75: 119.
SMITH, B. G., 1929. The history of the chromosomal vesicles in the segmenting
egg of Cryptobranchus allegheniensis. Jour. Morph., 47: 89.
STRANGEWAYS, T. S. P., 1922. Observations on the changes seen in living cells
during growth and division. Proc. Roy. Soc. London, Series B., 94: 137.
WASSERMANN, F., 1929. Wachstum und Vermehrung der lebendigen Masse.
Handb. der mikrosk. Anat., 1: 2.
WILSON, E. B., 1928. The Cell in Development and Heredity. MacMillan Co.,
New York.
THE EFFECTS OF LIGHT AND TEMPERATURE ON
THE MALE SEXUAL CYCLE IN FUNDULUS
SAMUEL A. MATTHEWS
(From the Thompson Biological Laboratory, Williams College}
Fundulus heteroditus is a teleost fish which breeds during the late
spring and early summer months. Its gonads undergo fairly definite
seasonal changes, reaching their greatest weight just before the breed-
ing season, falling off sharply just after spawning is completed in late
July, then undergoing a period of slow growth until the onset of rapid
maturation of germ cells prior to the next breeding season (Matthews,
1938). Several factors, acting independently or collectively, may be
concerned in the control of this gonad cycle. Of these factors the
pituitary gland as an internal factor and temperature and light among
the external factors might reasonably be supposed to possess some
degree of control. Evidence concerning the role that the pituitary
gland plays in the sexual cycle has already been obtained (Matthews,
1939). The following experiments are concerned with the effects of
light and temperature on the male sexual cycle.
From the experiments of a number of workers, particularly those
of Bissonnette (see his review article, 1936) it is clear that in some
birds and mammals light plays a dominant role and temperature a
subordinate one in controlling the seasonal cycle in the gonads. The
data on poikilothermous animals are not as conclusive. Clausen and
Poris (1937) in the case of Anolis and Burger (1937) in the case of
Pseudemys both believe that light is important in controlling the sexual
cycle. Bellerby (1938), on the other hand, finds no evidence "that
light is essential for the maintenance of reproductive activity in
Xenopus laevis or that seasonal variation in light intensity or wave
length plays any part in the control of the sexual cycle under natural
conditions." Turner (1919), in describing seasonal changes in the
spermary of the perch, pointed out that tremendous synthesis of
material in the testis occurs in August when the temperature of the
water has reached its peak and begun to decline, and expulsion of the
sperm occurs when the temperature begins to rise. No experiments
controlling the light factor were described. From his work with the
stickleback Craig-Bennitt (1930) also concludes that temperature is
the important factor in controlling the sexual cycle and that light is
92
LIGHT AND TEMPERATURE EFFECTS ON FUNDULUS 93
unimportant. More recently Hoover and Hubbard (1937) have shown
that a gradual increase in daily illumination followed by a gradual
decrease will cause brook trout (a fall breeding animal) to produce ripe
eggs and mature sperm several months earlier than normal.
To determine whether or not the absence of light exerts any in-
hibitory influence on spermatogenesis in Fundulus the following two
experiments were carried out. In the first, begun in December, 11
males were divided in such a way that 5 were maintained in an aquarium
subjected to ordinary daylight with no night illumination and the
other 6 were kept in a light-proof tank. They were fed daily with the
stock food used in all experiments, consisting of dried shrimp, puppy
biscuit and Mead's infant cereal, with occasional living food such as
worms or Daphnia. These animals were killed at five different in-
tervals over a period of 3 weeks. The average percentage of the body
weight formed by the testis of the animals in the light-proof tank was
0.56 per cent, that in the other aquarium 0.37 per cent, and microscopic
examination of the testes showed no significant differences in the state
of activity of the two groups. In the second experiment, begun in
March and extending into April, 4 males were placed in the lighted
aquarium, which in this instance was illuminated at night by a 50-watt
mazda bulb suspended above the tank, and 8 were kept in the dark.
The animals were killed over a period of 4 weeks. The percentage of
the body weight formed by the testis in the illuminated aquarium
averaged 1.76 per cent, in the darkened tank 1.88 per cent, and again
no structural differences were observed in the microscopic structure of
the two groups of testes. In these cases some of the animals killed
during April presented the white testis and numerous sperm asso-
ciated with a high degree of activity, and this occurred as early in the
darkened tank as in the illuminated one. From these experiments it
seems fairly clear that absence of light for at least 4 weeks prior to the
breeding season does not inhibit activation of the testis in Fundulus.
It should, of course, be noted that the animals which developed ripe
testes in the dark had been on a rising daylight curve for nearly three
months before they were placed in the dark. Whether or not this is a
significant factor in initiating the active phase in the testis cycle has
not been determined.
Experiments concerning the effect of temperature on activation of
the testis gave somewhat different results. Ten animals were kept in
a tank in which the temperature of the water averaged 21° C. (variation
19°-21.5°) and 10 animals were kept in a constant temperature room
with light conditions similar to those of the first group, the temperature
of the water in the aquarium here averaging 5.5° C. (variation 4°-7° C.).
94 SAMUEL A. MATTHEWS
In a series run during December the testis formed as large a percentage
of the body weight of animals in the cold room as in the normals
(0.53 per cent), but sections of the testes of animals after 23 days in the
cold room showed that these were retarded in development, particularly
in the later stages of spermatogenesis. In a series run during March
and April, moreover, the testes of those maintained at 5.5° C. averaged
only 1.16 per cent of the body weight as against 1.76 per cent for those
in the warmer room and in general animals maintained in the cold
produced sperm much later than did those in the warm room. The
retarding effect of the low temperature was noted in this series as early
as 9 days after the beginning of the experiment. Only one individual,
killed April 5 after 21 days in the lower temperature, showed a degree
of activity comparable with that of the control animals.
In brief, then, records have been obtained on 14 animals main-
tained in a light-proof tank from 4 to 55 days as compared with con-
trols subjected to daylight or to daylight and added night illumination.
The testes of these animals were like those of the controls and in cases
killed late in April those of both groups were whitish and filled with
sperm. Only one case, killed December 26 after 23 days in the dark,
showed a testis less developed than that of the control. Records have
also been obtained on 17 animals which were maintained at a tempera-
ture of 5.5° C. as compared with 13 control animals kept at 21° C.
After 9 to 23 days at the lower temperature spermatogenesis was
definitely retarded.
These experiments show that the presence of light is not essential
for complete activation of the testis of Fundulus; and that low tem-
peratures exert a retarding influence on maturation of the sperm.
Obviously no evidence is furnished concerning the effects of gradual
changes in the amount of light to which the animal is subjected daily,
which Hoover and Hubbard found of such importance in the sexual
cycle of the trout.
LITERATURE CITED
BELLERBY, C. W., 1938. Experimental studies on the sexual cycle of the South
African clawed toad (Xenopus laevis). II. Jour. Exper. Biol., 15: 82-90.
BISSONNETTE, T. H., 1936. Sexual photoperiodicity. Quart. Rev. Biol., 11 : 371-386.
BURGER, J. W., 1937. Experimental sexual photoperiodicity in the male turtle,
Pseudemys elegans (Wied). Am. Nat., 71: 481-487.
CLAUSEN, H. J., AND E. G. PORIS, 1937. The effect of light upon sexual activity in
the lizard, Anolis carolinensis, with especial reference to the pineal body.
Anat. Rec., 69: 39-54.
CRAIG-BENNITT, A., 1930. The reproductive cycle of the three-spined stickleback,
Gasterosteus aculeatus, Linn. Phil. Trans. Roy. Soc. London, Series B., 219:
197-279.
LIGHT AND TEMPERATURE EFFECTS ON FUNDULUS 95
HOOVER, E., AND H. E. HUBBARD, 1937. Modification of the sexual cycle in trout
by control of light. Copeia, 1937: 206-210.
MATTHEWS, S. A., 1938. The seasonal cycle in the gonads of Fundulus. Biol. Bull.,
75: 66-74.
MATTHEWS, S. A., 1939. The relationship between the pituitary gland and the
gonads in Fundulus. Biol. Bull., 76: 241-250.
TURNER, C. L., 1919. The seasonal cycle in the spermary of the perch. Jour.
Morph., 32: 681-711.
SOME EXPERIMENTS ON THE RELATION OF THE
EXTERNAL ENVIRONMENT TO THE SPERMATO-
GENETIC CYCLE OF FUNDULUS
HETEROCLITUS (L.) l
J. WENDELL BURGER
(From Trinity College, Hartford, Connecticut, and The Ml. Desert Island Marine
Biological Laboratory, Salsbury Cove, Maine)
INTRODUCTION
Within the last decade a considerable body of experimental work
has shown that the sexual cycles of many vertebrates of the north
temperate zone are regulated in part by the annual cycle of changes in
day-length. Little, however, is known about the relation of the ex-
ternal environment to the sexual cycle of cold water fish. Success in
modifying the piscine sexual cycle by light agencies has been reported
for the trout (Hoover, 1937; Hoover and Hubbard, 1937), for the
minnow, Phoxinus (Spaul cited from Rowan, 1938), and for the stickle-
back (Tinbergen cited from Rowan, 1938). Craig-Bennett (1930)
came to the conclusion that the sexual cycle of the stickleback was
regulated primarily by temperature. HoQver (private communication
to T. H. Bissonnette) has found that light is ineffective on yellow
perch which were kept in water below 44° F.
The normal sexual cycle of Fundulus has been described by Mat-
thews (1938). As in many cold-blooded vertebrates the sexual cycle
is a continuous process throughout, with no genuinely inactive phase,
although during the winter there is little or no spermatogenetic activity.
In the late summer and fall a limited production of spermatogonia
takes place. Vigorous spermiogenesis begins in the spring, with a
mating period during May and June. Thereafter occurs a gradual
deceleration of spermiogenesis with a concomitant testicular involution.
It is noticed that the major portion of the spermatogenetic activity
is present during the spring when the days are increasing in length, and
when the temperature of the water is rising. The experiments here
reported are to test the relation of light and temperature to the
spermatogenetic cycle of Fundulus.
1 Aided in part by a grant from the American Philosophical Society administered
by T. H. Bissonnette for 1938-39.
96
SPERMATOGENESIS IN FUNDULUS 97
MATERIALS AND METHODS
Over seven hundred newly captured adult male Fundulus were used
in four experiments. Two of these experiments were performed in
Maine, and two in Connecticut. Fish were secured on June 30 from a
tidal inlet on Mt. Desert Island, Maine, and were confined to laboratory
aquaria fed by sea water. One control aquarium was placed out-of-
doors in a well lighted spot. The fish therein were maintained on
natural daylight until August 27. Two other aquaria were placed in
a light-proof box, which was illuminated by two 50-watt lamps. From
June 30 to July 22 the daily light ration was reduced 20 minutes per
day from 15 hours to 8 hours. Then between July 22 and August 27
the daily light ration was increased 20 minutes per day from 8 hours to
20| hours. The temperature of the aquaria water ranged between
11° and 17° C., in general increasing in warmth from June to August.
On October 29 Fundulus were secured from a tidal inlet off Long
Island Sound near Niantic, Connecticut. These animals were placed
in fresh water aquaria at Hartford, Connecticut. The control fish
were exposed to daylight for natural day-lengths between October 29
and January 4. The experimental fish received in addition to natural
daylight illumination from a 100-watt lamp. The exposure to electric
light was increased every 5 days so that by December 1 1 the fish were
receiving 8| hours of electric illumination added to normal daylight.
After this date no further increases in the length of time of exposure
to light were made. The temperature of the fresh water aquaria
ranged between 11° and 18° C., decreasing in warmth from October to
January.
In a final experiment fish were captured from a tidal inlet at Old
Lyme, Connecticut, on February 25, and were confined to fresh water
aquaria at Hartford. All aquaria were made light-proof. The fish
never received more than 1| hours of light per day during the experi-
mental period which was from February 25 to March 25. This limited
exposure to light was necessary for feeding. One group of males were
kept in cold water which varied in temperature between 6° and 10° C.
Another group was kept in warmer water which ranged between 14°
and 20° C. There was always at least 6° C. difference in temperature
between the two groups.
Fish were also sampled from the wild at various intervals. The
animals were fed almost daily on either chopped livers or clams.
Only healthy, fungus-free fish were killed for histological study.
98 J. WENDELL BURGER
RESULTS
Confinement to aquaria and to fresh water had no deleterious effects
on the testes. Judging from the condition of the internal organs, the
diet was more than adequate for the maintenance of good health.
In all the experiments with light, and where there was no significant
difference in temperature between controls and experimentals, no dif-
ference in the state of the testes was found between control and experi-
mental fish. The experimental light rations were: 21 days of gradually
decreased lighting between June 30 and July 22; 37 days of gradually
increased lighting between July 22 and August 27; and 68 days of
gradually increased lighting between October 29 and January 4.
On June 30 at the start of the first experiment, spermiogenesis was
at its peak. As can be seen in Fig. 1, there exists at this time a broad
zone of cortically located spermatogonia etc. surrounding medullary
tubules which are filled with sperm. During the summer there occurs
a gradual decrease in the proliferation of spermatogonia, together with
a loss of sperm in the tubules. The rate of this testicular involution
can be seen by comparing Figs. 1,2, and 4. Figures 2 and 4 are from
laboratory control fish for July 22 and August 27 respectively.
That the fish which received 21 days of gradually decreased lighting
showed no differences in testicular state when compared with control
fish, is illustrated in Figs. 2 and 3. Figure 2 is from a control testis,
and Fig. 3 from an experimental fish whose light ration was gradually
reduced to 8 hours per day. Likewise a comparison of Figs. 4 and 5
shows that there was no difference in testicular state between control
fish (Fig. 4) and experimental fish (Fig. 5) after 37 days of gradually
increased lighting applied to Fundulus which previously experienced
21 days of decreased lighting.
These results indicate that the testicular involution which normally
occurs during the summer when the day-lengths are naturally shortened
cannot be hastened by 21 days of decreased lighting. Moreover, 37
days of subsequent increased lighting does not change the rate of
testicular involution, nor does it induce a precocious new spermato-
genetic cycle. Since many animals are refractory to photoperiodic
manipulations at the end of their sexual cycle, the above experiments
are no test for the efficacy of photoperiodic manipulations on the
sexual cycle.
The fish which were lighted for 68 days between October 29 and
January 4 offer a fair test, however, as to whether or not the sexual
cycle of Fundulus can be influenced by light. When this experiment was
begun, the cortical zone of the testis was slowly proliferating spermato-
gonia, while the medullary system of tubules was involuted and devoid
PLATE I
C-, **•
'- «F^ ;;'•'. rt-,
5 xv;'m^ ?
All figures are unretouched photomicrographs, X 80.
FIG. 1. Section of a testis from a fish captured 6/30/38. The cortical zone
(at the top of the figure) of spermatogonia, etc. is broad; the medullary zone of
tubules is black with sperm.
FIG. 2. Section of a testis from a laboratory control, 7/22/38. The spermato-
gonia are fewer than in Fig. 1, and the tubules contain fewer sperms.
FIG. 3. Section of a testis, 7/22/38 after 21 days of shortened day-lengths.
The testis is in the same condition as that of the controls (Fig. 2).
FIG. 4. Section of a testis from a laboratory control, 8/27/38. Spermiogencsis
is almost finished; the tubules have markedly involuted.
FIG. 5. Section of a testis, 8/27/38 after 37 days of increased lighting. No
significant difference is found between this and the control (Fig. 4).
100 J. WENDELL BURGER
of sperm. This condition can be seen in Fig. 6, which is a section of
a testis on October 29.
During this experiment sperm were produced both by the control
and experimental fish. These sperm can be seen in Fig. 7 which is
from a control fish on January 4, and in Fig. 8 which is from an experi-
mentally lighted fish on January 4. In the control fish, sperm were
formed while the days were decreasing in length, as indicated by fish
sampled in December. In the experimental fish, sperm were formed
no more abundantly when the day-lengths were increased in length by
means of 8| hours of electric light added at night. Thus for fish at
the threshold of a new spermatogenetic cycle, the application of
increased or decreased daily rations of light does not modify the rate
of the subsequent formation of sperm.
A comparison of laboratory fish and fish from nature in early
January showed that the fish in their natural habitat do not form
sperm at this time as did the laboratory fish. This statement needs
to be qualified slightly for there are Fundulus in nature which during
the winter form a very few spermatozoa. However, the general
condition for winter fish at least up until early March is similar to
that shown in Fig. 6. The most obvious difference between the
laboratory fish and the fish from nature is the difference in water tem-
peratures. The laboratory fish lived in water between 11° and 18° C.,
while the fish in nature during the winter lived in water whose tem-
perature was near 0° C.
The experiment with temperature where the daily light ration was
only long enough for feeding the fish indicates that spermatogenesis is
responsive to temperature manipulations. Figure 9 is a section from
a testis from a fish after 29 days in water whose temperature varied
between 6° and 10° C. These temperatures were somewhat higher
than those experienced by the fish in nature at the time of capture and
during the experimental period. This testis is a winter testis and shows
no transformations of spermatozoa. However, there did occur a slight
multiplication of spermatogonia so that the testis was not completely
inactive during this period. Figure 10 is a section of a testis from a
fish after the same 29 days in water whose temperature varied between
14° and 20° C. Here spermatozoa have been formed in large numbers.
This effect of higher temperature can readily be seen by comparing
Figs. 9 and 10. This result was uniform for all fish. It should be
emphasized that these fish never had more than 1| hours of light per
day during the experimental period, and usually not more than one-
half hour.
PLATE II
!l
'
IMYftS
^4pi«75?--v"~ • • -,~ —
. «fc^ r-~ .-*'•. «_ jc-^ •,
FIG. 6. Section of a testis from a fish captured 10/29/38. The bulk of the
testis consists of spermatogonia. Sperms are absent and the tubular system is
greatly reduced.
FIG. 7. A section from a laboratory control, 1/4/39. The black areas are
tubules which contain sperm.
FIG. 8. A section of a testis, 1/4/39 after 68 days of increased lighting. Sperms
while abundant are no more numerous than in the controls (Fig. 7).
FIG. 9. A section of a testis, 3/25/39 after 29 days in almost complete darkness
in water whose temperature varied between 6° and 10° C. This is essentially a winter
testis consisting of spermatogonia (compare with Fig. 6). Sperms are absent.
FIG. 10. A section of a testis, 3/25/39 after 29 days in almost complete darkness
in water whose temperature varied between 14° and 20° C. The black areas show
the large numbers of sperms that have formed.
102 J. WENDELL BURGER
DISCUSSION
These experiments indicate that light as such is of no importance
in the spermatogenesis of Fundulus. Spermatozoa can be formed in
almost complete darkness, and will form in equal abundance when the
days are either increasing or decreasing in length. Temperature
appears as the important factor of the external environment which
modifies spermatogenesis. In cold water spermatogenesis is retarded
or inhibited, while in warm water spermatogenesis is rapidly completed.
These experiments give no exact data on the critical temperatures
involved, but from our observations both on experimental fish and on
fish in nature a general scheme seems clear. At temperatures near
0° C. the testis is inactivated. As the temperature rises toward or
around 10° C. spermatogonial multiplications occur. Still higher tem-
peratures permit the transformations of sperm to take place.
Marine temperatures show a very orderly annual cycle. Dr. R. A.
Goffin kindly gave us the mean daily sea water temperatures for 1938
at Woods Hole, Massachusetts. Dr. V. L. Loosanoff also referred us
to his paper (Loosanoff, 1937) which gives the shallow water tempera-
tures for over three years at Charles Island, Long Island Sound. Both
sets of data show a low point in February followed by a continued rise
beginning in March and reaching a maximum in August. From August
to September a continued drop takes place.
The spermatogenetic cycle of Fundulus fits nicely into this annual
temperature curve. In the fall as the temperature drops spermato-
gonial multiplications take place. During the winter the testis is
relatively inactive. In fact, fish captured in late February show
slightly less testicular activity than those captured in early January.
The spring rise in water temperature is accompanied by increased
spermatogenetic activity. It should be remembered that in nature
the beginning of active spermatogenesis coincides with the warming
of the water, and not with the increased lengthening of the days which
began three months previously.
SUMMARY AND CONCLUSIONS
1. No differences in the velocity of the spermatogenetic cycle of
adult male Fundulus were found between control and experimental
fish kept in water of the same temperature when treated as follows:
(a) 21 days of gradually decreased day-lengths between June 30 and
July 22, (b) 37 days of gradually increased lighting subsequent to
treatment as in (a) between July 22 and August 27, (c) 68 days of in-
creased lighting between October 29 and January 4.
2. Laboratory fish kept during the late fall and early winter in
SPERMATOGENESIS IN FUNDULUS 103
water whose temperature was higher than that experienced by fish
in nature showed an acceleration of spermatogenesis.
3. Laboratory fish which received no more than 1| hours of light
per day and which were kept in water of from 6° to 10° C. between
February 25 and March 25 remained inactive sexually. Fish which
received no more than 1^ hours of light per day and which were kept
in water whose temperature varied between 14° and 20° C. formed
large numbers of sperm within this same period of time.
4. It is concluded: (a) that the spermatogenetic stages of the
annual sexual cycle are not affected by light as light; (b) that the
temperature of the water is the important factor of the external environ-
ment regulating spermatogenesis in Fundulus.
5. It is suggested that at temperatures near 0° C. sexual activity
is inhibited. As the temperature rises toward or near 10° C. sperma-
togonial multiplications occur. Still higher temperatures produce
complete spermatogenesis.
LITERATURE CITED
CRAIG-BENNETT, A., 1930. The reproductive cycle of the three-spined stickleback,
Gasterosteus aculeatus, Linn. Phil. Trans. Soc., Series B, 219: 197-279.
HOOVER, E. E., 1937. Experimental modification of the sexual cycle in trout by
control of light. Science, 86: 425-426.
HOOVER, E. E., AND H. E. HUBBARD, 1937. Modification of the sexual cycle in
trout by control of light. Copeia, 1937: 206-210.
LOOSANOFF, V. L., 1937. Spawning of Venus mercenarius (L). Ecology, 18:506-515.
MATTHEWS, S. A., 1938. The seasonal cycle in the gonads of Fundulus. Biol. Bull.,
75: 66-74.
ROWAN, WM., 1938. Light and seasonal reproduction in animals. Biol. Rev., 13:
374-402.
INFLUENCE OF THE SINUSGLAND OF CRUSTACEANS
ON NORMAL VIABILITY AND ECDYSIS1
F. A. BROWN, JR., AND ONA CUNNINGHAM
(From the Zoological Laboratory, Northwestern University)
Since the work of Perkins and of Roller in 1928, who independently
described the presence of a substance in eyestalk extract which exer-
cises a very potent effect upon the chromatophores of crustaceans,
there has been much interest shown in the crustacean eyestalk func-
tion. The picture has been rendered even more interesting as a result
of the work of Brown (1935) and of Kleinholz (1938), demonstrating
that humoral activity in this group of animals is by no means a simple
one, but that several hormonal substances are normally functioning.
Hanstrom (1935) performed experiments in which he showed that the
portion of the eyestalk which was active in affecting chromatophores
always contained, among other things, a tissue which he has termed
the sinusgland. This has given rather good evidence indicating which
tissue of the eyestalk is the active one in this regard. The cells of this
tissue were shown to be secretory in nature and to contain a rich
supply of secretory granules. The more recent work of Hanstrom
(1936), Stahl (1938) and others have shown the sinusgland to be
present in some degree in all the crustaceans that have been examined
in detail. Its occurrence appears to be quite independent of the state
of development of a chromatophore system. Functionally it appears
to have common properties with the corpora allata of insects since an
extract of the latter organ in many cases serves as an activator of
crustacean chromatophores. Abramowitz (1936, 1938) has demon-
strated that the chromatophorotropic substance from the sinusgland
and the intermedin of the vertebrates have certain common chemical
and physiological properties.
Koller (1930) was the first to demonstrate that the eyestalk sub-
stance has another function in addition to the control of chromato-
phores. He found that animals from which the eyestalks had been
removed failed to deposit calcium in their exoskeletons to the same
extent as normal animals. He interpreted this to be the result of
removal of the source of a controlling hormone. Welsh (1937) found
that when he perfused an exposed crayfish heart with eyestalk extract
1 This investigation was supported by a research grant from the Graduate
School of Northwestern University.
104
CRUSTACEAN SINUSGLAND AND VIABILITY 105
there was a pronounced speeding up of that organ. Brown (1938)
demonstrated that removal of the eyestalks appreciably shortened the
life of the individual and that the shortening thus induced could be
compensated for in part by implantation of eyestalk tissue into the
ventral abdomen. This shortening of the life of the animal has been
called a "viability effect " of an eyestalk hormone, though it is fully
realized that this is a function described in far too general terms. It
is hoped that this "viability effect" can soon be analyzed into the
particular phenomena responsible for the shorter life.
There has frequently been suggestion of a "molting effect" of the
eyestalk substances, though no adequate data have yet been published
to establish such a function. The only grounds for such a belief are
that several investigators have mentioned that eyestalkless animals
appear to molt more frequently than normal ones. No reason has
been advanced for thinking the effect is due to anything other than
the injury caused by the operation of eyestalk removal (indicated by
Darby, 1938).
The following research has been conducted in continuation of that
of Brown (1938) with the intention of discovering just what tissue of
the eyestalk is responsible for the "viability effect" of this organ.
There is included here the first direct evidence for an endocrine activity
of the sinusgland of the crustacean. Hitherto its functioning had been
supposed upon the grounds of the best of circumstantial evidence.
During these experiments the sinusgland has been dissected out and
implanted into the ventral abdominal sinus of eyestalkless animals.
Direct physiological evidence of its endocrine function has been dem-
onstrated. Furthermore, it is quite well established as a result of
these experiments that this gland is the one responsible for the normal
continuation of life of the animal and also that it has a functional
activity in the control of molting. The possibility of explaining the
viability effect of eyestalk hormones in terms of molt control will be
discussed.
METHODS AND MATERIALS
All the crayfishes used in these experiments were small individuals
(carapace lengths 15-30 mm.) of the species Cambarus immunis, with
the exception of certain large individuals (Cambarus virilis, C. blan-
dingii, and C. immunis of carapace lengths 30-40 mm.) which were
used as the source of the sinusgland for implantation. The animals
were brought into the laboratory a few days before the beginning of
an experiment. It was our purpose to use experimental extirpation
and implantation to determine the normal functions of the eyestalk
gland within the body.
106 F. A. BROWN, JR., AND ONA CUNNINGHAM
The method of extirpation was simple : the eyestalks were removed
as a whole and the wound sealed with an electric cautery. By so
sealing the wound, less than 10 per cent of the animals died as a result
of the operation. It is fully realized that such a method of gland
extirpation removed much tissue in addition to that of the sinusgland.
In the first experiment to be described the implantation consisted
of all the eyestalk tissue. The eyestalks were removed from an animal
and dropped into amphibian Ringer's solution. With the aid of a
dissecting microscope the exoskeleton of the eye was cut away. The
soft parts of the eyestalk were easily removed with fine forceps. This
tissue was then teased into minute fragments and injected by means
of a glass capillary pipette into the ventral sinus of the abdomen.
The glass pipette proved to be especially satisfactory since it was
possible to ascertain that all of the tissue entered the animal and none
was left adhering to the walls of the pipette.
In those experiments in which the sinusgland by itself was to be
implanted the gland was carefully dissected out in the following man-
ner: the eyestalk was removed from a large crayfish and dropped into
a watchglass containing amphibian Ringer's solution or a balanced
salt solution based on Griffeths' analysis of Astacus blood (which will
henceforth be referred to as Griffeths' solution). With a pair of sharp
pointed scissors the chitinous exoskeleton was clipped to free the dorsal
half of the stalk skeleton from the ventral half. The contents of the
stalk were then picked out with fine pointed watch-maker's forceps
and the dorsal tissue was teased away in the direct light of a strong
lamp. The sinusgland tissue stood out quite conspicuously as a seem-
ingly fibrous and granular bluish tissue. This mass of tissue was
easily torn away from the adjacent nerve tissue. All the adhering
tissue was teased away and the gland rinsed in amphibian Ringer's
or Griffeths' solution. With forceps the gland was next pushed
through an opening made in the ventral side of the abdomen. The
clear exoskeleton in this region made it possible to ascertain that the
minute gland was actually left in place upon removal of the forceps.
In order to determine the exact location of the tissue removed
from the eyestalk, sections were made of the bluish gland-like tissue
that was removed, and also of all the remaining portions of the eye-
stalk. In addition, longitudinal sagittal sections of the complete
eyestalk were made as a control. By study of these three sets of
sections it was readily determined just what tissue was being implanted.
It was discovered that the implant tissue in histological section ap-
peared to be definitely glandular in nature and occupied a position
wedged between the medulla externa and the medulla interna. Con-
CRUSTACEAN SINUSGLAND AND VIABILITY
107
sidering its position and the fact that its cytoplasm was richly charged
with eosinophilic inclusions, it seemed highly probable that this gland
was the same as that described by Hanstrom (1936) as the sinusgland.
The accompanying photographs show this gland as it occurs in Cam-
barus virilis. The first photograph is a median sagittal section of the
^rm
t %•„*•
*%
*
FIG. 1. Sagittal sections through the cyestalk of Cambanis virilis (6 micra thick
and stained with Delafield's haematoxylin and eosin). A. At a magnification of
80 X, showing the sinusgland as a somewhat triangular section of tissue located
dorsally to a point intermediate between the medulla externa and the medulla
interna. B. A higher magnification (360 X) of the central region of the sinusgland.
108 F. A. BROWN, JR., AND ONA CUNNINGHAM
eyestalk at a magnification of approximately 80 X and the second is
a higher power magnification (about 360 X) in the central region of
the gland.
During the experimental period all the animals were kept in indi-
vidual glass finger bowls in water not quite deep enough to cover the
carapace. These finger bowls were covered loosely with glass plates
to minimize evaporation of the water but still to permit circulation of
air over the water surface.
The experiments performed included extirpation and implanta-
tion, with appropriate controls, and observations were made upon
viability and molt behavior.
100
10 15 £0 25 30 35 40 45
TIME IN DAYS
FIG. 2. The relation between the percentage of animals dead and the number
of post-operative days for eyestalkless crayfishes, (O); eyestalkless crayfishes with
a heteroplastic implant of sinusglandless eyestalk tissue, ( o) ; and eyestalkless
crayfishes with only a heteroplastically implanted sinusgland, (3 ).
EXPERIMENTS ON VIABILITY EFFECTS
Experiment I
The animals of this experiment, all Cambarus immunis with both
eyestalks removed and the stubs cauterized, were divided into three
lots. In the first lot were 7 animals with no further treatment. The
second lot of 17 animals had a sinusgland taken from a single eyestalk
of a large Cambarus virilis or Cambarus bland ingii acutus implanted
into the ventral sinus of their abdomens. The third lot of 18 animals
had an abdominal implantation consisting of all the eyestalk tissue of
a single eyestalk of Cambarus virilis or Cambarus blandingii acutus,
from which the gland had been carefully removed.
The results of this experiment are best shown in the form of a
graph (Fig. 2) in which the percentage of animals dead is plotted
CRUSTACEAN SINUSGLAND AND VIABILITY 109
against the post-operational day. This graph demonstrates clearly
that eyestalkless animals without abdominal implants live significantly
shorter lengths of time than eyestalkless animals into which eyestalk
tissue minus the sinusgland has been implanted. Similarly, eyestalk-
less animals which have received abdominal implants of the minute
sinusgland by itself, live very significantly longer than those animals
into which the remaining portion of the eyestalk tissue was implanted.
Comparing only the instance of sinusgland implant with the case of
no implant, we can conclude definitely that the minute sinusgland
lengthens the post-operative life of the animal considerably. It is well
to bear in mind that these two latter groups have been subjected to
operations of different degrees of severity, in which the animals which
100
10 15 20 25 30 35 40 45
FIG. 3. The relation between the percentage of animals dead and the number
of post-operative days for eyestalkless crayfishes, (O); eyestalkless crayfishes with
a homoplastic implant of sinusglandless eyestalk tissue, ( €)); and eyestalkless cray-
fishes with only a homoplastically implanted sinusgland, (3 ).
live longer have been subjected to more severe operative injury, the
animals of the latter group having their abdomens punctured as well
as having both eyes removed. A logical explanation of the inter-
mediate length of post-operative life in the instance of those animals
with the glandless stalk tissue implants is that there is present in the
blood spaces of the general eyestalk tissue a product that has arisen
from the sinusgland. During its removal, a bluish liquid is seen to
diffuse out of the gland and infiltrate into the surrounding tissues.
The general stalk tissue is frequently filled with a homogeneous blue
liquid which, in all probability, comes from the same origin. We
believe, therefore, that this additional substance is responsible for
permitting these animals to live longer than those in which no implant
110 F. A. BROWN, JR., AND ONA CUNNINGHAM
is made. It is also possible that fragments of the gland itself still
remain which were not removed at the time of operation.
The implants in this experiment are heteroplastic, while in an
experiment to be described later all the implantations were autoplastic
as was the case with those observations published by Brown (1938).
It becomes doubly interesting that the sinusgland has a definite effect
not only upon the length of post-operative life in the same species of
animal, but that the tissue from one species is capable of working
effectively within the body of another species to the same end. Thus
these substances,- or this substance, is inter-specifically active.
Experiment II
In this experiment, like the preceding one, eyestalkless Cambarus
immunis were divided into three lots. In the first lot, consisting of
6 large animals, there was no further treatment. A single sinusgland
from an eyestalk of a large animal of the same species was abdominally
implanted into each of the 20 small animals of the second lot. Each
of the 20 animals of the third group received an abdominal implant
consisting of the tissue from a single large eyestalk from which the
gland had been removed.
The results of this experiment are shown in Fig. 2.
This experiment confirms the influence of the sinusgland on via-
bility demonstrated in Experiment I. Here the implantations were
homoplastic, from large Cambarus immunis to small Cambarus im-
munis. As in Experiment I, the animals without any implant lived
a much shorter time than those with sinusgland implants, and animals
in which sinusglandless eyestalk tissue was implanted lived for an
intermediate length of time.
EXPERIMENTS ON THE MOLTING CONTROL FUNCTION OF THE SINUS-
GLAND
Experiment I
This experiment was intended to discover any differences that
might occur in the molting process among animals from which both
sinusglands had been removed, one sinusgland removed, both sinus-
glands removed but with them autoplastically implanted into the
ventral abdominal sinus, and finally, completely normal animals.
In this experiment four lots of animals were isolated. The first
lot of 34 animals was left in perfectly normal condition, though placed
in the usual individual glass finger bowls with covers. The second lot
of 48 animals was subjected to removal of one eye each. A third lot
of 79 animals had both eyestalks removed in the usual manner. The
CRUSTACEAN SINUSGLAND AND VIABILITY
111
fourth lot of 44 animals had both eyestalks removed and the contents
of their own eyestalks in amphibian Ringer's solution injected into the
ventral sinus of the abdomen. Observations were made only with
regard to actual molting. The results that were obtained are sum-
marized in Table I.
TABLE I
Data indicating the extent of molting in crayfishes
under different experimental conditions.
Normal
Animals
One Eye
Off
Two Eyes
Off
Two Eyes Off
(Implant)
Total no. examined ....
No. "molts"
34
9
48
19
79
23
44
3
Per cent "molts"
Per cent "molts " dying
in process
26
44
40
16
29
74
7
100
Per cent molt/av. life
span
2.0
3.4
5.75
1.0
All the records of molting in Table I indicate instances in which the
animal either completed the molt or was well along in the process at
the time of death. The most significant portion of the table is the
item "per cent molt/average life span" which gives the only true
figure of the relative rates of molt. The "per cent molts" fail to do
this inasmuch as the different lots of animals survived different lengths
of time; consequently such animals as normal animals and those with
one eyestalk off had a longer time in which molts could occur. On
this strictly relative behavior (per cent molt/average life span) the
figure for normal animals is 2. With one eye removed, the rate of
molt is increased by about 75 per cent, and with the removal of two
eyes the molting has been accelerated about 200 per cent. The
striking fact, however, is that when both eyes were removed and the
eyestalk tissue abdominally implanted, the figure indicating the molt-
ing rate is 1, or about half that of normal animals. Were it not for
the anomalous molting rate of this last group the results could be
interpreted as indicating that the rate of molting is a function of the
extent of injury. But, taking the data together, there appears to be
a more probable explanation. The eyestalk tissue, under nerve con-
trol, liberates a humoral substance into the blood which inhibits the
molt. With one eye removed, relatively less substance is liberated
and with two eyes removed none of the material, and we see molt
correspondingly going on at relatively greater rates. In these terms
the explanation of behavior of the last group of animals might be
that the implanted glandular tissue continuously liberates some anti-
molting substance and the animal is almost unable to molt.
112 F. A. BROWN, JR., AND ONA CUNNINGHAM
Some of the acceleration resulting after eyestalk removal may be
due to injury effects, but that they are not totally due to injury is
indicated by the implantation experiments.
Experiment II
This experiment points to the sinusgland in the eyestalk as the
actual tissue involved in the formation of the molt control humoral
substance. The data for this conclusion are taken from observations
on molting in the animals in Experiment II on viability.
A consideration of the ratio of percentage of completed or nearly
completed molts to average survival period, shows that the implan-
tation of the sinusgland reduces the molting rate to about one-fifth
of that which occurs in the controls with the glandless stalk tissue
implants. The conclusions of the former experiment are confirmed
and it is further indicated that the sinusgland is the effective tissue
in molt control. The results of this experiment are summarized in
Table II.
TABLE II
Data indicating the extent of molting in crayfishes
under different experimental conditions.
Two Eyes Off
Two Eyes Off (Implant)
Total no. examined 20 20
No. "molts" 4 1
Per cent molts 20 5
Per cent molts/av. life span 1.57 .31
In the course of this experiment all the animals were carefully
watched, not only for completed molts but also for the slightest symp-
toms of the beginnings of molt. The early signs of molt were usually
indicated by a visible separation between the carapace and the first
abdominal tergite. Practically all of the eyestalkless animals, regard-
less of the type of implant, showed this separation from three hours
to three or four days prior to their death. This was so definite that
it was possible to predict the death of any animal within these limits.
In many instances this separation was followed by a completed molt,
though in the majority of cases the animals died before further steps
in the molting process. It is admitted that some other factors, such
as change in general tone of the abdominal musculature or upset in
the water metabolism of the animal, might be operating in inducing
the separation of these two skeletal elements. Superficially, however,
we are unable to differentiate between the initiation of the normal
molt and its induction by other causes. Furthermore, many of the
animals showing this apparent initiation in the molt process showed
CRUSTACEAN SINUSGLAND AND VIABILITY 113
muscular activity of the body such as is usually associated with the
normal molting process.
Those animals from which the eyestalks had been removed and
which received the glandless eyestalk implantation, all showed the
apparent initiation of molt or completed the molt prior to their death.
In three cases the animals completed the molt before death, in one
case dying within a day of the molt and in the other cases living two
and four days, respectively, after molting. In a fourth case the animal
died when well along in the molting process. These facts would indi-
cate that even without the eyestalks the animals are physiologically
able to complete the molt. But the fact that the eyestalkless animals
sometimes continue to live several days after molting and then die
without showing further signs of molt, indicates that the sinusgland
has a function in addition to molt control.
The majority of the animals with sinusgland implants also showed
the beginnings of molting prior to their death, just as did the first lot.
The only difference between the lots seemed to be that the molting
activity was postponed in the case of the implanted animals. These
animals seldom do more than show this first sign of molt, scarcely ever
proceeding far into the molt or completing it. ' A possible explanation
of this is that these animals are prevented from molting by action
of the implant until the absence of the eyestalk has worked other
degenerating effects upon the organisms to the extent that they no
longer have the power to go far with the molt, in spite of removal of
the inhibitor through loss of function of the implant. In this regard
it would be interesting to trace the rate of degeneration of the im-
planted tissue to see if there may be any correspondence between the
time of oncome of the molt and the structural degeneration of the
implanted cells.
It may be possible to interpret the data of Roller (1930) in terms
of molt control activity. Animals molting more frequently as a result
of absence of a hormone from the sinusgland might well be expected to
have less calcium salts in their exoskeleton than normally.
SUMMARY
1. Direct evidence for an endocrine activity of the crustacean sinus-
gland is given. This evidence has originated from implantation
experiments.
2. Removal of the sinusgland significantly shortens the life of the
animals, and conversely the length of life of animals with sinusglands
removed can be significantly lengthened by implantation of the gland.
3. The sinusgland is readily dissected out in fresh eyestalk tissue
114 F. A. BROWN, JR., AND ONA CUNNINGHAM
in strong reflected light. It has a distinctly bluish cast. It is a
definite organ which can be readily teased away from the surrounding
tissue and removed as a whole.
4. Certain evidence suggests very strongly that a substance con-
cerned with the control of molting is elaborated in this gland. The
most probable action of this substance is that of inhibiting molt.
5. The action of the sinusgland in molt control appears to be
insufficient to explain the viability effect entirely.
LITERATURE CITED
ABRAMOWITZ, A. A., 1936a. Action of crustacean eye-stalk extract on melanophores
of hypophysectomized fishes, amphibians, and reptiles. Proc. Soc. Exp.
Biol. and Med., pp. 714-716.
ABRAMOWITZ, A. A., 19366. The action of intermedin on crustacean melanophores
and of the crustacean hormone on elasmobranch melanophores. Proc. Nat,.
Acad. Sci., Washington, 22: 521-523.
ABRAMOWITZ, A. A., 1938. The similarity between the hypophyseal chromatophoro-
tropic hormone and the chromatophorotropic hormone of the crustacean
eyestalk. Physiol. Zool., 11: 299-310.
BROWN, FRANK A., JR., 1935. Control of pigment migration within the chromato-
phores of Palaemonetes. Jour. Exper. Zool., 71: 1-15.
BROWN, FRANK A., 1938. An internal secretion affecting viability in Crustacea.
Proc. Nat. Acad. Sci., Washington, 24: 551-555.
DARBY, HUGH H., 1938. Moulting in the Crustacean, Crangon armillatus. Anat.
Rec., 72: (Suppl.) 78.
HANSTROM, B., 1935. Preliminary report on the probable connection between the
blood gland and the chromatophore activator in decapod crustaceans.
Proc. Nat. Acad. Sci., Washington, 21: 584-585.
HANSTROM, B., 1937. Die Sinusdriise und der hormonal bedingte Farbwechsel der
Crustaceen. Kungl. Svenska Vetenskap. Handl., Ser. 3, 16 (3): 1-99.
HANSTROM, B., 1937. Vermischte Beobachtungen iiber die chromatophoraktivieren-
den Substanzen der Augenstiele der Crustaceen und des Kopfes der Insekten.
Kungl. Fys. Sdllsk. Handl., 47 (8): 3-11.
KLEINHOLZ, L. H., 1938. Studies in the pigmentary system of Crustacea. IV. The
unitary versus the multiple hormone hypothesis of control. Biol. Bull., 75:
510-532.
ROLLER, G., 1928. Versuche iiber die inkretorischen vorgange beim Garneelen-
farbwechsel. Zeitschr.f. vergl. Physiol., 8: 601-612.
ROLLER, G., 1930. Weitere Untersuchungen iiber Farbwechsel und Farbwechsel-
hormone bei Crangon vulgaris. Zeitschr.f. vergl. Physiol., 12: 632-667.
PERKINS, E. B., 1928. Color changes in crustaceans, especially in Palaemonetes.
Jour. Exper. Zool., 50: 71-103.
STAHL, FILIP, 1938a. Preliminary report on the colour changes and the incretory
organs in the heads of some crustaceans. Arkiv.fdr Zoologi, 30B: 1-3.
STAHL, FILIP, 19386. Uber das Vorkommen von inkretorischen Organen und Farb-
wechselhormonen im Ropf einiger Crustaceen. Kungl. Fys. Sdllsk. Handl.,
49 (12): 3-20.
WELSH, J. H., 1937. The eyestalk hormone and rate of heart beat in crustaceans.
Proc. Nat. Acad. Sci., Washington, 23: 458-460.
THE METHOD OF FEEDING OF CHAETOPTERUS
G. E. MACGINITIE
(From the, William G. Kerckhoff Marine Laboratory of the California Institute of
Technology, Corona del Mar, California)
INTRODUCTION
Ciliated currents present on the surface of animals, when examined
under artificial conditions, are seldom, if ever, typical of the animal
in its natural environment. Failure to recognize this fact and failure
to observe the presence of mucus and note its importance in the feeding
process have given rise to many erroneous descriptions of the feeding
mechanism of various marine invertebrates. In conformity with the
statement made in Science (MacGinitie, 1937), the feeding activities
of many marine invertebrates have been investigated (including tuni-
cates, pelecypods, gastropods, annelids and coelenterates) , and descrip-
tions of the feeding activities of these animals will follow as soon as
they can be prepared for publication. This paper will deal with the
feeding of the annelid Chaetopterus variopedatus Renier et Claparede.
Because of its wide distribution and its usefulness as a source of
embryological material, Chaetopterus is well known both abroad and
in this country. Also, because of its unusual and somewhat bizarre
structure, it has created a great deal of interest from both an anatomi-
cal and a natural history point of view (Laffuie, 1890; Enders, 1909).
However, no paper that I have seen has given the correct method of
feeding of this animal.
FEEDING METHOD
The structures concerned with the feeding activities of Chaetopterus
are the peristomial funnel with its lips, the mouth, the dorsal ciliated
groove, which ends in the dorsal cupule of the thirteenth segment, the
pair of aliform notopodia of the twelfth segment, and the three fans
of the fourteenth, fifteenth and sixteenth segments (see Fig. 1).
In preparing to feed, Chaetopterus approaches one or the other end
of the leathery U-shaped tube in which it lives and spreads its aliform
notopodia out against the sides of the tube. It then begins to secrete
mucus from the inner walls of these notopodia, the secretion beginning
at the distal ends and proceeding inward toward the body. The cilia
of the inner surface of the notopodia carry the mucus across the
115
116
G. E. MACGINITIE
opening in a sheet from the distal ends to the body of the worm,
whence it is carried posteriorly as a bag by the ciliated groove to the
dorsal cupule, where the closed end of the mucous bag is taken into
the cup or concave surface of this organ. This creates an elongated
bag of mucus, the anterior end of which is fastened to or continuous
with the glands lining the inner surface of the aliform notopodia, and
the closed posterior end of which is held by and rolled up within the
dorsal cupule.
A current of water is now maintained through the burrow by the
activity of the three fans just posterior to the dorsal cupule. Since
vs.
FIG. 1. A, Chaetopterus variopedatus within its tube, feeding; B, dorsal surface
of anterior portion of worm, a.n., aliform notopodium; c., cirrus;/., fans;/.6., food
ball being rolled up within the dorsal cupule; d.g., dorsal ciliated groove; m., mouth;
m.b., mucous bag; p.f., peristomial funnel; v.s., ventral suckers.
the walls of the burrow are completely in contact with the body of the
animal and the aliform notopodia at the anterior end of the mucous
bag, it is necessary for the current of water to pass into the bag, out
through its sides, and thence along the body of the worm, and ulti-
mately to issue from the burrow at th& opposite end. While the
current is being maintained by the fans, mucus is continuously secreted
at the anterior end of the bag, and, at the same rate, the posterior end
is rolled into a ball within the dorsal cupule by the cilia of its inner
surface. Since all water entering the burrow while a mucous bag is
FEEDING OF CHAETOPTERUS 117
present passes through the walls of the bag, the mucus removes from
the current all solid particles, whatever their size. It is these particles
which lodge on the inner surface of the mucous bag that constitute
the food of Chaetopterus. It consists mainly of detritus (organic
debris and bacteria) stirred up from the surface of the ocean or estu-
arine bottom by wave action, currents, other animals, etc.
Because the entrances to the tube of Chaetopterus are considerably
constricted, no very large particles find their way in with the feeding
current. Such that do are usually detected by the peristomial cilia
of the worm and are passed out at the sides of the worm anterior to
the aliform notopodia, which are lifted to allow the material to pass,
and so do not find lodgment in the mucous bag. Since the mucus of
the bag is being secreted continuously, and at the same time the
posterior end is being rolled into a ball in the dorsal cupule, it is evi-
dent that the entire bag is constantly being renewed, and that the
posterior portion is much more heavily laden with food than is the
anterior.
When the ball of mucus and food in the dorsal cupule reaches a
certain size, the anterior end of the mucous bag is cut off from the
notopodia, and the dorsal cupule continues to rotate the ball until the
remainder of the bag is completely (or, occasionally, only partly)
rolled up. The dorsal cupule is then turned anteriorly and stretched
forward somewhat to expel the ball of food onto the posterior end of
the dorsal groove. At the same time the action of the cilia of the
groove is reversed, and the bolus of mucus with its entrapped food is
carried forward along it to the mouth, where the bolus is enveloped
by the lips and swallowed.
The size of the bolus of food depends upon the size of the dorsal
cupule, and, therefore, upon the size of the animal. For a Chaetopterus
about 6 inches long the food ball averages about 3 mm. in diameter.
When Chaetopterus is feeding there is some variation in the length of
its body, particularly in that portion between the head and the dorsal
cupule, and, therefore, the length of the mucous bag will vary in the
same animal at different times.
The following figures are given for a worm 142 mm. in length,
measured during a time when the animal was feeding. Fifteen milli-
meters posterior to its point of origin, the width of the mucous bag
was 6 mm., and the dorso- ventral diameter at the same point was
7 mm. The length of the mucous bag was 37 mm. The rate of
secretion of this bag was approximately 1 mm. per second. While the
worm was feeding the number of beats for any one of the three fans
was 64 per minute, and this rate was the same for this particular
118 G. E. MACGINITIE
worm as observed on successive days over a period of several weeks.
Although the rate of beating of the fans is quite uniform for any one
worm, it varies with individuals, for another worm maintained a rate
of 52 beats per minute. From the beginning of the spinning of the
mucous bag to the ingestion of the bolus of food required, on the
average, 17 minutes, and varied only plus or minus 1 minute from
this average.
LITERATURE CITED
ENDERS, HOWARD EDWIN, 1909. A study of the life history and habits of Chaetop-
terus variopedatus, Renier et Claparede. Jour. Morph., 20: 479-531.
LAFFUIE, J. J., 1890. Etude monographique du Chetoptere (Chaetopterus vario-
pedatus Renier), Arch, de Zool. Exp. et Gen., Ser. 2, 8: 245-360.
MACGINITIE, G. E., 1937. The use of mucus by marine plankton feeders. Science,
86: 398-399.
THE ACTION OF EYE-STALK EXTRACTS ON RETINAL
PIGMENT MIGRATION IN THE CRAYFISH,
CAMBARUS BARTON I
JOHN H. WELSH
(From the Biological Laboratories, Harvard University)
I
Pigment cells of the retina of the vertebrate eye and pigment cells
of the compound eye of arthropods have long been known to lack
motor innervation. Hence there has been much speculation regard-
ing the nature of the mechanisms controlling the movements of these
cells or the pigment within them. As recently as 1932 when Parker
reviewed the literature on retinal pigments there was no direct evi-
dence as to the nature of the control, but considerable indirect evidence
suggested that hormonal agents were responsible for initiating and
maintaining retinal pigment migration. The first successful attempt
to demonstrate the existence of a hormone acting on the retinal pig-
ments of arthropods was made by Kleinholz (1934; 1936). He found
that the injection of an active principle from eye-stalks of Palae-
monetes into dark-adapted individuals of the same species caused the
movement of the distal and reflecting pigments to positions charac-
teristic of the light.
Studies of the persistence, under constant external conditions, of
24-hour cycles of pigment migration in the compound eye had led to
one of the earlier suggestions that there was a hormonal control of
retinal pigment (Welsh, 1930; see also Welsh, 1938, for review of the
literature pertaining to diurnal rhythms). The extension of these
studies to the eye of Cambarus made necessary an investigation of
hormone factors in the control of retinal pigment migration in this
crustacean. Certain of the results obtained will be presented in this
paper.
II
The majority of observations were made on eyes of Cambarus
bartoni but eye-stalks of C. clarkii and C. limosus were sometimes used
as sources of the pigment-activating substance.
The approximate positions of the three sets of pigment (distal,
proximal and reflecting) were determined by briefly illuminating the
119
120 JOHN H. WELSH
eye of an animal, in the dark, by a bright beam of light, and observing
the amount of light reflected from the eye. This method has been
employed by Day (1911). Exact determinations of pigment positions
were first made by sectioning the eyes but this is a time-consuming
procedure and a rapid method was developed as follows. Animals
were killed by dipping in water at 80° C. for 10-15 seconds. The
eyes were then removed and split in halves. When these halves were
examined under a binocular, using bright reflected light, it was possible
to measure the positions of the pigments quite as accurately as in
sections.
The active substance from the eye-stalk, which may be identical
with the chromatophorotropic hormone (Abramowitz and Abramo-
witz, 1938), was prepared by grinding 20 eyes and eye-stalks of
medium-sized crayfishes in 1 cc. of cold-blooded Ringer, then heating
to 100° C. and filtering. The injection of an appropriate volume of
the filtrate made it possible to administer the active material from a
fraction of an eye-stalk or from one or more eye-stalks as a given dose.
Appropriate control injections of Ringer's fluid and of extracts of
ventral nerve cord were made and always with negative results.
Ill
The first experiments to be reported were done to test the effect
of the eye-stalk extract on light-adapted eyes. Several C. bartoni were
allowed to adapt for several hours in bright diffuse sunlight. At
10:30 A.M. 0.05 cc. of eye-stalk extract (containing the active material
from one eye-stalk) was injected in the ventral abdominal musculature
of each of half the individuals. At 2:00 P.M. the entire lot was killed
with hot water and the eyes removed, split and examined.
In Fig. 1 may be seen the distribution of the pigments of a light-
adapted eye. The distal pigment forms a sheath around the cone
and the process leading from the cone to the rhabdome, but a portion
of each distal pigment cell next to the retinular or proximal pigment
cells is not filled with pigment. Most of the proximal pigment sur-
rounds the rhabdome, but some remains below the basement mem-
brane. The reflecting pigment in crayfish eyes does not migrate as
it does in some crustaceans (Welsh, 1932). The appearance of an eye
of a light-adapted animal, when viewed by reflected light, is shown
in Fig. la. The positions of the black screening pigments are such
that light cannot reach the reflecting pigment layer nor can light rays,
except those which are parallel to the main axis of an ommatidium,
reach the rhabdome or light-sensitive element of the eye. Such an
eye is called an apposition eye since a given rhabdome receives light
RETINAL PIGMENT MIGRATION IN THE CRAYFISH
121
only from its adjacent lens system and is not acted on by light enter-
ing at an angle through neighboring ommatidia.
-- b". rn.
EXPLANATION OF FIGURES
con. = cone
b.m. = basement membrane
d.p.c. = distal pigment cell
p. p.c. = proximal pigment cell
rh. = rhabdome
r.p.c. = reflecting pigment cell
FIG. 1. An ommatidium of a typical light-adapted eye showing the positions
of the eye pigments. This and the following figures of ommatidia show the situation
as seen in thin sections of the eye. In the intact light-adapted eye each cone, cone
process and rhabdome is almost completely surrounded by a cylinder of pigment.
FIG. la. Showing the appearance of an intact eye with the pigment distribu-
tion seen in Fig. 1 when viewed, in the dark, by bright reflected light.
FIG. 2. An ommatidium showing the effect on the pigments of injection of eye-
stalk extract into a light-adapted animal.
FIG. 2b. The intact eye has essentially the same appearance as does the normal
light-adapted eye.
122 JOHN H. WELSH
An ommatidium from a typical eye of a light-adapted animal,
injected with eye-stalk extract and left in the light, is represented by
Fig. 2. The distal pigment is in a more extreme proximal position
and all of the proximal pigment is above the basement membrane.
It is as though the effect of the injected material were added to the
effect of light, which probably acts by causing the release of the active
material or hormone. The intact eye of such an animal has essen-
tially the same appearance as does the normal light-adapted eye
(Fig. 2&), although it may not be as black. This is due to the distance
of the distal pigment from the surface of the eye.
IV
When specimens of C. bartoni were placed in the dark, the typical
dark-adapted condition in the eye was seen after two hours or less.
It is known, however, that there is a diurnal migration of proximal
retinal pigment in crayfishes which are kept in continuous darkness
(Bennitt, 1932), so in order to assure uniform conditions in all experi-
ments on dark-adapted animals the majority of observations were
made in the early evening.
The positions normally occupied by pigments in a dark-adapted
eye are shown in Fig. 3. The distal pigment forms a collar surround-
ing the cone and the proximal pigment is all below the basement
membrane. In such a condition the rhabdome of a given ommatidium
may receive light from neighboring ommatidia. Such an eye is re-
ferred to as a superposition eye and is commonly found in those insects
and crustaceans which are active at night.
The intact eye of a dark-adapted crayfish has a brilliant orange-
red center when viewed by reflected light, due to the mirror-like
property of the exposed reflecting or tapetal layer (Fig. 3c). The
color is due to the visual red of the rhabdomes.
When dark-adapted C. bartoni were injected with eye-stalk ex-
tracts, and left in the dark, varying effects on the pigment were seen
depending on the amount injected, and the interval between the time
of injection and the time of observation. Animals which were dark-
adapted for several hours and injected in the early evening with an
amount of material equivalent to that obtained from one-fourth to one
eye-stalk showed, after three hours, a migration of the distal pigment
to or toward the light position. The proximal pigment was not
affected (Fig. 4). When such eyes are viewed by reflected light they
appear gray rather than black and very little light is reflected from
the tapetal layer (Fig. 4d).
The injection of 0.1 cc. of the extract (= the extractible material
from two eye-stalks) had a distinct effect on the proximal as well as
RETINAL PIGMENT MIGRATION IN THE CRAYFISH
123
the distal pigment. After three hours both pigments were found to
occupy positions more or less typical of light adaptation (Fig. 5). The
intact eye when viewed by reflected light had the same appearance as
the normal light-adapted eye (cf. Fig. 5e with la).
FIG. 3. Ommatidium of a typical dark-adapted eye showing the positions of
the pigments.
FIG. 3c. The intact eye of a dark-adapted animal has a bright orange-red
center when viewed by reflected light.
FIG. 4. The injection of the active material from one eye-stalk into a dark-
adapted animal causes the migration of the distal pigment to the light position.
FIG. 4d. The intact eye of such an animal may have a small reflecting central
area.
FIG. 5. The injection of the active material from two eye-stalks into a dark-
adapted animal causes the migration of both distal and proximal pigments to their
light positions.
FIG. 5e. The intact eye of such an animal has the same appearance as that
of a light-adapted animal.
124 JOHN H. WELSH
V
It has been demonstrated that it is possible, by means of a simple
extraction process, to obtain from two eye-stalks of a crayfish an
amount of retinal pigment activator, or hormone, equivalent to that
normally released by the animal during the process of light adaptation.
From one eye-stalk the amount of hormone is sufficient only to acti-
vate the distal pigment cells; thus indicating that they have a lower
threshold than do the proximal pigment cells. Such threshold differ-
ences between the three sets of pigments in a given species may
account for such a situation as was first seen in the eye of Macro-
brachium (Welsh, 1930), where under continuous 'illumination the
distal pigment cells migrate toward the periphery of the eye at the
time of sunset and return to a proximal position at the time of sunrise,
while the proximal pigment remains in a constant light position (see
also Welsh, 1935, 1936; and Kleinholz, 1937, 1938).
The injection of eye-stalk extracts into dark-adapted crayfishes
makes it possible to obtain a "light-adapted" eye, as regards the
positions of the screening pigments, while the "dark-adapted" level
of the light-sensitive substance of the retina remains unaffected. This
enables one to study the effect of pigment position on visual acuity
and response to flicker and has been employed by Crozier and Wolf
(1939).
SUMMARY
A substance similar to, or identical with, the eye-stalk or chroma-
tophorotropic hormone may be obtained from the eye-stalks of cray-
fishes. When injected, in proper amount, into light-adapted cray-
fishes it causes the distal and proximal pigments to migrate to more
extreme "light positions" than normal. When injected into dark-
adapted crayfishes which are allowed to remain in the dark it causes
the migration of one or both sets of screening pigment to their "light
positions." The distal pigment has a lower threshold than the proxi-
mal pigment, as it is affected by lower concentrations of the active
substance. It is suggested that such threshold differences may ac-
count, in part, for the unusual pigment responses which have been
observed in compound eyes in studies of 24-hour cycles in pigment
migration.
LITERATURE CITED
ABRAMOWITZ, A. A., AND R. K. ABRAMOWITZ, 1938. On the specificity and related
properties of the crustacean chromatophorotropic hormone. Biol. Bull.,
74: 278.
BENNITT, R., 1932. Diurnal rhythm in the proximal pigment cells of the crayfish
retina. Physiol. Zool., 5: 65.
RETINAL PIGMENT MIGRATION IN THE CRAYFISH 125
CROZIER, W. J., AND E. WOLF, 1939. The flicker-response contour for the crayfish.
II. Biol. Bull., 77: \26.
DAY, E. C., 191 1. The effect of colored light on pigment-migration in the eye of the
crayfish. Bull. Mus. Comp. Zoo!., 53: 305.
KLEINHOLZ, L. H., 1934. Eye-stalk hormone and the movement of distal retinal
pigment in Palaemonetes. Proc. Nat. Acad. Sci., 20: 659.
KLEINHOLZ, L. H., 1936. Crustacean eye-stalk hormone and retinal pigment migra-
tion. Biol. Bull, 70: 159.
KLEINHOLZ, L. H., 1937. Studies in the pigmentary system of Crustacea. II.
Diurnal movements of the retinal pigments of Bermudan decapods. Biol.
Bull., 72: 176.
KLEINHOLZ, L. H., 1938. Studies in the pigmentary system of Crustacea. IV.
The unitary versus the multiple hormone theory of control. Biol. Bull.,
75: 510.
PARKER, G. H., 1932. The movements of the retinal pigment. Ergbn. der Biol., 9:
239.
WELSH, J. H., 1930. Diurnal rhythm of the distal pigment cells in the eyes of certain
crustaceans. Proc. Nat. Acad. Sci., 16: 386.
WELSH, J. H., 1932. The nature and movement of the reflecting pigment in the
eyes of crustaceans. Jour. Exper. Zool., 62: 173.
WELSH, J. H., 1935. Further evidence of a diurnal rhythm in the movement of
pigment cells in eyes of crustaceans. Biol. Bull., 68: 247.
WELSH, J. H., 1936. Diurnal movements of the eye pigments of Anchistioides.
Biol. Bull., 70: 217.
WELSH, J. H., 1938. Diurnal rhythms. Quart. Rev. Biol., 13: 123.
THE FLICKER-RESPONSE CONTOUR FOR THE CRAYFISH
II. RETINAL PIGMENT AND THE THEORY OF THE
ASYMMETRY OF THE CURVE
W. J. CROZIER AND ERNST WOLF
(From the Biological Laboratories, Harvard University, Cambridge)
I
•
The flicker-response contour (F • - log /) for the crayfish Cambarus
bartoni resembles that for other arthropods having markedly convex
eyes (see Crozier and Wolf, in press). Only its very uppermost part
can be fitted by a probability integral. Over its lower part the slope
increases too rapidly, so that the whole curve is quite asymmetrical.
This departure from the rule observed in the responses of vertebrates
(see Crozier and Wolf, 1937a and b, 1938a) has been accounted for
(Crozier and Wolf, 1937 c, 19386) by the shape of the optic surface in
the majority of arthropods. With increasing flash-intensities the
retinal area effectively involved is increased, which results in a higher
F; this is due to the greater chance of exciting ommatidia toward the
circumference of the curved eye. Confirmation of this view, consistent
with the consequences of changing the light-time fraction in the flash-
cycle (Crozier and Wolf, 1937c, 19386), is given by the fact that an
arthropod with sufficiently flat optic surfaces, the isopod Asellus
(Crozier and Wolf, 1939), gives a flicker-response contour which is a
perfectly symmetrical probability integral. The asymmetry of the
curve with Anax is appropriately reduced by blocking out all but a
central area of the eye (Crozier and Wolf, 1937c, 19386), and in a form
with still more markedly curved optic surfaces (Cambarus) (see
Crozier and Wolf, in press) the asymmetry is much more extreme.
In our experiments with Anax (Crozier and Wolf, 1937c, 19386)
the limitation of the increase of effective retinal area with increase of
illumination by painting portions of the eyes was recognized to be im-
perfect. A certain amount of leakage of light near the margins of a
cap of enamel, and under its edge, cannot be prevented. A neater
method of accomplishing the purpose is to use the migrations of retinal
pigment cells. The flicker-response contours we have discussed were
determined with animals previously dark-adapted. For such a crusta-
cean as Cambarus this means that the proximal retinal pigment is
below the level of the receptive retinulae, the distal pigment cells well
126
FLICKER-RESPONSE CONTOUR FOR THE CRAYFISH 127
out toward the surface of the eye around the crystalline cones. The
retinulae are completely unshielded from laterally spreading light, and
the condition is that for the "superposition " type of eye (Exner, 1891).
In the eye well light-adapted the forward migration of the proximal
pigment shields the retinulae, while the inward movement of the distal
pigment forms around each ommatidium an opaque tube of pigment
along the length of the crystalline lens and down to the proximate
pigment (Bernhards, 1916; Day, 1911; Parker, 1932). The effective
isolation of each recipient unit from light other than that proceeding
down the axis of the ommatidium then produces the condition for the
"apposition eye" (Exner, 1891).
For our purposes, however, no use could very well be made of the
control of retinal pigment migration by light. The process of light
adaptation involves not only movements of the retinal pigment cells,
but also, it must be presumed, the intrinsic photic adaption of the
visual response system itself. At the same time, if some other pro-
cedure could be found to cause the retinal melanophores to assume the '
"light-adapted" condition, it should serve admirably for a test of
certain properties of the Cambarus flicker-contour. It should also
give some direct behavioral evidence as to the functional role of the
retinal pigment and its movements, as well as providing material for
a logical approach to the method of estimating the time-course of visual
light-and-dark-adaptation in such animals.
It was pointed out to us by Dr. J. H. Welsh that extracts containing
the "eyestalk hormone" from the optic peduncle produce an effect on
the melanophores and also on the movement of retinal pigment
(Kleinholz, 1934, 1936, 1938; Welsh, 1939) in dark-adapted eyes of
Cambarus, so that injection of sufficient extract into a dark-adapted
animal leads to the migration of retinal pigment into positions charac-
teristic of the normal light-adapted state. This we have verified in
C. bartoni.
II
The observational procedure was identical with that employed
in measuring the flicker-response contour for dark-adapted Cambarus
(Crozier and Wolf, in press): temperature 21.5°, 50 per cent light-time
in the flash cycle. To keep the handling of the animals uniform with
respect to time after injection and the like, a lot of 5 rather than of 10
was used. The eyestalks from 10 Cambarus bartoni were extracted in
Ringer solution. Into each crayfish prepared for observation there
was injected into the abdomen 0.08 ml. of extract, the equivalent of
2 eyestalks. After 75 to 90 minutes in the dark the crayfish are
128
\V. J. CROZIER AND ERNST WOLF
bluish in body color and by means of a beam of light directed into the
eye the retinal pigment is seen to be in the position characteristic of
light adaptation. Sectioned eyestalks fixed in hot water at this stage
show the condition clearly under the ultrapak microscope. In the
normal dark-adapted eye the proximal pigment is retracted below the
basement membrane, while the distal pigment is out between the
crystal cones. There is no detectable pigment between the om-
matidial units. After about 90 minutes in darkness subsequent to
injection of eye-stalk extract, the proximal pigment surrounds the
TABLE I
Data for the flicker-response contour of the crayfish Cambanis bartoni, with
eye-pigment in the "light adapted " state as result of injection of eye-stalk hormone.
N = 5 individuals, n — 3 observations on each; the same individuals used through-
out; t° = 21.5° C.; IL = to. See Fig. 1. 7 in millilamberts, F in flashes per second.
P.E.i = P.E. of the dispersions.
F
Fm
P.E.1Fl
loglm
log P.£.i/!
2
3.3404
5.9958
5
2.0777
5.3334
8
2.2584
5.4806
12
2.5249
3.8155
16
2.6674
5.9737
20
2.7771
3.0398
25
2.9254
3.8291
30
1.0730
3.2355
35
1.2865
3.6169
40
1.5937
3.8785
41.79
0.289
0.00
42
0.0233
2.3664
43
1.0077
1.2823
43.10
0.357
0.50
43.23
0.361
1.00
43.66
0.282
1.25
44
1.5231
0.1464
44.05
0.130
1.50
retinulae, while the distal pigment now envelopes each ommatidial
unit down to its base. The condition is one of quite complete shielding
of each ommatidium by a dense layer of black pigment, more extreme
than is the case in ordinary light adaptation.
Ill
The determinations of mean critical flash-intensity and mean criti-
cal flash-frequency for response (Crozier and Wolf, in press) to visual
flicker are given in Table I. Comparison with the results for normally
FLICKER-RESPONSE CONTOUR FOR THE CRAYFISH
129
dark-adapted Cambarus bartoni (Crozier and Wolf, in press) shows that
there is a pronounced (reversible) effect of the injection of eyestalk
hormone upon the properties of the flicker-response contour. This
cannot reasonably be traced to an effect of the eye-stalk extract upon
the intrinsic processes of photic excitability, for several reasons. In
normal
•with E.S.E
FIG. 1. The variation of I\ for normal Cambarus bartoni and after injection with
eye-stalk extract (E.S.E.); Table I; see text.
the first place injection of ca. 0.06 ml. of the eyestalk extract into
A nax (dragon fly) nymphs produces no detectable effect either on
pigment migration or on the flicker-response curve, as the following
observations showed (tests on 5 individuals) :
F
20
30
Normal
log /„,
2.473
2.749
2.741
Normal + eye-
stalk extract
log /m
2.478
2.745
2.750
Any effect of this sort would thus have to be specific. In the second
place, the results of adapting Cambarus are rapidly apparent even
when the retinal pigment is already fully advanced into the "light"
position, as subsequently shown (§ IV). Finally the various modifica-
130
W. J. CROZIER AND ERNST WOLF
tions of the flicker-response contour are those to be expected as the
result of the optical shielding of the ommatidia, so that no specific
effect on excitability need be invoked.
For any given level of flash-intensity the variation of I\ among the
individuals used is statistically of the same magnitude as for the normal
group previously examined (Crozier and Wolf, in press). The 5 indi-
viduals giving the data of Table I were in the lot of 10 providing the
normal curve for this species (Crozier and Wolf, in press). The scatter
of the variation indices (P.E.iJ is even a little less than might have
been expected in view of the smaller number of readings in the eyestalk
injection series (Fig. 1).
The effects to be expected if the "dark" position of the retinal
pigment shields ommatidia from all but light parallel to the retinular
axis, and if this is to prevent the recruitment of optic impulses from
a larger retinal surface as flash intensity is increased, are the following:
(1) the total achievable sensory effect (= Fmax.} must be reduced; (2)
at given /, F must be less; (3) the asymmetry of the F - - log / curve
must be markedly reduced ; and (4) it would not be surprising to find
the slope of the "fundamental " curve increased (i.e., a'\os /, for the ideal
frequency distribution of log / thresholds, reduced), owing to the
mechanical exclusion of a large proportion of the otherwise marginal!}
excitable units.
FIG. 2. F — log / curves for dark-adapted Cambarus and under the same conditions
for individuals injected with eye-stalk extract (E.S.E.); Table I.
Figure 2 shows that the F - - log / curve with Cambarus dark-
adapted but under the influence of eye-stalk extract is moved toward
higher intensities and exhibits a lower maximum. These are the
FLICKER-RESPONSE CONTOUR FOR THE CRAYFISH
131
100
80
60
Cambarus
° normal
20
FIG. 3. The curves of Fig. 2 brought to the same Fmil*. (= 100 per cent), to show
change of shape.
results of a decrease in the total number of excitable elements (Crozier
and Wolf, 1937c, 19386), as expected.
The asymmetry of the curve is also decreased (Fig. 3). The
50
40
2,0
10
0
FIG. 4. The curves of Fig. 2 with probability integrals adjusted to the upper
portions (cf. Crozier and Wolf, 1937f, 1938&, 1939, and paper in press), to show that
the flicker-response contour after injection of eye-stalk extract departs less than the
normal; see text.
132
W. J. CROZIER AND ERNST WOLF
sheathing of the ommatidia by pigment materially reduces the chance
of photic action on additional elements as intensity increases, hence the
slope of the F - - log / curve cannot increase so rapidly.
It is to be presumed that in the absence of comparatively free
passage of light through the eye (as in the dark-adapted state), the
actual intensity at each receptor locus will be decreased. This cannot
be a major factor in the changes shown in Fig. 2, else increase of in-
tensity would find the F - - log / curve continuously rising at its upper
end.
The diffusion of light within the substance of the eye cannot be
ignored, however. Figure 4 shows that the asymmetry of the flicker-
FIG. 5. Portions of the curves in Fig. 2 for dark-adapted Cambarus with
(D + E.S.E.) and without (D) injection of eyestalk extract, and the first (1) and
second (?) sets of readings (Table II) during the progress of dark adaptation after
light adaptation.
response contour has been decreased (cf. Fig. 3), but not abolished. In
view of the proximal movement of the distal retinal pigment under the
influence of eye-stalk extract (Kleinholz, 1934, 1936, 1938; Welsh,
1939), this is not surprising. It probably explains the slight but
detectable rise of the curve at the highest intensities used (Table I ;
Fig. 2), particularly when Fm is determined at constant flash-intensity;
this cannot be accounted for by light adaptation (§ IV).
With allowance for this effect, a reasonable adjustment of an ideal
probability integral can be made to the upper part of the curve (Fig. 4).
FLICKER-RESPONSE CONTOUR FOR THE CRAYFISH
133
Comparison with the normal, in the same figure, shows that <j'iog / is, as
expected, much reduced.
IV
Light adaptation of Cambarus reduces the F - - log / curve (Table
II, Fig. 5) ; with even brief residence in darkness the curve rises toward
TABLE II
Critical flash-frequencies at two flash-intensities for Cambarus: (1) very shortly
after light adaptation to bright daylight; (2) after ca. 10 minutes in darkness; 3 ob-
servations on each of the same 4 individuals at all points; 21°. 5, IL = to- See text,
and Fig. 4.
(1)
(2)
log/
T.50
0.00
1.00
0.50
17.5
38.3
14.5
44.5
P.E,Fl
0.491
0.371
1.52
4.81
the position typical for dark adaptation (Fig. 5). Obviously, for a
quantitative investigation of the kinetics of photic excitation, the
effect of the migration of retinal pigment as governed by light and
darkness must be ruled out. The present data supply the first evi-
dence of a functional role of the position of the retinal pigment in
matters of visual response. The result of light adaptation, as with
certain other forms, is to reduce F at fixed /, but to follow by this
means the recovery of excitability during subsequent darkness is made
difficult by the fact that the retinal pigment also changes position.
Either the pigment must be held in a fixed position throughout, by
suitable repeated injection of eye-stalk extract, or else a procedure
found for extrapolation to a constant condition of the pigment.1 The
latter could perhaps be achieved by determining the relation between
the position of the F • - log / curve and various known positions of the
pigment; in any event the whole course of the function must be known.
SUMMARY
Injection of Cambarus bartoni with extract of eyestalks of this
species forces migration of retinal pigments of individuals kept in
darkness into positions characteristic of the light-adapted eye. In
this condition the receptor elements of each ommatidium are effectively
shielded from light passing through their neighbors. The flicker-
response contour then differs in four particulars from that found when
the retinal pigment is in the "dark" position, for which effective screen-
1 For a somewhat analogous case of changing sensitivity during the interval of
observation, a technic of this kind was used with Agriolimax (Crozier, W. ]., and
Wolf, E., 1928-29, Jour. Gen. Physiol., 12: 83).
134 W. J. CROZIER AND ERNST WOLF
ing of the ommatidia is not present : Fmax. is lowered ; the whole curve is
moved to higher intensities; the spread of the log / thresholds for the
cumulative population of sensory effects is lessened; and the asym-
metry of the F - - log / curve is markedly reduced. It is pointed out
that these results are to be expected if the asymmetry of the curve in
normal dark-adaptation is due to the relation between flash-intensity
and the curvature of the optic surface and divergence of the ommatidial
axes.
CITATIONS
BERNHARDS, H., 1916. Zeitschr.f. wiss. Zool., 116: 649.
CROZIER, W. J., AND E. WOLF, 1939. Jour. Gen. Physiol., 22: 451.
CROZIER, W. J., AND E. WOLF. Jour. Gen. Physiol., in press (Cambarus, I).
CROZIER, W. J., E. WOLF, AND G. ZERRAHN-WOLF, 1937a. Proc. Nat. Acad. Sci.,
23: 516.
CROZIER, W J., E. WOLF, AND G. ZERRAHN-WOLF, 19376. Jour. Gen. Physiol., 21:
17, 203.
CROZIER, W. J., E. WOLF, AND G. ZERRAHN-WOLF, 1937c. Jour. Gen. Physiol.,
21: 223.
CROZIER, W. J., E. WOLF, AND G. ZERRAHN-WOLF, 1938a. Proc. Nat. Acad. Sci.,
24: 125.
CROZIER, W. J., E. WOLF, AND G. ZERRAHN-WOLF, 1938&. Jour. Gen. Physiol., 21:
463.
DAY, E. C., 1911. Butt. Museum Compar. Zool., 53: 305.
EXNER, S., 1891. Die Physiologic der facettierten Augen von Krcbsen und Insekten.
Leipzig u. Wien, 206 pp.
KLEINHOLZ, L. H., 1934. Proc. Nat. Acad. Sci., 20: 659.
KLEINHOLZ, L. H., 1936. Biol. Bull., 70: 159.
KLEINHOLZ, L. H., 1938. Biol. Bull., 75: 510.
PARKER, G. H., 1932. Ergebn. Biol., 9: 239.
WELSH, J. H., 1939. Biol. Bull., 77: 119.
THE SIGNIFICANCE OF GERMARIA IN DIFFEREN-
TIATION OF OVARIOLES IN FEMALE APHIDS
CHESTER A. LAWSON
(From the Department of Biology, Wittenberg College, Springfield, Ohio)
INTRODUCTION
Recognition of the fact that winged parthenogenetic female aphids
produce both parthenogenetic and gamic female offspring differing in
part in the structure of the ovarioles, invites an understanding of the
mechanism that controls the development of the ovarioles. Such an
undertaking may aid in determining how genes, presumably identical,
can produce two types of individuals.
In studies on the development of aphids (Lawson, 1939), it was
noted that the germaria are the first embryonic structures to mark a
distinction between gamic and parthenogenetic females. Other dif-
ferentiating characters do not appear until after birth. As determina-
tion of all differentiating characters occurs before birth (Shull, 1930a),
it is possible that the germaria are instrumental in determining the
adult nature of the individual at least in so far as the ovarioles are
concerned.
In female aphids the essential reproductive organs consist of a pair
of ovaries in which the eggs are developed, and an oviduct leading
from each ovary to an external opening. Each ovary is made up of a
number of loosely parallel ovarian tubes (ovarioles) which open into
the oviduct. Three different regions are recognized in an ovariole
(Fig. 1), — the terminal filament, the germarium, and the vitellarium.
The terminal filament is a thread-like structure at the end of the
ovariole farthest from the oviduct, which attaches the ovariole to the
body wall. Next behind the terminal filament is the germarium which
contains the germ cells from which the eggs develop, and nurse cells
whose function is to furnish nutriment to the developing eggs. The
vitellarium is a tubular structure which extends from the germarium
to the oviduct and contains developing eggs in a gamic female and
both eggs and embryos in a parthenogenetic female. A nutritive
thread or yolk stream extends from the nurse cells to the youngest
growing oocyte in the vitellarium.
DIFFERENCES BETWEEN GAMIC AND PARTHENOGENETIC FEMALES
Gamic females are described by Shull (19306) as follows: "Gamic
females of this species of aphid have a wax yellow body color, dark
135
136
CHESTER A. LAWSON
TF
FIG. 1. Diagrams of aphid ovarioles, gamic at left, parthenogenetic at right.
E, egg; Em, embryo; G, germarium; GC, germ cell; NC, nurse cell; 0V, oviduct;
TF, terminal filament; YS, yolk stream.
brown antennae, and greatly swollen hind tibiae of dark brown color,
covered with hundreds of sensoria. Their reproductive systems con-
sist of a vagina on which are borne a pair of colleterial glands and a
seminal receptacle, a pair of short oviducts formed as branches of the
vagina, and a variable number (usually ten) of ovarioles branching
DIFFERENTIATION OVARIOLES IN FEMALE APHIDS 137
from the oviducts. Each ovariole, in a mature female, contains
usually one mature or nearly mature egg, distinctly opaque and of
very regular ovoid form ; beyond this often an pocyte, in early growth
stage, hence long and slender and not very opaque; and lastly a large,
spherical germarium forming a conspicuous knob at the end of the
ovariole. In old gamic females, especially those that have not been
laying eggs, the second reproductive cell from the base of the ovariole
may be large and opaque and regular in form, and is then presumably
mature like the one posterior to it. Almost never, however, in typical
gamic females (that is, those produced at low temperature by winged
females whose other daughters are practically all gamic), are there
more than two oocytes in any stage in one ovariole. An ovariole of
a gamic female may therefore be regarded as regularly consisting
of a tube containing one or two eggs or oocytes, and a large round
germarium."
In stained sections of adult gamic females the ovarioles (Fig. 1)
are prominent in the abdominal cavity. The germaria usually lie
anterior to the large yolk-laden eggs. Each germarium is surrounded
by the closed end of an ovariole tube composed of a single layer of thin,
squamous epithelial cells. Posterior to the germarium the tube con-
stricts, forming a short neck in which the lumen is quite narrow. The
narrowing of the lumen is due in part to contraction of the tube, but
also to an increase in the height of the cells which change from a
squamous to a columnar type in the neck region. A constriction of
the tube likewise occurs between the eggs contained in the vitellarium,
but this is due to contraction only as there is no change in cell shape.
Around the young growing oocytes the ovariole wall is constructed
of a single layer of cuboidal cells. In the posterior part of the ovariole
the cells are rectangular with the greatest width parallel to the surface
of the egg.
The germarium is composed of two types of cells, a round ball of
large nurse cells and a small group of germ cells. Each nurse cell is
roughly pyramidal in shape (triangular in section) with the base at
the periphery of the germarium and the apex in the center. The nurse
cells fit closely together and form a ball, in the center of which is a sub-
stance secreted by the nurse cells. This substance flows from the
central area through the neck of the ovariole into the growing oocyte
within the vitellarium. The exact nature of this substance is unknown,
however, as it flows directly into the growing oocyte; it might be yolk.
No better term is available, hence the term "yolk" is used to facilitate
discussion, and the term "yolk stream" is used to indicate the distinct
cord, or string of substance which passes from the germarium into
the growing oocyte.
138 CHESTER A. LAWSON
Germ cells, smaller and fewer in number than the nurse cells, lie
between the ball of nurse cells and the neck of the ovariole. Posterior
to the neck the vitellarium contains ovoid oocytes in various stages of
growth. Because only young adults were used for this study, it is
likely that no mature eggs were examined. Each oocyte consists of
a central mass of yolk surrounded by a thin peripheral layer of cyto-
plasm. Outside of this cytoplasm is a single egg membrane. In the
smaller younger oocytes the nucleus occupies the center of the cell,
but in older ones it lies at the periphery halfway between the two ends
of the egg. The nucleus is large and clear and contains but a few small
bodies which stain heavily.
Parthenogenetic females are described by Shull (19306) as follows:
"The parthenogenetic females have bright green body color, antennae
quite pale except in the distal segment, and very slender, pale hind
tibiae bearing no sensoria. The reproductive system consists of a
vagina, without collegerial glands or seminal receptacle, two short ovi-
ducts branching from the vagina, and a variable number (apparently
up to ten) of ovarioles branching from the oviducts. Each ovariole is
a very delicate tube containing, in healthy individuals, usually six to
nine embryos or eggs or oocytes, and bearing at the end a very small
germarium which is usually not much larger, and is often smaller,
than the oocyte or egg next behind it. All of these reproductive ele-
ments except one (the one next to the germarium) are as a rule em-
bryos in some stage of development. They are all translucent or
transparent, unless dead, and if dead they have a clouded appearance
not at all like the opaque gamic eggs. Only the smaller embryos are
ellipsoidal; the medium and larger ones always possess angles which
correspond to the form of the young aphids. The six to nine embryos
or eggs in one ovariole, in a typical healthy female, are of regularly
decreasing size from oviduct to germarium, so that they resemble a
tapering string of beads."
In stained sections of parthenogenetic female aphids the entire
abdominal cavity is crowded with embryos of varying sizes and stages
of development. In general, the larger more developed embryos lie in
the posterior region while the smaller less developed embryos lie
anterior to them. The germaria (Fig. 1), small and difficult to locate,
usually lie in the anterior abdominal region squeezed among young
embryos or between the embryos and the lateral body wall. The wall
of the ovariole tube around the germarium is a single squamous epi-
thelium which constricts, forming a neck just posterior to the ger-
marium. The cells become somewhat cuboidal in this region. Ex-
tending posteriad from the neck the ovariole wall encloses embryos,
DIFFERENTIATION OVARIOLES IN FEMALE APHIDS 139
and is composed of a thin single layer of squamous cells. The tube is
always constricted between the embryos within any one ovariole.
The parthenogenetic germarium consists of two types of cells, a round
ball of nurse cells and a small group of germ cells. Each nurse cell is
roughly pyramidal in shape with the base at the periphery of the
germarium and the apex in the center. The nurse cells fit closely
together and form a ball. This ball of nurse cells contains yolk, and
a yolk stream extends from the nurse cells through the ovariole neck
into the youngest growing oocyte. The germ cells lie in the germarium
between the ball of nurse cells and the neck of the ovariole. Young
oocytes are found immediately posterior to the germarium. Progress-
ing caudad, the next in line is usually an egg in cleavage followed in
turn by a young developing embryo. Thereafter each succeeding
embryo is larger and more fully developed.
MAJOR DIFFERENCES BETWEEN ADULT GAMIC AND
PARTHENOGENETIC OVARIOLES
The primary difference between gamic female and parthenogenetic
female ovarioles is in the development of the germ cells. In gamic
female ovarioles development of germ cells consists of growth through
accumulation of yolk and possibly meiosis, though no divisions have
been observed in this species of aphid. In the parthenogenetic female
ovarioles the germ cells are stimulated to develop parthenogenetically.
The size difference in the germaria presumably is secondary to and
correlated with the germ cell difference. Gamic female ovarioles con-
tain eggs that undergo embryonic development outside of the mother's
body, are dependent on a large yolk supply and consequently accumu-
late this yolk supply during growth in the ovariole. The large size of
the gamic female germarium (about three times larger than a partheno-
genetic female germarium in fixed material) is evidently correlated
with the necessity of producing much yolk. Eggs produced by par-
thenogenetic females develop within the body of the mother, and it is
probable that nourishment for this growth and development is supplied
directly by the mother by body fluid. Thus a large quantity of yolk is
unnecessary for parthenogenetic eggs, and the small size of partheno-
genetic female germaria may be correlated with this decreased secretory
activity.
OVARIOLES OF GAMIC FEMALE AND PARTHENOGENETIC
FEMALE EMBRYOS
The ovarioles of gamic female and parthenogenetic female embryos
were studied and compared in those embryos of both types which
140
CHESTER A. LAWSON
showed the greatest degree of development. These embryos were well
developed, occupied the posterior abdominal region of the mother and,
presumably, would have been born very shortly had the mother been
allowed to live.
Each ovariole in gamic embryos of this late stage of development
consists of a terminal filament, a germarium and a vitellarium (Fig. 2).
Each germarium contains nurse cells and germ cells. The nurse cells
are roughly pyramidal in shape and form a ball at the end of the
FIG. 2. Photomicrograph of an embryonic ovariole of a gamic female aphid.
G, germarium; GC, germ cell; NC, nurse cell; V, vitellarium.
ovariole. This is similar to the adult condition although the cells seem
to be more loosely packed and the entire germarium is more elongated
than in the adult. The center of the ball of nurse cells contains yolk,
but there is no yolk stream. Germ cells occupy the region of the
germarium posterior to the nurse cells. The ovariole tube surrounds
the germarium as a simple squamous epithelium and continues
posteriad as a small tubular vitellarium with a narrow lumen. The
vitellarium contains no eggs or oocytes.
In parthenogenetic female embryos of the same degree of develop-
DIFFERENTIATION OVARIOLES IN FEMALE APHIDS 141
ment the ovariole consists of a terminal filament, a germarium, a
vitellarium, and parthenogenetically developing germ cells (Fig. 3).
Each germarium consists of nurse cells and germ cells. The nurse cells
form a ball of cells at the tip of the ovariole tube within which is found
a small amount of yolk. A yolk stream extends from the center of the
ball of nurse cells through the neck of the ovariole into the youngest
growing oocyte. The germ cells lie just behind the nurse cells. Each
germarium is surrounded by the closed end of the ovariole tube which
FIG. 3. Photomicrograph of an embryonic ovariole of a parthenogenetic female
aphid. E, egg undergoing cleavage; G, germarium; O, oocyte.
consists of a thin squamous epithelium. The ovariole tube continues
posteriad of the germarium as a thin-walled vitellarium which in-
variably contains a growing oocyte just behind the germarium and an
egg undergoing cleavage behind the oocyte.
To insure that embryos identified as gamic females were actually
gamic, parthenogenetic winged females were fixed when they were
producing gamic females only. The parthenogenetic female embryos
were studied only in wingless parthenogenetic females which seldom
produce gamic females. The offspring of both types of parents pro-
duced before fixation were reared and examined.
142 CHESTER A. LAWSON
Do GERMARIA CONTROL DIFFERENTIATION OF OVARIOLES?
The possibility that germaria play a significant role in the dif-
ferentiation of ovarioles is indicated by three observations. (1) Adult
ovariole differences are due primarily to the type of egg development
that occurs within the ovariole and to the size of the germarium. (2)
Germaria contain and produce the germ cells that develop within the
ovarioles. (3) Germaria are the first reproductive structures to be
differentiated in the embryo (Law^son, 1939).
Determination of an embryo into either a gamic or parthenogenetic
female must occur sometime during parthenogenetic development
between the growth of the oocyte and the differentiation of the
germaria. This determination may affect the embryo in two ways.
(1) It may include the entire embryo in its effect so that the differen-
tiation of the germaria would simply represent the first differential
reaction of a general condition throughout the embryo. This would
exclude the germaria from any significance in future development.
(2) It may occur at the time that germaria are developing and affect
them only. According to this assumption the embryo wrould be po-
tentially capable of developing into either a gamic or a parthenogenetic
female prior to determination of the germaria. After this event the
aphid (more specifically the ovarioles) would become gamic or par-
thenogenetic depending on the nature of the germaria.
The study of normal gamic and parthenogenetic female aphids
offers no choice between the two ways in which determination may
occur. However, aphids intermediate between gamic and partheno-
genetic are occasionally produced and an analysis of these interme-
diates gives us a choice.
These intermediates, described by Shull (1930&), show the inter-
mediacy in several structures of which the ovarioles only are to be
considered here. The intermediacy is expressed in the ovarioles in a
very irregular fashion so that no two aphids with intermediate ovarioles
are necessarily identical. An intermediate aphid may be a mosaic
with respect to the ovarioles, in that one or more of the ovarioles are
strictly gamic while the others are strictly parthenogenetic. Any one
ovariole may be intermediate in that the germaria are smaller than
normal gamic germaria, but larger than normal parthenogenetic
germaria. The contents of the vitellarium may be intermediate in
three different ways. (1) Eggs may vary from gamic in being less
opaque than strictly gamic eggs. (2) Gamic eggs may occur in
greater numbers than is normal for a gamic ovariole. (3) Embryos
characteristic of parthenogenetic ovarioles may be abnormal. Ac-
cording to Shull's description, it is possible to have any combination
of the above conditions in one aphid.
DIFFERENTIATION OVARIOLES IN FEMALE APHIDS 143
The first type of intermediate mentioned here in which one indi-
vidual contains both gamic and parthenogenetic female ovarioles
could not be produced unless the ovarioles are able to develop within
the aphid independently of one another. As the germaria appear in
the embryo prior to other ovariole structures, it follows that the
germaria also must develop and be determined independently of one
another.
The second type of intermediate in which the germaria are inter-
mediate in size between gamic female and parthenogenetic female
germaria indicates that the mechanism of determination is such that
intermediate germaria are determined and differentiated within the
embryo as well as germaria that are strictly gamic or parthenogenetic.
The mechanism of determination suggested by Shull (1930a), in
which a high level of some substance within the embryo produces one
type while a low level of the same substance produces the opposite
type, could very easily account for intermediate germaria. These
intermediate germaria could result from a condition in which deter-
mination of the germaria occurred when the level of concentration of
the determining substance was intermediate between the high and
low extremes.
The remaining types of intermediate aphids show the intermediacy
in the ovariole contents. All of these can be explained by assuming
that the germarium attached to the end of each intermediate ovariole is
intermediate. As gamic female germaria produce much yolk while
parthenogenetic female germaria produce little yolk, it would be
expected that intermediate germaria would produce an amount of yolk
intermediate between the gamic and parthenogenetic extremes.
One of the types of intermediate ovarioles described by Shull had
eggs that were gamic but less opaque than strictly gamic eggs. Such
eggs could be produced by a germarium that was gamic with respect
to the type of eggs produced but intermediate with respect to yolk
production. The decreased amount of yolk in the eggs might make
them less opaque than normal gamic eggs.
Another type of intermediate ovariole had gamic eggs in greater
numbers than the typical one or two of strictly gamic ovarioles. One
characteristic of gamic germaria is that they produce no more than
two eggs while parthenogenetic germaria produce many more than
two. The above intermediate ovariole could have resulted from a
germarium that was intermediate with respect to the number of eggs
produced, while at the same time it was gamic with respect to the type
of eggs produced.
The last type of intermediate described by Shull contained par-
144 CHESTER A. LAWSON
thenogenetic ovarioles in which the embryos were abnormal. The
details of the abnormality were not described but it is possible that
the abnormality could have been due to an intermediate germarium in
which parthenogenetic oocytes or eggs were produced plus a quantity
of yolk greater than is normal for a parthenogenetic germarium.
This abnormal amount of yolk very likely would interfere with normal
embryonic development and produce abnormal embryos.
The theory is proposed that germaria are determined independently
of one another and also of the rest of the aphid embryo, that a single
germarium may be caused to develop into a gamic female type, a
parthenogenetic female type or a type intermediate between the gamic
and parthenogenetic types. It is proposed, further, that the germar-
ium once determined, controls the differentiation of the ovariole to
which it is attached and thus controls, in part, the development of the
adult aphid type.
SUMMARY
Winged parthenogenetic female aphids produce both partheno-
genetic female and gamic female aphids.
The ovarioles of adult gamic female and parthenogenetic female
aphids differ primarily in the nature of the eggs developing within
them and secondarily in the size and secretory activity of the germaria.
Gamic female germaria are large and secrete much yolk; partheno-
genetic female germaria are small and secrete little yolk.
The ovariole differences apparent in the adult aphids are also evi-
dent in the embryos. In parthenogenetic female embryos of a late
stage of development the embryonic ovarioles already contain oocytes
and eggs undergoing parthenogenetic development while in the gamic
female embryos of the same stage of development the germ cells have
not yet entered the vitellarium. The germaria of parthenogenetic
female embryos are smaller than the germaria of the gamic embryos
of the same stage of development.
In both gamic and parthenogenetic female embryos the germaria are
the first reproductive structures to develop.
The theory is proposed that determination of the ovariole type
(either gamic female or parthenogenetic female) affects the germaria
only. Each germarium, thereafter, controls the development of the
ovariole to which it is attached.
Aphids intermediate between gamic female and parthenogenetic
female aphids with respect to the ovarioles are described and analyzed
to support the above theory.
DIFFERENTIATION OVARIOLES IN FEMALE APHIDS 145
BIBLIOGRAPHY
LAWSON, C. A., 1939. Order of differentiation in relation to order of determination
in gamic female aphids. Am. Nat., 73: 69.
SHULL, A. F., 1930a. Control of gamic and parthenogenetic reproduction in winged
aphids by temperature and light. Zeitschr.f. ind. Abst. u. Vererb., 55: 108.
SHULL, A. F., 19306. Order of embryonic determination of the differential features
of gamic and parthenogenetic aphids. Zeitschr. f. ind. Abst. u. Vererb.,
57: 92.
Vol. LXXVII, No. 2 October, 1939
THE
BIOLOGICAL BULLETIN
PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY
ACTIVITY-PREVENTING AND EGG-SEA-WATER
NEUTRALIZING SUBSTANCES FROM
SPERMATOZOA OF ECHINOMETRA
SUBANGULARIS
WALTER E. SOUTHWICK
(From the Bermuda Biological Station for Research l)
INTRODUCTION
A spermatozoon, like all highly specialized biological units, has
many utterly unique, but very significant characteristics. Of these,
probably the most fundamental is the finiteness of its period of life.
As has been clearly demonstrated by Lillie (1919), Gray (1928) and
others, mature spermatozoa, except possibly those of certain arthro-
pods, are unlike all other biological units in that they do not at any
time ingest or otherwise assimilate substances from which energy may
be derived. Once, therefore, that a spermatozoon has been liberated
from the testis, its every power and activity must be accomplished on
the basis of substances that were located in its structure at the time
of its liberation. Studies to determine what different substances
might be located in a spermatozoon, the location, nature, and ap-
proximate amounts of such substances, the changes in location and
state which they might undergo, and the factors which might in-
fluence, affect, or effect such processes are, therefore, especially
important for an understanding of spermatozoan physiology. A
series of studies which were made at the Bermuda Biological Station
for Research have provided some observations concerning such
substances.
MATERIALS AND PROCEDURES
The studies were made with the common reef urchin, Echinometra
subangularis, individuals of which were obtained from the reefs on
the eastern end of St. David's Island. The trip to the reefs was made
by bicycle, and the urchins were brought to the laboratory in a pail
with a small volume of water and covered with seaweed. The water
1 Laboratory space for this investigation was provided by the Bermuda Bio-
logical Station for Research, Inc. during July, 1938.
147
148 WALTER E. SOUTH WICK
was continually shaken, and so kept reasonably well aerated during
the trip, a period of approximately twenty minutes.
Upon arrival at the laboratory, the urchins were immediately
transferred to vessels in which a large volume of air was continually
bubbled through a comparatively small volume of circulating sea-
water. In such vessels, the urchins remained in good condition for
approximately three days, and so trips were made regularly every two
or three days in order to maintain a constant supply of freshly collected
urchins.
The animals were opened by means of the usual circumferential
cut, and the body fluids and intestines carefully removed. Every
precaution was taken to avoid contamination, either of eggs with
sperm, or of the sperm with egg secretions, and the gonads were re-
moved from the test with glass needles. The ovaries were divided
into small pieces in a small volume of sea-water with glass needles,
and the mass strained through several thicknesses of unbleached
cheesecloth. The supernatant fluid from the eggs was removed ten
minutes later and fresh sea-water added. After two or three washings
in this way, the supernatant fluid was allowed to stand for thirty
minutes, at which time it would be capable of producing clear-cut and
definite agglutinations with fresh sperm. The egg-sea-water was kept
separate from the eggs during all subsequent tests. The testes were
removed to a clean, dry Syracuse watch crystal, and, as fresh sperm
was needed, the tubules were broken with a glass needle and the exud-
ing dry sperm collected with a moderately fine pipette.2 The watch
crystal with the testes and the containers with the eggs or egg-secre-
tions were kept covered at all times except when the materials were
being withdrawn.
MOVEMENTS OF THE SPERMATOZOA
When dry sperm is examined immediately under the microscope,
the whole mass may be seen to be in a state of most intense vibratory
activity. In such spermatozoa, however, due probably to the com-
pactness of the mass, the active vibrations of the tails serve but to
cause a rapid milling about of waving heads. Progressive movements
are entirely absent, and the same group of spermatozoa sway back and
forth, in constant, rapid vibration, but always in the same position,
relative to each other and to the field of the microscope.
In such dry sperm, this motion lasts for from 50 to 120 seconds,
2 Insemination tests made with such spermatozoa in a dilution proportion of one
drop of dry sperm to 20 cc. sea-water, one drop of this suspension to one drop of eggs
in 7 cc. sea-water gave, consistently, a fertilization percentage of from 92 to 100
per cent.
EGG-WATER NEUTRALIZING SUBSTANCES 149
and then all movement ceases. The cessation in activity, once it
has started, spreads rapidly, so that the entire transition from uni-
versal activity to complete quiescence is accomplished within about
15 seconds. Such spermatozoa do not show motion when they are
redistributed mechanically by means of pressures on the cover glass.
When, however, a drop of sea-water is brought in contact with a
drop of dry sperm, the edge of the drop of dry sperm "frays" slightly.
After about one minute, the spermatozoa in this frayed edge begin to
move slowly. Gradually, they come to move more rapidly, more
spermatozoa become active, and they move slowly out into the drop
of sea-water. In about two minutes, the whole mass, or as much of
it as is reached by the diffusing sea-water, becomes intensely active.
In the denser portions of this mass, as in the original drop of dry
sperm, the motion is essentially a rapid milling about of waving heads
with progressive movements entirely absent. In the less dense
portions, however, progressive movements do occur.
When the dilution of one drop of dry sperm to one drop of sea-water
is made upon a glass slide where such changes in the supporting
medium as might be produced by evaporation are minimized by means
of placing pure petroleum jelly around the edges of the cover glass,
the activity continues, with gradually diminishing intensity, for about
three or four hours, when all motion ceases. If another drop of sea-
water then be added to the suspension under the cover glass, the
spermatozoa again become active, and this activity continues with
gradually decreasing intensity for about one and one-half hours, when
all motion again ceases. Such spermatozoa can be reactivated again
by the addition of another drop or two of sea-water, but the length
of time during which the spermatozoa remain active with each suc-
cessive reactivation progressively decreases until finally, after about
eight such reactivations, no further activation is obtained. This
phenomenon is shown more clearly in Table I which gives the actual
observations of a typical experiment of this nature.
If the reactivation obtained from the addition of fresh sea-water
to a suspension of inactivated spermatozoa were due to dilution
effects, then one might expect a substance to occur in the suspensory
fluid of an inactivated suspension of spermatozoa which would have
the property of preventing dry sperm from becoming active. In
order to test this possibility, a suspension of dry sperm was centrifuged
for 30 minutes at 4,000 revolutions per minute. When this centrifug-
ing had been completed, a clear, slightly opalescent, bluish fluid com-
prising about 11 per cent of the total volume of the original dry sperm
had separated from the mass of spermatozoa. Utmost care had been
150
WALTER E. SOUTHWICK
used to have the centrifuge tubes clean and perfectly dry before the
spermatozoa were added, and great care was now used to have pipettes,
glass slides, and cover glasses all perfectly dry. A drop of the clear
supernatant fluid was removed from the centrifuge tube and carefully
TABLE I
Changes in the activity of a suspension of spermatozoa with time, and the effects
of repeated additions of fresh sea -water upon the duration and changes in the activity
of the spermatozoa in such suspensions.
Time
Observations on the Activity
Treatment of the
Suspension
Number
of the
Sea-water
Addi-
tions
Time Interval
between Sea-water
Additions
10:45
1 dr. dry sperm
0
10:45
Intensely active
10:47
All sperm utterly motionless
1 dr. sea-water
1
10:48
Intensely active
11:02
Active
11:13
Some motion
11:29
Some motion
11:44
A little motion
12:05
A little motion
12:26
A little motion
1:21
Still a little motion
1:48
Just a little motion
2:12
No motion
1 dr. sea-water
2
3 hours 27 min.
2:13
Intensely active
2:39
Active
2:49
Active
3:27
Some activity
3:53
Motionless
Sea-water added
3
1 hour 40 min.
3:57
Activation
4:50
Motionless
Sea-water added
4
57 min.
4:52
Some activity
5:12
Active
5:51
Motionless
Sea -water added
5
61 min.
5:55
Some activity
6:25
Active
7:03
Motionless
Sea-water added
6
72 min.
7:05
Some activity
7:47
Motionless
Sea-water added
7
44 min.
7:51
A little activity
8:22
Motionless
Sea-water added
8
35 min.
8:30
No further activation
brought in contact with a drop of dry sperm. The whole process of
fusion of the two drops was carefully watched with the microscope,
but there was no slightest sign of any activation whatsoever at any
time. Even when the spermatozoa were thoroughly distributed
throughout the supernatant sperm fluid by means of stirring with a
EGG-WATER NEUTRALIZING SUBSTANCES 151
clean dry glass needle, there was no activation, and even examinations
with a magnification of 950 X failed to show any activity whatsoever.
Dilution of dry spermatozoa from the same testis tubules with ordinary
sea-water gave perfectly typical activation.
Two preparations with the supernatant fluid were made permanent
with the edges of the cover glass sealed with petroleum jelly, one at
12:35 P.M., and one at 1:55 P.M. The spermatozoa in the former
were still motionless at 2:21 P.M. but when fresh sea-water was added
at that time, the spermatozoa became intensely active by 2:25 P.M.
In the latter, the spermatozoa remained motionless until 7:30 P.M.,
but when fresh sea-water was added at that time, became intensely
active by 7:37 P.M. The residual spermatozoa after centrifuging
showed normal activation in all cases when ordinary sea-water was added.
This experiment definitely indicated that the suspensory fluid of
dry spermatozoa presents a condition which serves to prevent fresh
dry sperm from becoming active. This condition occurs also, in the
supernatant fluid, when 3" of dry sperm is centrifuged for 30 minutes
at 2,500 revolutions per minute through \Yz' of fresh sea-water. If
the supernatant fluid from such centrifuging be removed and replaced
with fresh sea-water, and the spermatozoa of the first centrifuging
be centrifuged through this sea-water for 45 minutes at 2,500 revolu-
tions per minute, the supernatant fluid from this centrifuging, too, will
prevent the activation of dry sperm.
All gradations occur. All supernatant fluid obtained by any sys-
tem of centrifuging dry sperm will prevent activation of fresh sperm.
When more dry sperm than sea-water is present, one passage of the
spermatozoa through the sea-water is sufficient to render the super-
natant fluid capable of preventing activation of dry sperm. When,
however, more sea-water than dry sperm is present, several washings,
the number depending upon the condition of the spermatozoa and the
relative proportions of sperm and sea-water, are necessary, and there
are several experiments which demonstrate the relationship between
the relative amount of dry sperm and sea-water present to the amount
of centrifuging necessary to cause the supernatant fluid to prevent
activity in dry sperm. Thus, in one typical experiment, dry sperm to
sea-water in the proportions of */%" : 3%", 1&" : 2%", 2%" : 2^",
and 1%" : V/£' were each washed five times, being centrifuged ap-
proximately 10 minutes at 3,550 revolutions per minute for each wash-
ing. The supernatant fluid from the %" '• 3/4" proportion did not
stop dry sperm, whereas the supernatant fluid from each of the other
proportions stopped dry sperm completely. In another typical experi-
ment, the proportion of dry sperm to sea-water was made the same
152 WALTER E. SOUTHWICK
in all four tubes, namely }/<£' : 3", and a tube removed after 5, 10, 15,
and 20 washings of approximately four minutes at 3,250 revolutions
per minute for each washing. The supernatant fluid after five wash-
ings did not stop dry sperm, the fluid obtained after 10 washings
allowed "just a little activity" in dry sperm, while the supernatant
fluid obtained after 15 and 20 washings completely prevented activity
in dry sperm.
In order to make certain that the occurrence of the condition in the
supernatant fluid was developed by the centrifuging, an exactly similar
proportion of dry sperm and sea-water was made and thoroughly
mixed at the same time that the other tubes were prepared. This
suspension of spermatozoa was kept in a tube until the centrifuging
of the other tubes had been completed. The sperm in this control
tube were then separated from the suspensory fluid by means of one
centrifuging at 3,250 revolutions per minute for 8 minutes, and the
resulting supernatant fluid tested. It was found to be utterly in-
capable of preventing or reducing activity in dry sperm to any extent
that could be optically determined.
These experiments indicate that some substance or condition de-
velops or appears in suspensions of spermatozoa which has the prop-
erty of preventing activity in the spermatozoa of that or of a fresh
suspension of dry sperm. This substance or condition is rendered
ineffective, or is reduced to sub-threshold concentration or intensity
by the addition of fresh sea -water, but its concentration or intensity
may be definitely and markedly increased by washing the spermatozoa
through the suspensory fluid by means of the centrifuge.
OTHER PROPERTIES OF SUPERNATANT SPERM FLUID
When a drop of supernatant fluid which will definitely prevent
activity in dry sperm is thoroughly mixed, by means of a clean, dry
glass needle, with one drop of an egg-sea-water which will produce
definite and clear-cut agglutination clumps with dry sperm and this
mixture, in the form of a drop on a clean, dry glass slide, is brought
into contact with a drop of dry sperm from the same tubules, and the
whole process watched carefully with the microscope, the spermatozoa
will be seen to become active in a manner exactly similar to that which
occurs when a dilution is made with ordinary sea-water, but no slightest
sign of any form or degree of agglutination whatsoever occurs. This
test was repeated several times with many different preparations, and
with many different lots of gametes, but always with the same result.
In all cases when the supernatant sperm fluid would prevent activity
in dry sperm, it would neutralize or destroy in some way the ag-
EGG-WATER NEUTRALIZING SUBSTANCES 153
glutinating power of egg-sea-water, but reciprocally, its power to
prevent activity in dry spermatozoa was similarly destroyed, and the
mixture of supernatant fluid plus egg-sea-water served to activate
spermatozoa in a manner exactly similar to that of ordinary sea-water.
This phenomenon was even more vividly shown when a small drop
of the supernatant sperm fluid was added, with a fine capillary pipette,
to the middle of the field where the agglutinated clumps, produced by
the addition of egg-sea-water to dry sperm, could everywhere be seen,
and the whole process watched continuously under the microscope.
The response was almost instantaneous. The agglutination clumps
everywhere within the area of the added drop immediately dispersed,
while outside the boundary of the drop and on all sides, clumps
persisted in a perfectly typical and unaffected arrangement. Within
the drop, the spermatozoa instantly became inactive, while along the
boundary of the drop, some activity and motion could be seen.
The phenomenon, too, was readily demonstrated by making prep-
arations on one slide which could be observed successively and
sequentially, and directly compared, and which would consist of dry
sperm mixed with (1) ordinary sea-water, (2) egg-sea-water, (3) super-
natant sperm fluid, and (4) one drop egg-sea-water plus one drop
supernatant sperm fluid. (1) and (4) showed typical activation, but
no agglutination, (2) showed distinct and definite agglutination, while
the spermatozoa in (3) were utterly inactive.
There seems to be a parallelism between ability to prevent activity
of dry sperm and ability to neutralize egg-sea-water. All supernatant
fluid that would prevent activity in dry sperm would also neutralize
the agglutinating power of egg-sea-water. When the supernatant
fluid allowed "just a little activity," the mixture of supernatant sperm
fluid and egg-sea-water allowed "slight and evanescent tendencies"
towards the formation of agglutination clumps, while finally, the super-
natant fluid obtained from a sperm suspension that had been diluted
for 2% hours, but that had not been washed by centrifuging, would
not prevent activity of dry sperm, and would not prevent egg-sea-
water from causing agglutination. These observations seem to
indicate that the activity-preventing and the egg-sea-water neutralizing
properties of supernatant sperm fluid are due, either to the same sub-
stance or condition, or to two or more substances which, however,
arise or occur together in the supernatant fluid under all the treatments
where the effects have been observed.
DISCUSSION
Attempts to extract such substances from the spermatozoon as
would have a relationship to the fertilization reaction have been.-
V I '
154 WALTER E. SOUTH WICK
made by Sampson (1926), Hibbard (1928), Wintrebert (1929, 1930a,
1933), Einsele (1930), and Parat (1933a). Sampson was unable to
obtain any substances in sperm nitrates and dialysates which would
activate "fertilizin" in the egg-sea-water so as to make it an efficient
parthenogenetic agent, or would combine with the agglutinating
substance in egg-sea-water so as to destroy its power to agglutinate
fresh sperm suspensions, though she did obtain substances which would
initiate development of mature ova of the same species. Hibbard
and Wintrebert found that solutions of macerated spermatozoa would
digest egg membranes, while Einsele and Parat found that filtered
ether dialyzates and extracts of entire testes would, when injected
into the egg with a micropipette, give an activation of the eggs in 60
per cent of the cases. Parat found that the development was much
more regular, and that many more of the gastrulae would form larvae
when the eggs were activated parthenogenetically by means of the
introduction of the acrosome of spermatozoa removed and injected
by means of microdissection needles and pipettes. Both Parat and
Einsele have obtained evidence that the substance concerned in such
parthenogenetic activations is a proteolytic enzyme.3 Histo-anatomi-
cal studies of spermatozoa have been made by Bowen (1924), Popa
(1927), Wintrebert (19306), Parat (1928, 19336) and others, and some
of these studies indicate the presence of substances of secretory origin
in the acrosomal region of spermatozoa.
Since egg-sea-water, reciprocally, will neutralize or destroy the
activity-preventing or inhibiting substance of a sperm suspension, the
activating property of egg-sea-water, as described by Lillie (1913),
hereby receives an explanation. The power of the substances in the
supernatant sperm fluid to neutralize the agglutinating power of egg-
sea-water also conforms very readily with the fertilizin theory of
Lillie (1919), and with the observations of Lillie (1919) and others with
Arbacia punctulata that it was not possible at any time to regain
agglutinating substances that had once been used in order to cause
an agglutination of spermatozoa.
SUMMARY
•
1. Dry sperm of Echinometra subangularis is intensely active im-
mediately after its removal from the testes tubules, but this motion
3 Recently (1939), Frank has described a sperm extract of Arbacia punctulata
spermatozoa, obtained by heating the sperm suspension, which has the property of
neutralizing the fertilizin of egg-sea-water, and which has an agglutinating effect on
the cilia of Arbacia plutei. It is possible that the inactivating effect of the super-
natant fluid of Echinometra spermatozoa is due to a similar action upon the tails of
the sperm.
EGG-WATER NEUTRALIZING SUBSTANCES 155
lasts for but from 50 to 120 seconds, when all activity ceases. When,
however, a drop of sea-water is brought in contact with such a drop of
dry sperm, the spermatozoa again become active, and this activity
continues for from three to four hours. The addition of another
drop of sea-water will cause the spermatozoa again to become active,
and this activity continues with gradually decreasing intensity for
about one and one-half hours. Such spermatozoa can again be
reactivated, but the length of time during which they remain active
progressively decreases, until finally, no further activation is obtained.
2. This inactivation of sperm suspensions with time is caused,
probably, by the accumulation of a substance with time which has,
as its characteristic identificatory property, the prevention of sperma-
tozoa from activity. It occurs in the supernatant fluid obtained from
centrifuging dry sperm, and in the supernatant fluid obtained by wash-
ing dry sperm through ordinary sea-water several times with the
centrifuge, but the suspensory fluid of diluted sperm suspensions
through which the spermatozoa have not been washed does not contain
the substance in detectable amounts.
3. Supernatant fluid which contains this substance also, in all
cases, has the property of neutralizing the agglutinating power of
egg-sea-water, and there seems to be a parallelism between ability to
prevent activity of dry sperm and ability to neutralize egg-sea-water.
4. These observations indicate that the activity-preventing and the
egg-sea-water neutralizing properties of supernatant fluid are due,
either to the same substance, or to two substances which, however,
occur together under all the treatments where the effects have been
observed.
LITERATURE CITED
BOWEN, R. H., 1924. On the acrosome of the animal sperm. Anal. Rec., 28: 1-13.
EINSELE, W., 1930. Entwicklungserregung von Froscheiern durch Injektion
Zellfreier Organextrakte. Arch.f. Entw.-mech., 123: 279-300.
GRAY, J., 1928. The senescence of spermatozoa. Brit. Jour. Exper. Biol., 5: 345-
361.
HIBBARD, H., 1928. Contribution a 1'etude de 1'ovogenese, de la fecondation, et de
1'histogenese chez Discoglossus pictus Otth. Arch, de Biol., 38: 249-326.
LILLIE, F. R., 1913. Studies of fertilization. V. The behavior of the spermatozoa
of Nereis and Arbacia with special reference to egg-extractives. Jour.
Exper. Zool., 14: 515-574.
LILLIE, F. R., 1919. Problems of Fertilization. University of Chicago Press,
Chicago.
PARAT, M., 1928. Contribution a 1'etude morphologique et physiologique du
cytoplasme, chondriome, vacuome (appareil de Golgi), enclaves, etc. Arch.
d'anat. micros., 24: 73-357.
PARAT, M., 1933a. L'acrosome du spermatozoi'de dans la fecondation et la partheno-
genese experimentale. Compt. Rend. Soc. Biol., 112: 1134-1137.
156 WALTER E. SOUTHWICK
PARAT, M., 19336. Nomenclature, genese, structure et function de quelques elements
cytoplasmiques des cellules sexuelles males. Compt. Rend. Soc. Biol., 112:
1131-1134.
POPA, G. T., 1927. The distribution of substances in the spermatozoon (Arbacia
and Nereis). Biol. Bull, 52: 238-257.
SAMPSON, M. M., 1926. Sperm nitrates and dialyzates. Biol. Bull., 50: 301-338.
WINTREBERT, P., 1929. La digestion de 1'enveloppe tubaire interne de 1'oeuf par des
ferments issus des spermatozoides, et de 1 'ovule, chez Discoglossus pictus
Otth. Compt. Rend. Acad. Sci. Paris, 188: 97-100.
WINTREBERT, P., 1930a. Phenomenes d'attraction reciproque des gametes, de
captation et de reception du spermatozoide par 1 'ovule, chez Discoglossus
pictus Otth. Compt. Rend. Soc. Biol., 105: 520-524.
WINTREBERT, P., 19306. Les voies de rapprochement des pronuclei et le mode de
formation du premier noyau de segmentation dans 1'oeuf du Discoglossus
pictus Otth. Compt. Rend. Soc. Biol., 105: 764-769.
WINTREBERT, P., 1933. La fonction enzymatique de 1'acrosome spermien du
Discoglosse. Cvm.pt. Rend. Soc. Biol., 112: 1636-1640.
THE "AGGLUTINATION" PHENOMENON WITH
SPERMATOZOA OF CHITON
TUBERCULATUS
WALTER E. SOUTH WICK
(From the Bermuda Biological Station for Research J)
INTRODUCTION
It has been shown by Crozier (1922) that when sperm, diffusing
from a male individual of Chiton tuberculatus during the month of May
(fully a month before ripe eggs are seen), was taken up between the
ctenidia of a female, it issued from the posterior ends of the ctenidial
channels principally in the form of "numerous agglutinated masses
of active sperms" which persisted in sea-water for at least one half-
hour. He found similar "agglutination" when sperm had passed
through the ctenidial channels of males, and when it had been added
(1) to ovarian extracts from mature eggs in sea-water, or (2) to sea-
water into which ripe eggs had been shaken from an ovary and allowed
to stand for half an hour. He considered these conditions to indicate
that "mere evidence of sperm agglutination (cluster formation) may
well have no bearing on the fertilization reaction." This conclusion
is in distinct conflict with that formulated by Lillie (1919) on the basis
of his observations on Arbacia punctulata and Nereis limbata.
The observations of Crozier (1922) have been extended during a
series of observations made during the summers of 1933 and 1938 at
The Bermuda Biological Station for Research.
OBSERVATIONS
Dry spermatozoa 2 from a mature male Chiton tuberculatus are
homogeneously motile. When such dry sperm is introduced into the
mantle cavity either of a male, or of a female which does not shed eggs,
in a way such that it is caught up in the ctenidial current and carried
1 This investigation was supported in part by a grant from the Porter fund.
Laboratory space was provided in part by Harvard University and in part by the
Bermuda Biological Station for Research.
2 When a drop of such sperm is brought into contact with a drop of mature eggs
on a glass slide, the egg contents, within about one minute, shrink visibly so that a
clear area is produced around the egg and between the egg and the chorion. When
such eggs are transferred to Syracuse watch crystals containing approximately 10 cc.
sea-water, about 98 per cent of them will have cleaved, within approximately one and
one-half hours, to form the two-cell stage.
157
158 WALTER E. SOUTHWICK
through the gills to be discharged at the posterior end, this discharged
sperm, if allowed to collect in the dish, or to rest undisturbed on a glass
slide, can be seen to form macroscopically visible white masses in a
short time, as Crozier observed. If, on the other hand, the discharged
sperm is collected with a clean pipette, transferred to a glass slide, and
examined immediately, a very interesting series of changes can be
observed.
During the first few seconds, the spermatozoa swim about actively,
freely, homogeneously. During the next few seconds, they come
together and form small clumps of spermatozoa, but the point of attach-
ment is the tail, while the heads remain perfectly free. Sometimes the
group may consist of but ten or twelve spermatozoa, in which case
the clumps resemble bouquets of flowers, tufts of grass, or even more
appropriately, a handful of balloons waving in the breeze. Soon these
clumps fuse into larger masses, either in such a way as to form complete
spherical masses, as shown in Fig. 1, or much more frequently, to form
strands of spermatozoa. These strands, at first, are slender and com-
paratively short, and are often but slightly branched, but soon they
elongate, thicken and branch, as shown in Fig. 2. Soon these strands
fuse with others, in a way such that within about three minutes
extensive networks appear, as may be seen from Fig. 3. These net-
\vorks soon become so large that they are macroscopically visible.
Careful observation of the mode of formation of these strands
reveals that the process is quite comparable to a braiding of the tails
of the spermatozoa. Always at the end of the strand, a tuft of
spermatozoa with entirely free heads may be seen. These heads are
continually waving back and forth, in and out, moved by the whipping
motion of the spermatozoon tail. In a short time, the latitude of the
motion becomes restricted, and the restriction progressively increases
until finally only the head is free. It can then be seen, still actively
waving, from the side of the strand. Often, however, the head is
included in the braid.
As the sperm become more and more bound in the strand, other
spermatozoa get caught and soon these are inescapably bound while
others are caught. This continues until all of the free-swimming
spermatozoa are bound, when the terminal tufts then persist for long
periods of time, probably permanently. These tufts have been
examined with especial care, and in every case, the sperm head has
been found to be absolutely distinct and completely separate from its
neighbors.
When, however, a drop of dry sperm on a glass slide is brought
in contact with a drop of sea-water in a way such that the spermatozoa
AGGLUTINATION PHENOMENON IN CHITON 159
are carried into the sea-water by the resultant currents, it frequently
happens that the sperm suspension is simply swept into the sea-water
drop. Unlike the condition with most forms, the sperm mass tends
to remain intact, and though it generally exhibits a slight increase in
homogeneous motility, it shows no slightest sign of the clumping phe-
nomenon. Occasionally, however, under such conditions, tufts of
spermatozoa may form along the contacting edge, as may be seen in
Fig. 4.
On the other hand, when the two drops are fused in a way such that
the spermatozoa are distributed widely and rapidly into the sea-water,
all stages that were observed with the sperm that had passed through
the ctenidial channels could be seen repeated under such conditions.
The phenomenon was first noted when the sperm drop was brought
into contact, in the usual way, with a drop of sea-water to which a little
ether had been added. The markedly reduced surface tension pro-
duced violent currents which served rapidly to carry the sperm to all
parts of the drop. The ether evaporated rapidly, and by so doing
probably produced still more currents, but did not appear to affect
the motility of the sperm in any way. Later it was possible to repeat
exactly the same series of changes with non-etherized, normal sea-
water by the proper regulation of the relative sizes of the drops.
When the two drops are fused with the aid of a clean glass needle in a
way such that the sperm suspension is spread widely throughout the
drop of sea-water, the typical strands, as shown in Fig. 2, form im-
mediately and everywhere, and these soon anastomose to form the
extensive net-works shown in Fig. 3. In these nets, the sperm heads
frequently project into the interstices of the net and there continue
to wave actively for a long period of time. Sometimes the network
formation may be so extensive that distinct membrane-like structures
are produced, which readily curl up and may readily be caused to wave
in a manner typical of any such membrane, if the slide be moved or
shaken gently under the microscope.
When a drop of dry sperm is introduced suddenly into 8 or 10 cc.
of sea-water in a small beaker, the sperm mass may be seen to drop to
the bottom of the beaker in the form of a much-folded membrane,
resembling in every respect a piece of silk allowed to fall lightly on a
table. Microscopic examination of this mass shows that it is composed
of a still intact mass of homogeneously distributed spermatozoa.
Though this membrane of spermatozoa is very delicate, it is possible
to lift it, as a membrane, with a fine glass needle, and to fold it back upon
itself, or to roll it into a markedly more compact mass. Tufts, in due
time, appear along the edges of the mass, and circular clumps may
160
WALTER E. SOUTH WICK
• /;*?r -**^
.--'*" ""^
.,,-^'-
fe& rife*' ^
PLATE I
FIG. 1. The structure of the spherical masses that are formed by the sperma-
tozoa of Chiton tuberculatus after dilution with sea-water, or after passage through
the ctenidial chambers of a male or immature female. Sometimes the masses formed
are more compact, but the structure, with the tails bound and the heads free, is the
same in all cases. X 440.
FIG. 2. The elongating, thickened, branching strands that form when the
spermatozoa of Chiton tuberculatus is passed through the ctenidial chambers of a
male or immature female, or is diluted with sea-water. X 440.
FIG. 3. The nature of the network that forms by the fusion of the strands
produced by the spermatozoa of Chiton tuberculatus when diluted with sea-water, or
passed through the ctenidial chambers of a male or immature female. X 100.
FIG. 4. Photomicrograph to show the retention of mass integrity, with oc-
casional formation of tufts along the contacting edge, when a drop of dry spermatozoa
of Chiton tuberculatus is gently brought into contact with a drop of sea-water. X 440.
AGGLUTINATION PHENOMENON IN CHITON 161
appear separated from, but near the margins of, the membrane.
Rarely, strands may also appear. When, however, the original
sperm mass becomes spread very thin, widespread formation of the
network usually results.
If, however, some of the sperm be separated from the main mass,
either by means of a glass needle, or by mechanical agitation, typical
strands and networks are immediately formed. Any mechanical
disturbance of the drop of dry sperm in order to obtain a uniform,
homogeneous, diluted sperm suspension results only in a complete
transformation of the sperm mass into strands, and a microscopic
examination of sperm suspensions of one drop of dry sperm to 5, 10, 15,
25, 50, 100, and 150 cc. sea-water have all shown the presence of such
strands, and the complete absence, in any case, of completely free
swimming spermatozoa. These strands slowly settle to the bottom
of the container, the rate depending upon their size, where they form
into networks, and, if the concentration be sufficient, into more or
less homogeneous membranes. These membranes can again be broken
up into strands, and the process can be repeated several times. The
strands in sea-water persist indefinitely, or as long as the spermatozoon
tail is intact. In the more dilute suspensions, however, the strands
remain quite uniformly distributed in the suspension for some time.
On the other hand, when extracts from the ovaries of ripe or spent
females, obtained by cutting the ovary into small pieces and washing
it thoroughly through cheese-cloth with about 25 cc. of sea-water,
are used instead of sea-water, no dumping in any form occurs. Instead,
the spermatozoa in the advancing edge of the drop of dry sperm move
freely, and the sperm mass progresses steadily until the far side of the
drop is reached. At no time do tufts, strands, or networks appear,
and, furthermore, if spermatozoa be drawn from the mass by means
of a glass needle, they simply disperse and soon merge imperceptibly
with the other spermatozoa as the main mass advances upon and
closes in about them.
Dilution of the ovarian extracts appears to lessen their effectiveness.
A dilution of one drop of extract to ten drops of sea-water is often quite
as effective as the full strength extract. A 1 : 25 dilution, however,
prevents the formation of tufts and other similar structures, but does
not remove the restriction to a free movement of the sperm through
the drop of ovarian extract; while a 1 : 50 dilution allows the formation
of tufts, clumps, strands, and networks just as though it were pure
sea-water. These figures, however, are of relative value only, since
there is a wide variation between the extracts from the different
ovaries, as seen in the fact that one of those tested gave a perfectly
162 WALTER E. SOUTH WICK
typical clumping reaction with a 1 : 10 dilution. Clumps of spermato-
zoa formed by dilution of dry sperm with sea-water can be caused to
disperse by the addition of ovarian extract.
When egg-sea-water, made by allowing the eggs of one female to
stand in 25 cc. sea-water for half an hour, was used, exactly similar
effects were obtained to those obtained with the ovarian extracts. Of
course, dilution of this egg-sea-water reduced the effect, just as was
the case with the ovarian extract. When, too, a drop of dry sperm was
brought in contact with a drop of eggs and watched under the micro-
scope, the sperm could be seen to move freely across the open spaces,
and to gather about the eggs, but there was no sign whatsoever of a
clumping reaction. These latter observations are in accord with,
and to an extent provide an explanation of, the observations of
Crozier (1922) that "during natural fecundation, however, no sperm-
balls are formed. The thick, glutinous stream of spermatozoa passes
under the girdle of the female, is somewhat diluted with sea-water by
the tractive current, and emerges posteriorly in company with numer-
ous large greenish eggs, about which, under the microscope, it can be
seen that many sperms are gathered. But no real 'cluster formation'
takes place."
Other substances that prevent clumping include the body juices
of a mature male, body juices of a mature female, as was also noted by
Crozier (1922), sublethal solutions of saponin in sea-water,3 similar
solutions of sodium taurocholate in sea-water, saturated and somewhat
diluted solutions of trypsin in sea-water, and possibly by other sub-
stances. On the other hand, no prevention of clumping was obtained
with acetone, ether, methyl alcohol, ethyl alcohol, adrenaline chloride
(1 : 1000, 1 : 2000, or 1 : 5000), or carbon disulphide.
DISCUSSION
It is obvious that in Chiton tuberculatus, the clumping reaction is
different in every way from the agglutination reaction which has been
described by Lillie (1913) for Arbacia punctulata, by Loeb (1914) for
Strongylocentrotus purpuratus and 5. franciscanus, by Just (1919) for
Echinarachnius parma, and later (1929) for Paracentrotus lividus and
Echinus microtuberculalus, by Carter (1932) for Echinus esculentus,
3 The sources of the substances used for these tests were as follows:
Saponin: Eimer and Amend, New York, " A-61 Purified."
Sodium taurocholate: Eimer and Amend, New York, "A-61 Purified."
Trypsin: Eimer and Amend, New York, "A-61" "Pure."
Acetone: U. S. P., J. T. Baker Chemical Co., Lot No. 92237.
Adrenaline Chloride: Parke, Davis and Co., Detroit, Mich., U. S. A.
Ether, methyl alcohol, ethyl alcohol, and carbon disulphide : Usual laboratory supplies.
AGGLUTINATION PHENOMENON IN CHITON 163
by Lillie (1913) for Nereis limbata, by Just (1915) for Platy nereis mega-
lops, and by other workers with other forms. With the agglutination
reaction in these cases, the agglutination is between the heads of the
spermatozoa, while the tails are apparently unaffected; the agglutina-
tion reaction is spontaneously reversible, cannot be repeated, and
generally is produced only by substances secreted by eggs of the same
species. With the clumping reaction in Chiton tuberculatus , on the
other hand, the agglutination is between the tails while the heads are
apparently utterly free and unaffected; the clumps, when once formed,
persist indefinitely unless dispersed by means of the addition of certain
substances, or by mechanical means. In the latter case, the clumps
will reappear with an almost endless number of repetitions. The
clumping forms as a perfectly natural and normal result of dilution
with ordinary sea-water, and is, in addition, a phenomenon which
can be prevented by means of body juices of the same form, egg and
ovarian secretions and extracts, and certain lytic substances, such as
saponin and sodium taurocholate.
With Chiton tuberculatus, it is unlikely that the clumping reaction,
first described by Crozier, and described in detail herein, has any
direct relation to the fertilization reaction as such. Instead, it is
probably a mechanism by means of which masses of dry sperm may be
transferred, in an intact manner, from the male to the mature female.
This mass of dry sperm, thus transferred in an essentially intact condi-
tion, comes in contact with substances in the ctenidial channels of the
female which destroy the substance which causes the tails to stick
together and thus form clumps. The spermatozoa thus become freed
from each other and are then able, by their own individual and utterly
independent movements, to activate the all-environing eggs. The
reason for transferring the spermatozoa to the female in an intact
mass, however, might possibly reside in the need to preserve sub-
stances which might be essential for the actual fertilization reaction,
and which might be rapidly lost from the spermatozoon in less con-
centrated suspensions.
The fact that these clumps can be dispersed by means of the
proteolytic enzyme, trypsin, and by the lytic substances saponin and
sodium taurocholate indicates that the clumping reaction in this form
rests, fundamentally, upon the presence, on the outside surface of the
spermatozoon tails at the time they are liberated from the testes, of a
substance which (1) is distinctly sticky in nature, and which (2) can
be dissolved or destroyed by the above-mentioned substances. Since
lytic substances, as suggested by Ponder (1930), might act by the de-
struction of the structure of proteins, a process which is also hastened
164 WALTER E. SOUTHWICK
by trypsin, it is possible that the substance on the tails of the spermato-
zoa of dry sperm suspensions might be a sticky protein of some sort.
On the other hand, Fieser (1937) has suggested that the hemolytic
effect, of saponin at least, might be produced by a combination with
cholesterol or lecithin of the cell membrane in a way such as to render
the membrane permeable to hemoglobin, and evidence in partial sup-
port of this suggestion has been provided by Ransom (1901) in that he
has shown with certainty that a combination of saponin and cholesterol
is possible and that treatment of a saponin solution with cholesterol
destroys its hemolytic activity. Popa (1927) has obtained evidence
that the tails of the spermatozoa of Arbacia punctulata and Nereis
limbata are enveloped by a large amount of lipoid substance.
SUMMARY
1. When dry sperm of a mature male Chiton tuberculatus is intro-
duced into the mantle cavity of a male or immature female and the
discharged sperm is collected and examined immediately with a micro-
scope, the spermatozoa will be seen to come together and form small
clumps. The point of attachment of these clumps is the tail, while the
heads remain perfectly free.
2. These clumps fuse readily to form either large spherical masses,
or strands, which, in turn, soon fuse with other strands to form ex-
tensive networks. Such structures also form, readily and extensively,
when the two drops are fused with the aid of a glass needle in a way
such that the sperm suspension is distributed widely throughout the
drop of sea water.
3. On the other hand, when extracts from the ovaries of ripe or
spent females, egg-sea-water, body juices of a mature male, or of a
mature female, or sublethal solutions of the lytic substances, saponin
or sodium taurocholate, are used, no clumping in any form occurs.
Instead, the spermatozoa in the advancing edge of the drop of dry
sperm move freely, and the sperm mass progresses steadily until the
far side of the drop of diluting fluid is reached.
4. These observations indicate that, in Chiton tuberculatus, the
clumping reaction rests fundamentally upon the presence, on the out-
side surface of the spermatozoon tails, of a substance which (1) is
distinctly sticky in nature, and which (2) can be dissolved or destroyed
by certain substances.
LITERATURE CITED
CARTER, G. S., 1932. Iodine compounds and fertilisation. VI. Physiological
properties of extracts of the ovaries and testes of Echinus esculentus. Part
I. Jour. Exper. Biol., 9: 253-263.
AGGLUTINATION PHENOMENON IN CHITON 165
CROZIER, W. J., 1922. An observation on the "cluster-formation" of the sperms
of Chiton. Am. Nat., 56: 478-480.
FIESER, L. F., 1937. The Chemistry of Natural Products Related to Phenanthrene.
Reinhold Publishing Corporation, New York.
JUST, E. E., 1915. An experimental analysis of fertilization in Platynereis megalops.
Biol. Bull., 28:93-114.
JUST, E. E., 1919. The fertilization reaction in Echinarachnius parma. II. The
role of fertilizin in straight and cross fertilization. Biol. Bull., 36: 11-38.
JUST, E. E., 1929. The fertilization reaction in eggs of Paracentrotus and Echinus.
Biol. Bull., 57: 326-331.
LILLIE, F. R., 1913. Studies of fertilization. V. The behavior of the spermatozoa
of Nereis and Arbacia with special reference to egg-extractives. Jour.
Exper. Zool., 14: 515-574.
LILLIE, F. R., 1919. Problems in Fertilization. University of Chicago Press,
Chicago.
LOEB, J., 1914. Cluster formation of spermatozoa caused by specific substances
from eggs. Jour. Exper. Zool., 17: 123-140.
PONDER, E., 1930. The form of the frequency distribution of red cell resistances
to saponin. Proc. Roy. Soc. London, 106B: 543-559.
POPA, G. T., 1927. The distribution of substances in the spermatozoon (Arbacia
and Nereis). Biol. Bull., 52: 238-257.
RANSOM, F., 1901. Saponin und sein Gegengift. Deutsch. med. Wochschr., 27: 194-
196.
THE LUMINESCENCE OF A NEMERTEAN,
EMPLECTONEMA KANDAI, KATO
SAKYO KANDA
(From the Institute of Physical and Chemical Research, Hongo, Tokyo, Japan)
Introduction
A great many species in the five phyla, Plathelminthes, Nemertea,
Trochelminthes, Nemathelminthes and Chaetognatha, are closely
allied to one another. Among these species, no luminous form has
been previously recognized. I found, however, a number of luminous
nemerteans, when I visited the Marine Biological Station of the
Tohoku Imperial University at Asamusi, Aomori, Japan in the summer
of 1936.
These nemerteans had coiled up on Chelyosoma, which were col-
lected from the bottom of Aomori Bay between Natutomari and
Aburame at a depth of about 35-40 meters, and were placed in the
laboratory for study. They were identified by Koziro Kato (paper in
preparation) as Emplectonema kandai sp. nov.
It is an extraordinary fact that among so large a number of species
of the five phyla, only one is found to be luminous. Emplectonema is
a genus of the nemerteans which is widely distributed in America, the
Atlantic and Pacific Oceans, the Mediterranean Sea, the White Sea and
Japan. It may be expected, therefore, that more luminous species of
the same genus, at least, will be observed somewhere in the future.
I made some experiments on Emplectonema kandai during the three
summers of 1936-38 at the Station mentioned above. The results are
given in the present paper.
I wish to express my sincere appreciation of the facilities afforded
me there by Professors S. Hatai (1937) and S. Hozawa (1938), Directors
of the Laboratory. I would also like to acknowledge my indebtedness
to Messrs. N. Abe, K. Atoda and K. Kato, without whose aid this
paper would not have been completed.
Material
As already stated, these luminous nemerteans coiled up on Chelyo-
soma. It is necessary, therefore, to collect the latter, which are
dredged (by three fishermen) from the bottom of Aomori Bay, about
1 A preliminary note in Japanese was published in the Rigakukai, 35 (1937): 5-11.
166
LUMINESCENCE OF A NEMERTEAN 167
15 km. off the Station. But the nemerteans are not abundant. Only
six or seven individuals at best, or sometimes only one or two indi-
viduals, are obtained on about two hundred Chelyosoma, which are
collected by the fishermen as the result of one day's work.
The nemerteans are reddish orange in color. They have many
eyes. They vary in length, from 53-115 cm., and are about 0.5-0.7
mm. in diameter, when they are stretched. I found one individual
10 cm. long, but one so short is extremely rare. The female animal is
readily distinguished during the summer season because it is full of
eggs or enlarged gonads, but I could not distinguish the males. The
animals coil up on the wall of a large vat of running sea water or on the
bottom, attached by the slime which is abundantly secreted from the
surface of its body (Fig. 1). They remain there quietly for two
months or more, if they are not disturbed.
FIG. 1. The living and coiled whole Emplectonema kandai about 115 cm. long.
About natural size. (Photographed by N. Abe at my request.)
The animals flash brilliantly only on stimulation. The stimulus
may be mechanical, chemical, thermal or electrical. The light may
appear on all parts of the body, but it disappears in one or two seconds.
It is whitish green in color.
Mechanical Stimuli
The animal flashes when a glass rod or a finger is gently touched
to the surface or surfaces of the coiled body. The light does not spread
very far from the place or places of the contact, and lasts for only one
or two seconds. Its intensity varies, depending on the strength of
the contact. I thought at first that some luminous material was
thrown into the sea water mixed in a slime discharged from the surface
of the animal body, but this observation turned out to be incorrect.
If the coiled animal is strongly rubbed between the fingers, a
brilliant light appears, but it is not observed that any luminous
material comes off which adheres to the fingers. If the animal is
suddenly extended, without being broken, between two hands, the
168 SAKYO KAN DA
head in one hand and the tail in the other, the brilliant light also
appears through the whole surface of the long body except the tip of
the head. This luminescence is a most beautiful sight.
Chemical and Osmotic Stimuli
If the sea water containing the nemertean is acidified with a very
dilute HC1 or acetic acid, the animal gives a bright light. The acid
should not be too strong, or it will kill the animal too quickly. The
dead or dying nemerteans produce light continuously, until all the
luminous material is probably exhausted. The addition of dilute
NaOH or NH4OH to the sea water produces the same effect, although
it precipitates the Ca and Mg of the sea water.
The best way to test the luminescence of the animal is to add dilute
H2O2 to the sea water. This action is not injurious and is reversible.
If 1 to 2 cc. each of ^ NaCl, ^ KC1, y NH4C1, ~ MgCl2)
^y MgSO4, ^y Na2SO4, or ~ (NH4)2SO4 solution are added to 100 cc. of
Zt £ £
sea water which contains a nemertean, no luminescence is observed.
In 0.5 cc. -y CaCl2 plus 100 cc. sea water, however, the animal begins
Zi
to flash occasionally after about 10 minutes. The intervals of its
flashing become quite regular after about 20 minutes, resembling those
of a firefly. Besides these flashes, there is a very faint and continuous
light in other parts of the body. If this treated animal is removed to
normal sea water after about 40 minutes, it lives normally. In 1 or 2
M
cc. — CaCl2 plus 100 cc. sea water, the intervals of the flashing are
very slow and somewhat irregular.
MM M
In pure — KC1, — CaCl2 or— Na2SO4 solution, practically isotomc
£t ft Zt
with sea water, the animal gives a bright light. But if the animal is
kept too long in the solution, it will be killed. A mass of slime is
secreted into each solution, but no luminous material is observed in it.
M
In pure -y MgSO4 solution, the animal flashes after about 8 minutes.
M
In pure — NH4C1 solution, it gives a faint light after about 20 minutes.
It would seem that K, Ca, Na, Mg, or NH4 ions cause the luminescence
of the animal.
In pure — NaCl or — MgCl2 solution, isotonic with sea water, the
Li _
LUMINESCENCE OF A NEMERTEAN 169
M
animal gives no light. In pure— (NH4)2SO4 solution, also, no lumines-
L*
cence appears. In these cases, no cation, Na, Mg or NH4, seems to
cause any light whatever. It is a little difficult to decide from these
experiments whether cation or anion stimulates the animal to become
luminous since no luminescence is observed in NaCl, MgCl2 or
(NH4)2SO4 solution, whereas some luminescence occurs in Na2SO4,
MgSO4 or NH4C1 solution.
If crystals of NaCl, KC1, NH4C1, CaCl2, MgCl2, MgSO4, Na2SO4,
or (NH4)2SO4 are added to 20 cc. of sea water, which contains the
nemertean, a brilliant light is always observed. If a large amount of
salt is used, the light is continuous and fades gradually, due to the
death of the animal. On addition of fresh water or distilled water to
the sea water, a bright light appears also. Saponin acts in the same
way. The increase or decrease of osmotic pressure plays, of course, a
distinct role in each case.
Temperature and Electrical Stimuli
If the sea water at 20° C., which contains the animal, is heated to
32-33° C., or is cooled to about 1° C., the animal produces light.
With induced currents, the animal also gives light.
Luciferin and Lucif erase
If the animals are placed on a heavy blotting paper, they give a
bright light immediately. When they are dried over P2O5, light is
still observed during the drying and the dried, dead animals give light
when again moistened with water. When these moistened ones are
dried again over P2O5, however, they produce no more light on being
moistened again with water. The animal is slender and all parts of the
body are covered by a simple, thin epithelium, where the luminous cells
are located, as are the cells of other luminous animals. Evidently the
luminous cells of the nemertean are not large, as the cross-section of
the animal indicates (Fig. 2). This may explain why the amount of
luminous material secreted by the cells is comparatively small.
The existence of luciferin and luciferase cannot be demonstrated in
the usual way with either the fresh or the dried animals, which are
ground with sand in a mortar and are extracted with hot or cold water.
Methyl or ethyl alcohol extracts of the fresh and dried nemerteans also
give no light with cold or hot water extracts. The cold water extract
of the nemertean gives no light with Cypridina luciferin, nor does the
hot water extract of the nemertean give light with Cypridina luciferase.
170 SAKYO KANDA
Potassium Cyanide
Since the luciferin-luciferase reaction cannot be demonstrated,
the question may be asked: Is not the luminescence of the nemertean
due to the symbiosis of luminous bacteria? Pierantoni (1918) holds
that the light of all animals is due to symbiotic luminous bacteria. I
did not attempt to raise luminous bacteria from the nemertean on an
artificial culture medium, but studied the effect of KCN. According
to Harvey (1921), the light of marine luminous bacteria disappears in
M
4 minutes, if they are treated with — KCN solution, namely 0.325
M
per cent solution, and in 6 minutes, if treated with — KCN or 0.162 per
cent solution. He also shows that the light of an emulsion of the
luminous organ of a fish, Photoblepharon, which is suspected to be
symbiotic, disappears in about 20 minutes, if it is treated with 0.25
per cent KCN solution, and in about 30 minutes, if treated with 0.125
per cent KCN solution. I have found that the nemertean gives light
immediately and that the light continues for about 110 minutes, if 1 cc.
M
of the aqueous solution of -y KCN is added to 10 cc. of sea water which
£t
contains the animal. The animal begins to give light after 50 minutes
. M
and continues for about 140 minutes, if 0.5 cc. of — KCN solution is
added to 10 cc. of sea water. In both cases, the animal dies, not from
dilution of sea water, but from KCN, since the worm can live indefi-
nitely without evident injury in 100 cc. of sea water plus 20 cc. of
distilled water.
These facts indicate that KCN, which inhibits most cell oxidations
instantly, has very little effect on the luminescence of the nemertean.
They would seem, also, to show that the light of the animal is not of
bacterial origin. The failure to prove the presence of luciferin and
luciferase in the animal does not necessarily indicate the symbiosis of
luminous bacteria. On the contrary, I believe that luminescence in
this nemertean arises from a chemical luminous material secreted in
its luminous cells.
FIG. 2. Microscopic photograph of portion of a transverse section of the body
surface of Emplectonema kandai, snowing mainly the mucin-secreting cells (m. c.}.
About X 500. (Section prepared by K. Atoda at my request.)
FIG. 3. Microscopic photograph of portion of a transverse section of the body
surface of Emplectonema kandai, showing some light-producing cells (/. c.) and mucin-
secreting cells (m. c.). About X 500. (Section prepared by K. Atoda at my
request.)
FIGS. 2 and 3.
172
SAKYO KAN DA
Histology
I have studied the transverse and longitudinal sections of this worm,
which had been kindly prepared by K. Atoda and K. Kato. The
epithelium of the worm is very simple, though it is comparatively wide.
In general, there appear in the epithelium, two kinds of glandular cells
which stain with Delafied's haemotoxylin and eosin. Those which
stain blue with haemotoxylin are large and open through the cuticle
of the epithelium (Fig. 2). They are apparently the mucin-secreting
FIG. 4. Microscopic photograph of portion of a longitudinal section of the head-
tip surface of Rmplectonema kandai, where no light cells show. About X 500.
(Section prepared by K. Kato at my request.)
cells. In some preparations, however, a great many cells are almost
devoid of slime, which was probably discharged while the worm was
being narcotized with menthol.
The cells staining with eosin appear to consist of two types, al-
though this is not always evident. Those of one type, which stain red
with eosin, though not very deeply, show a small nucleus at the base,
are elongate and open through the cuticle. They are rilled with
granules. These cells are most common throughout all preparations
LUMINESCENCE OF A NEMERTEAN 173
studied. The cells of the other type are especially evident when
Mallory's stain is used. They stain deeply with eosin. Under a high
power of the microscope they are seen to contain fine granules and in
some cells their content is homogeneous. I assume that these cells
are merely the young, unripe ones of the second type.
I believe that all the cells which stain in eosin are the light cells of
the worm (Fig. 3). It is interesting to note that the tip of the head of
the worm, where no light appears, as already stated, shows none of the
eosinophil cells at all (Fig. 4.) In the head or anterior part farther
from the tip, however, there appear some cells, which stain deeply
with eosin and also contain the granules typical of the light cells.
The number of such cells increases gradually towards the middle of the
worm.
As I have already stated, the luminous material of the worm has
not been observed to separate from the cuticle. But this does not
mean that the glandular structure of the ducts has no opening or pore
in the cuticle. On the contrary, all the ducts appear to have openings,
as the sections show. The luminous secretion should be very small,
however, as the light cells are also small, and the light production may
take place at the instant of discharge, or the light-giving action may
take place in the cells before the substances reach the openings of the
ducts in the cuticle.
This work was aided by a grant from the Foundation for the Pro-
motion of Scientific and Industrial Research of Japan.
Summary
The luminescence of a marine nemertean worm, Emplectonema
kandai, living on Chelyosoma, is described. Light appears from the
whole of the body, except a small region at the head end, in response to
mechanical, chemical, thermal or osmotic stimulation. The effect of
salts has been studied.
The photogenic cells are in the epithelium, stain with eosin, and
appear to have openings in the cuticle, but no extracellular luminous
secretion could be demonstrated. Histological sections are figured.
Luciferin and luciferase could not be demonstrated, but since KCN
does not inhibit luminescence, the origin of the light is thought to be
the gland cells of the worm and not symbiotic bacteria.
LITERATURE
HARVEY, E. NEWTON, 1921. The production of light by the fishes, Photoblepharon
and Anomalops. Carnegie Inst. Wash., p. 43.
PIERANTONI, UMBERTO, 1918. I microrganismi fisiologica e la luminescenza degli
animali. Scientia, 23: 102.
ABSENCE OF THE EPITHELIAL HYPOPHYSIS IN A FETAL
DOGFISH ASSOCIATED WITH ABNORMALITIES
OF THE HEAD AND OF PIGMENTATION
DON WAYNE FAWCETT
(From the Department of Anatomy, Harvard Medical School)
The specimen herein described is an albino fetus of the spiny dog-
fish (Squalus acanthias) which presents malformations of the head
including cyclopia and astomia. Associated with these is the very
rare anomaly — absence of the epithelial hypophysis. It is well known
that there are abnormalities of the pituitary in human anencephalic
fetuses, but these involve absence of the neural lobe. Covell (1927),
in making a quantitative study of such abnormal human fetuses,
reviewed the literature and reported personal observations comprising
in all nearly a hundred cases. He concluded that an hypophysis is
always present, although the lobus nervosus is lacking in the majority
of specimens. The pars anterior he found constitutes most of the
gland volume and in some cases the total volume. He mentions no
case of absence of the anterior lobe. The only instance hitherto
described of spontaneously occurring absence of the anterior pituitary,
in any animal, is the case reported by Evelyn Holt (1921) of absence of
the pars buccalis in a 40-mm. pig.
The study of the present specimen of an elasmobranch fetus has
been undertaken not only on account of its rarity but because of the
evidence of interdependence in development of the separate lobes of
the pituitary gland and the effect of absence of the oral components
of the gland on pigmentation.
MATERIAL AND METHODS
The fetus was discovered among a great number of normal Squalus
pups, at the David Richardson Laboratory, Bailey Island, Maine.
Because its abnormalities escaped notice when it was removed from
the uterus, normal littermates are not available for study as controls.
Two normal pups, from other uteri, but of a comparable stage of de-
velopment, were selected, instead, as controls. The three fetuses
were fixed in 7 per cent formalin. The heads were subsequently im-
bedded in celloidin, cut serially in 35 fj. sections and stained with
haematoxylin and eosin.
174
ABSENCE OF EPITHELIAL HYPOPHYSIS 175
DESCRIPTION
The specimen is silvery white with the exception of the tip of the
dorsal fin and the tip of the tail where in each case a small area is deep
grey, approaching in intensity the color of a normal pup. The other-
wise translucent skin is faintly clouded with light grey over the dorsum
of the body, suggesting that the albinism is not due to complete absence
of pigmentation. Aside from this general albinism, the obvious
external deviations from the normal are limited to the region rostral
to the first gill slits; the rest of the .body is of normal configuration.
The eyes give the appearance of having been drawn from their usual
lateral position ventrally and medially to the midline where they are
fused into a single dumbbell-shaped eye with two discrete lenses.
There are no external nares; only a midventral prominence indicates
where the olfactory bulbs have coalesced. The mouth is represented
by a dimple-like depression and a narrow fold formed by a shallow,
rostrally directed invagination of the integument (Fig. 1). In addi-
tion to these abnormalities of the head, it is noted that the yolk sac is
exceptionally large as compared to that of normal fetuses.
Examination of the sections reveals radical departures from the
normal structure of the cartilaginous cranium, a description of which
requires frequent reference to the normal processes of development in
order to understand and interpret them correctly. The base of the
elasmobranch chondrocranium normally develops from two pairs of
cartilaginous bars. The posterior or parachordal cartilages constitute
the caudal part of the basis cranii. In the anterior or prechordal
region, the cranial floor develops from paired trabeculae cranii whose
rostral ends fuse in the midline to form the interorbital plate and the
more anterior rostral plate. Between the diverging posterior ends of
the trabeculae and the anterior edge of the basal plate is a median
space, the fenestra hypophyseos, through which the hypophysis and
carotid arteries gain entrance to the cranial cavity. The original
hypophyseal connection with the oral cavity is gradually obliterated
by centripetal growth of these cartilages. In the present malformed
cyclopean fetus the abnormalities of the chondrocranium itself are
limited almost entirely to the prechordal region. Cyclopean terato-
genesis is generally believed to occur very early in embryonic develop-
ment of the eyes and is attributed to a local arrest of growth ventrally,
with fusion of the elements of the primary optic vesicles and normal
growth of dorsal parts. It is commonly thought that the cyclopean
eye obstructs the path of forward growth of the cranial trabeculae —
and this appears to have occurred in the present specimen. As one
can observe in Figs. 7 and 8, there is an amorphous horizontal plate
176 DON WAYNE FAWCETT
of cartilage which overlaps the back of the posterior edge of the eye
and extends caudally to the level of the spiracle. Even though this
cartilage does not unite with the basal plate at any point, it seems
probable that it represents a fusion of the trabeculae in a somewhat
abortive attempt to grow forward past the obstructing eye. This
cartilage does not approach the basal plate caudally, as might be
expected, because of the interposition of jaw elements. The primor-
dium of the mandibular arch normally takes the form of an inverted
U at each corner of the mouth. In this specimen the dorsal parts of
the head have unfolded normally, carrying lateralward the pterygo-
quadrate limb of the ^/-shaped cartilages while at the same time arrest
of growth in the midventral line has caused the mandibular limb
(Meckel's cartilages) to be crowded against the median basihyoid
plate. Subsequently there appears to have been more or less fusion
of these cartilages such that, at the level of Fig. 9, they constitute a
single plate of cartilage in which the pterygoquadrate components are
represented most laterally, Meckel's cartilages next, and the basihyoid
cartilage in the middle. The homologies ascribed to the abnormal
cartilaginous elements seem justified because the conspicuous adductor
mandibuli muscle complex which normally surrounds the angle of the
jaw between pterygoquadrate and mandibular elements (Fig. 9-a) is
oriented with respect to the abnormal fused mass of cartilage precisely
as would be expected if the above explanation were correct (Fig. 9).
The brain is for the most part normal save for minor readjustments
in relation to the misplaced olfactory bulbs and retinae. In the
normal dogfish the floor of the diencephalon bulges ventrocaudally
forming the infundibulum which consists of two hollow oval lobes —
the inferior lobes. A long tongue of glandular tissue consisting of the
pars distalis and pars medialis of the pituitary extends anteriorly
from the pars intermedia and may be seen lying in the groove between
the inferior lobes (Fig. 5-a). At the level of the emergence of the
oculomotor nerves the inferior lobes of the infundibulum are normally
continuous with a thin-walled vascular outgrowth, the saccus vascu-
losus (Fig. 6-a), which is connected posteroventrally with the glandular
intermediate lobe. At this point, in the normal animal, nervous tissue
from the thickened floor of the saccus vasculosus is commingled with
PLATE I
FIG. 1. Ventral view of the abnormal Squalus acanthias fetus (natural size)-
The yolk-sac has been removed.
FIG. 2. Ventral view of a normal dogfish fetus.
FIG. 3. Dorsal view of the abnormal albino dogfish fetus.
FIG. 4. Dorsal view of a normal dogfish fetus.
ABSENCE OF EPITHELIAL HYPOPHYSIS
177
PLATE I
178
DON WAYNE FAWCETT
the glandular cords of the pars intermedia. The nervous tissue around
this area of contact is usually thought to be the functional pars neuralis.
The pars neuralis undergoes but little differentiation, the cells around
6- a
-Inf. L.
.A.L.
.jUnfd.
PLATE I
FIG. 5-o. A section of the head of a normal fetus showing the pars distalis of
the anterior lobe (A.L.), lying in the groove between the inferior lobes of the infun-
dibulum (Inf.L.}. Photomicrograph (hematoxylin and eosin; X 4).
FIG. 5. A corresponding section of the head of the anomalous fetus showing no
anterior lobe and a deformed infundibular process (Infd. pr.) not supported beneath
by an interorbital plate of cartilage. Photomicrograph; X 4.
the infundibular lumen retaining much of the character of the em-
bryonic ependymal layer (Butcher, 1936).
In the present abnormal specimen, on the contrary, the infundib-
ulum extends posteriorly in a long conical process which, due to
ABSENCE OF EPITHELIAL HYPOPHYSIS 179
absence of the anterior part of the cranial floor, is for some distance
not supported beneath by cartilage but traverses the groove between
the two dorsal convexities of the malformed eye (Fig. 5). This at-
tenuated infundibulum becomes gradually more slender as it passes
out of the cranial cavity beneath the anterior edge of the basal plate.
The basal plate dorsally, the pterygoquadrate and mandibular rudi-
ments laterally, and a ventral plate of cartilage (possibly the basihyoid)
are all partially fused about a tubular space in which the narrow in-
fundibulum is lodged (Fig. 7). Caudal to this point these fused
cartilages separate from the base of the chondrocranium providing
much more space for the infundibular process (Fig. 8). In consequence
of this, the infundibulum expands at its caudal tip into a bulbous
enlargement which ends blindly at the level of the spiracle in contact
with the blind end of the entodermal pharynx (Fig. 9). The in-
fundibular recess which extends from the third ventricle into the
infundibular stalk is nearly obliterated at its narrowest point but
finally terminates in a conspicuous cavity within the terminal bulbous
enlargement of the neurohypophysis. At no place does the infun-
dibular process show any tendency to differentiate into a saccus
vasculosus.
The most striking abnormality of the pituitary is the total absence
of the epithelial portions of the gland. No trace can be found of the
parts of the pituitary deriving from the embryonic stomodeum — pars
distalis, pars medialis, pars intermedia, and pars ventralis.
DISCUSSION
The pigmentary deficiencies, the absence of all buccal components
of the pituitary, and the aberrant infundibular process exhibited by
this anomalous fetus are of interest in connection with the findings of
P. E. Smith in albino tadpoles produced by early ablation of the pars
buccalis of the hypophysis. It was shown by him that atypically
placed buccal epithelium would induce hypertrophy of adjacent ner-
vous tissue whereas in complete absence of the buccal hypophysis, the
neural lobe did not attain its normal size, shape, or histological develop-
ment. Evelyn Holt's 40-mm. pig, while lacking entirely the oral
portion of the hypophysis, is described as possessing a pars neuralis
"normal in position, extent, and structure." This, she points out, is
contradictory to Smith's findings but it is noteworthy that her speci-
men is from a relatively early stage of development. If the pig fetus
had had an opportunity to continue its intrauterine life, the further
growth of the pars neuralis might well have been retarded or modified.
180
DON WAYNE FAWCETT
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182 DON WAYNE FAWCETT
Furthermore, the interdependence in development of one part of the
pituitary upon another may be quite different in mammals than in
amphibia and fishes. The present case of pituitary agenesis in a lower
vertebrate seems to bear out Smith's observations, for here, as in his
tadpoles, in the total absence of the buccal ectoderm, the pars neuralis
has not attained its normal shape nor has it undergone its typical
differentiation into a saccus vasculosus.
Tadpoles in which the buccal hypophysis has been removed at
an early stage of development display albinism in which the epidermal
melanophores are diminished in number and pigment content besides
remaining in a persistent state of contraction. These conditions in
the tadpole are closely paralleled by this albino dogfish fetus in which
the melanophores are less numerous, contracted, and noticeably with-
drawn from the surface. Lundstrom and Bard, in a study of the
effects of ablation of various parts of the brain of the dogfish (Mustelis
canis}, first discovered the hypophyseal control of the cutaneous
pigmentation in the elasmobranch fishes. They found that removal
of the neuro-intermediate lobe invariably resulted in pallor of the skin.
The present anomalous specimen constitutes an interesting confirma-
tion of their work. Evidently there has been a spontaneous suppres-
sion of the oral hypophysis equivalent to actual experimental ablation.
Because of the intermingling of the elements of the pars neuralis and
pars intermedia of the dogfish pituitary, it has so far been impossible
to accomplish a complete operative separation of these two parts.
The presence of the neuro-hypophysis in the present specimen, but the
total absence of the oral pituitary (including the pars intermedia)
indicates that the humoral agent affecting pigmentation is a derivative
of the oral components of the gland. Presumably in that portion of
the gland referred to as the neuro-intermediate lobe it is the buccal
elements that are responsible for the chromatophore-expanding factor.
Observations in many cases of human anencephaly (Covell, 1927)
make it apparent that aberrant formative processes involving defective
closure in the dorsal midline may result in agenesis of the neural
lobe of the pituitary. Cyclopia and astomia are occasionally found
together in human monsters. It appears from the present observa-
tions that, in the dogfish, anomalous development in the ventral mid-
line with imperfect separation of symmetrical parts and consequent
cyclopia and astomia may result in agenesis of the oral hypophysis.
ABSENCE OF EPITHELIAL HYPOPHYSIS 183
SUMMARY
1. An anomalous fetus of the spiny dogfish (Sqiialus acanthias)
is described in which there are malformations of the head comprising
cyclopia, astomia, and abnormalities of the hypophysis.
2. The abnormalities of the hyophysis involve: —
(a) The total absence of the oral components of the gland, and
(b) A neural lobe which is deformed and possesses no saccus
vasculosus. The conclusion is drawn that the neural lobe
has not undergone normal differentiation because it has
been deprived of its usual association with the buccal
hypophysis.
3. The specimen is albino, displaying a diminished number of
chromatophores in a state of persistent contraction. This finding
indicates that the melanophore-controlling principle in the dogfish
is a derivative of the buccal components of the pituitary.
4. Only one other instance of total spontaneous suppression of the
oral hypophysis is described in the literature, namely, in a pig fetus
(Holt, 1921). In human fetuses anencephaly occurs not infrequently
but is associated with suppression of the neuro-hypophysis instead of
with the adenohypophysis.
BIBLIOGRAPHY
BUTCHER, E. O., 1936. Histology of the pituitaries of several fish. Bull. Mt. Desert
Island Biol. Lab., pp. 18-20.
COVELL, W. P., 1927. A quantitative study of the hypophysis of the human an-
encephalic fetus. Am. Jour. Path., 3: 17-28.
HOLT, E., 1921. Absence of the pars buccalis of the hypophysis in a 40-mm. pig.
Anat. Rec., 22: 207-216.
LUNDSTROM, H., AND P. BARD, 1932. Hypophyseal control of cutaneous pigmenta-
tion in an elasmobranch fish. Biol. Bull., 62: 1-9.
SMITH, P. E., 1920. The pigmentary, growth, and endocrine disturbances induced
in the anuran tadpole by the early ablation of the pars buccalis of the
hypophysis. Am. Anat. Mem., No. 11, pp. 5-151.
VARIATIONS OF COLOR PATTERN IN HYBRIDS OF
THE GOLDFISH, CARASSIUS AURATUS
H. B. GOODRICH AND PRISCILLA L. ANDERSON1
(From the Department of Biology, Wesleyan University]
This paper gives an account not only of the differences between
fish arising from the same genetic cross but also of the variations of
color pattern taking place during the life of individual fish.
The cross between the common goldfish and the transparent
shubunkin which are both varieties of the species Carassius auratus
was first subjected to genetic analysis by Berndt (1925 and 1928) and
Chen (1925 and 1928). The results indicated that the two parental
types are genetically distinguishable by a single gene difference. The
formulae as denoted by Chen are : common goldfish TT, the transparent
shubunkin T'T', and the hybrid TT'. This hybrid is known to the
fanciers as the calico shubunkin. The common goldfish, which is
quite brown or black during youth, changes to the familiar orange or
red type by destruction of part or nearly all of its melanophores
(Berndt, 1925; Goodrich and Hansen, 1931). This type also carries
at least two layers of reflecting tissue, one beneath the scale layer and
the other backing each individual scale. The transparent shubunkin
has lost most of the chromatophores (both melanophores and xantho-
phores) and also most of the reflecting tissue. The heterozygous type,
or calico fish, shows great variability in the distribution of both melano-
phores and xanthophores and there is no bilateral symmetry of pattern.
A deep abdominal layer of reflecting tissue is present and a few scales
are also backed with the tissue. For full details, papers by Chen
(1928) or Goodrich and Hansen (1931) may be consulted.
Goodrich and Hansen (1931) made a detailed comparative study
of the history of the melanophores of the three phenotypes covering
the first eight weeks after hatching during which period the fish grew
from 4.5 mm. to about 33 mm. in length. It was found that the history
of the three types was similar for the first week (to 9 mm.) showing
a uniform rate of multiplication of the chromatophores. After this
the three types diverged. The normal goldfish showed a very rapid
1 This paper is published as part of a research program at Wesleyan University
supported by the Denison Foundation for Biological Research. The authors wish
to acknowledge their indebtedness to Miss Marian Hedenburg for carrying on the
program during the last half-year.
184
VARIATIONS OF COLOR PATTERN IN HYBRID GOLDFISH 185
and uniform increase in number of chromatophores. In the trans-
parent shubunkin the melanophores began to disintegrate until nearly
all were destroyed. The hybrid, however, was found to be highly
variable, showing great diversity between individuals. New cells
appeared; others were destroyed. It gave the impression of a conflict
between the cell proliferation and cell destruction.
MATERIAL AND METHODS
This paper continues the observations on the melanophore pattern
of the heterozygous type beginning where the previous study was
discontinued. The work was begun during the summer of 1937 with
fish varying from 23 to 36 mm. in length (tip of mouth to base of
caudal fin). The fish were chiefly obtained from the Grassyfork
Fisheries of Martinsville, Indiana, to which institution we are greatly
indebted. The hybrid fish were obtained directly from the hatchery
which raises them regularly for the market. Records were made by
photographing one side of the fish at intervals of approximately one
month, but the periods were lengthened to longer intervals during the
last six months. Ten of the fish are still under observation at this
time, one year and six months after the start of the work. They vary
from 47 to 58 mm. in length. All others that were started died.
Anaesthetization, necessary for photography, proves to be fatal in
some cases.
The individuals differ markedly from each other. For purposes
of description twro types, A and B, may be recognized, but it should
be understood that there are intermediate gradations. Type A shows
a relatively uniform distribution of melanophores on the dorsal half
of the body and extending variably below the lateral line (Figs.
1 and 2). In Type B, the distribution of melanophores is much more
uneven. They tend to be aggregated in clusters (Figs. 4 and 5).
Xanthophores are present in both types and are unevenly distributed,
but are not studied in this paper as it is very difficult to distinguish
and identify the individual cells.
HISTORY OF COLOR PATTERNS
Type A
It is possible with these fish to enumerate and reidentify from time
to time all cells of large areas on the photographed side of the body.
Except in the cases where wholesale destruction of melanophores
occurs, it is found that few cells are lost and that individual cells have
long life. An example may be taken (our fish number MG-3) on which
907 cells were enumerated and located on the side of the body (see
186
H. B. GOODRICH AND PRISCILLA L. ANDERSON
Table I for this and other references to cell counts). The first photo-
graph was taken August 2, 1937 and the last February 17, 1939 making
a total series of 18 photographs. During this time, 50 of the 907 cells
disappeared and three new ones appeared. Figure 1 is the photograph
TABLE I
This table gives records of photographs of four of the fish studied. The dates
are accurate only for MG-3 as it was not always possible to take all photographs on
the same day.
MG-3
MG-4
MG-IS
MG-16
Calico
Type A
Calico Type B
Transparent
Calico
Type .4
907
cells
97
cells, 2 cl.
21
cells
613
cells
D
A
D
A
D
A
D
A
July
17, 1937
0
0
0
0(4)
Aug.
2
0
0(1)
9
1 cl
0
0
0
0
Aug.
26
1
0
6
2
0
0
6
0
1 cl
Sept.
20
5
0
2
0
0
0
1
0
Oct.
18
5
0
0
0
1
0
0
1 cl
Nov.
16
5
0
7
0
1
0
(6)
0
Dec.
15
4
0
4
1 cl
0
0
0
Jan.
26, 1938
2
0
3
4cl
0
0
0
Feb.
23
4
0
7
2cl
0
0
0
Mar.
28
1
0
1
3cl
0
0
0
May
2
4
1
1
0
1
0
0
June
7
9
1
1
0
1
0
(7)
0
July
16
2
1
0
0
0
0
0
Aug.
5
2
0
1
0
0
0
0
Sept
13
-
-
8
0
0
0
0
Oct.
12
4
0
1
0
0
0
0
Nov
14
0
0
0
0
0
0
0
Jan.
4, 1939
0
0(2)
0
0(5)
0
0
0
Feb.
17
2
0
0
0
0
0
0
Totals
50
3
51
2
4
0
All
1 cl
12 cl
D — number of cells that disappeared since preceding photograph.
—number of new cells appearing since preceding photograph,
cl — cell cluster or spot.
(1). (2), (4), (5) indicate pictures reproduced in Figs. 1, 2, 4, and 5.
(6) — time of beginning of wholesale destruction of melanophores.
(7) — time at which all melanophores were destroyed.
taken August 2, 1937 and Fig. 2 that of January 4, 1939. The dotted
lines outline arbitrarily delimited areas marked on the prints to facili-
tate the counting and identification of cells. The small circles indicate
the former location of cells that have disappeared. Figure 2 is taken
at a lower magnification than Fig. 1 and fish had grown from 26 mm.
VARIATIONS OF COLOR PATTERN IN HYBRID GOLDFISH 187
to 58 mm. in length (exclusive of caudal fin). Figure 3, however,
shows the rectangular area of Fig. 2 raised to the same magnification
as Fig. 1.
-
•
• i *
If 1 :* * *
*. 9 .•
, I
* • *
FIG. 1. Fish MG-3. Photograph taken August 2, 1937. X5}2- This is
a "Type A" calico shubunkin; 907 cells are located in outlined areas.
FIG. 2. Fish MG-3. Photograph taken January 4, 1939. X 11A. Fifty-two
cells have been lost and 3 new cells appeared since record of Fig. 1 . Dotted circles
indicate location of cells that have disappeared.
FIG. 3. Section outlined by dashes in Fig. 2 enlarged to same magnification as
in Fig. 1, showing increase in size of area and of cells.
188
H. B. GOODRICH AND PRISCILLA L. ANDERSON
TypeB
These fish show the irregular mottling which is prized by the
fanciers. The dark spots are usually clusters of small melanophores
too densely crowded to count. Of 97 selected on the first photograph
of MG-4 on July 18, 1937, 46 remained on January 1, 1939 (Figs.
4 and 5). In the meanwhile, however, others have appeared and
ff
*
^
FIG. 4. Fish MG-4. Photograph taken July 18, 1937. X
FIG. 5. JlfG-4. Photograph taken January 4, 1939. X2*2. Fifty-one cells
disappeared and 12 new cell clusters appeared in area under observation.
there has been a notable eruption of spots, or clusters of melanophores—
12 altogether on the left side. These spots are first recognized as
one or a few minute melanophores which rapidly increase in number.
A spot for a time is often bounded by the posterior edge of a scale.
Indeterminate Types
In many cases the clusters of small cells appear among, or super-
ficial to, cells uniformly distributed and in this way combine character-
VARIATIONS OF COLOR PATTERN IN HYBRID GOLDFISH 189
istics of Types A and B. An example is MG-5, where it was possible
to identify cells only in a small area. Ten disappeared out of 164 in
this area, but six new clusters of cells have arisen similar to those
discussed under Type B.
Extensive Cell Destruction
Occasionally a sweeping destruction of melanophores occurs within
a few weeks. This is similar to the process in the ordinary goldfish,
which is gold because melanophores but not xanthophores have been
destroyed. This change most frequently takes place in ordinary
goldfish at about three months of age but may occur much later
(Cf. Berndt, 1925; Chen, 1925; and Goodrich and Hansen, 1931). It
occurred in two of the calico shubunkins which we had under observa-
tion in this series. The history of one of these, MG-16, is given in
Table I and in this the breakdown occurred at about eight months
of age.
INCREASE IN SIZE OF CELLS
As mentioned above, Fig. 3 shows the rectangular area marked
on Fig. 2 enlarged to the same magnification as Fig. 1. The compari-
son of Figs. 1 and 3 then shows the actual increase in size of the area
outlined. It also shows that the individual cells, which for the most
part show approximately the same degree of melanin dispersion in
both pictures, have definitely increased in size. It, therefore, appears
that, in so far as the melanophores are concerned, the increase of body
size has involved an enlargement of cells rather than a multiplication
of cells.
DISCUSSION
These observations not only show that there is much variation
among individuals of these hybrids but also that each individual is
variable in respect to color patterns displayed during its life cycle.
The heterozygous type, as noted for earlier developmental stage by
Goodrich and Hansen (1931), continues in later stages to be in a con-
dition of unstable equilibrium between opposing tendencies — those of
cell multiplication and cell destruction.
Fukui (1927 and 1930) has shown that the destruction of melano-
phores in the ordinary goldfish tends to take place in definitely bounded
areas, giving rise to some degree of uniformity of pattern in black- and
goldfish. These areas, he believes, correspond to regions of looser
subcutaneous tissue bounded by more dense tissue. In effect, these
may be perhaps regarded as sinuses filled with tissue fluids or lymph.
190 H. B. GOODRICH AND PRISCILLA L. ANDERSON
His experiments with injection of adrenalin showed a restoration of
pigment which tended to be circumscribed in such areas. These
results suggest that endocrine factors operating on such a region bring
about under certain conditions the destruction of chromatophores and
under other conditions the production of pigment. Fukui suggests
that pigment destruction is due to a higher metabolic rate in these
areas, but this might be stimulated by the chemical environment.
In contrast to the above, the origin of new spots or cell clusters is
entirely irregular, having no relation to the areas described by Fukui.
It, therefore, seems unlikely that their location can be due to endocrinal
conditions.
It then seems probable that the goldfish presents a new example
of the dual gene control such as has been suggested in the plum-
age of birds. In the case here described the direct gene action
may control cell multiplication, resulting in the formation of cell
clusters or spots, while remote gene control of "endocrinal regulation"
may cause the destruction of cells (see Danforth, 1932, p. 33).
A discussion of the developmental origin of cell clusters will be
presented in the companion paper, Goodrich and Trinkaus (p. 188).
SUMMARY
1. The FI heterozygous types from the cross of the common goldfish
with the transparent shubunkin (both of the species Carassius auratus)
show not only a great range of variability between individuals, but
frequently the pattern of a single individual changes markedly during
the life cycle. This is due to destruction and emergence of chromato-
phores producing a varying pattern. It is suggested that the multipli-
cation of cells is an example of "direct gene control" and the destruc-
tion is due to "endocrinal regulation" or remote gene control.
2. Many individual melanophores are long-lived, having been iden-
tified at the beginning and end of the 19-month period of observation.
3. Such long-lived melanophores gradually increase in size during
the growth of the fish.
BIBLIOGRAPHY
BERNDT, WILHELM, 1925. Verererbungstudien an Goldfischrassen. Zeitsclir. f.
Indukt. Abst. u. Vererb., 36: 161-349.
BERNDT, WILHELM, 1928. Wildform und Zierrassen bei der Karausche. Zool.
Jahrb., Abt. Allgem. Zool. u. Physiol., 45: 841-972.
CHEN, SHISAN C., 1925. Variation in the external characters of goldfish, Carassius
auratus. Contr. Biol. Lab. Sci. Soc. China., 1: 1-65.
CHEN, SHISAN C., 1928. Transparency and mottling, a case of mendelian inheritance
in the goldfish, Carassius auratus. Genetics, 13: 434-452.
DANFORTH, C. H., 1932. In Allen, E. Sex and Internal Secretions, pp. 12-54.
Baltimore.
VARIATIONS OF COLOR PATTERN IN HYBRID GOLDFISH 191
FUKUI, KEN'ICHI, 1927. On the color pattern produced by various agents in the
goldfish. Folia Anal. Japan., 5: 257-302.
FUKUI, KEN'ICHI, 1930. The definite localization of the color pattern in the goldfish.
Folia Anat. Japan., 8: 283-312.
GOODRICH, H. B., AND I. B. HANSEN, 1931. The postembryonic development of
mendelian characters in the goldfish, Carassius auratus. Jour. Exper. Zool.,
59: 337-358.
GOODRICH, H. B., AND J. P. TRINKAUS, 1939. The differential effect of radiations
on mendelian phenotypes of the goldfish, Carassius auratus. Biol. Bull.,
77: 188-195.
THE DIFFERENTIAL EFFECT OF RADIATIONS ON
MENDELIAN PHENOTYPES OF THE GOLD-
FISH, CARASSIUS AURATUS1
H. B. GOODRICH AND J. P. TRINKAUS
(From the Department of Biology, Wesley an University)
The types of goldfish used in the following experiments are those
described in the companion paper by Goodrich and Anderson (1939).
These are the common goldfish, the transparent shubunkin, and the
hybrid between these two known as the calico shubunkin. Genetic
analysis has shown that this is a monohybrid cross and the formulae
assigned have been: ordinary goldfish TT, the transparent shubunkin
FT', and the calico fish TT'.
The original purpose of the ultraviolet treatment was to destroy
certain parts of the color pattern in the calico fish and to study its
regeneration. It was, however, soon discovered that lighter treatment
than that needed to destroy the chromatophores apparently induced
the formation of new pigmented areas. Consequently a more careful
program of experimentation was outlined to verify these preliminary
findings.
METHODS
The source of illumination has been a small laboratory mercury
lamp obtained from the Hanovia Company (their model E). The
quartz tube is 16 mm. in diameter, has a length of arc of 50 mm., and
operates on 110-volt circuit. For purposes of destruction of melano-
phores, treatments frequently of 30 minutes or more were administered,
but for stimulation of pigment formation most treatments were of 10
minutes duration at distances varying from 2 cm. to 6 cm. from the
lamp. Only a small area was irradiated on each fish. Other parts of
the body within the zone of illumination were protected. The areas
treated varied from about 0.2 to 0.9 sq. cm. in size. These were
delimited by pieces of wet filter paper over which were placed pieces
of tin foil, which in turn were held in place by more filter paper. Wet
cotton was put over the head and the operculum and over the rear of
1 This paper is published as part of a research program at Wesleyan University
supported by the Denison Foundation for Biological Research. The authors wish
to acknowledge their indebtedness to Miss Priscilla Anderson who performed the
preliminary experiments.
192
EFFECT OF RADIATIONS ON MENDELIAN PHENOTYPES 193
the body and caudal fin. This kept the fish moist and helped to hold
it in place. The fishes were anesthetized in a 1 per cent urethane
solution and were placed on a paraffin block modeled to hold the fishes
nearly upright. During irradiation the spot treated was kept wet
with distilled water to prevent drying of the tissue. Photographs of
both sides of the fish were taken before treatment. The irradiated
areas were outlined on the photographic prints and later the location
of new spots was marked on these prints, or additional photographs
taken if thought desirable. The fish were inspected at weekly intervals
for the first three months after the treatment and those fishes that
survived were observed at longer intervals for the succeeding six
months.
EXPERIMENTS
After the preliminary experiments, it was first planned to treat
approximately equal numbers of the three Mendelian types. Ac-
cordingly, ten of each were irradiated. Later, the numbers treated
were increased, especially of the hybrid type which was the only form
which gave a positive reaction. The final lot of fish irradiated included
24 of the ordinary goldfish TT, 17 of the transparent shubunkin T'T',
and 52 of the hybrids TT', giving a total of 93 fish treated.2 Areas
with few or no melanophores were selected for irradiation. The
essential result from the comparative study was that the hybrids alone
showed a positive reaction by development of new melanophores, while
in the two parental types no melanophores were formed. Most fish
in all these groups exhibited inflammation and sometimes necrosis of
tissues. In the goldfish TT the xanthophores and guanin crystals
(of the reflecting tissue) were frequently destroyed. Spots or cell
clusters appeared only in the hybrids. These were first observed as
small faintly grayish chromatophores, having long delicate processes.
The number of cells increased and in about eight weeks these cells
became typical mature goldfish melanophores. (Figure 3 shows the
inflammation following irradiation, and Figs. 4 and 5 the development
of a cell cluster in the same spot.) Figure 1 is a photograph of a
hybrid TT' taken on March 29 just before radiation and the area
irradiated is outlined. Figure 2 is of the same fish on May 27. Three
new spots, one small and two large, have appeared in the radiated
area and one outside (in dotted circle). All but one of the new spots
2 Eight fish of doubtful classification are excluded from these totals. Inspection
of pattern indicated that they probably were one normal goldfish and seven trans-
parent shubunkins. All gave negative reactions. Even if the presumed transparent
types were transferred to the list of 52 calico shubunkins the essential results as
indicated by the graphs, Figs. 6 and 7, would not be altered.
194
H. B. GOODRICH AND J. P. TRINKAUS
were located in the dermis superficial to the scales. This one excep-
tional spot was beneath the scales.
The companion paper (Goodrich and Anderson, 1939) has shown
that the hybrid or calico fish is characterized by an irregular mottling
and, moreover, that this pattern is subject to change during the life
of the individual. It therefore seemed possible that the appearance
of new spots after radiation might be nothing more than the normal
sequence of events. On this account, many more of the hybrids were
-**T'
v
FIG. 1. Photograph of a hybrid TT' taken on March 29 just before radiation-
The area later irradiated is outlined with dotted line. X 1/4-
FIG. 2. Photograph of same fish as in Fig. 1 taken on May 27. Three new
spots (one small and two large) have appeared in the radiated area and one outside
(in dotted circle). X 1J4-
FIGS. 3, 4, 5. Successive photographs of the same area on a hybrid fish. X 6.
Irradiation Nov. 13, 1937. Fig. 3, appearance Nov. 27; congestion of capillaries in
center (an older spot at right). Fig. 4, Jan. 2, 1938. Fig. 5, Jan. 25, 1938.
treated and the results subjected to analysis. This has shown that
the irradiated areas produced a significantly greater number of spots
or cell clusters than appeared on non-radiated areas. It was also
found that the new spots appeared chiefly from three to six weeks
after treatment with the maximum number arising during the fifth
week (see charts, Figs. 6 and 7). In 24 cases two or more cell clusters
appeared within the radiated area, in 17 cases only one new spot and
EFFECT OF RADIATIONS ON MENDELIAN PHENOTYPES 195
none were recorded in 11 cases. These results have been compared
with the total number of cell clusters appearing on both sides of the
body (exclusive of head and fins). The results appear significant even
14.
WEEKS AFTER IRRADIATION
FIG. 6. Graph of numbers of spots X10 appearing in successive weeks after
irradiation. Dotted line, irradiated area. Solid line, other parts of body (head and
fins not included).
§4
WEEKS AFTEB IRRADIATION
FIG. 7. Graph showing same data as Fig. 6 corrected for relative size of areas.
Dotted line, numbers in irradiated area X20. Solid line, numbers on other parts
of body X-l.
196 H. B. GOODRICH AND J. P. TRINKAUS
when no correction is made for the difference in areas compared.
When, however, such a comparison is made it is found that the total
non-radiated surface examined was approximately twenty times that
of the average irradiated area. A graph, incorporating this correction,
is shown (Fig. 7) and indicates a notable excess of development of
spots in the irradiated areas. During the observational periods, from
treatment until 14 weeks thereafter, there appeared a total number of
62 new spots or cell clusters within the irradiated areas and 32 outside
of these areas. If we multiply by the factor 20 (20 X 62 = 1240),
it appears that had spots appeared at a similar rate in the non-radiated
region there would have been 1240 spots, whereas there were only 32.
This proportion of nearly 38 : 1 is then an index of the increased reac-
tion of the radiated region. It is not impossible that this is an under-
estimate. The areas chosen for treatment were frequently below the
lateral line, because this region was more clear of melanophores, and
it is possible that the ventral region is one having less inherent capacity
for production of melanophores.
The new cells recorded in the above experiments were in all respects
similar to normal melanophores present elsewhere on the fish. Two
sets of subsidiary experiments were carried out which, incidentally,
gave further confirmation that these cells were normal melanophores.
(1) It was found that the melanophores of the hybrid responded very
irregularly to an illuminated white environment. In some cells the
pigment became concentrated and in others it remained dispersed.
New cells arising in the irradiated areas showed this same variability of
reaction. (2) Ten scales bearing new cell clusters were transplanted
to other parts of the fish as had previously been done by Goodrich
and Nichols (1933) with non-radiated fish. The results were similar.
The cells lived and in four cases increased, spreading over adjoining
scales.
DISCUSSION
The observations presented in this and the preceding paper (Good-
rich and Anderson, 1939) show that the hybrid or calico shubunkin
retains the potentiality to produce irregularly situated spots during a
considerable part of the life cycle. The radiation appears to stimulate
a precocious development of the spots in the areas treated. The
question then arises as to what developmental or other conditions con-
trol the appearance of these spots or cell clusters. Goodrich (1927),
working on the Japanese fish Oryzias latipes, suggested that the varie-
gated pattern could be explained by the ameboid migration of pre-
determined melanoblasts of two types — that producing the maximum
EFFECT OF RADIATIONS ON MENDELIAN PHENOTYPES 197
amount of melanin and the other such a small amount that they re-
mained virtually colorless. Recent investigations such as those of
DuShane (1935) and Twitty (1936) on amphibia have tended to con-
firm the hypothesis of an early determination of wandering chromato-
blasts. The paper by Willier and Rawles (1938) on the chick opens
the possibility of cell determination and migration in forms where
hormones have been shown to be largely operative in other phases
of pigment control. The observations of Apgar (1935) on Triturus
have suggested the concept of a widespread distribution of colorless
chromatoblasts. It, therefore, seems not improbable that we may
consider the calico shubunkin (especially Type B of the companion
paper) to be invisibly spotted during development with colorless
chromatoblasts — singly or in nests — and that these multiply and
differentiate independently at irregular intervals to form the spots or
clusters of melanophores. In some respects this hypothesis resembles
the old theory of embryonic cell rests advanced to explain the cause
of cancer.
In certain individual fish a wave of destruction takes place, possibly
due to some hormone action, which destroys all melanophores and
possibly all melanoblasts in the affected areas. We have never ob-
served the appearance of new spots in a region which has suffered
such wholesale destruction.
Attention should be called to the production in goldfish of pigment
cells by X-rays (Smith, 1932). The cells appeared within a few days
after treatment and disappeared a few weeks later. They did not
seem to be homologous to the pattern-producing cells and resembled
cells that had previously been observed arising after various mechanical
injuries to the tissues (Smith, 1931). In our own experiments we have
noted three cases of the formation of such cells. They were seen on
the normal goldfish TT after unusually severe radiation from the
mercury arc lamp and the appearance and history of these cells were
similar to those noted by Smith.
The contrasting reactions of the three genotypes indicate that the
hybrid or calico fish retains in adult condition a far greater potency to
produce melanophores than either parental form. Goodrich and Han-
sen (1931) have pointed out that all three types form melanophores in
early development. The ordinary goldfish loses these by wholesale
destruction usually at about three months of age, while in the trans-
parent shubunkin relatively few ever appear. Neither of these two
parental forms produced typical melanophores when irradiated and
it may be suggested that melanoblasts have also been destroyed or are
198 H. B. GOODRICH AND J. P. TRINKAUS
largely absent. In contrast, the heterozygous type retains the
melanoblasts.
No attempt is made in this paper to determine what wave-lengths
have produced the observed effect. The mercury vapor arc produces
a wide range of wave-lengths. The extensive literature on effects of
ultraviolet light shows that both stimulating and destructive effects
have been observed. Sperti, Loofbourow, and Dwyer (1937), working
on yeast cells, have suggested that cells when injured by ultraviolet
liberate some growth-promoting substance, thus indicating a possible
interrelation of injurious and stimulating effects. The treatments
used in our experiments have been relatively more severe than those
which have produced primarily stimulating effects on isolated cells.
Ultraviolet light penetrates but a few millimeters through animal
tissues. Sato (1933) has shown that the ultraviolet light bands charac-
teristic of the mercury arc will pass through fish scales. The effect
produced in our experiments may well be due chiefly to the regenerative
processes following the inflammation and destruction of tissue.
SUMMARY
1. Radiation from a mercury vapor lamp produced differing reac-
tions in three Mendelian phenotypes. Two parental forms, the
ordinary goldfish and the transparent shubunkin, do not develop
melanophores as a result of the treatment. The FI hybrid, or calico
shubunkin, does respond by an acceleration in the production of new
spots or clusters of melanophores.
2. It is suggested that the hybrid during development becomes
supplied with colorless chromatoblasts throughout the dermis which
are stimulated to precocious multiplication and differentiation as a
result of the radiation.
BIBLIOGRAPHY
APGAR, B. D., 1935. A study of the reappearance of melanophores and the formation
of melanophore aggregations (spots) in regenerated ventral skin of the
common newt, Triturus viridescens. Jour. Morph., 58: 439-461.
DuSHANE, G. P., 1935. An experimental study of the origin of pigment cells in
Amphibia. Jour. Exper. Zool., 72: 1-32.
GOODRICH, H. B., 1927. A study of the development of mendelian characters in
Oryzias latipes. Jour. Exper. Zool., 49: 261-287.
GOODRICH, H. B., AND P. L. ANDERSON, 1939. Variations of color pattern in hybrids
of the goldfish, Carassius auratus. Biol. Bull., 77: 180-187.
GOODRICH, H. B., AND I. B. Hansen, 1931. The postembryonic development of
mendelian characters in the goldfish, Carassius auratus. Jour. Exper. Zool.,
59: 337-358.
GOODRICH, H. B., AND ROWENA NICHOLS, 1933. Scale transplantation in the gold-
fish, Carassius auratus. Biol. Bull., 65: 253-265.
SATO, N., 1933. Light passing through the scales of fish. Ada Dermatologica, 22: 45.
EFFECT OF RADIATIONS ON MENDELIAN PHENOTYPES 199
SMITH, G. M., 1931. The occurrence of melanophores in certain experimental
wounds of the goldfish (Carassius auratus). Biol. Bull., 61: 73-84.
SMITH, G. M., 1932. Melanophores induced by X-ray compared with those existing
in patterns as seen in Carassius auratus. Biol. Bull., 63: 484-491.
SPERTI, G. S., J. R. LOOFBOUROW, AND SR. C. M. DWYER, 1937. Proliferation-
promoting factors from ultra-violet injured cells. Stud. Instil. Divi Thomae,
1: 163-191.
TWITTY, VICTOR C., 1936. Correlated genetic and embryological experiments on
Triturus. I and II. Jour. Exper. Zool., 74: 239-302.
WILLIER, B. H., AND MARY E. RAWLES, 1938. Feather characterization as studied
in host-graft combinations between chick embryos of different breeds.
Proc. Nat. Acad. Sci., 24: 446-452.
THE REACTIONS OF THE PLANKTONIC COPEPOD,
CENTROPAGES TYPICUS, TO LIGHT
AND GRAVITY1
W. H. JOHNSON AND J. E. G. RAYMONT
(From the Department of Physiology, McGi.ll University, the Biological
Laboratories, Harvard University, and the Woods Hole
Oceanographic Institution)
INTRODUCTION
Field investigations on the vertical distribution of the plankton
carried out by many different workers in recent years have established
the occurrence of a diurnal vertical migration for most species of the
zooplankton. Since most investigators agree in considering light as
an important controlling factor, it seemed desirable, following the
work of Esterly, Spooner, Clarke and others, to attempt to study the
light responses of a single planktonic species under controlled labora-
tory conditions.
Centropages typicus is a neritic copepod, extremely abundant off
Woods Hole at certain times of the year. Clarke (1933) states that
the adults show a diurnal vertical migration correlated with changes
in the submarine illumination. A few preliminary observations in the
laboratory showed us that the adult females are very definitely
affected by light. It was therefore decided to conduct experiments on
the phototropic and geotropic responses of these animals. Our choice
was fortunate in that it was possible to obtain the copepods quickly
and easily off Woods Hole, and to keep them at a conveniently low
temperature in the laboratory to ensure their healthy existence for at
least a few days.
PHOTOTROPISM : EXPERIMENTS WITH TUBES HORIZONTAL
In order to separate the phototropic from possible geotropic re-
sponses, it seemed advisable to test first the reactions of the copepods
to light in a horizontal direction.
Methods
The experimental animals were obtained in Vineyard Sound by
towing a scrim plankton net horizontally near the surface for about
fifteen minutes. The animals, collected in the glass jar attached to
1 Contribution No. 207 from the Woods Hole Oceanographic Institution.
200
REACTIONS OF COPE POD TO LIGHT AND GRAVITY 201
the net, were poured into 3 liters of sea water and transported im-
mediately to the laboratory. The adult female Centropages typicus
were selected in diffuse daylight using a wide-mouthed pipette and a
binocular microscope. Usually 20 healthy appearing copepods were
placed in each of two glass tubes (13 X 2|"), each of which was sealed
at one end with a glass plate. The open ends of the tubes were then
sealed with similar glass plates, and the tubes arranged in constant
temperature tanks maintained at 12° C. in the darkroom.
The two experimental tubes could be separated from each other by
a distance of 21 feet. It was thus possible to obtain a wide range of
light intensities for any one source, by varying the distance between
the light source and the tubes. The intensities of the various inside
frosted bulbs employed were as follows : 2
Wattage Approximate Intensity at 1 foot
15 13.5 foot-candles
25 25.0
40 43.0
60 75.0
100 150.0
It should be borne in mind that all the light intensities mentioned in the
text are only approximate figures.
The lowest intensities used were obtained by means of neutral
filters in the form of opal discs and white paper, the percentage
absorptions of which were obtained by means of a photoelectric cell.
Since several filters were used together at the very lowest intensities,
corrections were made for diffusion and back-scattering.
Each experimental tube was marked off into quarter-lengths, and
the distribution of the animals at any time, under any one condition
of light, was expressed as the numbers in each section. At the very
low light intensities, counting of the copepods was facilitated by
lighting the tubes from behind for a moment with a weak red lamp.
Preliminary tests made with this lamp showed that it had no effect
on the distribution of the animals.3
In all the experiments, unless otherwise noted, the distribution of
the animals was observed at the end of each time interval shown in the
tables. After each observation, the tubes were changed, end for end,
by turning them slowly in a horizontal plane. This procedure forced
the animals to orientate afresh, and to redistribute themselves accord -
2 On the advice of Mr. Eddie Kline, electrical engineer of the Canadian Laco
Lamp Co., these can be considered as accurate only within 20 per cent, due to voltage
fluctuation.
3 Dr. Horton of the Department of Physics, McGill University, kindly made a
spectroscopic photograph of the light emitted and found that the transmission begins
o o
at 6402 A, and continues beyond 8600 A.
202
W. H. JOHNSON AND J. E. G. RAYMONT
ing to the tropistic responses actually in operation during that time
interval. Enough time was allowed for the animals to establish their
TABLE I
Experiment commenced at 4:00 P.M., August 19.
Tubes A and B set at distance of 5 ft. and 1 ft. respectively, from source.
At 12:00 noon, August 20, tube A moved to 10 ft.
At 4:00 P.M., August 20, tube A moved to 20 ft.
Tube B was kept at 1 ft. throughout.
Source: 60-\vatt lamp.
Time
Distance
Intensity
(Positive) *
I
II
(Negative)
III IV
Aug. 19
4:10 P.M.
5ft.
3.0 f.c.
20
-
-
-
1 ft.
75.0 f.c.
20
—
—
—
4:40 P.M.
5 ft.
20
1 ft.
20
-
-
-
6:45 P.M.
5 ft.
16 and 4
—
_
1 ft.
20
-
-
-
9:15 P.M.
5 ft.
10 and 6
2
3
1 ft.
10 and 3
3
-
1
9:30 P.M.
5ft.
8 and 6
2
2
1 ft.
3 and 7
3
3
2
Aug. 20
11:30 P.M.
5 ft.
10 and 3
1
4
3
1 ft.
4 and 8
3
1
1
2:30 P.M.
10ft.
0.75 f.c.
8 and 3
3
3
1 ft.
75.0 f.c.
5 and 9
3
1
1
4:00 P.M.
10ft.
8 and 4
4
4
_. _
1 ft.
3 and 7
4
1
1
4:45 P.M.
20ft.
0.19 f.c.
9 and 6
_
3
_
1 ft.
75.0 f.c.
5 and 10
3
2
1
8:00 P.M.
20ft.
6 and 9
2
2
1 ft.
3 and 9
4
1
2
*Two numbers are sometimes given under Section I (e.g. 16 and 4). This
distinguishes those copepods right at the positive end (16), from those still in this
section but apparently less strongly attracted.
new distribution before a second record was taken, so that their final
position was unaffected by the configuration of the previous time
interval.
Observations
A series of tests (Table I) was first carried out in order to determine :
REACTIONS OF COPEPOD TO LIGHT AND GRAVITY
203
(1) The normal responses of the copepods to various light intensities
within limits found in nature.
(2) The effect of continued exposure to constant light intensities over
the range studied.
The results obtained (Table I) showed that the copepods were
positive to all illuminations, and remained largely so after exposure.
A number of experiments was then carried out to determine the
range of light intensities to which the copepods were sensitive, and to
investigate the possibility of the existence of critical light intensities
at which the phototropic sign might become reversed.
The copepods were found to be positive to low light intensities, the
lowest to which they were attracted being ca. 0.005 f.c. (Table II).
TABLE II
Responses to low light intensities. Distance of experimental tube from source:
20 ft. throughout.*
Intensity
Time
(Positive)
I
II
in
(Negative)
IV
0.06 f.c.
Aug.
20 9:30
P
M.
5
and 4
—
2
3
< i
Aug.
21 8:30
A
.M.
4
and 6
1
2
1
0.015
10:45
A
.M.
0
and 12
-
3
1
( (
12:00
Noon
2
and 12
1
2
3
t (
3:30
P
.M.
4
and 5
1
4
—
0.008
Aug.
24 12:40
P
.M.
14
1
3
2 (New
animals)
1 (
2:00
I'
.M.
1
and 10
2
2
3
i 1
3:00
P
.M.
3
and 9
1
3
3
0.006
3:30
P
.M.
11
2
1
3
t t
4:50
P
.M.
6
5
-
7
t i
7:00
P
.M.
11
2
2
1
I 1
8:00
P
.M.
10
-
2
7
u
10:50
P
.M.
13
-
4
1
i t
Aug.
25 8:30
A
.M.
15
1
-
-
1 (
9:30
A
.M.
14
-
2
1
0.005
Aug.
26 12:30
P
.M.
9
5
4
1
t t
1:55
P
.M.
10
4
3
-
t (
2:25
P
.M.
3
7
6
3
0.003
Aug.
28 2:30
P
.M.
3
4
7
6 (New
animals)
1 1
4:45
P
.M.
9
4
4
3
1 1
Aug.
29 9:40
A
.M.
11
6
2
-
1 1
11:50
A
.M.
4
6
3
6
i 1
6:45
P
.M.
8
3
3
3
* Each time the light intensity was changed, it was done immediately following
the preceding observation.
On continued exposure to the much higher light intensities of 150 and
600 f.c. (Table III), the majority of animals on the whole exhibited
a positive phototropism, although at times there were more animals in
the darker half of the tube and some of the animals apparently became
negative on prolonged exposure.
204
W. H. JOHNSON AND J. E. G. RAYMONT
It seemed desirable to determine whether the copepods would be
repelled by the still higher light intensity (11,380 f.c.) approximating
to that of bright sunlight. As a check on the results, other copepods
which had been collected at the same time were subjected to a much
lower intensity of 4 f.c. The results (Table IV) show that, at least
after a short exposure to this very high intensity, half of the animals
became negatively phototropic, while the others remained positive.
TABLE III
Responses to high light intensities
Source: 100-watt lamp. Intensity at i ft. : 600 f.c.
Intensity at 1 ft. : 150 f.c.
Time
Distance
(Positive)
II
ill
(Negative)
IV
Aug. 30 5:00 P.M.
1ft.
15
-
-
3
1ft.
14
—
2
3
5:30 P.M.
ift.
13
1
1
3
1 ft.
13
-
1
6
6:45 P.M.
I ft.
14
1
2
1
1 ft.
10
1
-
8
9:00 P.M.
I ft.
14
-
1
2
1 ft.
14
1
2
3
10:15 P.M.
ift.
14
2
2
-
1 ft.
10
-
1
8
Aug. 31 9:10 A.M.
ift.
5
1
2
10
1ft.
12
3
3
2
10:20 A.M.
ift.
8
1
3
7
1ft.
10
-
2
8
11:15 A.M.
ift.
11
2
1
4
1 ft.
9
-
2
8
12:15 P.M.
ift.
9
1
1
8
1 ft.
10
2
3
6
1:20 P.M.
ift.
12
2
-
4
1ft.
12
2
2
4
4:00 P.M.
ift.
12
2
-
4
1ft.
8
2
2
8
5:00 P.M.
ift.
12
1
3
3
1 ft.
11
2
—
7
It was rarely that all the animals displayed an invariable reaction
(either positive or negative) to any one condition of light. It was
possible then that some of the animals were negatively phototropic
even though the majority were positive; or again, perhaps some were
indifferent. To gain evidence on these points, observations were made
on individuals, one being sealed within a tube. At first, observations
were made for the most part once every hour, using three widely
separated intensities: 3.0, 150, and 600 f.c.
REACTIONS OF COPEPOD TO LIGHT AND GRAVITY
205
At the lowest intensity (3.0 f.c.), an individual remained photo-
positive for four hours, but appeared to become indifferent after
exposure overnight. A second individual was indifferent from the
first, and remained so for 15 hours. This behaviour was not modified
if the individual was left in darkness, and then exposed to the light.
TABLE IV
Source: 1000-watt lamp. Tube at 4J inches from source.
Control tube at 20 feet.
Ice added to aquarium to offset intense heat from source.
Time
Intensity
(Positive)
I
II
ill
(Negative)
IV
11:40 A.M.
11,380 f.c.
10
—
2
6
4 f.c.
8
4
1
6
11:55 A.M.
11, 380 f.c.
12
—
—
6
4 f.c.
11
2
—
5
12:05 P.M.
11, 380 f.c.
8
1
-
9
12:20 P.M.
11, 380 f.c.
10
_
4
6
4 f.c.
13
—
2
3
1:20 P.M.
11,380 f.c.
8
1
2
10
4 f.c.
11
2
2
3
1:35 P.M.
11,380 f.c.
9
1
3
8
1:45 P.M.
11,380 f.c.
8
1
3
8
4 f.c.
15
2
2
-
2:00 P.M.
11,380 f.c.
9
-
-
9
2:10 P.M.
11,380 f.c.
10
1
1
7
4 f.c.
14
2
1
1
2:25 P.M.
11, 380 f.c.
11
1
-
7
2:40 P.M.
11, 380 f.c.
8
1
—
11
4 f.c.
14
2
1
1
The responses of two individuals at an intensity of 150 f.c., and of
two others at 600 f.c. were such that one individual at each intensity
remained positive for 24 hours, while the other individuals were posi-
tive for the first 5 hours but apparently became indifferent after
exposure overnight.
More extensive experiments on individuals were carried out, mak-
ing observations every ten minutes, so long as it was possible to do so,
206 W. H. JOHNSON AND J. E. G RAYMONT
over a long period of time, and at a wide range of intensities (600,
150, 75, 67, 33, 13.5, 2.4, 0.87, 0.03, 0.006, and 0.002 f.c.). Of four
individuals (A, B, X, and F), specimens B and F were strongly and
constantly photopositive to all the above intensities; indeed, specimen
B was never recorded outside Section I. Individual A was in the main
attracted although less so at intensities above 75 f.c. Individual X,
although less consistent, was generally attracted by the light, but
occasionally at both high and low intensities it was found at the
negative end of the tube, even from the beginning of the experiment.
Having studied the effects of continued exposure to different
intensities, it was decided to determine the effect of changing light
intensity — a condition which is more like that which occurs in nature.
The changes in intensity were obtained by varying the position of the
source relative to the two experimental tubes. Thus the quality of
the light remained unchanged, and two experiments could be carried
on at once.
Successive experiments were carried out by moving the source
first 1 foot, then 2, 5, 10, and finally 20 feet every ten minutes (owing to
difficulties in counting, 15-minute intervals were sometimes unavoid-
able). The intensities ranged from 11,380 to 4 f.c. Before the
experiments were commenced, the tube at the maximum intensity
was left exposed to light until a considerable percentage of the animals
exhibited repulsion.
Regarding the one-foot changes: On increasing the intensity from
4 to 11,380 f.c., the animals remained continually attracted showing
always at least 80 per cent in the positive half of the tube. However,
after continued exposure for one hour at the highest intensity, only
40 per cent were still attracted. In the opposite tube, 55 per cent of
the copepods were repelled at the beginning when the intensity was
11,380 f.c., and it was necessary to decrease the intensity to 64 f.c.
before 80 per cent of the animals were attracted.
Considering the results of the 2 ft. changes, it was found that
essentially similar conclusions could be reached. In the increasing
intensity experiment, actually 100 per cent of the animals exhibited
constant positive phototropism. Decreasing the intensity resulted
in progressive attraction down to 16 f.c., when about 80 per cent of the
animals were in Sections I and II. Further decreases caused little
change.
The 5, 10 and 20-foot changes may be considered together. Re-
garding the increasing intensities, it is striking that none of the changes
had any effect on altering the original distribution of the animals. The
numbers of animals in each half of each tube remained almost perfectly
REACTIONS OF COPEPOD TO LIGHT AND GRAVITY 207
constant with the ten-minute intervals allowed, and it was only after
prolonged exposure (45 to 60 minutes) at 11,380 f.c. that repulsion was
brought about. Of the decreasing intensity experiments, in the 5-ft.
changes progressive attraction resulted in 80 per cent of the animals
being positive at an intensity of 7 f.c. Progressive attraction also
resulted in the other experiments, with 70 per cent of the animals being
attracted in the 10-ft. changes at the minimum intensity of 4 f.c.
(After one hour at 4 f.c., 80 per cent were positive.)
All these experiments on different magnitudes of decrease, each
occurring with 10-minute intervals, would seem to indicate that the
greater the magnitude of change, the lower the intensity at which a
large number of the copepods became positively phototropic. This
statement may be misinterpreted unless it be remembered that un-
doubtedly 80 per cent, or more, of the copepods would have migrated
to the positive half of the tube at much higher intensities had more
time been allowed before the next change was made. (There would
thus appear to be a "time-lag" effect.)
The above experiments show the effects of different magnitudes of
increase and decrease with a constant time interval of 10 minutes.
The percentage relationship between any one intensity and that which
immediately preceded it is not by any means constant during any one
succession of changes. Thus experiments were next conducted
similar to the foregoing, except that there was a constant percentage
increase or decrease throughout each series of changes. The actual
rates of change used were such as may occur in nature. (The values
chosen were the maximal changes observed by Clarke (1933) at one
station in the Gulf of Maine.)
Increases and decreases of 10 per cent per hour were first tried,
through a range of high intensities (11,380 to 2,840 f.c.), and then
through a low intensity range (9.5 to 6.2 f.c.). Considering first the
decreasing intensities, through the high range there was progressive
attraction, while through the low range there was practically no
alteration in the distribution. As regards the increasing intensities
experiment, there was little observable change, but, if anything, a
rather larger percentage of animals was attracted with time. The
same result was obtained with the low intensity range. Decreases
and increases of 20 per cent per hour, through both high and low
intensity ranges, gave similar results.
PHOTOTROPISM AND GEOTROPISM: EXPERIMENTS WITH TUBES
VERTICAL
Parker, Dice, Esterly, Clarke and others have demonstrated that
geotropism is frequently an important factor in the vertical migration
208 W. H. JOHNSON AND J. E. G. RAYMONT
of plankton. It seemed desirable, therefore, to carry out experiments
using vertical tubes to ascertain whether the light responses would be
different, and to test for the occurrence of a true geotropic reaction.
Methods
The aquaria were replaced by two large bell-jars held upright by
specially constructed wooden stands. The same experimental tubes
were used, but they stood vertically in all the following experiments.
A lamp was suspended over each tube, and, by means of a pulley
system, the distance between the lamp and the tube could be quickly
altered. The maximum distance thus obtainable was 4| feet. When-
ever it was desired to illuminate the animals from below, the tubes
were simply placed upright on an iron tripod, and the lamp placed
underneath.
Observations
It was decided to find the effects of various rates of change of light
intensity, and to compare the results with those obtained in the
horizontal experiments. Unfortunately the 1,000-watt lamp burned
out and as it was impossible to replace it in the short time remaining,
it was necessary to confine the indoor experiments to the lower light
intensities (0.67 to 240 f.c.). A wide variety of rates of change was
used: 25 per cent per hour, and 25, 50, 100, 300, and 700 per cent per
half-hour.
Considering the experiments on increasing light intensities the
following conclusions were reached. Within the range of intensities
used, it seemed that, in general, increasing the light at a variety of
rates does not bring about repulsion. One experiment, however,
using 25 per cent increases per half-hour, through a range from 7.4
to 19.1 f.c. did cause repulsion: — 70 per cent of the copepods were
attracted initially, but as the intensity increased, fewer remained
positive until only 16 per cent were attracted at 19.1 f.c. A large
number of other experiments, however, at intensities near 7.4 to 19.1
f.c. (also at higher and lower ranges, and at rates from 10 per cent to
several hundred per cent) was carried out, and in no other case was
this repulsion observed. In the great majority of cases the distribution
remained almost constant. It may be then, that this single case of
repulsion does not demonstrate the normal behaviour of these animals,
at least under laboratory conditions.
In the experiments on decreasing light intensities, with the excep-
tion of a single experiment, decrease in intensity at all rates, and
through all the ranges of intensity employed, resulted in more and more
of the animals swimming to the top of the tube as the light diminished.
REACTIONS OF COPEPOD TO LIGHT AND GRAVITY 209
This progressive attraction was sometimes very great. For example,
in two experiments only 25 per cent of the copepods were positive at
the beginning, and nearly 90 per cent at the end. The exceptional
experiment was the only one employing so low a rate of decrease as
10 per cent. It is possible that such changes are too slow to be per-
ceptible to the animals (below threshold).
It was thought desirable to determine the effect of increasing light
intensity, using direct sunlight, so that a very high intensity range
would be available. The experiment was conducted in the open behind
the Oceanographic Institution. An inverted bell-jar was used as in
the darkroom, with the experimental tube placed inside it, standing
vertically. Since it was here impossible to circulate cooled water
through the bell-jar, it was simply refilled with cold sea water whenever
the temperature rose. The stand holding the bell-jar was completely
TABLE V
Reactions to direct sunlight
Time
No. of
Opals
Relative
Sunlight
Approximate Int.
in Tube (f.c.)
(Top)
II
III
(Bottom)
IV
per cent
12 Noon
4
100
1,080
12:30 P.M.
4
100
1,080
11
1
4
3
1:00 P.M.
3
98
1,400
11
1
3
3
1:30 P.M.
2
93.6
2,000
12
1
1
4
2:00 P.M.
1
88
2,640
19
-
-
1
2:30 P.M.
0
79
9,470
11
2
1
5
3:00 P.M.
0
73
8,760
11
' -
1
5
3:30 P.M.
4
59
636
12
-
3
3
4:00 P.M.
2
43.6
935
5
2
6
4
4:45 P.M.
0
27
3,240
7
1
3
7
covered with black tar-paper. A small aperture cut in the top allowed
a beam of sunlight to fall on the top of the experimental tube. On
one side of the stand, the tar-paper formed a moveable flap which
could be lifted, and the necessary counts made. Four opal diffusing
discs were placed over the aperture to reduce the light; these were
removed at intervals. In the first experiment, they were removed one
at a time, in the second two at a time, and in the last experiment all
four were removed together. Each disc alone transmits 25 per cent
of the light falling upon it. The beam of sunlight was directed on to
the aperture above the tube by means of a simple plane mirror which
could be turned as the sun changed its elevation. The light intensity
was measured by means of a Weston Photronic Cell.
The results of the experiment (Table V) show that when the light
had increased from about 1,000 to about 9,000 f.c. over a period of two
210 W. H. JOHNSON AND J. E. G. RAYMONT
hours, the animals were at all times strongly photopositive. How-
ever, increases starting at lower intensities resulted in a majority of the
animals in the lower half of each tube. Is there also a negative geo-
tropism which becomes stronger with increase in light intensity?
Certainly the results indicate that mere rate and direction of change of
light alone cannot account completely for the movements of Centropages
typicus.
Thus experiments were next carried out in order to test the possi-
bility that the copepods might react to gravity, and that the above
results were only partially due to phototropic responses.
The experimental tubes were placed vertically in the bell-jars in the
normal way. The animals were then left in darkness, and counts
made later with the red lamp. For example, the tubes were left for
1^ hours in darkness and subsequent counts gave the following results
(Tubes A and B were treated identically to furnish checks on each
other) :
(Top) (Bottom)
I II III IV
Tube A 14 4 1
Tube B 64 8
The tubes were then reversed vertically end for end. After one-
half hour the following results were obtained :
(Top) (Bottom)
I II III IV
Tube A 12 5 3
TubeS 12 3 3
The tubes were again reversed. After one-half hour the following
results were obtained :
(Top) (Bottom)
I II III IV
Tube A 11 3 1 2
Tube B 15 1 3
The above results clearly show that the animals are on the whole
negatively geotropic in darkness. Careful observation showed that
the animals sink rapidly if they cease swimming. Hence actual
effort was necessary for them to remain at the tops of the tubes, and
the geotropism must then be quite strong.
The relation between geotropism and phototropism was then
tested by taking the above animals from darkness and illuminating
them from below, with the following results:
(Top) (Bottom)
I II III IV
Tube A (15-watt lamp below) 7 4 3 4
Tube B (100-watt lamp below) 1 - 19
REACTIONS OF COPEPOD TO LIGHT AND GRAVITY 211
The results would indicate that negative geotropism is stronger
than positive phototropism when the light is weak, while positive
phototropism is overwhelmingly strong when the intensity is high.
Tube B was returned to darkness and a count 15 minutes later
showed that the majority of animals were in Section I.
These results verified the negative geotropism. A 60-watt lamp
was then placed below the tubes and the following results obtained :
(Top) (Bottom)
I II III IV
Tube B (60-watt lamp below the tube) 1 19
Both tubes were again returned to darkness and a count 45 minutes
later again showed a large majority exhibiting negative geotropism.
A 25-watt lamp was then placed below the tubes:
(Top) (Bottom)
I II III IV
Tube B (25-watt lamp below the tube) 4 1 3 9
The experiment was repeated. The animals again showed negative
geotropism in darkness. With a 25-watt lamp below the tubes the
results were as follows:
(Top) (Bottom)
I II III IV
Tube B (25-watt lamp below the tube) 4 1 12
Finally it was decided to determine the effect of replacing a low light
intensity by a high one, when the geotropism and phototropism were
in opposition. It has been shown that after exhibiting negative
geotropism in darkness, on exposure to a 25-watt bulb from below the
distribution was:
(Top) (Bottom)
I II III IV
Tube B (25-watt lamp below) 4 1 12
This lamp was then replaced by a 100-watt lamp. A count after 15
minutes showed :
(Top) (Bottom)
I II III IV
Tube B (100-watt lamp below) - 17
All the above experiments definitely establish that the adult female
Centropages is primarily negatively geotropic and positively photo-
tropic. When the two are acting in opposition, the positive photo-
tropism becomes progressively stronger as the light intensity increases.
DISCUSSION
It is still a controversial matter how far laboratory experiments of
the type conducted are applicable to conditions in nature. Through-
out all the experiments, however, it was our aim to avoid "shock"
212 W. H. JOHNSON AND J. E. G. RAYMONT
conditions, and the use of surface tow-nettings avoided large changes
in light intensity during the collections.
It would seem from the experiments with artificial light, that adult
female Centropages typicus should be right at the surface during most
of the day, since they are strongly positively phototropic to a very
wide range of light intensities, and it does not seem that continual
decreases are always necessary to cause a majority to remain positive,
such as was found to be the case with Acartia clausi (Johnson, 1938).
However, repulsion does occur to some extent on prolonged exposure
to very high intensities, and also in the experiments using direct sun-
light (Table V) when the illumination increased through such ranges
of low intensities as may occur in the early morning. Hence, after
considerable exposure to strong sunlight (about midday in summer)
and possibly also when the light is increasing in the early morning,
Centropages might be expected to be a little lower in the water.
G. L. Clarke (1933) however, found that these copepods have a
maximum of about 13 m. during most of the day in the Gulf of Maine.
Some hauls made in August, 1935, near Woods Hole, were examined
and these in general confirmed this finding, although there were cases
when the majority were at the surface. (Clarke also did find, for two
stations, the majority at the surface.)
In considering this difference it must be remembered that there
are other factors acting in nature. Thus, especially at the surface,
turbulence may carry the copepods to somewhat lower depths.
Further, the possibility of muscular fatigue must not be overlooked.
As has been mentioned, Centropages will sink rapidly as soon as it
ceases swimming, and thus some will tend to sink below the surface,
though positively phototropic. This probably accounts for the
observation, that, although using the same intensities, a considerably
larger percentage of animals is found in the negative half of the tube
in the vertical experiments than in the horizontal ones. It should also
be noticed that Clarke did find a secondary maximum of Centropages
at the surface.
The rise to the surface at night, observed by Clarke and others,
is explainable since Centropages is always very strongly attracted
when the light intensity is diminished. The negative geotropism,
evident at least during and just after exposure to darkness, will aid the
rise.
Parker (1901) found that female Labidocera migrate surfacewards
at night due to positive phototropism and negative geotropism, and
Dice (1914) considered geotropism the major factor in the migration
of Daphnia. However, the recent findings of Kikuchi (1938) exemplify
REACTIONS OF COPEPOD TO LIGHT AND GRAVITY 213
the fact that the actual r61e played by geotropism probably varies from
species to species.
Since Centropages is positively phototropic to very low intensities,
the upward migration will presumably continue when the light is
exceedingly weak. Further, when the animals have reached the
surface, they will tend to remain there during darkness owing to the
negative geotropism, and they will not take up a more or less uniform
distribution, as Russell has supposed for some planktonic species.
As regards the downward migration in the morning, we were gen-
erally unable to demonstrate repulsion with increase in light intensity
using electric light in the laboratory. However, in the experiments
using direct sunlight, it was shown that increase in intensity at a low
intensity range from about 700 to 3,500 f.c., did cause repulsion, and
this range of light change might be expected in the early morning.
It is possible that exposure to darkness during the night might also
tend to render the animals more sensitive to light, but there is the
opposing geotropism to consider. This has been shown, however,
to be definitely weaker for average light intensities. Further experi-
ments, however, are desirable in this connection.
Although no experiments were conducted to test specifically Ester-
ly's theory of a diurnal rhythm (Esterly, 1917, 1919), it would seem
from an examination of our readings at different times of the day that
such a rhythm is absent in Centropages. Rose considers that a species
exhibiting diurnal vertical migration is adapted to a certain optimum
light intensity (Rose, 1925). Many investigators have been unable
to demonstrate such optima in the laboratory. Esterly, for example,
found Calanus negative to all intensities used, provided the tempera-
ture was above 10° C. Rose believed that if a wide range of intensities
was employed in the experiments, the optima would be demonstrable.
We therefore used a very wide range in our experiments, but did not
find any such optimum for Centropages.
Reversal of phototropic signs with absolute intensity of light was
also difficult to obtain, though Loeb, Parker, Rose, etc. have demon-
strated this for many planktonic species. It should be noted that
Clarke also found there was no evidence from his experiments for an
optimum light intensity in Daphnia. He also found that reversal of
phototropic sign could not be brought about by absolute light intensity
in this form. (Clarke, 1930 and 1932.)
Various authors have frequently pointed out the complexity of the
problem of vertical migration by showing differences in behaviour
between different species (e.g. Clarke, 1933), between the sexes of a
single species (e.g. Russell, 1928), and between ages of the same sex
214 W. H. JOHNSON AND J. E. G. RAYMONT
of one species (e.g. Gardiner, 1933). The observations of the authors
of the present paper further illustrate that although the majority of
adult female Centropages typicus do behave in a similar manner, varia-
tion in vertical distribution between individuals may be expected
even when they are of the same species, sex and age. This is in agree-
ment with field studies.
SUMMARY
Experiments on phototropism and geotropism in adult female
Centropages typicus were conducted. The following conclusions were
indicated :
A. Experiments with experimental tube horizontal.
1. The copepods are primarily photopositive and constant exposure
does not modify this reaction except at very high intensities in the
neighborhood of that of bright sunlight (ca. 12,000 foot-candles) when
a large number exhibited negative phototropism after continual
exposure for about an hour.
2. The lowest intensity at which there were always more copepods
in the brighter than the darker half of the tube was ca. 0.005 f.c.
3. There are two types of individuals. One type, after continuous
exposure to light, becomes indifferent. In the other type, the animals
are persistently attracted.
4. Decrease in light intensity, at a variety of rates and at a wide
range of intensities, always results in increased attraction.
5. Increase in light intensity, at a variety of rates and at a wide
range of intensities, has no effect on the behaviour. Only prolonged
exposure at high intensities repels the animals.
B. Experiments with experimental tube vertical.
1. With the light from above the animals stay mainly at the top
of the tube through a wide range of intensities, a distribution which is
probably the result of positive phototropism, negative geotropism, or
both.
2. Increases in intensity have no effect on the animals except when
sunlight is used. A fair percentage of the animals is then repelled.
3. With the exception of decreases as low as 10 per cent per hour,
decreases in intensity result in increased attraction.
4. The animals are strongly negatively geotropic in darkness.
When geotropism and phototropism are opposed, the reactions depend
upon the intensity of the light.
5. The possible bearing of these conclusions on the vertical distribu-
tion and diurnal vertical migration of adult female Centropages typicus
is discussed.
REACTIONS OF COPEPOD TO LIGHT AND GRAVITY 215
BIBLIOGRAPHY
CLARKE, G. L., 1930. Change of phototropic and geotropic signs in Daphnia
induced by changes of light intensity. Jour. Exper. Biol., 7: 109-131.
— , — . - — ., 1932. Quantitative aspects of the change of phototropic sign in
Daphnia. Jour. Exper. Biol., 9: 180-211.
CLARKE, G. L., 1933. Diurnal migrations of plankton in the Gulf of Maine and its
correlation with changes in submarine irradiation. Biol. Bull., 65: 402-
436.
DICE, L. R., 1914. The factors determining the vertical movements of Daphnia.
Jour. Animal Behavior, 4: 229-265.
ESTERLY, C. O., 1917. The occurrence of a rhythm in the geotropism of two species
of plankton copepods when certain recurring external conditions are
absent. Univ. Calif. Publ. Zool., 16: 393-400.
, — . — ., 1919. Reactions of various plankton animals with reference to their
diurnal migrations. Univ. Calif. Publ. Zool., 19: 1-83.
GARDINER, A. C., 1932. Vertical distribution in Calanus finmarchicus. Jour. Mar.
Biol. Ass'n., N.S., 18: 575-628.
JOHNSON, W. H., 1938. The effect of light on the vertical movements of Acartia
clausi (Giesbrecht). Biol. Bull., 75: 106-118.
KIKUCHI, K., 1938. Studies on the vertical distribution of the plankton Crustacea.
Records of Oceanogr. Works in Japan, 10: 17-41.
LOEB, J., 1908. Uber Heliotropismus und die periodischen Tiefenbewegungen
pelagischer Tiere. Biol. Zentral., 28: 732-736.
PARKER, G. H., 1901. The reactions of copepods to various stimuli and the bearing
of this on daily depth-migrations. Bull. U.S. Bur. Fish., 21: 103-123.
RUSSELL, F. S., 1928. The vertical distribution of marine macroplankton. VII.
Observations on the behavior of Calanus finmarchicus. Jour. Mar. Biol.
Ass'n., N.S., 15:429-454.
ROSE, M., 1925. Contribution a 1'etude de la biologie du plankton. Le probleme
des migrations verticales journalieres. Arch, de Zool. exper. et gen., 64:
387-542.
EMBRYONIC INDUCTION IN THE ASCIDIA
S. MERYL ROSE
(From the Department of Zoology, Columbia University, and the
Marine Biological Laboratory, Woods Hole, Massachusetts)
INTRODUCTION
The Ascidia are grouped with those animals whose early develop-
ment is termed mosaic. Yet, in the closely related Vertebrata, organs
form as the result of interaction between those cells which become the
definitive organ in question and neighboring cells, whose descendants
take no part in the actual formation of the organ.. The independent
differentiation of organs in the Ascidia and the dependent differentia-
tion of the same organs in the Vertebrata presents a problem.
It is believed that the Ascidia and the Vertebrata are descendants
of a common ancestor which contained organs similar to those now
common to both groups. The nerve cord, for example, in the two
groups is thought to be homologous. The common ancestor must have
had a nerve cord which arose either under the influence of inductors or
independently as a mosaic piece. It seems strange that in the course
of evolution the vertebrate nerve cord and the ascidian nerve cord
could have remained such similar embryonic structures, when their
modes of origin were diverging so greatly that the one now forms under
the influence of inductors and the other quite independently of induc-
tors. Possibly this difference in mode of development between the two
groups is more apparent than real.
The injury experiments of Conklin (1905&) established the fact
that in Styela partita surviving blastomeres do not deviate from their
prospective potency by regulating to form more morphological units
than they normally form as parts of a whole embryo. Conklin's
work further showed that differences in protoplasmic appearance and
cleavage peculiarities develop in the uninjured blastomeres just as they
would were the blastomeres part of an intact embryo. These differ-
ences are numerous enough to allow a careful observer to differentiate
the presumptive tissues and organs before the formation of the defini-
tive structures. This fact, I think, is the basis for the belief that the
ascidian egg is a mosaic of self-differentiating parts. The isolated
parts certainly self-differentiate into what are recognizably distinct
presumptive regions, but the question is whether any or all of these
isolated presumptive regions are capable of further self-differentiation
into embryonic organs.
216
EMBRYONIC INDUCTION IN ASCIDIA 217
The problem, then, is to determine whether or not inductive influ-
ences are present in the developing ascidian embryo, and, if they are
present, which cells release inductors and which structures develop in
dependent fashion. The answers have been sought with the aid of iso-
lation and transplantation techniques.
I wish to express deep gratitude to Dr. Barth for his valuable sug-
gestions during the course of this work.
MATERIALS AND METHODS
The animal chosen for these experiments was Styela partita. This
particular animal was used because its normal embryonic development
is comprehensively portrayed and because the mapping of presumptive
regions is complete (Conklin, 1905a). A further reason for using the
egg of Styela is that much of the experimental work on early ascidian
development with which the present work must be compared was done
on this egg.
Fertilized eggs were obtained in two ways. In the early part of this
work several animals were cut in two and the eggs and sperm removed
from the gonads and ducts in a pipette and mixed in sea water. Only
a small percentage of eggs was fertilized. These were recognized by
the cap of concentrated yellow pigment which forms after fertilization
and were sorted out for use. This method is laborious and leaves little
time before the first cleavage occurs in which to prepare the eggs for
operations. Fertilized eggs are obtained more easily and quickly from
spawning animals. Usually Styela spawn in the laboratory some time
between 4 and 7 P.M. However, they may be induced to spawn at
any time of day or night by subjecting them to light for eleven or
twelve hours preceding the desired time of spawning. The animals
were kept in running sea water aquaria where the light was controlled
with an opaque oil-cloth cover and an electric light. Bulbs of 40 and
150 watts placed directly over the tank and about eighteen inches from
the animals were found to be equally effective. As a rule the aquarium
was shaded with the oil-cloth during the afternoon and evening and the
light turned on at about 10 P.M. The animals then started to spawn
the next morning between nine and ten. The time between the
spawning of the first animal and the last varied from fifteen minutes to
several hours. During the longer spawning periods eggs were collected
for use several times. The same group of animals could be induced to
shed clouds of sperm and eggs on four or five successive days, by con-
trolling the illumination.
There is something released into the water by the spawning animals
218 S. MERYL ROSE
which induces others to spawn, provided that the latter have had almost
the necessary eleven or twelve hours of light. This knowledge was
used occasionally in causing the animals of one tank to spawn several
hours before the expected time by adding some water from another
tank in which spawning had ceased shortly before.
Eggs were carried in small pipettes through eight washes of pasteur-
ized sea water. The water had been heated to 70° C. and maintained
at that temperature for five or ten minutes. After cooling it was
aerated by shaking and used immediately or kept in the refrigerator
overnight until shortly before use the next day. The operating dishes
were 20 mm. Stender dishes. These were flamed with a Bunsen
burner each time before use and a hot 1.5 per cent agar solution in sea
water was permitted to cool and solidify upon the bottom of each dish.
The smooth agar surface prevented the eggs from adhering to the
glass. The operating solution was 0.4 per cent 0. IN HC1 in pasteurized
sea water, which changes the pH to approximately 7.6. The sea water
was slightly acidified because most of the eggs, after removal from the
membranes in ordinary sea water, cleaved abnormally and often
cleavage furrows disappeared although nuclear divisions continued,
very much as is the case when Arbacia eggs are treated with alkaline
or acid sea water (Smith and Clowes, 1924). Acid rather than base
was tried because Child (1927) had found more normal development of
Corella willmeriana embryos outside of the atrium when the CC>2
tension was increased. Child found the pH of the atrium to be ap-
proximately 7.4. In the acidified sea water injury from manipulation
was much less frequent. The eggs seemed more viable and, without
membranes, could develop into normal tadpoles not distinguishable
from those grown within the protective membranes. Pasteurized sea
water and semi-sterile precautions with operating dishes and instru-
ments were employed because survival with good differentiation was
increased from about 10 per cent to over 90 per cent by so doing.
Instruments were dipped in alcohol between operations and the pipette
shaft flamed each time after use.
Membranes were removed from the eggs in operating dishes with
the aid of fine watchmakers' forceps. The denuded eggs were then
transferred to other operating dishes in finely tipped pipettes. Blasto-
meres were separated with Spemann glass needles. Transplantations
were accomplished by simply bringing one cell or group of cells to rest
upon another group with which combination was desired. The cells
of the cleavage stages are quite sticky and adhere readily.
Embryos were fixed in Bouin's fluid. After fixation they were
transferred to a 1.2 per cent agar solution as it was cooling. After
solidification of the agar, blocks containing the embryos were cut out
EMBRYONIC INDUCTION IN ASCIDIA
219
and passed through the alcohols. The 95 per cent alcohol through
which the blocks were passed during dehydration contained some
water-soluble eosin. The blocks and the embryos were stained enough
so that they might be seen more easily during clearing and imbedding.
This is a modification of a technique employed by Dalcq (1932) for the
manipulation of ascidian embryos. Sections of seven micra were cut
and then stained a few minutes in Heidenhain's haematoxylin at
45° C. after a previous mordanting of twenty minutes in 4 per cent
ferric alum. Further staining for three minutes in 1 per cent light
green after treatment in 0.5 per cent phosphotungstic acid for five
minutes was sufficient to stain the yolk material. A 0.5 per cent solu-
TABLE I
Stage
Presumptive Value and Cell Lineage of Cells Used
in Experiments
16
43—
44.1—
45.1—
32
"46.1
46.2
45.2—
"46.3
46.4
c4.2—
"a5.3
a5.4
B3—
'54.1
M.2
Presumptive
Value
Endoderm
Notochord
and
Spinal cord
Endoderm
and
Mesenchyme
Notochord
and
Spinal cord
Cerebral
vesicle
and
Epidermis
Endoderm
and
Mesoderm
Epidermis
tion of eosin in slightly acidified 95 per cent alcohol counterstained
sufficiently in thirty seconds.
The photomicrographs were taken through an oil immersion lens.
I wish to thank Mr. J. Godrich for his part in the preparation of the
photographs and plates.
EXPERIMENTAL SECTION
In Table I the presumptive value and cell lineage of the cells used
in the experiments to be described are given. The presumptive value
and cell lineage were worked out by Conklin (1905o.). The relative
positions of the cells described in Table I may be seen in Figs. 1-4.
220
S. MERYL ROSE
In both the table and figures the cell notations are given for only one
side of the embryo, since the cleavage pattern is bilaterally symmetrical.
When reference is made to corresponding cells of both sides of the
embryo, the figure 2 is placed before the cell lineage notation. A
figure greater than 2 indicates that corresponding cells of more than
one embryo have been used.
PLATE I
Abbreviations: A, anterior; An animal pole; P, posterior; Veg, vegetal pole.
The cell lineage notations may be understood by referring to Table I.
FIG. 1. A vegetal view of a four-cell stage.
FIG. 2. A right side view of an eight-cell stage.
FIG. 3. A vegetal view of the eight vegetal cells of a sixteen-cell stage.
FIG. 4. A vegetal view of the sixteen vegetal cells of a thirty-two cell stage.
FIGS. 5-8. Surface views of anterior half, 2A3, embryos. Supernumerary
pigment spots are present in all and a bare notochord is shown in Fig. 6.
FIG. 9. A surface view of an anterior quarter embryo, 1.43.
FIG. 10. A surface view of an anterior vegetal quarter embryo, 2^14.1. A
notochord is present.
The early cleavages allow an experimental isolation of the pre-
sumptive regions and combinations of various regions in order that
the normal interactions may be ascertained.
Comparison of Anterior and Posterior Half Embryos
Separation of the yellow, B3, and gray cells, A3, in the four-cell
stage serves to test to what extent the two may differentiate inde-
EMBRYONIC INDUCTION IN ASCIDIA 221
pendently of each other. Both parts have been shown, by Chabry
(1887) in Ascidiella, and by Conklin (19056) in $tyela, to undergo
partial cleavage and to gastrulate. The anterior or gray cells may
form a notochord, neural structures including the pigmented sensory
cells (otolith and eye-spot), and endoderm which in some cases becomes
arranged in the form of a gut with lumen. Figure 13 is a drawing of a
section of an anterior half embryo showing the above-mentioned
features. The high degree of differentiation of the anterior partial
embryos is in agreement with the results of Chabry and Conklin.
A peculiarity shown by approximately half of these anterior half
embryos, grown from 2^13 cells, is the presence of more than the
normal two sensory spots. Figures 5-8 show surface sketches of four
such embryos. Figure 9 shows an anterior quarter embryo grown from
1^43. The greatest number of pigment spots observed in the anterior
half embryos was nine. Often pigment formed in cells widely sepa-
rated, sometimes on opposite sides of the embryo. In many of the
anterior embryos the neural plate did not fold over to form a cerebral
vesicle, but, instead, remained on the surface of the embryo. This
was usually the case when supernumerary sensory spots were formed.
Figures 15 and 16 are adjoining sections of a 2^43 embryo which has an
infolded embryonic nervous system. One of the pigment spots is ex-
ternal and three are internal. One of the internal sensory cells was
cut in such a way as to be included in both sections. It is readily seen
that more pigment is produced by anterior half embryos than would
be produced by such cells when part of a whole embryo. The presence
of extra sensory cells has also been observed in unoperated embryos.
Here, however, their occurrence is rare, and never more than four have
been seen in one embryo. The phenomenon of supernumerary pigment
spots will be further discussed below.
Contrasted with the rather complete differentiation of the anterior
half embryo is the unorganized condition of the posterior half. Gastru-
lation occurs and the embryos survive past the time when the controls
become swimming tadpoles, but the presumptive muscle cells remain
large and almost round, never elongating nor taking on the fibrous
appearance of muscle cells. Figure 11 is a drawing of a section of a
posterior half embryo showing the absence of differentiation. Chabry
(1887) cultured posterior half embryos of Ascidiella and found poorer
development of posterior than of anterior halves. No mention was
made of muscles. Conklin (1905a, p. 52, footnote), employing the
convention of calling a cell a "muscle cell" if, in normal development,
it would give rise to nothing but muscle, designated these undifferen-
tiated cells of the partial embryos, muscle cells. Since this work is
222
S. MERYL ROSE
II
12
13
PLATE II
Abbreviations: £5, eye-spot; Ep, epidermis; E, endoderm; G, gut; M, mesoderm;
N, neural tissue; No, notochord; 0, otolith.
FIG. 11. A drawing of a section of a posterior half embryo, 2B3, containing
epidermis, mesoderm and endoderm.
FIG. 12. A drawing of a section of a posterior vegetal plus anterior animal
embryo, 254. 1 + 2a4.2, containing epidermis, mesoderm and endoderm.
FIG. 13. A drawing of a section of an anterior half embryo, 2.43, containing
epidermis, gut, notochord and neural tissue with otolith and eye-spot.
EMBRYONIC INDUCTION IN ASCIDIA
concerned with the problem of differentiation, such cells are considered
as presumptive, and the term "muscle cell" is reserved for those cells
which attain the stage of differentiation found in contractile tissue and
acquire myonbrillae.
The isolation and study of twenty-seven anterior and posterior
embryos have shown that the gray cells, the A3, contain within them-
selves the ability to self-differentiate, whereas the yellow cells, the B3,
lack something which would enable them to differentiate.
Animal and Vegetal Embryos
It is possible to observe the development of presumptive epidermal
and cerebral vesicle cells isolated from mesodermal and endodermal
cells by separating the animal from the vegetal blastomeres in the
eight-cell stage. In forty-five such cases there was never evidence of
neural differentiation in either the animal or vegetal half. Nothing
like a neural tube formed, nor did sensory cells develop. The picture
one obtains from sections of partial embryos of the animal region is
one of undifferentiated cells showing no cerebral vesicle (Figs. 17-19).
Instead of a row of epidermal cells surrounding a vesicle of neural
tissue bearing pigment spots in two of the cells, the isolated animal
embryos show nothing but a group of closely packed similar cells
usually arranged about a cavity. This cavity formed between the
dividing cells before the time of formation of neural tissue at the time
when control embryos were gastrulae. Some of the animal embryos
have a wrinkled appearance and instead of a single cavity, contain
several.
Tung (1934), performing the same operation in Ascidiella scabm,
obtained animal embryos, some of which he believed contained neural
tissue. These embryos showed folds or depressions, the cells of which
stained more heavily with eosin than did the other cells, or contained
a few cells grouped together making a small tube. Since the cerebral
vesicle in normal embryos stains more readily with eosin than do the
other tissues, and since neural structures arise through a folding proc-
ess, Tung thought his animal embryos possessed neural tissue. The
stain criterion may be reasonably doubted. Tung shows that the
presumptive neural cells in the gastrula stage are already eosinophil.
At this time the cells are undifferentiated. It seems inadvisable,
therefore, to use the eosinophil nature of the cells as a criterion of
neural differentiation.
Conklin (19056) also recognized neural tissue in isolated anterior
animal cells, but used different criteria. His criteria were that the
cells in question in the living condition were very clear cells, as are the
224 S. MERYL ROSE
neural plate cells of a whole embryo, and, further, that their cell lineage
and size were the same as the neural plate cells of a whole embryo.
These criteria of neural differentiation seem valid for only the very
beginning of differentiation of neural cells. A better criterion would be
the formation of a structure more like the normal cerebral vesicle, a
vesicle bearing an otolith or an eye-spot. Such has never been recorded
from isolated animal cells. Never in past work (Chabry, Conklin,
Tung), nor in the present work have sensory structures been seen to
develop in isolated animal cells. It seems, then, that there must be
some factor extrinsic to the presumptive brain cells which enables them
to differentiate.
Isolation of the vegetal quartet of blastomeres, 2^44.1 -f- 254.1, in
the eight-cell stage should test whether vegetal cells are able to self-
differentiate. Few vegetal half embryos survived until the time when
differentiated structures might be expected. The great majority con-
tinued to cleave until gastrulation time. Then the embryos became
loosely adhering masses of cells which soon disintegrated. One, how-
ever, remained intact long enough to produce a differentiated noto-
chord. The vegetal embryos of Ascidiella produced by Tung (1934)
show a higher degree of differentiation.
Notochordal cells have also been seen to form in quarter embryos
derived from the anterior vegetal cells alone, the 2^44.1. Figure 9 is
a surface view of such an embryo showing the bare notochord.
Abbreviations: A, cup of animal cells; M, myofibrillae; No, notochord; Ot,
otolith; V, plug of vegetal cells.
FIG. 14. A section through the cerebral vesicle of an unoperated tadpole,
showing the size of the larger pigment spot, the otolith.
FIG. 15. A section through the pigment spot region of an anterior half embryo,
2.43.
FIG. 16. An adjoining section of the same embryo shown in Fig. 15.
FIG. 17. A section through an animal half embryo, 2a4.2 -+- 264.2, showing
several cavities.
FIG. 18. A section through an animal half embryo, showing the unorganized
nature of the embryo.
FIG. 19. A section through an anterior animal quarter embryo, 2a4.2, showing
epidermal vesicle formation.
FIG. 20. A section of a 2/14.1 + 264.2 embryo through the induced cerebral
vesicle and otolith.
FIG. 21. A section through a 2.44.1 + 264.2 embryo, showing the induced
pigment spot.
FIG. 22. A section through a 2a4.2 + 1^15.2 embryo. The otolith is attached
to the cup of cells which arose from the 2a4.2. Inserted in the concavity of the cup
may be seen the plug of cells derived from the .45.2 cell.
PIG. 23. The section passes through a cerebral vesicle containing a typical
otolith in a 2a4.2 + 2.45.1 combination.
FIG. 24. A tail section of an unoperated embryo. The central notochord is
flanked by the rows of dark myofibrillae.
PIG. 25. A tail section of a posterior three-quarter embryo, showing the noto-
chord flanked by rows of dark myofibrillae.
15
19
20
I
Ot,
vt
4
^
21
ot
V
22
23
PLATE III
226 S. MERYL ROSE
The isolation experiments seem to indicate that factors or inductors
necessary for the differentiation of other parts of the embryo are
located in the anterior vegetal region. The evidence for this is the ab-
sence of differentiation in embryos which lack this region and the higher
degree of differentiation of embryos which contain the anterior vegetal
material.
Transplantations
A more striking and positive demonstration of an inductor is ob-
tained when the inductor region in combination with cells incapable of
self-differentiation causes those cells to form a structure which neither
the inducing nor the reacting cells would form in normal development.
Combinations of 2^44.1 + 2M.2 from the eight-cell stage have led to
the development of partial embryos possessing cerebral vesicles and
sensory cells. Figures 20 and 21 are sections of two such embryos
through the otolith region. The number of ,44.1 -f &4.2 combinations
which produced embryos containing pigmented sensory cells was fifteen
out of forty-six. In these embryos presumptive epidermis cells have
replaced presumptive cerebral vesicle cells, and, in combination with
inductor, have formed cerebral vesicles and the pigmented sensory cells.
An attempt to determine the extent of the cerebral vesicle inductor
in the anterior vegetal quadrant has been made. The ^45.1 and the
A5.2 cells of the sixteen-cell stage and the A6A and ,46.3 and the A6.2
and ,46.4 cells of the thirty-two cell stage have been combined with
animal cells of the eight-cell stage. Both the .45.1 cells and the A5.2
cells have induced cerebral vesicles and sensory cells. Figure 23
shows a well-formed otolith in a cerebral vesicle. This embryo arose
from a 2a4.2 + 2,45.1 combination. Figure 22 is a section through a
2a4.2 + 1A5.2 embryo. It is of interest because it shows that the
otolith formed from an animal cell which was in direct contact with
,45.2 derivatives. The animal cells are seen in the form of a cup with
a plug of vegetal cells protruding from the concavity of the cup. A
few combinations were made in which one member of the pair was
stained with Nile Blue Sulphate. In five instances gastrulation was
incomplete and in these the sensory cells formed on the surface at the
boundary between the stained and unstained portions. The proximity
of the inducing vegetal cells and the reacting animal cells suggests a
direct transfer of inducing substance from the vegetal to the animal
cells.
The extent of the inductor in the thirty-two cell stage is less clear.
One thirty-two cell embryo from which the 2,46.2 + 2,46.4 cells were
EMBRYONIC INDUCTION IN ASCIDIA 227
removed produced a sensory pigment cell. This was the only operation
of this type performed. In this embryo the only anterior vegetal cells
present were derivatives of the 2A6.1 and 2.46.3 cells, presumptive for
endoderm and mesenchyme. The presumptive notochord and spinal
cord cells were removed. A few other operations testing for the
presence of neural inductor in the .46.1 and ^46.3 derivatives were per-
formed late in the operating season during September when sensory
pigment was not forming in the operated embryos, or even in a number
of the control embryos. The combination was .46.1 + ^46.3 + 2a4.2.
Of four successful combinations, two showed evidence of neural in-
vagination. One of these two contained a solid internal rod of neural
type cells. Of four embryos resulting from ^46.2 + ^46.4 + 2a4.2
combinations, none showed any evidence of neural invagination. The
negative cases are so few here and the criteria of neural differentiation
so limited that our knowledge of the neural inducing ability of the
^46.2 and .46.4 cells remains uncertain. The ^46.1 and -46. 3 cells in
the whole embryo give rise to the endodermal cells which directly
underlie the cerebral vesicle. It is probably they, in normal develop-
ment, which induce the cerebral vesicle.
Twenty combinations of 254.1 -f- 2a4.2 gave no evidence of neural
differentiation (Fig. 12). The embryos are very similar in appearance
to posterior half embryos, 2B3 (Fig. 11). This result indicates that
the neural inductor is limited to the anterior vegetal region and does
not spread over the entire vegetal region.
The extrinsic factors functioning in muscle differentiation will be
described in a future paper. At present, it may be said that the pre-
sumptive muscle cells, when they are part of a posterior half embryo, do
not self-differentiate (Fig. 11). Neither do they differentiate when
combined with anterior animal material, 254.1 + 2a4.2 (Fig. 12).
Functional tail muscles do form, however, in posterior three-quarter
embryos. The operations were performed in the thirty-two cell stage
when the 2.45.1 + 2a5.3 cells were removed, leaving the 2a5.4 and
2y45.2 cells in combination with the posterior half of the embryo.
Figure 11 is a photograph of a section of an unoperated embryo's tail,
and Fig. 12 is a similar section of a tail of a posterior three-quarter
embryo. Myofibrillae may be seen in both sections.
Potency to Respond to Cerebral Inductor
The relative potency to respond to the cerebral inductor has been
found to differ in various parts of the embryo. Table II is a summary
of the data. The normal number of sensory pigmented cells found in
the cerebral vesicle of Styela is two. Rarely four appear. Blasto-
228
S. MERYL ROSE
mere combinations which are predominantly of anterior materials
regularly produce sensory cells. These cells, which form in normal
development in the brain, may be considered to be evidence of the
presence of neural differentiation, even though in many cases a neural
tube has not formed. The number of sensory cells which developed
TABLE II
Reaction to Cerebral Inductor
Combination
Presumptive Value
No. with
Sensory
Cells
No. without
Sensory
Cells
No. of
Sensory
Cells
2.43
Ep, CV, Not, SC, G,
22
2
1-9
Mes.
2o4.2+L45.ri
2a4.2 + 2/45.1J
Ep, CV, Not, SC, G.
2
4
0
0
1-4
1-4
2a4.2 + 2/15.2l
2fl4.2 + L45.2J
Ep, CV, Not, SC, G,
Mes.
5
1
1
0
1-4
1
2a4.2+264.2 + 2/45.ll
2a4.2+264.2+L45.1J
Ep, CV, Not, SC, G.
2
1
3
0
1-2
2
2a4.2 + 264.2 + 2/45.2\
2a4.2+264.2+L45.2J
Ep, CV, Not, SC, G,
Mes.
3
1
1
2
1^
2
2/13 + 154.1
Ep, CV, Not, SC, G,
Mes, Mus.
3
2
2-3
164.2+244.1"
3
0
1-2
264.2 + 2,44.1
7
35
1-2
264.2 + 1/44.1 -
Ep, Not, SC, G, Mes.
3
6
1-2
464.2+2,44.1
1
0
2
564.2+244.L
1
0
1
264.2 + 1,45.1)
264.2 + 2/15. 1J
Ep, Not, SC, G.
0
0
1
3
-
264.2 + 1,45.21
0
1
—
264.2+2/15.2
Ep, Not, SC, G, Mes.
0
3
-
164.2 + 2,45.2]
1
0
1
254.1 + 264.2 + 1/14.1
Ep, Not, SC, G, Mes,
Mus.
0
8
—
CV, cerebral vesicle; Ep, epidermis; G, gut; Mes, mesenchyme; Mus, muscle;
Not, notochord; SC, spinal cord.
in the anterior embryos was often greater than two and the amount of
pigment was greater than in whole tadpoles. The numbers of pigment
cells range from one to nine, the average being 3.8 for the anterior half
embryos. The a4.2 + .45.1 or .45.2 combinations also regularly pro-
duce neural tissue. Supernumerary sensory cells may also appear in
EMBRYONIC INDUCTION IN ASCIDIA 229
these embryos. The sensory cell production in a4.2 + ^45.1 or ^45.2
embryos may be contrasted with that of embryos whose animal ma-
terial comes from the posterior region, 64.2 -f- .45.1 or ^45.2. The a4.2
material has responded positively in twelve of thirteen cases, whereas
the 64.2 material gave a negative response in eight of nine cases. This
comparison is of embryos from the same batches of eggs. The response
of posterior animal cells is somewhat better when the inductor cells are
the .44.1 cells of the eight-cell stage. In this case both .45.1 and ^45.2
materials are represented. The positive responses with 64.2 + .44.1
were fifteen of fifty-six. This is in spite of the fact that most of the
64.2 + ^44.1 operations were performed before the introduction of the
semi-sterile technique.
Not only do anterior animal cells respond more often than posterior
animal cells to the same inductor, but also the anterior cells produce
more sensory structures. Never have the posterior animal cells pro-
duced more than two sensory cells. The number is usually one.
A further result obtained from the transplantation experiments is
that the addition of posterior cells to combinations which alone would
produce neural material decreases the frequency of its appearance.
When the a4.2 cells alone were in combination with .45.1 or .45.2,
twelve of thirteen embryos contained sensory cells. When 64.2 + a4.2
cells were host to .45.1 or ^45.2, only seven of thirteen produced
sensory cells. Similarly, when the 254.1 material was added to a
264.2 + 1^44.1 combination, there were no sensory cells produced in
eight cases. Alone the 264.2 + 1^44.1 combination had been shown
to form sensory cells in three of nine cases.
Although in some of the individual experiments the cases are too
few, the combined data seem to allow the following conclusions: (1)
Anterior animal cells have greater potency to form cerebral structures
than do posterior animal cells. (2) Posterior cells tend to inhibit the
formation of sensory structures in embryos containing competent
materials.
DISCUSSION
The classical works of Conklin (1905&, 19056) on Styela demon-
strated that early in development there is a segregation of ooplasmic
materials. These visible cytoplasmic materials are correlated in
normal development with particular embryonic organs or regions.
However, some of these substances may be centrifugally displaced and
come to lie in foreign organs (Conklin, 1931). In a sense, the segre-
gation of visible ooplasmic materials is differentiation. Further,
230 S. MERYL ROSE
isolated blastomeres differentiate in respect to cleavage patterns.
But differentiation also includes the establishment of the various
functional structures. The present work indicates that the anterior
vegetal region is necessary for this latter type of differentiation.
The earlier idea that the ascidian egg is a strict mosaic has been
altered in recent years. Schmidt (1931) has found that lateral half
embryos of dona intestinalis and Phallusia mammillata may sometimes
form the normal three adhesive papillae. Cohen and Berrill (1936)
obtained some rather normal appearing larvae from lateral half em-
bryos of Ascidiella aspersa. They, however, interpreted the regulation
as a mechanical regulation of an original mosaic pattern. Recently,
von Ubisch (1938) has described a case in which two fused two-cell
embryos of Ascidiella aspersa regulated to form a single individual.
Dalcq (1932, 1938) has shown that lateral, or animal, or vegetal
portions may be removed from the egg before fertilization without
resulting depletion of organs in the larvae which develop from the egg.
Reverberi (1931) obtained larvae very similar to normal larvae from
fragments of fertilized Ciona eggs. The results of Dalcq and Reverberi
plainly show that the egg is not a determined mosaic before completion
of the first cleavage.
Tung (1934) suggested the possibility that adhesive papillae and
sensory cells might be dependent upon extrinsic factors, since they did
not form in the isolated presumptive cells. The present work indi-
cates that induction of organs is more general in the ascidian embryo.
It appears that all cells outside of the anterior vegetal region differen-
tiate dependently. This anterior vegetal region, presumptive for
notochord, spinal cord, endoderm and some mesoderm, is similar in
function to the corresponding region of the amphibian embryo, the
organizer region. It is capable of self-differentiation and supplies
necessary developmental factors to other regions. The great difference
between amphibian dorsal embryos (Ruud, 1925) and ascidian anterior
embryos is that the former regulate and form more than they would as
parts of intact embryos, while the latter offer no evidence of regulation.
The recent work of Reverberi (1937) demonstrates that both animal
and vegetal materials must be present in egg fragments of Ciona
intestinalis in order that the sensory cells may differentiate. Rever-
beri's work and the present work suggest a possible interpretation.
There are fundamental regional differences in the egg. Materials
necessary for the differentiation of endoderm and notochord and for
the production of inducing substances are in highest concentration in
the anterior vegetal region. Materials which react with the cerebral
inducing substances, or materials which produce the reacting sub-
EMBRYONIC INDUCTION IN ASCIDIA 231
stances, are more concentrated in the animal region, especially the
anterior animal region. The contiguity of original animal and vegetal
regions established during gastrulation enables the interaction of
anterior vegetal inducing substance or substances and the reacting
animal material, which process leads to the differentiation of cerebral
vesicle.
CONCLUSIONS
1. Blastomeres from the animal region of the eight-cell stage are
incapable of self-differentiation.
2. Posterior blastomeres of the four-cell stage are also unable to
self-differentiate.
3. The anterior vegetal blastomeres of the eight-cell stage are
capable of self-differentiation.
4. The anterior vegetal region is necessary for the differentiation of
other regions.
5. The cerebral inductor is confined to the anterior vegetal region.
6. Presumptive epidermis may form brain under the influence of
the inductor.
7. Anterior animal cells have greater potency to form cerebral
structures than do posterior animal cells.
8. Posterior cells inhibit the formation of cerebral structures in
embryos containing competent materials.
LITERATURE CITED
CHABRY, L., 1887. Contribution a 1'Embryologie Normale et Teratologique des
Ascidies Simples. Jour, de I'Anat. et Physiol., 23: 167.
CHILD, C. M., 1927. Developmental modification and elimination of the larval
stage in the ascidian, Corella willmeriana. Jour. Morph., 44: 467.
COHEN, A., AND N. J. BERRILL, 1936. The development of isolated blastomeres of
the ascidian egg. Jour. Exper. Zool., 74: 91.
CONKLIN, E. G., 1905o. The organization and cell lineage of the ascidian egg.
Jour. Acad. Nat. Sci., Philadelphia, 13: 1.
CONKLIN, E. G., 1905i. Mosaic development in ascidian eggs. Jour. Exper. Zool.,
2: 145.
CONKLIN, E. G., 1931. The development of centrifuged eggs of ascidians. Jour.
Exper. Zool., 60: 1.
DALCQ, A., 1932. Etudes des Localisation Germinales dans 1'Oeuf Vierge d'Ascidie
par des Experiences de Merogonie. Arch, d'anat. Micros., 28: 223.
DALCQ, A., 1938. Etude micrographique et quantitative de la Merogonie double
chez Ascidiella scabra. Arch, de Biol., 49: 397.
REVERBERI, G., 1931. Studi sperimentali sull 'uovo di Ascidie. PuU. Staz. Zool.
Napoli, 11: 168.
REVERBERI, G., 1937. Richerche sperimentali sulla struttura dell'uovo fecondato
delle ascidie. Commentationes Pontif. Acad. Scient., 1: 135.
232
S. MERYL ROSE
RUUD, G., 1925. Die Entwicklung isolierter Keimfragmente frtihester Stadien
von Triton taeniatus. Roux' Arch., 105: 209.
SCHMIDT, G. A., 1931. Die Entwicklung der Palpen bei Ascidienhalbeilarven.
Arch. Zool. Hal. (Torino), 16: 490.
SMITH, HOMER W., AND G. H. A. CLOWES, 1924. The influence of hydrogen ion
concentration on the development of normally fertilized Arbacia and
Asterias eggs. Biol. Bull., 47: 323.
TUNG, Ti-CHOW, 1934. Recherches sur les potentialites des Blastomeres chez
Ascidiella scabra. Arch, d'anat. Micros., 30: 381.
VON UBISCH, L., 1938. Uber Keimschmelzungen an Ascidiella aspersa. Roux'
Arch., 138: 18.
ANDROGENETIC DEVELOPMENT OF THE EGG
OF RANA PIPIENS1
K. R. PORTER
(From the Biological Laboratories, Harvard University and the
Department of Biology, Princeton University)
INTRODUCTION
The aim of the investigator in seeking to initiate androgenetic
development is to remove or inactivate the female pronucleus, at the
same time leaving undisturbed the male pronucleus (if it is within the
egg), the cytoplasm, and conditions essential for activation and first
cleavage. To achieve this, especially by mechanical means, it is
important that the egg be large, that the position of the egg chromatin
be detectable, and that development proceed under laboratory condi-
tions. It is, therefore, not surprising that the amphibian egg has been
generally used.
G. Hertwig, in 1911, treated the eggs of Rana fusca with radium
emanations, then fertilized them, and obtained androgenetic develop-
ment for what appears to be the first time. Since then a variety of
methods have been used to remove or inactivate the egg nucleus (see
below). These have been applied to various European species of frogs
(G. Hertwig, 1911; P. Hertwig, 1923; Dalcq, 1932) and toads (G.
Hertwig, 1913; P. Hertwig, 1923), to various species of Triton (P.
Hertwig, 1916, 1923; G. Hertwig, 1927; Curry, 1931, 1936; Baltzer,
1933; Baltzer and de Roche, 1936; Hadorn, 1934) and to one American
species, Triturus viridescens (Kaylor, 1937).
None of these experiments has produced an adult haploid. In
general, with androgenetic haploids as with haploids produced by
parthenogenesis, gynogenesis and merogany, development ceases after
a few days or in some cases a few weeks, is always abnormal, and where
it continues to the larval stages produces an animal which is inactive
and edematous.
Despite their abnormalities, these haploids offer numerous possi-
bilities for the study of nucleo-cytoplasmic relationships. Indeed,
the abnormalities in themselves are not without interest, for an experi-
1 Part of data previously presented in thesis submitted to the faculty of Harvard
University in partial fulfilment of the requirements for the degree of Doctor of
Philosophy, June, 1938; part of data from experiments performed during tenure of
National Research Fellowship at Princeton University.
233
234 K. R. PORTER
mental demonstration of their cause should throw considerable light
on the problems of differentiation. To be most serviceable as an
experimental material, it seems essential that the haploids and the
methods by which they are produced should possess certain positive
characteristics. Their development should be fairly normal and con-
tinue to an advanced stage of differentiation; the peculiarities of
haploid development should be uniformly displayed by all animals;
the haploid nuclear condition should remain unchanged; and the
operative technique should be simple, effective, and capable of pro-
ducing relatively large numbers. Haploids produced from eggs of
various species of amphibia and by a variety of methods have satisfied
these criteria to varying degrees, in no case perfectly. In view of this
fact it is important to experiment further with new materials and
methods.
The report which follows presents the results of such experiments.
An effective technique for the removal of the egg chromatin from the
egg of Rana pipiens is described; the development which results from
these operated eggs is described and compared with the normal diploid ;
it is shown that the great majority of these animals develop as haploids;
and certain cytological observations are presented which are of possible
importance in explaining the abnormalities of haploid development.
I should like to express my sincere gratitude to Professor Leigh
Hoadley for his aid and advice during early investigations of this
material. I am also indebted to Professor G. Fankhauser for valued
suggestions in more recent studies.
MATERIALS AND METHODS
The eggs of the frog, Rana pipiens, secured from the state of
Vermont were used in these experiments. Ovulation was induced
by injecting water extracts of the anterior lobe of the frog pituitary
following in general the method described by Rugh (1934). Such
eggs when inseminated usually give a high percentage of fertilization
and since the development which follows is perfectly normal there is
little reason for considering the eggs so obtained as inadequate for
experimental purposes.
The operation, which results in the removal of the maternal
chromatin, is simple and effective. Since it is in part original to these
investigations and since its successful application depends on an under-
standing of events taking place within the egg, a rather complete
description follows.
At the time of insemination the egg of R. pipiens has undergone
the first maturation division and the second division is in metaphase
ANDROGENETIC DEVELOPMENT OF FROG EGG
235
awaiting the entrance of the sperm before continuing in the production
of the second polar body and the female pronucleus. Sections through
the egg in this stage of maturation reveal the relation of the spindle to
the egg surface. (Fig. 1). It is to be noted that it lies close to the
surface and is almost completely covered over by pigment granules.
As the second maturation division proceeds this relationship is altered.
z.
. :•;#:'.!••!#• . '•. ' %V\ \ ::: .• • •
:;ln; "#•
^^ <p0\Mr:'£
' <r2>v" *
4-.
FIGS. 1-4. Semi-diagrammatic representations of four stages in second polar
body formation of R. pipiens eggs. Drawings were made with camera lucida and
give exact distribution of pigment granules, yolk platelets and chromosomes, only
part of which are shown. Selected from considerable material sectioned at lOju.
(Eggs inseminated and kept at 12° C.) 1125 X.
Fig. 1. Division spindle as in egg at time of insemination.
Fig. 2. Anaphase of maturation division. Stage at which spindle can be seen
from exterior of egg as small black dot. Egg fixed 35 minutes after insemination.
Fig. 3. Early telophase. Egg fixed 50 minutes after insemination.
Fig. 4. Polar body just forming. Egg fixed 56 minutes after insemination.
Between 20 and 35 minutes after insemination the metaphase gives
way to anaphase and the pigment granules directly over the division
figure become widely dispersed (Fig. 2). If, at this time, the egg is
observed from the exterior under strong illumination and a magnifica-
tion of more than 25 or 30 diameters the location of the maturation
spindle can be detected as a small black dot. This appearance is
236
K. R. PORTER
doubtless due to the absence of light-reflecting pigment granules over
the spindle (Figs. 2 and 3). Many of these so-called black dots have
been watched and in all cases they have been observed to disappear
gradually (between 35 and 45 minutes after insemination) and to be
replaced by the small second polar body (Figs. 3 and 4).
The removal of the egg chromatin is accomplished by means of a
glass needle possessing a very fine but rigid point. While the location
of the maturation spindle is apparent the point of the needle is inserted
through the jelly capsule and into the cortex of the egg to one side of
and diagonally beneath the spindle (Fig. 5). A slight upward motion
of the needle then produces a small exovate which contains the spindle
and consequently all of the maternal chromosomes (Fig. 6). When
5.
6.
FIG. 5. Diagram of operative procedure, n, needle; ms, maturation spindle;
sn, sperm nucleus; iwz.vitelline membrane; jc, jelly capsule; ec, egg cytoplasm.
FIG. 6. Diagram of egg and exovate after operation, ex, exovate; others as
in Fig. 5.
the operation is performed slowly and carefully the small pellucid
spindle can occasionally be seen in the yolky cytoplasm which comes
out with the needle. Thus the egg is left otherwise intact with only
the male chromatin present to influence the development which
follows. The exovate which forms outside the vitelline membrane
is soon completely detached from the egg and generally no mark
remains on the embryo to mark the place of exovate origin and former
attachment.
The usefulness and value of such an operation are in part deter-
mined by the ease with which it can be executed and therefore the
ANDROGENETIC DEVELOPMENT OF FROG EGG 237
number of eggs which can be treated in a short length of time. Within
the 10 to 15 minutes during which the maturation spindles on a group
of eggs are apparent it is possible to operate on 25 or 30 eggs and
exercise considerable care in so doing. If the eggs are inseminated
in small quantities and at 15-minute intervals this number can be
increased several times and sufficient material is made available for
quantitative studies of a physiological as well as morphological
character.
The loss of the small amount of egg cytoplasm which forms the
exovate appears to have no harmful effect upon later development.
Evidence for this statement is drawn from the following sources: (a)
Experiments have been performed in which small exovates were pro-
duced on eggs in the immediate vicinity of, but not including, the
maturation spindle. These developed normally as far as could be
observed from external appearances and certainly displayed none of
the abnormalities characteristic of haploid embryos. (6) Occasionally
a normal appearing embryo arises from an operated egg (possibly as
result of unsuccessful operation). In two cases these have been
allowed to develop and have ultimately metamorphosed without
showing any notable deficiencies. Therefore, it seems justifiable to
conclude that the abnormal characteristics of the animals which result
from these operated eggs are due to an altered nucleus rather than to
an altered cytoplasm.
Various other methods have been applied to amphibian eggs to
bring about androgenesis. The egg chromatin has been rendered
inactive by radium emanations and x-rays (G. Hertwig, 1911, 1913,
1927; P. Hertwig, 1916, 1923; Dalcq, 1929, 1932), it has been removed
by pricking the egg of R. esculenta with heated and unheated needles
(Dalcq, 1932), and it has been destroyed with a needle and then with-
drawn by a micropipette (Curry, 1931, 1936; Baltzer, 1933; Baltzer
and deRoche, 1936; Hadorn, 1934; Kaylor, 1937).
A comparative evaluation of these various methods should be made
only by one who has tried them all. Furthermore, for different eggs,
different operations may be required. For example, with the egg of
Triturus viridescens it is necessary to use a micropipette to remove
the egg chromatin for an exovate is not formed by merely pricking the
egg. Therefore, whatever may be the merits or drawbacks of these
various methods, it is necessary in any evaluation to consider them in
conjunction with the egg to which they are applied.
Further comment should be given to Dalcq's method of pricking
the egg with heated and unheated needles. It is similar to the tech-
nique applied in these experiments to the egg of R. pipiens but from his
description it does not appear that he observed the exact location of
238 K. R. PORTER
the maturation spindle. Instead, he pricked the egg in the lighter
region in the centre of the animal pole where the maturation figure is
generally, but not always, located. That he did not always remove
the egg chromatin, as he himself suggests, is further indicated by the
presence of 5 diploid embryos in a group of 22 which developed from
operated eggs.
In all experiments to be reported, experimental animals and controls
were from the same female, were inseminated simultaneously, were
kept under identical conditions of temperature (generally constant to
± 0.05° C.), volume of water per animal, water change, etc. For
fixing, a corrosive sublimate, acetic, formalin mixture was generally
used. This has been found to be especially valuable for the younger,
yolky stages for it has little hardening effect. Harris haematoxylin
has been found most serviceable as a general stain. With it the nuclei
stain a deep blue and the yolk granules remain a purple, thus permitting
some degree of differentiation.
OBSERVATIONS
The Development of Androgenetic TLmbryos
The description of androgenetic development wrhich follows is
taken from observations on several groups of experimental animals.
Developmental rates and illustrations (Figs. 7-22), however, refer
to one particular group (Exp. 38-1) numbering 52 experimental
animals which were raised at 19.4° C. From this group and one other,
experimental animals were selected and fixed at 24-hour intervals as
recorded in Tables II and III. Controls were simultaneously pre-
served. In this way material was provided for an examination of
internal as well as external morphogenesis. While some variation is
shown among the members of a single group, especially in the older
stages, there is a majority which show the general features described
below.
Observations were normally begun at the time of first cleavage.
This may take place anywhere between 2 and 3 hours after insemina-
tion depending on the temperature at which the eggs have been in-
seminated. It is customary for between 90 per cent and 100 per cent
of the operated eggs to divide normally and to do so simultaneously
with the control eggs (Table I). This behavior, while typical for
these androgenetic frog eggs, is not typical for all amphibian eggs.
For example, Kaylor (1937) reports that a considerable proportion of
his androgenetic Triturus viridescens eggs cleaved abnormally or failed
to cleave at all and that 90 per cent of those for which cleavage records
were available showed a significant delay in the appearance of the first
cleavage furrow.
ANDROGENETIC DEVELOPMENT OF FROG EGG
239
Blastula development of androgenetic R. pipiens embryos is quite
normal. As the time for gastrulation approaches slight indentations
occasionally appear in the animal hemisphere of the blastula. Since
these later disappear, do not occur in all of the experimental embryos,
and have been noted in the controls, they are not considered a typical
abnormality of androgenetic development. Comparative examination
of androgenetic and control late blastulae reveals noticeably smaller
cells in the former. This observation made from the outside has been
verified from sections. Though cell counts have not as yet been
made, it seems probable that there are more cells in the androgenetic
blastulae and that they have resulted from a more rapid rate of cell
division.
Gastrulation begins approximately one hour earlier in the controls
than in the experimental animals. This constitutes the first clear
TABLE I
Record of first cleavage in several lots of operated eggs.
Exp. Number
Number of Eggs
Operated
Number that Cleaved
Percentage Cleavage
36-5
38
38
100
36-10
179
179
100
36-13
278
255
92
37-2
38
38
100
37-4
70
69
98
38-1
52
52
100
Note: Experiments 36-10, 37-2, 37-4, and 38-1, provided the data upon which
this report is based.
indication of a retardation of differentiation. This delay is a distinct
characteristic of amphibian androgenetic development and has been
reported by other investigators for a variety of species. It is more
clearly indicated at the end of 24 hours by a difference in the size of the
crescentic blastopore (smaller in the androgenetic embryos). In the
great majority of the experimental animals gastrulation proceeds
normally and by the end of 48 hours the yolk plug stage is reached
(Figs. 7 and 8). The larger yolk plug of the androgenetic embryo
provides evidence of retarded development.
During the formation of the neural tube on the third day it be-
comes clearly evident that androgenetic development is not simply
normal development, slightly delayed, but is abnormal as well as
delayed. For instance, the neural plate of the experimental animal
remains approximately one-third shorter than the neural plate of the
control; the neural folds stand up less prominently from the body of<
:o
240
K. R. PORTER
8
10
14
15
16
18
20
-
21
22
FIGS. 7-22. Photographs of typical androgenetic embryos and normal diploid
controls from a group of operated eggs (Exp. 38-1) raised at 19.4° C. Figs. 7,9, 11,
13, 15, 17, 19, 21 are respectively 2-, 3-, 4-, 5-, 6-, 8-, 10-, 12-day-old androgenetic
haploids. Figs. 8, 10, 12, 14, 16, 18, 20, and 22 are respectively 2-, 3-, 4-, 5-, 6-, 8-,
10-, 12-day-old controls. Photographs are of fixed animals, ca 5X.
ANDROGENETIC DEVELOPMENT OF FROG EGG 241
the embryo, and the neural groove is more shallow. There are
probably indiscernible abnormalities in gastrulation which contribute
to the above and in turn to the more pronounced departures from
the normal shown by the older animals. Closure of the neural folds
is completed between 2 and 3 hours later than in the controls which
indicates an increasing delay in differentiation.
The 3-day-old experimental animal depicts abnormal as well as
delayed differentiation (Figs. 9 and 10). The tail-bud is shorter, the
abdomen remains abnormally large and round, and the head is smaller
and apparently less differentiated. From the third day on develop-
ment is characteristic of the androgenetic embryos only, and exact
stages for stage comparisons with the controls are no longer possible.
The typical 4-day-old experimental animal is smaller than the
control, shows a pronounced bend in the back, a shorter and round
abdomen, and a head which does not show the normal downward
bend or cranial flexure (Figs. 11 and 12). The first indications of gill
filaments which appear at this time in the control do not appear in the
androgenetic animals until almost a day later.
Certain of these abnormalities persist on the fifth day and are
clearly shown in Fig. 13. The 5-day-old control possesses a pulsating
heart and a complete gill circulation whereas the experimental animals
do not clearly show these features until the end of the sixth day.
In the typical 6-day-old experimental animal (Fig. 15) the back
has straightened but in total length the animal is still considerably
shorter than the control. It is of interest to note that the head of this
animal (Fig. 15) more closely resembles that of the 5-day-old control
(Fig. 14) than it does the 6-day-old (Fig. 16). But even in this simi-
larity there are discrepancies as indicated by the position of the
olfactory pit. Generally more than one-half of the androgenetic
embryos of this age show a pulsating heart and of these fully one-
third can be expected to have a fairly normal gill circulation.
It is typical for a few of the 7-day-old animals to become edematous
and with each day thereafter the number of edematous animals in-
creases. This condition may become so extreme that not only the
body cavity but also the tissue spaces in the head become filled with
fluids (Fig. 21). When this extreme is reached death generally
ensues. Therefore, if the animals are to be saved, fixing agents are
applied. In the group of animals from which this description is
illustrated most of the animals were fixed on the ninth and tenth
days (Table II).
During the eighth day the operculum grows over whatever gill
filaments the animal may happen to have. This operculum develop-
ment is outstanding in that it takes place at the same time and rate
242 K. R. PORTER
as in the controls whereas other organs may be more than 24 hours
delayed.
TABLE II
A record of fixation and examination for chromosome numbers of animals which
developed from 52 operated eggs. (Temp. 19.4° C.)
Age at Time of Fixation Number Fixed Classification
1 day 3 3 haploids
2 days 4 2 haploids
1 normal diploid
1 abnormal diploid
3 days 5 3 typical haploids
2 atypical haploids
4 days 2 2 typical haploids
5 days 1 1 haploid
6 days 3 1 typical haploid
2 atypical haploids
7 days 2 2 typical haploids
8 days 1 1 haploid
9 days 11 11 edematous haploids, 5 of which show a few
diploid nuclei
10 days 8 8 edematous haploids. 7 of which show a few
diploid nuclei
1 1 days 3 3 edematous haploids, 2 of which show a few
diploid nuclei
12 days 6 4 edematous haploids showing a few diploid
nuclei
1 edematous haploid with several diploid
nuclei
1 diploid-triploid, developed more successfully
than the typical haploid
15 days 1 1 haploid-diploid, haploid on one side, diploid
on other side.
22 days 2 1 pure diploid of normal structure
1 triploid, appearance of normal diploid
Summary: 46 haploids, 3 diploids, 1 triploid, 1 haploid-diploid, 1 diploid-triploid;
89 per cent of population haploids.
After the eighth day there is slight change in the gross appearance
of the experimental animals except that the majority become in-
creasingly edematous (Figs. 19 and 21). Differentiation of some
ANDROGENETIC DEVELOPMENT OF FROG EGG 243
parts continues but a discussion of such differentiation is not essential
to this general description. It should be mentioned, however, that in
those cases where a circulation is established, at least for a short
time, differentiation is more successful and the animal lives over a
greater number of days.
The behavior of these animals can scarcely be called normal.
Most of the time they are rather inactive and lie on their sides on the
TABLE III
A record of fixation and examination for chromosome numbers of animals
which developed from 38 operated eggs. (Temp. 20.1° C.)
Age at Time of Fixation Number Fixed Classification
2 days 2 2 typical haploids
3 days 1 1 typical haploid
4 days 2 1 typical haploid
1 slightly atypical haploid
5 days 2 2 typical haploids
6 days 1 1 typical haploid
7 days 10 1 typical haploid
8 edematous haploids
1 very atypical haploid
8 days 4 4 edematous haploids
9 days 11 10 edematous haploids, 5 of which show a few
diploid nuclei
1 accidentally destroyed had developed as
haploid
10 days 5 1 edematous haploid
2 edematous haploids showing a few diploid
nuclei
1 haploid-tetraploid (Fig. 32)
1 died before fixation, had developed as
haploid
Summary: 37 haploids, 1 haploid-tetraploid; 97 per cent of population haploids.
bottom of the container. When sufficiently stimulated, however,
they will respond by swimming about in undirected circles.
Chromosome Numbers and Nuclear and Cell Size
That the embryos which develop from operated eggs are haploids
has been indicated, not only by the rather certain removal of the second
polar spindle, but also by the abnormalities which they show. For
further evidence, however, a cytological examination was made of
some part or parts of each animal of two different groups of operated
244 K. R. PORTER
eggs. For the younger animals this evidence was obtained from
sections; for the older animals, from tail tips clipped from fixed speci-
mens and made into whole mounts. In the case of each animal one
or more metaphase plates were examined in detail to establish the
chromosome number, and, in addition to this, a record was kept of the
total number of division figures which could be identified as haploid
or otherwise by brief examination only. In general, the quality of the
preparations permitted the examination of 25 or more (in some cases
many more) mitotic figures. The results of these studies are sum-
marized in Tables II and III and additional evidence is shown in Figs.
23 to 27.
It is clearly shown that the vast majority of these operated eggs
developed as haploids. For the exceptions there is at the present time
no definite explanation. There always remains the possibility that
they resulted from unsuccessful operations whereby the egg chromatin
remained within the egg. But even if this is the explanation, the
results indicate that at its worst the method is about 90 per cent
effective. The animals which did not develop as haploids were easily
detected for they showed either the characteristics of normal diploids
or other characteristics not typical for haploids.
It is of interest to compare these results with those reported by
Dalcq (1932) for androgenesis with the egg of R. esculenta and by
Parmenter (1933) for parthenogenesis with the egg of R. pipiens and
R. palustris. Out of 22 operated eggs in Dalcq's experiments 5
developed as diploids; out of 29 embryos which developed partheno-
genetically Parmenter reports 10 pure diploids. These results would
lead one to expect a larger number of diploids among these andro-
genetic R. pipiens embryos than have been found. In the case of
Dalcq's results, however, the high percentage may be due to a poor
localization of pricking and not to any marked instability of the frog
haploid nucleus. But failure of operative technique could scarcely
account for the large percentage of parthenogentic diploids. Several
explanations, which are reviewed by Parmenter, have been suggested.
It is possible that a study of very early cleavage stages will provide
an explanation for this difference between the results of partheno-
genetic and androgenetic experiments.
It has been noted (Tables II and III) that the tail tips of some of
the older haploids show a few diploid nuclei. These were identified
by their larger size and by the presence of two nucleoli (Fig. 32).
Since they occur solely within the tissues of haploids which have more
or less reached the end of their development, it would seem that some
condition or conditions within these animals are related to their origin.
But as to the mechanism of their origin, there is only slight evidence.
ANDROGENETIC DEVELOPMENT OF FROG EGG
245
In a very few cases monastral divisions of haploid nuclei have been
observed. The presence of scattered diploid nuclei in the older
stages is not a feature confined solely to these androgenetic haploids.
23
24 27
FIGS. 23-27. Camera lucida drawings of mitotic figures. 3250 X.
Fig. 23. Diploid metaphase from tail epithelium of 15-day-old control.
Fig. 24. Triploid metaphase from tail epithelium of 22-day-old triploid animal
which developed from an operated egg (Table II). Shows 36 chromosomes (triploid
39).
Fig. 25. Haploid metaphase from 1-day-old androgenetic haploid in early stages
of gastrulation.
Fig. 26. Haploid late prophase from tail epithelium of 7-day-old androgenetic
haploid.
Fig. 27. Haploid metaphase from cell in tail mesoderm of 10-day-old andro-
genetic haploid.
•
Dalcq discovered the same in his preparations and Parmenter located
a few diploid divisions in the tissues of some of his older animals which
were otherwise predominantly haploid.
246
K. R. PORTER
30
31
FIGS. 28-32. Camera lucida drawings of cells and nuclei from tail epithelia.
750 X.
Fig. 28. From 9-day-old diploid control.
Fig. 29. From 9-day-old androgenetic haploid.
Fig. 30. From 9-day-old androgenetic haploid; shows 3 haploid nuclei in one
large cell.
Fig. 31. From 10-day-old androgenetic embryo showing large tetraploid nuclei
and cells which predominate epithelium on one side of tail (Table III).
Fig 32. From 10-day-old androgenetic haploid showing diploid nucleus and
cell among haploid nuclei and cells.
ANDROGENETIC DEVELOPMENT OF FROG EGG 247
The nuclear and cell size in haploids has been repeatedly shown to
be smaller than in diploids and to this rule these androgenetic frog
haploids are no exceptions (Figs. 28 and 29). Observations on haploids
and diploids of all ages reveal that this relationship holds whether the
observed animals are one day or several days old. It has also been
noted that with an increase in chromosome number to triploid and
tetraploid there is a corresponding increase in nuclear and cell size
(Fig. 31).
There is a tendency in these haploids for several nuclei (as many
as seven have been counted) to occupy a single cell. With this
increase in number of nuclei, as with an increase in chromosome num-
ber, there is a corresponding increase in cell size (Fig. 30).
The Extent and Uniformity of Development
Studies of groups of androgenetic embryos involving the fixation
of representative types at regular intervals do not indicate accurately
the extent or uniformity which might be displayed by a total popula-
tion of such animals. A simple demonstration of these qualities was
obtained by allowing each member of a given population of 40 animals
to proceed as far as possible in its development. These animals were
kept in separate containers under uniform conditions (temperature
constant at 20.1° C.). While they were ultimately killed by fixing
agents, the same were not applied until the indications were very defi-
nite that life would not continue for many hours. The graph presented
in Fig. 33 summarizes the data of this experiment. As can readily be
seen, up until the fifth day all but two of the original animals were
living. From this time until the end of the eighth day there was only
a slight change. At this time, however, it was necessary to preserve
a large number of them because of their extreme edema. After this
pronounced drop the decline is more gradual until the eleventh day
after which only one animal remained alive. This one continued to
live for several weeks, but, as was expected, it proved to be part
haploid and part diploid. The other 39 animals were considered as
haploids on the basis of the development which they displayed.
Since these animals were killed by artificial means it seemed ad-
visable to examine some data from earlier groups of androgenetic
embryos which had been raised at temperatures averaging 20° C.
and in which death was caused by natural agents rather than fixing
agents. The data are summarized in Fig. 34 and it is clearly evident
that there was a sharp increase in the mortality rate after eight days
just as depicted in Fig. 33. Hence the first graph (Fig. 33) can be
considered as a correct representation of the survival value of a
248
K. R. PORTER
population of androgenetic R. pipiens haploids raised at a temperature
of 20° C.
It should not be concluded from these results that androgenetic
frog embryos are incapable of further development than that expressed
by a 10 or 12-day-old animal raised at 20° C. It is certainly true that
the vast majority never go beyond this stage, but the occasional
animal will continue longer and while showing abnormalities and a
slower rate of growth, it will nevertheless take food and live over
several weeks or months. Two animals of the group described in
Fig. 34 lived for five weeks and another, which developed from an
1- 6 8 10 II
AGE. IN DAYS
8 10
IN DAYS
16 - 24
FIG. 33. Graph depicting survival of population of 40 androgenetic haploids
which were fixed when it was judged that they could not survive many hours.
FIG. 34. Graph depicting survival of 3 different populations of androgenetic
haploids which were allowed to die of natural causes.
operated egg in more recent experiments, lived for sixteen weeks.
It developed into a sizeable tadpole with small hind limbs. Cyto-
logical examination of the tail epidermis has revealed that it was pre-
dominantly haploid.
The uniformity of a group of androgenetic embryos cannot, un-
fortunately, be measured by any known unit but must be left entirely
to the judgment of the investigator. The fact that the majority of
the animals live for eight days, suggests that early development is
quite normal and uniform from animal to animal. If, on the other
hand, a few animals had died each day and in all stages from cleavage
to tadpole, the development could be referred to as un-uniform. The
ANDROGENETIC DEVELOPMENT OF FROG EGG 249
individual animals of the group, the survival of which is described in
Fig. 33, were examined every day throughout the duration of the
experiment and by means of this examination were compared with
one another and with one of the group selected as type. From this
study the uniformity can be described as follows: until the end of the
third day it was practically perfect, from the third to the fifth day it
was fair and from the fifth day on it was rather poor, with differences
becoming more pronounced. In other words, as the complexity of
structure increased the uniformity of the population decreased.
It is difficult to compare the success (extent and uniformity) of
androgenetic development displayed by these R. pipiens with the same
development of other species. Investigators have used ages rather
than stages to describe their results and in using such a unit as days-
development, temperature variations become important. Among
species of frogs, the androgenetic development described by G. Hertwig
(1911) for R. fusca and by Dalcq (1932) for R. esculenta is no more
successful than that reported here for R. pipiens. In fact, as far as
uniformity is concerned, the results with R. pipiens seem to be better.
This may be due to the method of operation rather than the species
of egg. It has been stated that toad haploids develop better than
haploids from the larger frog eggs and that Triton haploids develop
better than the anurans (P. Hertwig, 1923). A comparative study of
amphibian haploidy made at the present time might produce cause to
qualify this statement. Until the haploid development of a greater
variety of amphibian eggs has been studied it will be impossible to
determine whether it is the species of egg, the egg size, the method
of initiating haploid development or some combination of these or
other factors that makes for greater success in some cases than in
others.
Internal Morphology and Development
The typical experimental animals fixed at various ages as recorded
in Tables II and III have been sectioned. The description which
follows is based on an examination of these sections.
Observations on internal morphogenesis support those on external
in showing that development is delayed and abnormal. These facts
can be illustrated by an examination of eye development in 3-, 4-, and
5-day-old haploids and controls (Figs. 35-40). In the 3-day-old
diploid (Fig. 35) the optic vesicles have extended to the head ectoderm
and are in a position to induce lens formation. In the haploid (Fig. 36)
the vesicles are smaller, have scarcely reached the head ectoderm and
therefore show delayed development. By the end of four days, the
control (Fig. 37) shows a well-formed optic cup and lens whereas the
haploid (Fig. 38) has advanced only slightly beyond the stage repre-
250 K. R. PORTER
sented by the 3-day-old control and shows only the beginning of lens
formation. The 5-day-old haploid (Fig. 40) compared with the control
of the same age (Fig. 39) shows an optic cup which is decidedly ab-
normal. Its dorsal half and the lens are quite similar to the same
structures in the 4-day-old control (Fig. 37), but the ventro-lateral lips
of the cup fail to grow out leaving a wide choroid fissure. It looks as
if the optic stalk in failing to elongate had held in the ventral portion
of the cup. Later development does not make up this deficiency in the
optic cup, and by a continued proliferation of cells in the retinal layer
the structure becomes increasingly abnormal. Only rarely is develop-
ment more nearly normal. Thus it is observed that while development
makes a fairly normal beginning as shown by the vesicle of the 3-day-
old, the results as indicated by the 5-day-old and older stages are
quite abnormal.
The following survey presents some further outstanding features of
haploid internal morphology and morphogenesis as observed from sec-
tions of the older stages. They represent observations on the typical
haploid.
Nervous System. — An examination of the anterior central nervous
system reveals in the oldest haploids a poorly developed brain. In
many cases the ventricles are almost entirely obliterated by a marked
proliferation of cells or nuclei and a resulting thickening of the brain
walls. This condition continues to the posterior end of the medulla.
The spinal cord, on the other hand, displays a persisting neurocoele
and in the caudal regions is a relatively normal structure. The nuclei
are more numerous than in the diploid and in the sections of the older
haploids they give way to a vacuolar type of picnosis. The fibre tracts
are always indefinite in limitations and have nuclei scattered through
them in an abnormal fashion.
The eye develops abnormally as indicated above. Lenses are ab-
sent in many cases and when present are considerably smaller than
normal.
FIGS. 35-40. Photomicrographs of sections through optic vesicles and optic
cups of haploids and controls, aged 3, 4 and 5 days. 38 X.
Fig. 35. From 3-day-old control.
Fig. 36. From 3-day-old haploid.
Fig. 37. From 4-day-old control.
Fig. 38. From 4-day-old haploid.
Fig. 39. From 5-day-old control.
Fig. 40. From 5-day-old haploid.
FIGS. 41 AND 42. Sections through the same region of the medulla of 5-day-old
control (Fig. 41) and androgenetic (Fig. 42) embryos. Yolk granules are very darkly
stained inclusions. 160 X.
FIGS. 43 AND 44. Sections through the same muscle in the pharyngeal region of
7-day-old control (Fig. 43) and androgenetic (Fig. 44) embryos. 160 X.
ANDROGENETIC DEVELOPMENT OF FROG EGG
251
44
FIGURES 35-40.
252 K. R. PORTER
The otocyst, unlike the optic cup, differentiates at more nearly the
normal rate, but does so abnormally. Instead of one vesicle being at
first formed, several develop within the mass of cells which originally
arises from the head ectoderm.
The Notochord. — -In striking contrast with the nervous system, the
notochord is among the best developed and differentiated structures in
the androgenetic larvae. By the end of the third day it is well formed
and displays a cross-sectional area approximately the same as that of
the controls. This same relative size generally persists and when the
cells become vacuolated they tend to be smaller and therefore more
numerous than in the diploid. Whether or not the more successful
differentiation of this structure is related to its early histogenesis is a
question of some interest.
The Pronephric Kidney. — This appears slightly later than in the
controls and shows fair development. The nephrostomes open into the
body cavity and though some difficulty is encountered in tracing the
course of the convoluted tubules, they appear to connect with the
common duct. This latter is patent and has been traced to an open
cloaca in edematous as well as in the more normal androgenetic larvae.
This has its interest in that an incomplete lumen in the pronephric
duct has been used to explain the edema common to these haploids
(Dalcq, 1932). It is evident that such could not be the cause in all
cases. The convolutions of the androgenetic kidney are less extensive
than in the control kidney of the same age, which suggests a delay in
elongation of the tubules. This earlier kidney is vascularized though
generally to no avail as the circulation is seldom functional. Evidence
for this latter fact often exists in the form of abnormal accumulations
of blood cells around the tubules.
Other mesodermal derivatives such as the somites and visceral
arches show fair though delayed differentiation. The somites tend to
be smaller in cross-sectional area and to be underdeveloped in the thin
dorsal extensions lateral to the nerve cord. The muscle cells are
smaller and less compactly grouped.
The Circulatory System. — The circulatory system is functional in
very few cases though the heart beats in many. The differentiation of
the heart is considerably delayed and is generally about 24 hours behind
the control in showing its first pulsations. The larger vessels can be
located and traced, but the development of capillary connectives is
doubtful. This latter failure is suggested by the patches of blood cells
which accumulate in various regions of the body not normally asso-
ciated with blood formation. Only in the occasional haploid can a
good capillary circulation be located in any part of the body. The
ANDROGENETIC DEVELOPMENT OF FROG EGG 253
blood cells are generally less numerous, are smaller, and contain more
yolk granules. They often contain 2 or 3 nuclei after the yolk platelets
have disappeared.
The Gut. — The gut is markedly retarded in its differentiation.
This is most emphatically shown by the fact that in a 9-day-old
edematous haploid the gut appears as an almost straight tube whereas
in the control of the same age it is considerably coiled. The walls of
this short gut are thicker and the cells are packed with yolk. The
derivatives of the gut likewise differentiate rather tardily. For
example, the lungs, arising from the fore-gut, are in about the same
stage of development on the ninth day as they were on the seventh
day in the controls. This 2-day delay in differentiation is, however,
not common to the whole animal.
The Ectoblast. — The ectoblast in its differentiation more closely
parallels the controls than any other part of the embryo. Oral suckers,
olfactory pits, mouth parts and operculum all differentiate quite
normally and at approximately the normal time. The ectoderm, at
first wrinkled and thicker than in the controls, becomes thinner as the
animal becomes edematous. Tumor-like proliferations of the ectoderm
occasionally appear, and are not unlike those shown by frog embryos
treated with weak solutions of 2,4-dinitrophenol (Dawson, 1938), or
with high temperatures (Hoadley, 1937), or developed from over-ripe
eggs (Witschi, 1930).
Yolk Supply
Only a brief examination of the sections of these haploids was neces-
sary to show that yolk disappears more slowly from the cells of the
haploid than from the diploid. Since it was felt that considerable im-
portance could be attached to this observation studies of yolk content
were made along with studies of morphology. These are considered
but the beginning of future studies which may throw some light on the
causes of haploid deformities.
Until the haploids and controls are 4 days old (20° C.) the yolk
content of the cells in all regions of the embryo is so great that micro-
scopic comparisons are without value. In animals varying from 4 to 7
days a comparative examination of the same organs in haploids and
diploids of the same age reveals a greater quantity of yolk in the cells of
the haploid (Figs. 41 to 44). Within these age limits this difference
holds for all tissues of the embryos though it is more apparent in some
than in others. In haploids older than 7 days the yolk supply of some
tissues (ectoderm of 8- and 9-day-olds) is completely exhausted while in
others (the gut) it is still possible to observe a greater quantity in the
cells of the haploid. It can be noted further that differentiation seems
254 K. R. PORTER
to be more delayed in regions most richly supplied with yolk. Further
observations, and if possible measurements of yolk content, are neces-
sary before it can be stated that the cells of a haploid tissue do not ap-
proach a normal stage of differentiation until their yolk supply has been
reduced to the normal extent. It is hoped that future experiments may
clear up this matter and provide a basis for definite conclusions.
DISCUSSION
The results of these experiments indicate that the operation by
which the maturation spindle is removed from the egg is successful.
Approximately 90 per cent of the operated eggs develop as haploids,
the haploid nucleus being that of the sperm. It has been shown that
under the influence of this nucleus development proceeds for 8-10 days
(20° C.) and produces a tadpole showing considerable differentiation.
This is abnormal, however, and only future experiments on other eggs
will indicate whether more normal haploid development is possible
among the Salientia. The uniformity displayed by these populations
of haploid embryos has been described as good over the first 3 days
and fair from the third to the fifth days. This degree of uniformity
appears to be a distinctive feature of this material for it is not clear
that similar results have been previously obtained with other eggs
and methods.
It is shown, therefore, that haploid embryos of suitable quality
are made available in sufficient numbers for physiological studies and
measurements. The abnormalities which they demonstrate occur in
sufficient uniformity to make the study of their cause attractive and
possibly productive. And, from another angle, they become particu-
larly valuable as a material for hybridization experiments involving
the mixing of the cytoplasm of one species with the nucleus of another.
The subject of special interest in connection with this report is the
abnormal retarded development and reduced viability of these hap-
loids, which, it is clear, must be related to the presence of only the
haploid chromosome complement. Recessive genes, lethal or other-
wise, unsuppressed by dominant allels would, if present in the sperm
nucleus, find definite expression in these haploids. It seems hardly
probable, however, that these would occur with such regularity within
the male chromosomes as to produce, for example, a similar reduction
in the length of the neural plate in almost every haploid in a population
of 40 experimental animals. It is more logical to associate such a
departure from the normal with the presence of a haploid nucleus within
a quantity of cytoplasm normally associated with a diploid nucleus.
Several hypotheses have been proposed to account for these
haploid abnormalities and Fankhauser (1937) finds in them a common
ANDROGENETIC DEVELOPMENT OF FROG EGG 255
idea: a disturbance of the metabolism of the haploid cells. As to the
nature of this disturbance there is no clear understanding, but it is
presumably due to a supply of yolk and cytoplasm excessive for the
haploid nucleus. There is some evidence in support of this hypothesis
in the results of these investigations. It has been noted that the yolk
supply disappears more slowly from the cells of the haploid than from
the diploid. It has also been noted that differentiation is delayed and
abnormal and that the delay appears to be more pronounced in tissues
containing the greatest amount of yolk. From these observations it
is not unreasonable to link excess yolk with delayed and abnormal
differentiation. Additional supporting evidence comes from experi-
ments on merogonic development. In the production of merogonic
haploids the quantity of cytoplasm is more or less reduced and the
normal karyoplasmic ratio tends to be restored. One such fragment
of a Triton taeneatus egg developed through metamorphosis and consti-
tutes the most successful case of amphibian haploidy on record
(Baltzer, 1922; Fankhauser, 1938). Thus a decrease in egg cytoplasm
to conform with the haploid nucleus may have permitted more normal
development.
While these observations suggest a cytoplasmic influence as being
responsible for the abnormalities, there is evidence which indicates
that the influence in some cases arises from the nucleus. For instance,
investigations of the early cleavage stages of merogonic egg fragments
of Triton palmatus and Triturus viridescens have shown an unequal
distribution of chromosomes (Fankhauser, 1932c and 1934c). This
has been held responsible for the high mortality rate which it is cus-
tomary for these merogonic embryos to show before or during gastrula-
tion. The same explanation has been extended to the non-viable
blastulae and gastrulae among Triturus viridescens embryos (Fank-
hauser and Kaylor, 1935). It is impossible, however, for any such
alteration in nuclear structure to be responsible for the abnormalities
of the typical frog haploid since a complete haploid complement of
chromosomes has been observed in all cases studied.
Only when these studies have been extended and more is known
concerning nucleo-cytoplasmic reactions whereby differentiation is
brought about will it be possible to state with any certainty the condi-
tions within the cytoplasm or nuclei of these haploids which make more
normal development impossible.
SUMMARY
1. A technique is described by which the second maturation spindle
and so all of the maternal chromatin can be removed from the egg of
R. pipiens following its activation and penetration by the sperm.
256 K. R. PORTER
2. The operation as applied to this egg is considered satisfactory for
a large number can be treated in a short time, the maturation spindle is
removed with certainty, and the slight amount of cytoplasm removed
has no destructive effect on the development which follows.
3. Between 90 per cent and 100 per cent (generally 100 per cent) of
the operated eggs undergo first cleavage simultaneously with the con-
trols, and of these the majority develop for eight days, a few con-
siderably longer.
4. The development of androgenetic haploids compared with
diploid controls of the same age is abnormal and delayed. Certain
features of external and internal morphogenesis are described.
5. It is shown that 90 per cent of the operated eggs can be expected
to develop as haploids. This haploid nuclear condition remains un-
changed until the final stages of development and then is altered only
by the presence of a very few diploid nuclei.
6. As is typical for haploids, the cells and nuclei of these andro-
genetic embryos tend to be smaller than those of the diploid controls.
7. The development shows a high degree of uniformity from animal
to animal over the first five days after which the differences become
more pronounced.
8. Yolk disappearance from the cells of the haploids is notably
delayed.
9. The ease of production and the success of development of R.
pipiens androgenetic embryos seems to provide one of the best possibil-
ities so far encountered for the study of haploid morphogenesis from
eggs which normally develop as diploids.
BIBLIOGRAPHY
BALTZER, F., 1922. Ueber die Herstellung und Aufzucht eines haploiden Triton
taeniatus. Verh. Schweiz. Natf. Ges., Bern, 103: 248-249.
BALTZER, F., 1933. Ueber die Entwicklung von Triton-Bastarden ohne Eikern.
Verhandl. d. Deutsch. Zool. Ges., 35: 119-126.
BALTZER, F., AND V. DE ROCHE, 1936. Ueber die Entwicklungsfahigkeit haploider
Triton alpestris-Keime etc. Rev. Suisse de Zool., 43: 495-506.
CURRY, H. A., 1931. Methode zur Entfernung des Eikerns bei normalbefruchteten
und bastardbefruchteten Triton-Eiern durch Anstich. Rev. Suisse d. Zool.,
38:401-404.
CURRY, H. A., 1936. Uber die Entkernung des Tritoneies durch Absaugen des
Eifleckes und die Entwicklung des Tritonmerogons Triton alpestris ( 9 )
x Triton cristatus (d1). Roux' Arch., 134: 694-715.
DALCQ, A., 1929. A propos des effets de 1'irradiation des gametes chez les Amphi-
biens. Arch. d. Anal. Micr., 25: 336-371.
DALCQ, A., 1932. Contribution a 'analyse des fonctions nucleaires dans 1'ontogenese
de la grenouille. IV. Modifications de la formule chromosomiale. Arch.
deBiol., 43:343-366.
DAWSON, A. B., 1938. Effects of 2, 4-dinitrophenol on the early development of
the frog, Rana pipiens. Jour. Exper. Zool., 78: 101-110.
ANDROGENETIC DEVELOPMENT OF FROG EGG 257
EAST E. M. 1934. The nucleus-plasma problem. Am. Nat., 68: 289-303; 402^39.
FANKHAUSER, G., 1932c. The role of the chromosomes in the early development of
merogonic embryos in Triturus viridescens. (Abstract.) Anat. Rec., 54:
suppl., 73-74.
FANKHAUSER, G., 1934c. Cytological studies on egg fragments of the salamander
Triton. V. Chromosome number and chromosome individuality in the
cleavage mitoses of merogonic fragments. Jour. Exper. Zool., 68: 1-57.
FANKHAUSER, G., 1937. The production and development of haploid salamander
larvae. Jour. Hered., 28: 2-15.
FANKHAUSER, G., 1938. The microscopical anatomy of metamorphosis in a haploid
salamander, Triton taeniatus Laur. Jour. Morph., 62: 393-413.
FANKHAUSER, G., AND C. T. KAYLOR, 1935. Chromosome numbers in androgenetic
embryos of Triturus viridescens. (Abstract.) Anat. Rec., 64: 41-42.
HADORN, E., 1934. Uber die Entwicklungsleistungen bastardmerogonischer Gewebe
von Triton palmatus (9) X Triton cristatus (d") ini Ganzkeim und als
Explantat in vitro. Roux' Arch., 131: 238-284.
HERTWIG, G., 1911. Radiumbestrahlung unbefruchteter Froscheier und ihre
Entwicklung nach Befruchtung mit normalem Samen. Arch. f. Mikr.
Anat., 77: (Abt. II) 165-209.
HERTWIG, G., 1913. Parthenogenesis bei Wirbeltieren hervorgerufen durch art-
fremden radiumbestrahlten Samen. Arch.f. Mikr. Anat., 81: 87-127.
HERTWIG, G., 1927. Beitrage zum Determinations- und Regenerationsproblem
mittels der Transplantation haploidkerniger Zellen. Roux' Arch., Ill:
292-316.
HERTWIG, P., 1916. Durch Radiumbestrahlung verursachte Entwicklung von
halbkernigen Triton- und Fischembryonen. Arch. f. Mikr. Anat., 87:
63-122.
HERTWIG, P., 1923. Bastardierungsversuche mit entkernten Amphibieneiern.
Roux' Arch., 100:41-60.
HOADLEY, L., 1937. In conversation.
KAYLOR, C. T., 1937. Experiments on androgenesis in the newt, Triturus viri-
descens. Jour. Exper. Zool., 76: 375-394.
PARMENTER, C. L., 1933. Haploid, diploid, triploid, and tetraploid chromosome
numbers, and their origin in parthenogenetically developed larvae and
frogs of Rana pipiens and R. palustris. Jour. Exper. Zool., 66: 409-453.
RUGH, R., 1934. Induced ovulation and artificial fertilization in the frog. Biol.
Bull., 66: 22-29.
WITSCHI, E., 1930. Experimentally produced neoplasms in the frog. Proc. Soc.
Exper. Biol. Med., 27: 475-477.
THE ILLUMINATION OF THE EYE NECESSARY FOR
DIFFERENT MELANOPHORIC RESPONSES OF
FUNDULUS HETEROCLITUS l
EARL O. BUTCHER
(From the Biological Laboratory, Hamilton College, and the Mount Desert
Island Biological Laboratory, Salisbury Cove, Maine)
It has been established by previous investigations (Butcher, 1938)
that the upper region of the retina of Fundulus is related to the paling
of the body and the lower region to the darkening of the fish. During
the course of these investigations many problems were encountered
and left unsolved. Among them were: (1) Why does illumination
of the lower region of the retina with a Mazda lamp cause most fishes
to darken, but illumination of the upper region with a Mazda lamp
induce only a few to pale? (2) How much of the regions have to be
illuminated to elicit the related melanophoric responses? (3) To
what extent does illumination of the upper region have to be eliminated
in order that darkening can be induced by illuminating the lower
region? (4) Is the paling response more easily elicited when light is
entirely eliminated from the lower region?
The cause of a fish assuming a paler shade in a shaded white box
than in a brightly lighted gray box was also investigated. It seemed
that the assumption of the shade of the background by the fish might
depend upon the ratio of the direct light coming from above and the
reflected light from below which enters the eye as Sumner (1911), and
Sumner and Keys (1929) have contended to be the case for the flounder.
The present investigations show that when a Mazda lamp is placed
above fishes in a black dish, the image of the lamp falls upon enough
retinal receptors in the lower region of the eye to induce darkening of
the body, but when the same lamp is placed below fishes and its image
falls upon the retinal receptors of the upper region, this image is not
large enough to induce the paling response. Illumination of a large
area of the upper region is, therefore, necessary to induce the paling
response. Paling is also more easily elicited when only the upper
region is illuminated. Illumination of the lower region induces darken-
ing only when there is very little illumination to the upper region.
The melanophore response elicited by illuminating the lower region
1 Reported before the American Society of Zoologists at the December, 1938,
meetings. Anal. Rec., vol. 72 (suppl. no. 4), p. 80.
258
EYE AND MELANOPHORIC RESPONSES 259
of the eye may be reduced by the simultaneous illumination of the
upper region. For instance, illumination from above to the lower
region of the eye causes darkening of the body when there is little
reflected light from the bottom of a black dish to the upper region.
If a gray bottom is used, a greater percentage of the light is reflected
to the upper region of the eye, a greater inhibitory reaction is induced,
and the degree of darkening of the body is reduced. If this gray back-
FIG. l.
ground is more intensely illuminated from above, then the reflected
light is greater to the upper region. The same ratio, however, persists
between the direct and the reflected light, a proportional inhibitory
effect is induced and the fish assumes the same shade as when the
intensity of the direct light is lower.
The Conditions Affecting the Paling Response
The sides of crystallizing dishes, 20 cm. in diameter, were lined
with black paper which reflected approximately 1 per cent of the light
striking it. Fishes were placed in these dishes containing water 4 cm.
deep, and the top of the dish was covered with black paper. When
the fishes were illuminated through the glass bottom by a 60-watt,
260
EARL O. BUTCHER
inside-frosted Mazda lamp, placed 18 cm. below the dish, a few of
them became slightly pale and the rest assumed an intermediate shade.
The fishes, in this instance, were receiving about 200 footcandles of
illumination (determined by a Weston photronic illuminometer) from
the lamp while the brightness of the lamp was approximately 58,000
footlamberts.2
If a piece of white paper or opal glass, as large as the bottom of the
dish, were inserted between the source of illumination from the Mazda
lamp and the bottom of the dish, the fishes readily paled even when the
brightness below them was 1 footlambert or less. The image of the
white bottom being larger than the image of the bulb alone fell on a
great many more retinal receptors. Paling, therefore, depends mainly
upon the size of the white area seen by the fishes.
TABLE I
Relation between body size and diameter of circle below fish
necessary to induce paling.
Number
examined
Length
of body
Width of body at
level of eyes
Diameter of circles and number paling
9 cm.
7 cm.
5 cm.
3 cm.
6
mm.
40-45
mm.
5-6
6
6
2
59
50-60
7-8
59
48
16
0
15
60-70
9-10
15
14
7
0
7
70-80
10
7
7
0
0
As a means of determining the size of the white area necessary for
inducing paling of the body, fishes were enclosed in glass tubes (16 mm.
inside diameter) which had small openings at both ends for the circula-
tion of water. These tubes with the fish inside were placed over
circles of white paper in such a way that the fish's head was above the
center of the circle (Fig. 1). The circles were then either illuminated
from above or from below.
To induce paling of fishes 50 mm. in length, circles 7 cm. in diameter
were usually necessary (Table I). When the fishes were over 3 cm.
circles, an intermediate shade was always assumed. Fishes paled
equally as well when the circles were exposed to 4.5 footcandles as to
450 footcandles from above.
2 The author is greatly indebted to Mr. Frank Benford of the General Electric
Company, Schenectady, N. Y. for determining the brightnesses with a Luckiesh-
Taylor Brightness-Meter.
EYE AND MELANOPHORIC RESPONSES 261
Figure 2, which is drawn to scale, shows approximately the size
of the image in the upper region of the eye when the fish was over the
various circles. It is evident that a large area of retinal receptors
must be stimulated before paling is induced. Images of 5 and 7 cm.
circles, being nearly the same size (Fig. 2), caused only slight differ-
ences in the degree of paling. It might have been better to use a
square tube to hold the fish, since a round tube probably acted as a
cylindrical lens, and the fish did not get an image quite like the white
circle and even illumination.
Fishes of various lengths were tested in tubes of the same size over
circles of white paper. Since the body of a small fish covered less of
the 3 cm. circle than did the body of a large fish, the small fish saw
more of the circle (Figs. 3 and 4), and assumed a paler shade over the
3 cm. circle than the large fish (Table I).
B
357
FIG. 2. Sizes of images in upper region of eye when fish was over circles with
various diameters. Dorsal ventral diameter of eye — 4 mm.; width of head — 8 mm.;
distance between eye and background — 6 mm.; B., background; 3, 5, 7, boundaries
of 3-, 5-, and 7-cm. circles.
Paling, therefore, depends greatly upon the size of the white area
below the fish.
In previous experiments (Butcher, 1938) where blinders were used
in covering the eyes, there was some evidence that illumination from
above tended to inhibit the paling response or that paling was more
easily elicited when the lower region of the eye was not simultaneously
illuminated. There is no way of confirming this observation with a
white background below and illumination from above because variation
in the illumination from above causes a proportional variation in the
reflected light from below. Likewise, if a white bottom is illuminated
from below, causing the fish to pale, then illumination cannot be added
from above in any way so that paling will not persist.
Whether or not light from above was inhibitory to the paling re-
sponse was investigated in the following way. A circle of white paper
262
EARL O. BUTCHER
which would induce paling when placed below a fish and illuminated
from above (Fig. 1) was cut into halves. The fish in the glass tube
was then placed over half of this circle in such a way that the axis of
the fish corresponded with the diameter of the circle (Fig. 5). Fishes
arranged in this manner failed to pale, because illumination of the
FIGS. 3 AND 4. These figures illustrate that more of the 3 cm. circle is seen by
the small fish than by the large fish. The image in the small fish covers approxi-
mately 15 per cent more of the upper region of the retina.
FIG. 3. Eye of fish 80 mm. long, and 10 mm. wide at eye level.
FIG. 4. Eye of fish 40 mm. long, and 6 mm. wide at eye level.
lower regions of both eyes was enough to inhibit any response elicited
by the reflected light to the upper region of one eye. Even if the
diameter of the circle was greatly increased, paling was not induced
in most instances. When the eye which was not over the white semi-
circle was enucleated, the fish immediately paled, for now the inhibition
EYE AND MELANOPHORIC RESPONSES 263
resulting from illuminating the lower region of one eye was not enough
to prevent the influence of the upper region of one eye. These experi-
ments definitely showed that the paling response was more easily
elicited when the lower regions of the eyes were not so intensely
illuminated.
The Conditions Necessary for Inducing the Darkening Response
To determine the intensity of illumination of the eye necessary
for inducing complete darkening of the body, fishes were placed in
glass dishes lined with black paper. These dishes were about 20 cm.
in diameter, 8 cm. deep, and contained water 4 cm. deep. A cylinder
FIG. 5. This figure shows how a fish was placed over a semi-circle so that the
upper region of only one eye was illuminated by reflected light from below.
lined with a light-proof, black paper enclosed the dishes. The top
of the cylinder was covered with opal glass, and a Mazda lamp, inside-
frosted, was suspended above the cylinder as the source of illumination.
The diffusing opal glass was 18 cm. from the surface of the water in the
dish. Two small openings were made in the side of the cylinder.
One was used for observing the fish and the other was large enough
for transferring fish in and out of the black dish. The temperature
of the water was kept at about 16° C. It was always ascertained if
fish would assume both pale and dark shades before they were used
for the experiments.
In investigating the effect of illuminating the lower region of the
retina with different intensities three or four fishes were placed in the
dish and allowed to remain for 20 minutes. Meanwhile, control
264 EARL O. BUTCHER
fishes in other black dishes outside of the cylinder were being exposed
to intensities from Mazda lamps which definitely induced maximum
darkness. At the termination of 20 minutes, one studied the experi-
mental fishes through the small hole in the side of the cylinder and
observed their shades. In order to determine more definitely how
many were completely dark and the correctness of the observations
made in the experimental dish, the observer then viewed the control
fishes, and without changing his field of vision, he quickly transferred
an experimental animal into the control dish. This method involved
only a few seconds and reduced the possibilities of error as much as any
method used. Fishes were tested only once and then discarded.
When the intensity of illumination reaching the fishes was reduced
to 2 footcandles, the majority of them failed to completely darken.
Exposing 60 fishes to this intensity, 40 per cent of them became com-
pletely dark, 20 per cent darkish, and 40 per cent intermediate. The
tables in the article by Brown (1936) show that an intensity of il-
lumination of 1.75 footcandles caused complete dispersion of melanin
in many Ericymba buccata Cope, the silver-mouthed minnow. Daniel-
son (1938) reports that complete melanophore change appeared to
occur at and above 1 footcandle in Nocomis biguttatus Kirtland.
There are undoubtedly variations between different species and varia-
tions in threshold between different individuals. When the intensity
was increased to 3.5 footcandles and 54 fish were tested, 45 became
completely dark, and the other 9 had a darkish appearance.
Since it was necessary to stimulate a large area of receptors in the
upper region of the retina in order to induce paling of the body, a
few investigations were undertaken to determine the size of the source
of light to the lower region necessary to cause the darkening response.
In place of the opal glass covering of the cylinder, a black lid was
substituted. This covering contained a central aperture, the size of
which could be varied. When this aperture was 1.5 cm. in diameter
and the fish were receiving 2 footcandles, about the same percentage
(40 per cent) became maximally dark as when they received 2 foot-
candles through opal glass. Only 25 per cent, however, definitely
assumed a maximum darkness when the diameter of the aperture was
reduced to 1 cm. and they received an intensity of 2 footcandles.
Some Funduli thus become maximally dark when receiving an
intensity of 2 footcandles from a source of light 1 cm. in diameter.
The diameter of the image formed by a source of light 1 cm. in diameter
and 18 cm. from the eye is only about .085 mm. or 85 micra. Whether
or not more than 25 per cent of the fish will be induced to darken when
receiving an intensity greater than 2 footcandles from a source 1 cm.
EYE AND MELANOPHORIC RESPONSES 265
in diameter has not been determined. At least, a much smaller image
induces darkening than the image necessary to elicit paling of the body.
There are also undoubtedly individual differences in threshold.
Evidence that the Shade of the Fish Depends upon the Ratio
between the Light from Above and the Light from Below
Entering the Eye
Observations made by Sumner (1911), and Mast (1916) show that
the shade of the flounder's body does not depend upon a visual com-
parison between its body surface and the background. It seems more
probable from their experiments and those of Sumner and Keys (1929)
that the ratio between the light coming from above and that reflected
from below supplies the stimulus to the eye which enables the fish to
assume a certain shade.
To learn if the ratio of light was responsible for the shade assumed
by Fundulus it was first necessary to secure backgrounds which ranged
in shade from white to black, to determine the response of the fish
with each background, and the ratio of the direct to the reflected light
in each instance. Various gray papers were used for these back-
grounds and these were placed in the bottoms of large crystallizing
dishes, the sides of which were lined with black paper. These crystal-
lizing dishes were held with clamps about two feet from a table in a
dark room. For illuminating the bottom of the dish, Mazda inside-
frosted lamps were placed both above and below the dishes.
The response of the fish, the kind of bottom, and the brightness of
the bottom when illuminated only from above are recorded in Table II.
The responses of 15 fish were usually determined in each of these
experiments. Gray 1, gray 2, and gray 3 were very close to neutral 6,
5, and 3, respectively, of the Munsell "Book of Color." 3
The higher intensities induced about half of the fish in the dish with
gray bottom 2 to pale. Reducing the brightness of the bottom to .1
footlambert when the intensity from above was .5 footcandle caused
only a few to pale (Table II). Apparently the percentage of reflected
light has to be greater than it is from bottom 2 (50 : 10) to induce all
to pale.
If the fish assumes the shade of the background below because of
the ratio of the light from above to the light from below entering the
eye, then fish should pale over gray 3 when its brightness is increased
by illumination from below, and a ratio is established which is known
to induce paling of the body. To test this hypothesis the Mazda
lamp, inside-frosted, was turned on under gray 3 while the intensity of
3 "The atlas of the Munsell Color System," Munsell Color Company, Inc.,
Baltimore, Maryland.
266
EARL O. BUTCHER
illumination from above remained 5 footcandles. When the bright-
ness of this paper was 3 footlamberts or a ratio (5 : 3) existed which
induced paling over a white background, the fish, likewise, paled over
this gray 3. With added illumination (400 footcandles) from above
so that the ratio was 50 : 3.3, an intermediate shade was quickly
assumed.
If either gray 1, 2, or 3 were illuminated only from below and their
brightness was 1 footlambert (ratio in this instance is 0 : 1), all fish
quickly paled. With no illumination from above fish could un-
doubtedly be induced to pale when the brightness of the bottom was
much less than 1 footlambert for they have paled when the background
was .2 footlambert in brightness and the intensity from above was .5
footcandle (Table II).
TABLE II
Light relations and shade of fish's body with different shades of paper
below fish. P., pale; SP., slightly pale; Int., intermediate.
Shade of paper
below fish (sides
of container
black)
Intensity of
light from
above
footcandles
Brightness of
background
below fish
footlamberts
Ratio of light
from above to
brightness of
background below
Shade assumed
by fish
White
400
220
50 27
P
10
5.5
50 27
P
5
2.75
50 27
P
.5
.275
50 27
P
Gray 1
400
10
160
4
50 20
50 20
P
P
5
2
50 20
P
.5
.2
50 20
P
Gray 2
400
10
5
.5
80
2
1
.1
50 10
50 10
50 10
50 10
50% P, 50% SP
50% P, 50% SP
50% P, 50% SP
20% P, 80% SP
Gray 3
400
10
24
.6
50 3
50 3
Int.
Int.
5
.3
50 3
Int.
.5
.03
50 3
Int.
Some fish, therefore, paled when the ratio of direct to reflected
light was 50 : 10. Fish failed to pale over a gray background below
when lighted from above because this background did not reflect
enough light in comparison to the light coming from above. If the
gray background were illuminated from below so that its brightness
was increased, then the fish paled.
EYE AND MELANOPHORIC RESPONSES 267
SUMMARY
Only a small area of the lower region of the eye of Fundulus needs
be illuminated to induce a darkening of the body, since as little light
as 2 footcandles coming from a source 1 cm. in diameter and 18 cm.
above the fish elicits the darkening response. Darkening cannot be
induced by illuminating the lower region when there is much illumina-
tion to the upper region of the eye.
For eliciting paling of the body, a large area of the upper region
of the eye must be illuminated. This is shown by experiments with
fish over circles. Regardless of the brightness of a circle 3 cm. in
diameter beneath the fish, those 50 mm. in length failed to pale. When
the illuminated circle was increased in size, fish paled readily. Paling,
therefore, depends greatly upon the size of the white area seen by the
fish. A Mazda lamp arranged below a fish so as to illuminate the
upper region of the eye thus fails to induce paling because its image
does not fall upon enough retinal receptors. Paling is more easily
elicited when the lower region of the eye is not illuminated at the same
time that the upper region is illuminated.
The ratio between the direct and the reflected light, known to
exist with a white background below the fish, has been created with
gray bottoms by illuminating them both from above and below. Gray
backgrounds illuminated in this way have caused fish to pale readily.
The shade assumed by Funduli, therefore, depends upon the ratio
between the direct and the reflected light entering the eye.
As the percentage of reflected light to the upper region of the eye
is increased, there is induced a proportional increase in the inhibitory
reaction which causes a reduction in the degree of darkening of the fish.
LITERATURE CITED
BROWN, F. A., JR., 1936. Light intensity and melanophore response in the minnow,
Ericymba buccata Cope. Biol. Bull., 70: 8-15.
BUTCHER, E. O., 1938. The structure of the retina of Fundulus heteroclitus and the
regions of the retina associated with the different chromatophoric responses.
Jour. Exper. Zool., 79: 275-297.
DANIELSON, R. N., 1938. Light intensity and melanophore response in a cyprinid
fish. Physiol. Zool., 11: 292-298.
MAST, S. O., 1916. Changes in shade, color, and pattern in fishes and their bearing
on the problems of adaptation and behavior, with especial reference to the
flounders Paralichthys and Ancylopsetta. Bull. U. S. Bur. Fish., 34:
173-238.
SUMNER, F. B., 1911. The adjustment of flatfishes to various backgrounds; a study
of adaptive color change. Jour. Exper. Zool., 10: 409-505.
SUMNER, F. B., AND A. B. KEYS, 1929. The effects of differences in the apparent
source of illumination upon the shade assumed by a flatfish on a given
background. Physiol. Zool., 2: 495-504.
OBSERVATIONS UPON AMPHIBIAN DEUTOPLASM
AND ITS RELATION TO EMBRYONIC AND
EARLY LARVAL DEVELOPMENT1
ARTHUR N. BRAGG
(From the Zoological Laboratory of the University of Oklahoma}
During early ontogeny, several distinct morphogenic processes
proceed more or less synchronously whereas others tend to alternate
(Richards, 1935). In the exponential period, described by Schmal-
hausen (1930), mitotic activity dominates; but with the onset of
gastrulation, the mitotic rate falls in close correlation with an increase
in differentiation (initiation of the parabolic period). It is at this
time, just as the primary caudo-cephalic axis is about to be laid down,
that the first embryonic organizers become evident in the dorsal
blastoporal lip (at least in Amphibia) and also that important mitotic
centers are set up which feed cells into specific regions where they
later differentiate into various anlagen, in some cases, at least, under
the influence of induction (Derrick, 1937; Self, 1937; Bragg, 1938;
Jones, 1939). Behind these more or less morphological manifestations
are the actions of the genes, inductors, possibly hormones, etc. which,
working through the visible morphological configurations of the cells
or their parts, actually are the basic underlying factors in the produc-
tion of the embryo, and hence of the adult body.
From these considerations, it is evident that the basic factors in
embryonic development are essentially physiological, rather than
morphological, in character. Studies of cell-migrations or of mor-
phogenic movements (Vogt, 1929; Wetzel, 1929; Graper, 1929;
Pasteels, 1936; etc.), or mitotic indices (Minot, 1908; Self, 1937;
Derrick, 1937; Bragg, 1938; Jones, 1939, etc.), and all similar attacks
upon the problem of embryological organization cannot, each method
of itself, explain morphogenesis. Such studies are valuable mostly as
indicating changes in the morphological configurations of parts which
in turn are indirect evidences of the basic physico-chemical changes in
the protoplasm, a detailed understanding of which can only be at-
tained by physiological methods. Sometime in the future, therefore,
we may expect a synthesis of the observations made by the various
methods now in use wherein the relationship between cell division, and
1 Contribution from the Zoological Laboratory of the University of Oklahoma,
No. 199.
268
AMPHIBIAN DEUTOPLASM AND DEVELOPMENT 269
mitotic centers, induction, cell-migration, increase in size, histological
differentiation, problems of cell-size and body-size, nucleo-cytoplasmic
ratios, the mode of genie action, etc. will all be correlated into one
basic biological principle, only fragmentary glimpses of which any one
of us now sees by the results of his own special method.
It is well established that the yolky materials in an egg of an animal
constitute reserve food which is utilized during some phase of ontogeny
as a source of energy, of building materials, or of both: but at just
what phase of development and for what processes they are utilized
by the animal has apparently received but slight attention (see, how-
ever, Saint-Halaire, 1914). During a recent study of the relation of
cell division to early embryonic organization of a toad (Bragg, 1938),
it was observed that the yolk granules maintained their initial sizes,
shapes, and appearances at least to the stage in which the neural tube
closed. From indirect evidence, it was also strongly suspected that
the embryo did not increase materially in protoplasmic mass up to
this stage of development. If these two conclusions were substantially
correct, this could only mean that the yolk was not used during the
exponential period nor even during the earlier portion of the parabolic
period wherein all of the anlagen of the major organ systems were laid
down. In other words, the yolk contributed neither energy for the
very actively katabolic process of cleavage nor materials for the in-
crease in the size of the body up to this stage of development in the
embryos of the species investigated.
Since these observations were somewhat incidental to the main
subject of the former paper, and, further, since the yolk must bear
important relationships to some of the ontogenetic processes indicated
above, it seemed wise to study the yolk in greater detail in order to
establish when and where its utilization begins and, so far as possible
by the methods used, for what embryological processes it is utilized.
It is also of interest to ascertain whether the species used in the former
study (Bufo cognatus] is peculiar in these matters or whether other
amphibian species manifest the same phenomena.
MATERIALS AND METHODS
The embryos used were those of Bufo cognatus Say, B. woodhousii
woodhousii (Girard), Rana sphenocephala (Cope), and Scaphiopus
hammondii Baird, all from the vicinity of Norman, Oklahoma.2 Pre-
2 1 am indebted to the following for the use of slides of embryos and larvae
prepared and owned by them: to Mr. Virgil Johnson for all stages of B. w. wood-
housii; to Dr. Minnie S. Trowbridge for embryos of Scaphiopus; and to Mr. Robert
Taylor for larvae of Scaphiopus.
The species of Scaphiopus used is the same as that called tentatively S. bom-
bifrons Cope by Trowbridge and Trowbridge (1937). In a forthcoming paper, Dr.
270 ARTHUR N. BRAGG
pared slides of early cleavage, of the blastula, and of the gastrula of
the California newt, Triturus torosus, were studied also for comparison
with the anuran embryos.
The methods were those commonly employed for embryological
work. Embryos and larvae were fixed in one of several different
fixing fluids (Smith's, Goldsmith's, and Bouin's, most commonly),
dehydrated with ethyl alcohol, embedded by the method of Hamlett,
and serially sectioned (6-12 micra). Heidenhain's haematoxylin,
alum haematoxylin, and alum cochineal were the principal stains used.
The exact procedure made little difference for the purpose of the study.
Observations upon living embryos and larvae of all species used except
the newt were also made.
Following the same method as earlier (Bragg, 1938), the yolk
granules in selected regions were drawn under oil-immersion lenses
by means of a camera lucida, all carefully to the same scale. The
pictures so obtained were then compared with each other and with
the details of structure as seen in the microscopic fields. The facts
Minnie S. Trowbridge and the author will show that the species name, bombifrons
is not a synonym for hammondii as assumed by Wright and Wright (1933) and that
the species in question here is 5. hammondii.
EXPLANATION OF FIGURES
All figures in the plates drawn by camera lucida and to the same scale in order
that they may be compared with one another directly. All are of complexes of yolk
granules characteristic of the region given for each except Figs. 44 to 49.
A. Blastula No. 12A1. comparable to Bragg, 1938, Stage A.
FIG. 1. Micromere.
FIG. 2. Intermediate zone.
FIG. 3. Macromere.
B. Gastrula No. 263A2, comparable to Stage C (Bragg, 1938).
FIG. 4. Dorsal ectoderm.
FIG. 5. Just inside the dorsal lip of the blastopore.
FIG. 6. Anterior ectoderm (opposite the yolk plug).
FIG. 7. Dorsal blastopore region. The blastoporal groove between a cell of
the dorsal lip (left) and a cell of the yolk plug.
FIG. 8. Condition a short distance inside the blastopore at the dorsal lip.
Condition of yolk intermediate between those shown in Figs. 5 and 10.
FIG. 9. Ventral lip of the blastopore.
FIG. 10. Inner yolk mass.
C. Stage of the crescentic blastopore, No. 54A4, comparable to Stage B (Bragg, 1938).
FIG. 11. Micromere near the animal pole.
FIG. 12. Innermost yolk cells.
FIG. 13. Region of the dorsal blastoporal groove; compare with Fig. 7.
D. Neural plate stage. No. 75XA1, comparable to Stage D (Bragg, 1938).
FIG. 14. Neural plate.
FIG. 15. Dorsal endoderm.
FIG. 16. Lateral mesoderm.
FIG. 17. Lateral ectoderm.
FIG. 18. Ventral ectoderm.
FIG. 19. Ventral yolk mass.
AMPHIBIAN DEUTO PLASM AND DEVELOPMENT
271
^zz-f* /IQK*%%\ J~^ rf *•*?•*
Tf ^«\ ,/. * •%«:*.
PLATE I.
cognatus
272 ARTHUR N. BRAGG
gathered in this manner were then correlated with the known stage of
development of the individual animals from which the slides had
originally been made.
OBSERVATIONS
The distribution and sizes of the yolk granules of all species used
followed the general pattern already described for Bufo cognatus
(Bragg, 1938). Briefly, a gradient of size exists, the smallest granules
being mostly located in the animal region, the largest in the vegetal
portion of the egg. Species differ in the absolute sizes of the granules
but the mode of distribution is the same in all. During cleavage,
three types of blastomeres become recognizable, each easily differ-
entiated from the others by the type of yolk granules contained.
EXPLANATION OF FIGURES — PLATE II
FIG. 20. Dorsal mesoderm.
FIG. 21. Notochord.
E. Neural tube not quite closed. No. 112A3, comparable to Stage E (Bragg,
1938).
FIG. 22. Dorsal mesoderm.
FIG. 23. Superficial lateral ectoderm.
FIG. 24. Neural tube.
FIG. 25. Lateral endoderm.
FIG. 26. Endo-chordo-mesoderm.
FIG. 27. Lateral mesoderm.
FIG. 28. Dorsal endoderm.
FIG. 29. Notochord.
F. Neurula. No. 273a.
FIG. 30. Optical vesicle.
FIG. 31. Ectoderm adjacent to the adhesive organ.
FIG. 32. Brain.
FIG. 33. Adhesive organ.
FIG. 34. Ventral ectoderm.
FIG. 35. Notochord.
FIG. 36. Mesenchyme of the head.
FIG. 37. Lateral mesoderm.
FIG. 38. Ventral yolk mass.
FIG. 39. Lateral ectoderm.
FIG. 40. Dorsal endoderm.
FIG. 41. Somite mesoderm.
FIG. 42. Ectoderm of the head.
FIG. 43. Nerve cord.
G. Larva of 3 mm. total length. No. 134 2-2.
FIG. 44. Ventral yolk mass.
FIG. 45. Mesoderm. Note that the yolk is being used.
FIG. 46. Superficial ectoderm. Yolk nearly gone.
H. Larva of 5 mm. total length. No.HlEl.
FIG. 47. Myomere of the tail.
FIG. 48. Section of the nerve cord dorsal to the yolk region (Fig. 44). Yolk
granules scattered and small.
FIG. 49. Outline of a fold of superficial ectoderm with only the yolk granules
shown.
AMPHIBIAN DEUTOPLASM AND DEVELOPMENT
273
^4
PLATE II. Bufo cognatus
274 ARTHUR N. BRAGG
These are (1) the micromeres which contain mostly small granules, (2)
the macromeres which contain mostly large granules, and (3) cells
located in a thick section between these which contain granules mixed
and intermediate as to size. This last-mentioned region has been
designated as the intermediate zone. These matters are illustrated
in Figs. 1-3, 50-52, and 67-68.
I wish to call particular attention to the fact that it is not the
absolute sizes of the yolk granules that is to be emphasized. Rather,
it is the general appearance of the complex of granules in each type of
cell. Some micromeres contain large granules intermingled with the
smaller ones and the converse is true in the macromeres. The ap-
pearance is due in part to a greater number of the one type of granules
or the other in any given cell and in part to the average sizes of the
granules. The average length of the granules from the regions of the
embryo of Bufo cognatus shown in Figs. 1, 2, 3, and the two portions of
Fig. 7, for example, bear approximately the following relationships to
one another: 1.0 : 1.2 : 1.6 : 1.1 : 2.2, the last two figures being for
the dorsal lip of the blastopore and the adjacent yolk plug, respectively.
During gastrulation, the yolk granules maintain their original
relationships as to size and appearance within each type of cell (Figs.
4-15). A striking contrast between the appearance of the complex of
yolk granules in the dorsal lip of the blastopore and that in the cells
EXPLANATION OF FIGURES — PLATE III
A. Blastula No. 1.2a.
FIG. 50. Micromere.
FIG. 51. Intermediate zone.
FIG. 52. Macromere.
B. Neural plate stage.
FIG. 53. Dorso-lateral endoderm.
FIG. 54. Ventral yolk mass.
FIG. 55. Dorso-lateral mesoderm.
FIG. 56. Ventral ectoderm.
FIG. 57. Neural plate.
FIG. 58. Notochord.
C. Open neural groove.
FIG. 59. Notochord.
FIG. 60. Lateral ectoderm.
FIG. 61. Neural fold.
FIG. 62. Somite mesoderm.
FIG. 63. Dorso-lateral endoderm.
FIG. 64. Lateral mesoderm.
FIG. 65. Ventral yolk mass.
D. Embryo just younger than that from which Figs. 59-65 were taken.
FIG. 66. Posterior ventral yolk mass.
E. Trilurus torosus, two-celled stage.
FIG. 67. Yolk complex near the animal pole.
FIG. 68. Yolk complex near the vegetal pole.
AMPHIBIAN DEUTOPLASM AND DEVELOPMENT
275
PLATE III. Bufo woodhousii woodhousii (except Figs. 67 and 68 which
are of Triturus).
of the yolk plug just across the blastoporal groove illustrates this fact.
(See Figs. 7 and 13.)
In later stages, the essentials in all of the species studied are also
as earlier found for Bufo cognatus. The micromeres of the blastula
pass under the dorsal lip of the blastopore for a short distance only,
just enough to cover the yolk plug as the blastopore closes. The
micromeres, therefore, give rise mostly only to ectoderm; mesodermal
and endodermal derivatives contain only complexes of yolk granules
276 ARTHUR N. BRAGG
characteristic of the macromeres of the blastula, except, possibly,
some characteristic of the intermediate zone. (See Figs. 14-43 and
59-66.) Hence, it may be concluded without question that in Bufo,
Rana, and Scaphiopus and probably also in Triturus (stages later than
gastrulae not studied) the micromeres differentiate into ectoderm and
the remainder of the embryo is derived from the macromeres and the
cells of the intermediate zone. Since two orders, four families, and
five species appear to agree so closely, it seems very probable that the
principles here discussed will be found to apply generally to Amphibia.
In order to determine whether the embryo increases in size during
early development, the measurements of embryos and larvae sum-
marized in Table I were made. Taking change in diameter for stages
through gastrulation and length thereafter as a measurement of
growth, the figures show an increase between the early cleavage stages
and the neural plate stage of 33 per cent and a width increase of 16
per cent. At the neural tube stage, the increase in length is 1 per cent
more but the width has decreased 16 per cent. Between the neurula
and the stage at hatching, the increase in length has reached 136.8
per cent of the diameter at early cleavage and the outer configurations
of the embryo have become so irregular that exact measurements of
width at any one level of the body-axis can have little meaning. A
summary of these facts is presented in graphic form in Text-fig. 1.
It is well known that amphibian embryos absorb water during
cleavage. Morgan (1906) found an increase in diameter of about
25 per cent between early cleavage and gastrulation in embryonic
frogs, about one-half of which was due to the development of the
blastocoel. The figure for Bufo cognatus is somewhat less than this
(18.9 per cent), probably due to interspecific differences. However
this may be, the increase in diameter is not too great to be accounted
for almost or quite entirely by the absorption of water during cleavage,
particularly if one consider the space occupied by the blastocoel. The
data, therefore, confirm the earlier conclusion that no increase in
protoplasmic mass occurs up to this stage, although the embryo does
actually increase in size. Measurements of living embryos of Rana
sphenocephala substantiate this general result.
In later stages, but prior to hatching, growth in length is quite
rapid but the increase in width is not comparable. Cavities (arch-
enteron, neurocoel, etc.) develop which take up space and the cells
become progressively smaller, particularly in areas of high mitotic
rate (Bragg, 1938; 1939). While by no means demonstrated, it seems
very probable from these considerations that most of the increase in
bulk prior to hatching takes place without material increase in funda-
AMPHIBIAN DEUTOPLASM AND DEVELOPMENT
277
mental protoplasmic constituents (except, of course, the ever present
water). This conclusion, moreover, is strengthened by a study of the
TABLE I
Bufo cognatus. Growth of embryos and larvae as measured by length and
width after preservation in 70 per cent alcohol. Measurements in mm. to the
nearest 0.01. Animals grown in the laboratory at room temperatures (approximately
18-22° C.) ; cultures maintained in tap water with algae added as food after hatching.
Increase
between
Total
Percentage of total
Stage
Age
(hrs.)
No.
used
Length
Width
stages
increase
increase
^ength
Width
Length
Width
Length
Width
Early
cleavage
(2-8
cells)
1-2
20
1.06
1.06
—
—
—
—
—
—
Mid-
cleavage
3-6
20
1.12
1.12
0.06
0.06
0.06
0.06
5.6 +
5.6 +
Gastrula-
tion
18-20
20
1.26
1.26
0.14
0.14
0.20
0.20
18.9-
18.9-
Neural
plate
stage
33-35
20
1.41
1.24
0.15
-0.02
0.35
0.18
33.0 +
16.0+
Neural
tube
stage
42-46
20
1.42
0.88
0.01
-0.36
0.36
-0.18
34.0-
-16.0+
Hatching
51-55
91
2.51
—
1.09
—
1.45
—
136.8-
—
Mouth a
shallow
pit
70-74
39
3.00
— •
0.49
—
1.94
—
181.5 +
—
Mouth a
deep pit
74-100
20
3.15
—
0.15
—
2.09
—
197.2-
—
Mouth
first
102-
func-
tional
106
52
4.91
—
1.76
- —
3.85
— •
363.2 +
—
Ready for
meta-
mor-
45
phosis
(days)
22
25.79
—
20.38
—
24.23
—
2285.8 +
—
yolk granules in most regions of the embryo, the exceptions occurring
during late embryonic life in those areas most active in differentiation.
278
ARTHUR N. BRAGG
This is particularly true of Scaphiopus: for example, in the adhesive
organ of this organism, the yolk granules of a late embryo are noticeably
smaller than those in cells from which the anlage of the organ was
derived. The same is true of the optic cup. But in the superficial
ectoderm of the head and in the brain of the same embryo, they remain
essentially unchanged.
From the foregoing observations, therefore, the following general
conclusions may be stated :
(1) The embryo increases in size during all phases of development.
(2) This increase does not take place at a constant rate till hatching,
after which it does so (at least as measured by length).
TEXT FIG. 1. Graph, increase in average length plotted against age in hours.
The figures above the graph are camera lucida drawings, all to the same scale, of
representative embryos and larvae of the stages indicated. The numbers above the
drawings are the lengths (mm.) of the examples drawn; the numbers below are the
widths of these same embryos.
(3) The increase up to the gastrula is due very largely, if not wholly,
to the absorption of water, correlated with the space occupied by the
development of the blastocoel.
(4) The development of the neurula from the neural plate stage
is accomplished with little or no increase in bulk (length increases but
width decreases).
(5) The most rapid growth in length occurs between the neurula
and the stage at hatching; since the yolk is not altered within most of
the cells during the greater part of this period, however, protoplasmic
substance is increased but little in the embryo as a whole, even though
the bulk of the embryo may increase due to the further absorption of
water.
AMPHIBIAN DEUTOPLASM AND DEVELOPMENT 279
If the yolk is not utilized during early ontogeny, when and for what
is it used? Observations upon late embryos and upon early to half-
developed larvae of Bufo cognatus and Scaphiopus indicate the follow-
ing: (1) The yolk granules begin to break up and to disappear in some
regions before they do so in others. (2) Their digestion begins, in
general, earliest in those embryonic regions which are first in histo-
logical differentiation. (3) Just prior to and during active digestion,
vacuoles often appear in the cytoplasm and the yolk granules come to
lie in these as though the vacuoles were formed around them: from this
it is thought probable that the yolk is digested in intracellular vacuoles
into which digestive enzymes pass from the cytoplasm much as in a
protozoon.
Figures 30-49 illustrate these processes in Bufo cognatus. A cell
of the optic vesicle in the neurula contains yolk granules comparable
to those in the blastular micromeres (compare Fig. 1 with Fig. 30).
In the ventral ectoderm of the same embryo (Fig. 31) they are smaller.
In the adhesive organ (Fig. 33) they are not only smaller but also some-
what irregular in shape. The lateral ectoderm contains some irregu-
larly shaped granules but the complexes of yolk in the mesoderm,
endoderm, brain, and notochord remain unchanged. (See Figs. 32
and 35-38.) From this it appears that the yolk is used first in ecto-
dermal structures, particularly those in the region of the anlage of the
adhesive organ which is soon to differentiate and to function at hatching
(Bragg, 1939a). In larval stages (Figs. 44-49) the yolk is disappearing
in all regions except the ventral yolk mass.
Embryos of Rana sphenocephala show similar trends. In late
embryos and early larvae, the yolk is beginning to be utilized in the
brain, notochord, and the optic vesicle. The superficial ectoderm is
probably just beginning to utilize the yolk but there has been no
visible change in the mesodermal and endodermal portions. The
more cephalic portions of the anlage of the central nervous system
begin the use of the yolk before the more posterior portions. This
illustrates the use of the yolk in correlation with anterior and cephalic
differentiation in general as opposed to posterior and ventral
differentiation.
These conceptions are further illustrated by the study of embryos
and larvae of Scaphiopus. Little if any yolk is utilized before the
neural tube is being formed. However, immediately after the neural
tube closes, the differentiation of anterior and dorsal structures is well
under way. This is especially noticeable in the adhesive organ but it
apparently starts in the mesenchyme of the head before it does in the
brain or superficial ectoderm in this species. The yolk granules in the
280 ARTHUR N. BRAGG
notochord appear slightly decreased in size but those of the posterior
and middle ectoderm, somite mesoderm, ventral yolk mass, and
endoderm are still unchanged.
Just before hatching, the relation of the disappearance of the yolk
to histological differentiation is still more striking. Ectodermal
structures and some parts of the mesoderm are losing yolk but endo-
dermal derivatives, for the most part, are not. The dorsal cephalic
ectoderm, the optic cup, and the adhesive organ have lost more of
the yolk than most of the other parts.
In a 78-hour larva (approximately nine millimeters in total length),
differentiation has already reached a functional state in many organs.
Some of the potential blood cells have no yolk granules whereas others
have a few enclosed in vacuoles. Many contain small particles of
yolk with no visible vacuoles around them and some have granules
which are apparently unchanged. The superficial ectoderm in all
parts of the body has lost much of its yolk. In one embryo, two cells
were observed in this layer each of which contained a large vacuole
in which were located small particles which stained like yolk. The
endodermal wall of the gut still largely retains its yolk although a few
of the granules are within vacuoles. The adjacent mesothelial wall
of the splanchopleure has relatively few granules, some still quite
large, others small. The myotomes of the tail are functional at this
time. Sections of this region show the yolk to be small in amount and
scattered. The cells of the ventral yolk mass contain granules of
various sizes, but since some are definitely located in vacuoles, digestion
of yolk has probably just begun in this region.
DISCUSSION
In an earlier paper (1938) it was noted that the mitotic centers
in the embryos of Bufo cognatus often do not correspond to the centers
of susceptibility described by Bellamy (1919) in the embryonic frog
and by Hyman (1921, 1926, 1927) and Rulon (1935) in other verte-
brates. It was also noted that if the interpretation by these authors of
the gradients of susceptibility as metabolic gradients be accepted, one
seems justified in thinking of the regions of greatest susceptibility as
regions where anabolic metabolism dominates katabolic metabolism.
If this be granted, then it follows that histological differentiation is
also dominated by anabolic as contrasted with katabolic processes,
a conclusion in accord with the distribution of mitotic centers and with
the Gesetzmassigkeit of Schmalhausen. The place where yolk first
begins to disappear in the embryo (dorsal and cephalic regions, es-
pecially where most active differentiation is occurring) gives further
AMPHIBIAN DEUTOPLASM AND DEVELOPMENT 281
evidence for this view, for some of these are the very regions which
were found by others to be most susceptible to a variety of harmful
influences; and they also tend to be the areas of lowest mitotic rate in
certain stages (see especially Bragg, 1938, Table V, p. 165).
During cleavage, katabolic processes dominate and the respiratory
relationships of the embryo require much oxygen (Bragg, 1939a).
The source of the energy used during this period is still unknown,
but the results of the study presented herein show clearly that the yolk
is not used for this function (nor, indeed, for anything else) during
this time.
At gastrulation, the metabolism of the embryo becomes differential,
dominated in some regions by anabolism, in others by katabolism.
This is shown both by the distribution of the mitotic indices at this
stage and by the fact that a secondary center of susceptibility is set up
in the dorsal lip of the blastopore (Bellamy, 1919). Since, however,
the mitotic rate drops very suddenly at this stage, the embryo as a
whole is likely dominated by constructive metabolism; but this is
probably only another way of saying that the embryo enters the
parabolic period of Schmalhausen. It seems probable, also, that
early induction is anabolic in character, since the organizer of Spemann
is located in the dorsal blastoporal lip and thus coincides with a center
of susceptibility. This is, of course, only what one might expect,
inasmuch as the fundamental function of induction seems to be the
stimulation of cells to construct embryonic parts which would not
arise, at least at a given time or place, without it. Furthermore, all of
this correlates nicely with the distribution of the mitotic centers in the
gastrula (Bragg, 1938, Table III, p. 161).
The time between early gastrulation and the formation of the
neural tube seems to be one of great reorganization. The size of the
embryo increases only insignificantly (Table I, Text Fig. 1) and the
yolk remains inert; but in this short period (about twenty-five hours
at ordinary temperatures in Bufo cognatus and probably even shorter
in Scaphiopus), bilaterality is established, the notochordal and meso-
dermal anlagen make their appearance and the fundament of the whole
central nervous system is formed. The distribution of the paths
of cell-migrations and other morphogenic movements (Vogt, 1929 and
others), as well as the places of greatest mitotic activity, seem best
interpreted to mean that this reorganization is brought about almost
wholly by cell-migration from specific centers of katabolic (mitotic)
activity at specific places and times.
Following closure of the neural folds no further data on the mitotic
indices in the amphibian embryo are available at the present time, but
282 ARTHUR N. BRAGG
one would expect from the work on other forms that the mitotic rate
in the embryo as a whole would progressively decline and that centers
of high mitotic index would continue to arise, particularly just prior
to the formation of specific anlagen (Derrick, 1937; Self, 1937; Jones,
1939). The rate of growth in length is greatly increased during the
period between the neurula and hatching (a period of about ten hours
in Bufo cognatus at room temperatures), but this involves the dis-
appearance of the yolk from the cells only in the later stages (except
in Scaphiopus}. It seems probable, therefore, that the increase in
mass is only slight and the apparent growth is due to the space taken
up, in part by the development of cavities within the embryo, in part
by decrease in width relative to length, and in part by further absorp-
tion of water. The yolk seems to be used at a slightly earlier period
by Scaphiopus than in any of the other forms studied and this may be
correlated with the exceptionally high rate of development which this
form has (Trowbridge and Trowbridge, 1937; Trowbridge, 1939).
However, even in Scaphiopus the yolk is used first by the regions of
most active differentiation, mostly dorsal and cephalic in the embryo.
Late in embryonic life, the curve of growth becomes a straight
line and from this time on the yolk progressively disappears from the
cells, being used last in the large yolk mass ventral to the lumen of
the gut.
Whether, in larval stages, the yolk is utilized primarily for histo-
logical differentiation or for increase in the bulk of the protoplasm
could not be ascertained with certainty, since these two anabolic proc-
esses occur together. The methods used in this study could not,
therefore, distinguish between them so far as their relations to the
disappearance of the yolk is concerned.
SUMMARY
Sections of embryos and of larvae of several Amphibia, repre-
senting two orders, four families, and five species indicate that the yolk
is carried passively in most cells till late in the embryonic period.
Just before hatching in most species, but somewhat earlier in Scaphi-
opus hammondii, disappearance of the yolk begins in the areas of earli-
est histological differentiation, mostly dorsal and cephalic in the
embryo. Since the regions of greatest susceptibility to injury reported
by others are often the ones of lowest mitotic rate, it seems probable
that histological differentiation is dominated by anabolic, rather than
by katabolic, processes, just as growth must be. Similar reasoning
shows that embryonic induction in the dorsal lip of the blastopore is
also predominantly anabolic. This is indicated by the correlation
AMPHIBIAN DEUTOPLASM AND DEVELOPMENT
of the results of four methods of attack; the Getzmdssigkeit of Schmal-
hausen, the mitotic index, studies of cell migration, and differential
susceptibility to injurious environments, as reported by various work-
ers, both in Europe and in America.
One interesting result for which no explanation is offered is that
the yolk is not used during early ontogeny by any of the forms studied.
This leaves no explanation for the source of the energy required by the
very actively katabolic process of cleavage. Little if any increase in
protoplasmic mass occurs before the yolk begins to be utilized. It is
still uncertain whether the yolk serves primarily for increase in proto-
plasmic mass, for histological differentiation, or for both, since these
predominantly anabolic processes proceed concurrently in the late
embryonic and larval periods. However, since the process of early
embryonic organization and the laying down of most of the fundaments
of the major organ systems occur before the yolk is used, it is clear that
all of the early morphological manifestations (whatever their individual
natures) proceed normally without the aid of the yolk.
LITERATURE CITED
BELLAMY, A. W., 1919. Differential susceptibility as a basis for modification and
control of early development in the frog. Biol. Bull., 37: 312-361.
BRAGG, ARTHUR N., 1938. The organization of the early embryo of Bufo cognatus
as revealed especially by the mitotic index. Zeitschr. f. Zellforsch. u.
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BRAGG, ARTHUR N., 1939. Some cytological phenomena in the embryo of Bufo
cognatus Say. Trans. Am. Micros. Soc., 58: 357-370.
BRAGG, ARTHUR N., 1939a. Observations upon the ecology and natural history of
Anura. I. Habits, habitat, and breeding of Bufo cognatus Say. Am.
Nat. (in press).
DERRICK, G. ETHEL, 1937. An analysis of the early development of the chick by
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GRAPER, L., 1929. Die Primitiventwicklung des Hiihnchens nach stereokinemato-
graphischen Untersuchungen, kontrolliert durch vitale Farbmarkierung
und verglichen mit der Entwicklung anderer Wirbeltiere. Arch. f. Entw.-
mech., 116: 382-429.
HYMAN, LIBBIE H., 1921. The metabolic gradients of vertebrate embryos. I.
Teleost embryos. Biol. Bull., 40: 32-72.
HYMAN, LIBBIE H., 1926. The metabolic gradients of vertebrate embryos. II.
The brook lamprey. Jour. Morph., 42: 111-141.
HYMAN, LIBBIE H., 1927. The metabolic gradients of vertebrate embryos. III.
The chick. Biol. Bull., 52: 1-32.
JONES, ROY W., 1939. Analysis of the development of fish embryos by means of the
mitotic index. V. The process of early differentiation of organs in Fundulus
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MINOT, C. S., 1908. The Problem of Age, Growth, and Death. Putnam, N. Y. C.
MORGAN, T. H., 1906. Experiments with frog's eggs. Biol. Bull., 11: 71-92.
PASTEELS, J., 1936. Etudes sur la gastrulation des vertebres meroblastiques. I.
Teleosteens. Arch, de Biol., 47: 205-308.
284 ARTHUR N. BRAGG
RICHARDS, A., 1935. Analysis of early development of fish embryos by means of
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FURTHER EXPERIMENTS ON THE DECOMPOSITION
AND REGENERATION OF NITROGENOUS
ORGANIC MATTER IN SEA WATER1
THEODOR VON BRAND, NORRIS W. RAKESTRAW
AND CHARLES E. RENN
(From the Woods Hole Oceanographic Institution, Woods Hole, Mass.]
In an earlier paper we reported (1937) that the cycle of decom-
position and regeneration of nitrogenous organic matter in sea water
can be reproduced experimentally. The main stages in this cycle are :
living organism — dead organism — ammonia — nitrite — nitrate — living
organism. In view of the importance of nitrogenous material in the
economy of the sea, it seemed worth while to carry these experiments
somewhat further, and especially to consider the following questions:
1. Is it possible to reproduce more than one cycle in the same
water?
2. Can the cycle be made shorter by eliminating certain stages?
3. Do successive cycles differ significantly in character or in rates
of development?
4. In what ways do anaerobic and aerobic decomposition differ?
5. How completely can the changes observed in the different forms
of nitrogen be accounted for in terms of each other; in other words,
how constant is the quantitative balance?
METHODS AND MATERIAL
The plan of the experiments was the same as in our previous in-
vestigation. Organic material was suspended in sea water in large
carboys and allowed to decompose in the dark, during which time
chemical analyses were made periodically. Artificial cultures of
Nitzschia Closterium were chosen as a source of organic matter, for our
previous experience had shown these diatoms to be more satisfactory
from an analytical standpoint than mixed plankton. Waksman,
Stokes and Butler (1937) also used them successfully for a somewhat
similar purpose. The diatoms were separated from the culture medium
by centrifugation, washed several times with nitrate-free sea water, and
finally suspended in a carboy of sea water which had been filtered
through No. 4 sintered-glass. All experiments were carried out at the
1 Contribution No. 222 from the Woods Hole Oceanographic Institution.
285
286 VON BRAND, RAKESTRAW AND RENN
uncontrolled room temperature, varying from 15° C. in the winter
to 25° in the summer. Before samples were removed for analysis the
carboys were shaken vigorously to distribute the suspended matter
evenly. When the latter showed any tendency to stick to the glass
it was loosened with a rubber-tipped glass rod before shaking, after
the rubber had been carefully cleaned to avoid contamination.
The methods for the determination of particulate nitrogen, am-
monia, nitrite and nitrate were the same as those used in our previous
experiments and have already been described in detail.
THE POSSIBILITY OF CONSECUTIVE CYCLES
In our previous investigation we found that the nitrate resulting
from plankton decomposition could be regenerated into diatom
protoplasm. This raised the question of the possibility of repeating
such a cycle of decomposition and regeneration more than once in the
same water. When the original plankton has undergone decomposi-
tion and the nitrate stage has been reached the water is inoculated
with fresh Nitzschia. After about a week in the light an abundant
growth is always observed. The nitrate drops to a minimum and
when placed in the dark this newly-developed plankton undergoes
decomposition again. In Series 12 three complete, successive cycles
were carried out in this way. In another similar series, for which the
data are not given, two cycles were completed, and in several others
a part of the second cycle.
A poor diatom growth was observed after the second cycle in
Series 12 (Table I), in which unidentified algae developed, among the
Nitzschia.
It was important to know if a regeneration of phytoplankton
material is possible in parts of the decomposition cycle other than the
nitrate stage. During the plankton decomposition in Series 23,
when the ammonia had reached its maximum, but before nitrite or
nitrate had appeared, a portion of the water was transferred to another
carboy (23A). After inoculation with Nitzschia and a week's exposure
to light the ammonia had almost entirely disappeared and a heavy
diatom growth had occurred. In a similar way Series 19A was re-
moved from No. 19 before the nitrate had reached its maximum.
Here, too, diatoms developed rapidly and abundantly and the soluble
nitrogen compounds disappeared almost quantitatively. At these
stages, at least, no toxic substances had been formed, or deficiencies
developed, which could inhibit the development of phytoplankton.
"Short-cuts" in the nitrogen cycle can evidently take place, and the
possibility of their occurrence in nature suggests an explanation for
ORGANIC DECOMPOSITION AND REGENERATION
287
Series 12.
Quiet, in dark.
TABLE I
Woods Hole harbor water. Fresh culture of Nitzschia Closterium.
Micrograms of nitrogen per liter.
Date
Particulate
Ammonia
Nitrite
Nitrate
+
Nitrate
Total
nitrogen
Diatom
count
V 103
Nitrite
detd.
/N »v/
per ml.
7- 5-37
256
31
0
14
14
301
174
7
225
65
0
14
14
134
11
107
215
0
12
17
92
0
19
18
3
23
78
0
11
11
<1
30
49
260
0
11
11
320
<1
8- 5
46
1
11
10
<1
13
45
5
25
19
<1
20
40
210
105
110
5
360
<1
24
70
27
41
30
265
365
0
435
<1
9- 2
31
13
350
355
5
402
12
390
395
5
25
25
370
390
20
10-28
37
61
0
305
305
413
31
Reinoculated with diatoms and put in light
11- 7
137
13
780
16
455
21
0
2
2
478
16
Put in dark
21
706
12- 1
164
40
24
59
0
2- 1-38
1
8
7
4
247
24
189
246
1
(440 ±)
5- 6
0
70
350
280
29
0
6-25
142
12
1
300
300
455
7- 5
Reinoculated with diatoms and put in light
12
408
80
80?
(490 ±)
15
10
18
489
0
0
40
40
529
18
Put in dark
*
29
304
115
0
25
25
445
8- 5
190
175
4
18
14
387
12
202
110
45
50
5
362
19
196
20
150
155
4
371
25
208
10
170
160
0
388
31
237
0
200
(160)
0
437
9-13
260
18
0
300?
300?
578
* Dissolved organic nitrogen = 370.
288
VON BRAND, RAKESTRAW AND RENN
N PER LITE
- PARTICULATE
AMMONIA
-NITRITE
•^-NITRITE
:^r
s-s-s
82
3 5' P
-PARTICULATE
-PARTICULATE
AMMONIA
^NITRITE
NITRATE
ORGANIC DECOMPOSITION AND REGENERATION 289
the rapid succession of great numbers of phytoplankton organisms
belonging to different species. In nature, all stages of the cycle must
be taking place simultaneously, and the momentary picture is simply
one of equilibrium.
CHEMICAL OBSERVATIONS
Decomposition of P articulate Nitrogen
The particulate nitrogen is that contained not only in diatoms,
but also in bacteria and miscellaneous debris. This material began
to decompose rapidly in all cases, as soon as the water was placed
in the dark. It never disappeared entirely, however, but reached
an apparently constant level after periods varying from two to six
weeks. This was true not only in the first decomposition cycle but
also in the subsequent ones. This residual material is very resistant
to further decomposition, and consequently the level of residual
particulate nitrogen is higher after each successive decomposition
cycle. In the first cycle of Series 12, for example, the residual particu-
late nitrogen was about 40 7 per liter, in the second cycle about 150 7,
and in the third 200 7. These amounts are about 16 per cent, 33 per
cent and 41 per cent of the particulate nitrogen present at the begin-
ning of each respective cycle. It seems not unlikely that under natural
conditions in the sea the plankton is incompletely decomposed, and a
large part of the particulate nitrogen found by von Brand (1938)
in the deeper levels may be contained in such resistant or slowly
decomposing plankton and bacterial residues. The occurrence of
bacteria and debris in this resistant fraction is indicated by the fact
that the quantity of particulate nitrogen does not consistently follow
the diatom count. While the sum of particulate and ammonia nitrogen
is fairly constant during the first part of the decomposition cycle, the
diatom count falls off much more rapidly than does the particulate
nitrogen. Nevertheless, in nature this refractory residue cannot be
entirely resistant to decomposition, otherwise the insoluble nitrog-
enous material, in the water or on the bottom, would increase without
limit.
A curious irregularity was observed in Series 25, which was aerated
by a constant stream of pure air. After about six weeks of normal
decomposition, accompanied by the appearance, first of ammonia
and then of nitrite, the particulate nitrogen rose abruptly to its initial
value and the ammonia and nitrite disappeared entirely. A micro-
scopic examination, carried out for us by Dr. Lois Lillick, showed the
presence of an enormous number of bacteria and a few flagellates. The
diatoms had disappeared completely. This phenomenon must be
290
VON BRAND, RAKESTRAW AND RENN
attributed to the development of a peculiar bacterial flora, since we
did not observe it in any other series, including No. 24, which contained
the same water and plankton as No. 25 but differed only in not being
agitated with an air-stream during the decomposition.
FIG. 2. Series No. 23 (above) and 23A (below). The disappearance of par-
ticulate nitrogen and the simultaneous changes in ammonia, nitrite and nitrate,
plotted against time. Source of organic matter, fresh culture of Nitzschia Closterium
suspended in sea water from the Sargasso Sea. The shaded area represents a re-
generation period, with the culture in the light; the remainder, decomposition in
the dark.
DAYS 25
50
Series No. 24
25 50
Series No. 25
25 50
Series No. 26
FIG. 3
The disappearance of particulate nitrogen (P) and the simultaneous changes
in ammonia and nitrite in cultures of Nitzschia Closterium in sea water from Woods
Hole harbor. The three cultures were identical at the start. Series No. 24 stood
quietly in the dark. Through No. 25 a continuous stream of purified air was
bubbled. The air was completely removed from Series No. 26 and a continuous
stream of purified hydrogen bubbled through it.
Ammonia
During the first decomposition cycle, in all cases, ammonia ap-
peared in the water rapidly and in such amounts as to exclude the
possibility of soluble nitrogen compounds intermediate between
particulate nitrogen and ammonia. This but confirms our previous
findings. But in the second cycle, although the particulate nitrogen
disappeared at about the same rate, ammonia was not formed as
ORGANIC DECOMPOSITION AND REGENERATION 291
rapidly as in the first. In Series 19A, for example, ammonia appeared
only after 30 days of decomposition in the dark, during which time the
particulate nitrogen had diminished by 160 7 per liter. The same
behavior is found in Series 23A and in the second cycle of No. 19,
and very likely also in the second cycle of Series 12, although the data
are incomplete in the latter. These cases indicate the formation of
soluble nitrogen compounds of higher molecular weight, intermediate
between dead protoplasm and ammonia. Although there are two or
three possible explanations for this lag in ammonia formation, we are
not yet inclined to urge the acceptance of any one of them. It is also
to be observed that the third cycle of Series 12 resembles the first in
its more rapid rate of ammonia appearance.
In the first cycle it generally required from 16 to 25 days for the
ammonia to reach its maximum, where it remained for a period of
from 21 to 50 days before dropping. A notable exception, however,
occurred in Series 22 and 23, in which the ammonia did not appear
until about the forty-eighth day, required three to four months to
reach its maximum, and remained for another two months before
disappearing entirely. This unusually long duration of the ammonia
stage would seem to be connected with the source of the water in the
experiments. That used in Series 22 came from the Caribbean, and
that in Series 23 from the Sargasso Sea, while in all other cases the
water was taken from near the shore. Since the diatoms in all experi-
ments were from persistent cultures the bacterial flora introduced
with them was presumably constant, but whether the difference in
decomposition is due to differences in the bacterial flora of the water
itself or to such properties of the water as might influence the growth
of bacteria, is not yet clear.
The lag in the oxidation of ammonia to nitrite in Series 22 and 23
was apparently not due to the absence of the necessary bacteria,
for portions of these cultures were inoculated with 1 ml. from Series
12, in the midst of its nitrite stage, without any resulting change in
the rate of nitrite formation.
Nitrite
In the first cycle nitrite began to appear in 31 to 58 days, corre-
sponding to the beginning of the disappearance of ammonia. It
reached its maximum when the ammonia had dropped to a minimum;
that is, in a period of from 46 to 74 days. The total duration of the
nitrite stage of the first cycle was quite irregular: two months from
beginning to end in Series 12, but more than six months in Series 13
(See Table II). The lag in the latter case may in some way be related
to the continuous aeration.
292
VON BRAND, RAKESTRAW AND RENN
Our data concerning nitrite in the second cycle are too scattered
to tell whether or not the rate of nitrite formation is the same in the
second cycle as in the first. In the third cycle of Series 12 nitrite, like
ammonia, developed rapidly.
TABLE II
Series 13. Woods Hole harbor water. Fresh culture Nitzschia Closterium
in dark, with ammonia-free air bubbling through. Micrograms of nitrogen per liter.
Date
Particulate
Ammonia
Nitrite
Nitrate
+
Nitrite
Nitrate
Total
nitrogen
detd.
Diatom
count
X 10'
per ml.
7- 8-37
269
43
1
11
10
234
10
380
11
0
17
17
407
274
13
115
0
14
280
74
19
109
240
0
11
11
360
2
24
86
300
0
11
11
<1
31
60
310
0
11
11
381
<1
8- 5
53
1
11
10
<1
13
29
0
11
11
<1
20
25
400?
0
9
9
434
<1
24
360
27
23
330
3
10
7
365
<1
9- 2
34
350
30
'35
5
419
12
330
340
10
25
15
330
350
20
10-28
7
82
345
(310)
?
11-18
321
2- 1-38
20
272
(260)
?
24
74
20
232
5- 6
0
400?
400?
6-25
120
25
1
7-15
118
74
7
230
225
422
7-18
Put in light, not reinoculated
8- 2
97
20
4
200
200
<1
4
Reinoculated with diatoms
10
389
9
0
10
10
408
*
* No diatoms present; unidentified algae.
Nitrate
Nitrate begins to appear only when nitrite disappears, and this
never seems to happen as long as a significant amount of ammonia
remains. Since one cannot rely upon a greater accuracy than 10
per cent in the analytical determination, any quantitative balance is
uncertain when large amounts of nitrate are involved.
ORGANIC DECOMPOSITION AND REGENERATION
293
TABLE III
Series 19. Woods Hole harbor water, collected 2-25-38. Fresh culture Nitz-
schia Closterium. Standing quiet, in dark. Micrograms of nitrogen per liter.
Date
Particulate
Ammonia
Nitrite
Nitrate
+
Nitrite
Nitrate
Total
nitrogen
deter-
mined
Diatom
count
X 10»
per ml.
2-25-38
316
26
12
0
10
10
338
275
5- 6
41
12
25
13
6-25
71
50
87
285
200
406
7- 6
15
48
5
15
12
1
350
(Series
350
; 19A sepa
414
rated)
7-18
Reinoculated with diatoms and put in light
•
29
17
8- 2
396
0
0
10
10
406
56
8- 2
Put in dark
10
451
0
0
15
15
466
240*
16
262
10
0
20
20
292
220f
23
201
30
0
15
15
246
30
304?
15
1
20
19
339
9-13
193
70
0
Series 19.4. Portion separated from Series 19 on 7-6-38.
with diatoms and put in light.
Reinoculated
7- 6
12
382
5
12
12
0
15
15
409
475
12
Put in dark
18
0
3
25
22
350
21
396
7
0
10
10
413
29
252
5
0
15
15
272
5
8- 5
221
90
0
15
15
326
12
141
175
0
10
10
326
20
208
186
0
10
10
404
25
187
250
0
15
15
452
31
201
250
0
20
20
471
9-13
171
250
0
10
10
430
* Few Nitzschia; mostly Skeletonema Costatum.
t Both Nitzschia and Skeletonema.
ANAEROBIC DECOMPOSITION
In addition to the experiments already described, which were
carried out under aerobic conditions, the anaerobic decomposition of
diatoms was also studied in two series. The carboys containing the
water and diatoms were first evacuated to remove all the air from the
water and the container. Then a slow, continuous stream of purified
294
VON BRAND, RAKESTRAW AND RENN
hydrogen was bubbled through the water. After decomposition in
the dark for some time a strong odor of hydrogen sulfide was observed.
In both cases the particulate nitrogen diminished very slowly, but
remained constant at a level very much higher than that in the aerobic
decompositions. The diatom counts also remained high; in fact,
when Series 26 was discontinued, after 10 weeks, living diatoms were
still found, which grew when placed in fresh culture medium.
TABLE IV
Series 22. Water from the Caribbean Sea. (18°-35' N; 79°-14' W); one year
old. Fresh culture Nitzschia Closlerium. Quiet, in dark. Micrograms of nitrogen
per liter.
Date
Particulate
Ammonia
Nitrite
Nitrate
+
Nitrite
Nitrate
Total
nitrogen
deter-
mined
Diatom
count
X 103
per ml.
3-18-38
123
0
0
125*
213
5- 6
25
0
10
10
6-25
41
113
12
10
0
166
7- 6
115
12
13
22
15
118
12
25
13
8- 3
30
150
11
12
1
191
13
145
12
(Series 22B separated)
23
23
150
16
20
4
193
30
30
125
30
30
0
185
9-13
20
90
(150±)
Series 22B. Portion separated from Series 22 on 8-13-38 and inoculated
with 1 ml. from Series 12. In dark.
8-13
145
12
20
205
22
25
3
160
30
210
36
35
0
245
9-13
220
50
(270±)
* Dissolved organic nitrogen = 93.
Ammonia also increased in this series for the first three weeks,
and then gradually disappeared. In the other series no appreciable
amount of ammonia was formed; on the contrary, a small amount of
that originally present disappeared. This ammonia was not recover-
able from the effluent hydrogen, nor are we able as yet to account for
the behavior of ammonia in either of these series. As might be ex-
pected, no nitrite was formed.
After about two months under anaerobic conditions a portion
from one of the cultures was aerated and kept henceforth aerobically.
ORGANIC DECOMPOSITION AND REGENERATION 295
Two months later a large amount of ammonia had been formed, but
no nitrite or nitrate had appeared by the time the experiment was
stopped.
QUANTITATIVE BALANCE
In our previous investigation we pointed out that in nearly every
case studied the total determined nitrogen in the system (that is, the
sum of the particulate nitrogen, ammonia, nitrite and nitrate) in-
creased continuously throughout the period. In the cases we are now
reporting the quantitative balance is much more satisfactory. In
Series 13, 22, 23, 24 and 25 the changes in total nitrogen are small and
probably explainable in terms of accumulated errors. In Series 12,
19, 19A, 23A and one other, the increase observed is too large to be
accounted for in this way, but is less, relatively, than the increase
noted in our first experiments. Previously, we discussed three pos-
sible explanations for this increase in total nitrogen : systematic errors
in the determination of particulate nitrogen, the participation of
dissolved organic nitrogen in the decomposition, and nitrogen fixation.
We were in no position to prefer any one of these explanations. The
fact that a good nitrogen balance was observed in half of our later
experiments, including one which extended over more than a year,
seems definitely to eliminate the possibility of systematic errors. The
difficulty of determining dissolved organic nitrogen with any accuracy
makes it almost impossible to test the second hypothesis directly.
(The one determination given in the data for Series 12 was made, with
some difficulty, by the method of Krogh, 1934.) However, we sought
to investigate this question indirectly, by trying to see whether am-
monia appears, on standing, in water devoid of gross particulate
matter, and if so, whether the process is related to the content of
dissolved organic matter. Two filtered samples of water, one from
Woods Hole harbor and one from the Sargasso Sea, were placed in the
dark and the usual determinations made periodically. Previous work
had shown that the harbor water had a higher organic nitrogen con-
tent than water from the open sea, but there was no difference in the
behavior of the two kinds of water. A small increase in nitrogen,
about 60 7 per liter, was observed in both. This could indeed be the
result of the participation of dissolved organic nitrogen, but we are
inclined to believe that it is due to an entirely different cause. All
the stored carboys were tightly stoppered, but before the removal of
samples they were vigorously shaken and opened. During this time
the water comes into contact with a rather considerable quantity of
air, from which it may take up ammonia. To test this possibility,
clean filtered air was aspirated for 12 hours through a sample of
296 VON BRAND, RAKESTRAW AND RENN
sterilized Sargasso water. This resulted in an increase of 50 7 of
ammonia-N and 20 7 of nitrate-N. Such a quantity of ammonia is
not surprising, in view of the amount of decomposing organic matter
in this laboratory and its vicinity. It seems quite possible, therefore,
that at least a large part of the increase in total nitrogen observed in
some of our experiments may be due to contamination from the air.
Nevertheless, this is not conclusive, and we are still in no position to
exclude the possibility of either nitrogen fixation or participation of
dissolved organic nitrogen. Atmospheric contamination was ruled
out in Series 13 and 25, through which purified air was aspirated, and
in these cases, indeed, the total nitrogen did not increase. Still, no
increase was observed in Series 22, 23 and 24, which were not aerated,
but which stood side by side with carboys in which nitrogen accumu-
lated. Further work is still being done in the effort to clear up these
discrepancies.
SUMMARY
1. Several consecutive cycles of decomposition and regeneration
were carried out in the same water.
2. It is confirmed that in the first cycle the main stages of decom-
position are: dead body — ammonia — nitrite — nitrate. In the second
cycle there is evidence of intermediate soluble substances between
dead body and ammonia.
3. Under anaerobic conditions the initial states of decomposition
take place more slowly than under aerobic conditions, and no nitrite
or nitrate is developed.
4. Regeneration of nitrogen into phytoplankton protoplasm is
possible not only in the nitrate stage but also in the ammonia stage
and before the nitrate has reached its maximum.
5. The quantitative nitrogen balance was better than that reported
in previous experiments, and possible reasons for the discrepancies
still present are discussed.
BIBLIOGRAPHY
VON BRAND, T., N. W. RAKESTRAW, AND C. E. RENN, 1937. The experimental
decomposition and regeneration of nitrogenous organic matter in sea water.
Biol. Bull., 72: 165-175.
VON BRAND, THEODOR, 1938. Quantitative determination of the nitrogen in the
particulate matter of the sea. Jour, du Conseil, 13: 187-197.
KROGH, AUGUST, AND ANCEL KEYS, 1934. Methods for the determination of
dissolved organic carbon and nitrogen in sea water. Biol. Bull., 67: 132-144.
WAKSMAN, S. A., J. L. STOKES, AND MARGARET R. BUTLER, 1937. The relation of
bacteria to diatoms in sea water. Jour. Mar. Biol. Ass'n., 22: 359-373.
PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS PRE-
SENTED AT THE MARINE BIOLOGICAL LABORATORY,
SUMMER OF 1939
JULY 5
The effect of biologically conditioned medium upon the growth of Colpidium
campylum. G. W. Kidder.
The effects of metabolic products upon a population has been the subject of
many investigations in the past. Decreased growth rate and population yield due to
the accumulated products of metabolism have been reported for many organisms,
especially micro-organisms. On the other hand, growth acceleration has been noted
in many instances and the effect ascribed to substances given off by like or different
species.
Recently we have been interested in making comparisons between some of the
phases of protozoan growth and those which have been reported for the bacteria and
yeasts. In these studies it has been possible to utilize many of the techniques
employed by the bacteriologist due to the fact that the species of protozoa used were
all bacteria-free and growing in non-particulate broth. The following results were
obtained from studies on the ciliate Colpidium campylum.
Manipulation of the conditioned media (2 per cent proteose-peptone-1 per cent
dextrose broth) was as follows: control — plain, fresh broth; conditioned — broth in
which Colpidium had grown for varying lengths of time and the ciliates centrifuged
out; and filtered conditioned — conditioned medium which had been passed through
a Seitz bacteriological filter. All experiments were performed with the three types
of media run in parallel series, at 26° C.
Growth in control flasks (inocula taken from the log phase) exhibited typical
logarithmic growth for 28 to 40 hours, depending upon the size of the inoculum.
After a very short "negative growth acceleration" period the curve tends to level off
and remains at a relatively high level for some weeks. Medium conditioned 60
hours and then inoculated with log ciliates shows a large and significant acceleration
during the early growth period. Negative growth acceleration occurs sooner than
in the control and the maximum yield is never as great. Filtered medium which
has been conditioned 60 hours produces a decided lag phase, indicating inhibition in
the early periods of growth. Maximum yield is similar, however, to that in the
conditioned (supernatant of a centrifugate). Increasing the length of the condition-
ing period lowers the curves for both the conditioned and the filtered conditioned
media. Slight acceleration in the former was obtained, however, after 4 weeks of
conditioning, while the lag period of the latter was increased. Acceleration over
the growth of the control was not found after 8 weeks of conditioning although the
difference between this curve and that produced in the filtered 8 weeks conditioned
was still of the same magnitude as those previously described.
From the results of these experiments it might be said that it appears that
substances are released into the medium by growing ciliates which produce a decided
effect upon subsequent growth of fresh ciliates. We can think of these substances as
falling into two categories, one a growth inhibitor which will pass through an asbestos
filter and the other a growth accelerator which will be absorbed on the filter. When
both are present together (as in the supernatant of a centrifugate) the accelerator
masks the effect of the inhibitor during the early growth phases. When the acceler-
ator has been removed (filtrate) the inhibitor action is marked. There appears to
297
298 PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS
be a differential as to time of production as the accelerator becomes increasingly less
effective as time of conditioning increases while the inhibitor becomes more effective.
Until more work is completed upon these very interesting substances the above
analysis can only be a conjecture.
Respiration in Chilomonas paramecium. John Hutchens.
The rate of consumption of oxygen and the respiratory quotient of chilomonads
grown in sterile, pure, mass cultures in a medium containing CH3COONa, NH4C1,
(NH4)2SO4, K2HPO4, MgCl2, and CaCl2 were measured at 25° C., using simple
Warburg respirometers. The rate of consumption of oxygen was found to vary
inversely with the age of the culture from which the organisms were taken, and also
with the strain of chilomonads used. The respiratory quotient was found to vary
directly with the rate of oxygen consumption, i.e., inversely with the age of the cul-
ture. The following table compares the rate of oxygen consumption and the respira-
tory quotient of chilomonads from two different strains at different times following
inoculation of the cultures.
The relation between the rate of consumption of oxygen by chilomonads
and the respiratory quotient
Age of
culture
in hours
Strain of
chilomonads
Rate of consumption of
oxygen in cu. mm. per
10,000 chilomonads
Respiratory
quotient
24
2
0.40
0.93
24
1
0.35
0.91
48
2
0.24
0.80
48
2
0.23
0.81
48
2
0.23
0.79
48
1
0.17
—
72
2
0.17
0.75
72
2
0.17
0.74
72
1
0.12
Temperature and starch and fat in Chilomonas paramecium. Jay A.
Smith.
Starch and fat in Chilomonas are contained in particles, and by proper staining
(Lugol's solution for starch and Sudan III for fat) the size of each particle and the
total volume of each substance can be ascertained.
Temperatures from 9.5° C. to 39° C. were used.
The solution, which contains inorganic salts and sodium acetate, was that
employed by Mast and Pace and co-workers.
Within viable temperatures, it was found that the volume of starch remains
constant, that the volume of fat decreases as the temperature rises, but that the
volume of boih starch and fat synthesized by the progeny of one chilomonad during
a period of 24 hours increases greatly. This indicates that the rate of synthesis
within the viable temperature range may be the factor that controls the frequency of
division.
At a lethal low temperature (9.5° C.) there is no division, the volume of starch
gradually decreases, but the volume of fat increases, which indicates that in the
absence of synthesis of starch, starch is transformed into fat.
At lethal high temperatures (32.5°-39° C.) the frequency of division decreases as
the temperature rises, and there is an accumulation of starch and fat that reaches a
maximum at 35° C. and then decreases. This indicates that the rate of synthesis
PRESENTED AT MARINE BIOLOGICAL LABORATORY 299
of starch and fat increases in the same manner that it did at viable temperatures,
and thus the accumulation of starch and fat is due to two interacting factors: the rate
of synthesis and the period the chilomonads live.
Thus, the decrease in the frequency of division at lethal high temperatures is
probably caused by the same factors that cause death, but there is no relation
between the death, the frequency of division and the rate of synthesis of starch
and fat.
JULY 11
The differentiation of isolated rudiments of the Amblystoma punctatum
embryo. Floyd Moser.
Using the technique which has been developed in Harrison's laboratory, a number
of structures associated with the embryo of Amblystoma punctatum have been isolated
and cultured in vitro under nearly aseptic conditions. The embryos were generally
in Harrison's Stage 29 at the time of operation, though in some cases Stages 27, 28
and 30 were also used. Among the structures isolated were the balancer rudiment,
gill rudiment, limb rudiment and tail bud. During the 10 or 11 stages immediately
following operation the explants keep pace both in rate and degree of differentiation
with the intact structures in unoperated control animals. This, while it is itself of
some interest, takes on more special significance in view of the fact that the explants
are entirely free of nervous and vascular connections. The tail bud explants alone
are exceptions to the latter statement in that they doubtless are well provided with
nervous structures.
Typically, the isolation of the ectoderm of the gill rudiment yielded nothing that
was gill-like in appearance, while the ectoderm plus the underlying layers of meso-
derm gave rise to a single gill. Isolations of gill rudiment consisting of ectoderm,
mesectoderm, mesoderm and entoderm gave rise to three gills, which in external
appearance, were not unlike those of the unoperated control animals.
Explanted balancer rudiment consisting of ectoderm and the underlying layers
of mesoderm and entoderm gave rise to what appeared to be perfect balancers, but
these were no better than those obtained from isolated balancer ectoderm alone.
Limb rudiment isolations consisted of the mesoderm and its overlying sheet of
ectoderm. As differentiation of the explanted structure takes place, it becomes
possible to tell whether the rudiment has come from the right or the left side because
of the presence of characteristic surface contours.
When the unoperated controls reach Stages 38 to 39, the isolated tails exhibit
function in the sense that they twitch when stimulated by means of slight pressure
with a hair-loop.
The present experiments, as well as others involving the fragmentation and
fusion of rudiments, and experiments in which isolated rudiments of various ages
have been grafted back to host embryos, will be reported at greater length elsewhere.
The production of duplicitas cruciata and multiple heads by regeneration
in Planaria. Robert H. Silber.
The observations reported were taken from a paper published in Physiol. Zool.
(vol. 12, No. 3, July, 1939, p. 285) by Robert H. Silber and Viktor Hamburger and
entitled: "The production of duplicitas cruciata and multiple heads by regeneration
in Euplanaria tigrina."
Neural differentiation without organizer. L. G. Barth.
Previous experiments have shown that the amphibian ectoderm in the gastrula
stage may very easily be stimulated to form a neural plate. This suggested that
under certain conditions the ectoderm might form a neural plate without the organizer
300 PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS
or any external stimulus. It was first found that the fusion of two to eight explants
of ectoderm would develop with neural tubes, while a single explant formed only a
mass of epidermis.
Following this a number of fusions between two pieces were made, some with
the antero-posterior axis coincident and some reversed. When the antero-posterior
axis of the two explants coincides a neural tube differentiates but when the axes are
reversed only epidermis results. Thus the polarity of explants must be preserved in
order that a neural tube may form without an organizer.
The neural tube differentiates from the anterior end of the explant since when
two explants are united by their anterior ends a neural tube appears in the middle.
Further when an anterior half explant is fused with the anterior end of a whole explant
the neural tube forms at the end. Other fusions also show that the anterior end
forms the neural tubes. There is then an antero-posterior polarity or gradient in
the isolated ectoderm and this polarity must be maintained in order to obtain neural
tubes without organizer.
This antero-posterior gradient exhibits itself further by differences in oxygen
consumption. The roof of the blastocoel was cut into four parts along the antero-
posterior axis from the dorsal lip to the ventral epidermis. The results show that the
oxygen consumption is high in the anterior pieces of ectoderm and low in posterior
pieces. The dorsal lip respires at about the same rate as the anterior end of the
ectoderm.
JULY 18
The effect of substrate concentration on the cyanide sensitivity of the oxygen
consumption of yeast. Kenneth C. Fisher.
This paper has already appeared in the Proc. Am. Physiol. Soc. under the title,
"The sensitivity of the oxygen consumption of yeast to cyanide " (Am. Jour. Physiol.,
126, pp. 491-492).
A comparison of cyanide and azide as inhibitors of cell respiration.
C. W. J. Armstrong.
This paper was published by C. W. J. Armstrong and Kenneth C. Fisher in the
Proc. Am. Physiol. Soc. under the title: "The effect of sodium azide on the frequency
of the embryonic fish heart" (Am. Jour. Physiol., 126, p. 423).
The relation of blood to the respiratory ability of fresh water fish . Laurence
Irving.
Fresh water which is habitable for fish may be well or poorly oxygenated and
may contain CO2 at practically zero or at a quite noticeable pressure. The properties
of the blood for transport of oxygen also vary. Oxygen dissociation curves for the
blood of seven common fish at 15° in the absence of CO2 are similar and differ only
in ease of combination with oxygen. The Po2 necessary for half saturation defines
the position of the curve for each fish and is as follows: catfish 1.4 mm.; carp 5 mm.;
bowfin 4 mm.; common sucker 12 mm.; brown trout 20 mm.; brook trout 22 mm.;
rainbow trout 22 mm.
Carbon dioxide increases the Po2 necessary for oxygenation. If the increase in
Po2 for half saturation be divided by the Pco2 which effects that change, then the
quotient distinguishes the blood of each fish. The quotients are: catfish 0.3; carp 1.0;
sucker 3.5 ; brook, brown and rainbow trout 6.0. The effect of CO2 on half saturation
with oxygen indicates the influence upon unloading of oxygen from blood into the
tissues.
Carbon dioxide also reduces the ability of blood to combine with oxygen at
150 mm. pressure of oxygen. The curves relating oxygen contained at Po2 = 150
PRESENTED AT MARINE BIOLOGICAL LABORATORY 301
mm. with increasing Pco2 are similar for the various fish and reach limits at Pco2 = 60
mm. At these pressures of CO2 the fraction of the oxygen capacity remaining in
the blood is as follows: catfish 1.00; carp 0.90; yellow perch 0.77; sucker 0.58; chain
pickerel 0.54; brook trout 0.50; rainbow trout 0.48; brown trout 0.50; lake trout
0.40. The species take the same order on the basis of either ease of saturation with
oxygen or magnitude of CO2 effect.
The effect of CO2, which must facilitate unloading of oxygen into the tissues,
should also hamper oxygenation if CO2 is present in the water passing over the gills.
The ability of the fish to utilize oxygen from water is cut down by CO2 and in the
same order for the species as the order of the CO2 effect. Trout are unable to
utilize oxygen in the presence of much CO2, but catfish tolerate large pressures of
CO2. While the -Pco2 necessary to prevent completely the utilization of oxygen is
larger than would occur naturally, the sensitivity of trout to CO2 is such that some
naturally occurring conditions would hamper oxygen utilization and hinder or
prevent respiration in such water. In this manner it is shown that the specific
properties of blood of fish which facilitate respiratory transport under some condi-
tions would serve as definite barriers to existence of the fish under other conditions.
JULY 25
The relation between fermentation and respiration in higher plants
(The Pasteur Effect).1 David R. Goddard.
The experimental material was thin slices of cortical root tissue of the carrot
(Daucus car ota). All measurements of gaseous exchange were conducted in the Fenn
micro-respirometer at 22° C.
It was found that carrot root respiration was strongly inhibited (75-85 per cent)
by 10~3 M HCN or NaN3 and about 65 per cent by 95 per cent CO. The CO in-
hibition was reversed by light. The partition coefficient of the oxidase for CO and O2
was 9. The results are strong evidence that the major part of the respiration is
catalyzed by cytochrome oxidase. Since these same poisons at the above concen-
tration (and 100 per cent CO) did not inhibit fermentation in carrot, these poisons
may be used to separate fermentation and respiration in the carrot under aerobic
conditions.
Unpoisoned carrot tissue did not show any aerobic fermentation, but the
anaerobic fermentation was high. These results demonstrate the existence of the
Pasteur effect. As the O2 pressure is lowered the rate of O2 consumption begins to
fall at about 5 per cent, but fermentation does not occur until 2| per cent or less.
In fact, a very considerable inhibition of respiration (about 45 per cent) may be
obtained before fermentation begins. With decreasing respiratory rate, the rate of
fermentation increases. Aerobic fermentation may be obtained by poisoning
respiration with HCN or NaN3, but no fermentation occurs until the respiratory
inhibition is 45 per cent or greater. In the presence of CO aerobic fermentation also
occurs, and this effect may be overcome by light. These experiments indicate that
the mechanism of oxygen inhibition of fermentation is through cytochrome oxidase.
In the carrot root it has been impossible to poison the Pasteur effect, that is, to obtain
aerobic fermentation without respiratory inhibition, with low O2 tensions, HCN,
NaN3, or ethyl carbylamine. These results are all consistent with Meyerhof's
oxidative resynthesis theory as an explanation of the Pasteur effect. They do not
prove this theory. The above experiments are consistent with Lipmann's explanation
only in the special case that the oxygen oxidation of the fermentation enzymes is
catalyzed by cytochrome oxidase.
1 The results reported here are based on experiments of Mr. Paul Marsh and the
author; and a full report will appear shortly in the Amer. Jour. Bot.
302 PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS
The role of bacteria in the fouling of submerged surfaces. Claude E.
ZoBell.
Studies on the sequence of events in the fouling of submerged surfaces reveal that
bacteria are the predominating primary film formers. Within an hour or two after
the immersion of clean glass slides in the sea, bacteria begin to attach thereto.
Some of the bacteria are so tenaciously attached that they resist dislodgement when
the slides are washed under running water during the staining procedures, while
others are only loosely associated with the primary film. The number of bacteria
increases geometrically with time until their abundance together with the simul-
taneous and subsequent attachment of other organisms and detritus defeat census
attempts. The adsorption and accumulation of organic matter on the submerged
surfaces which has been demonstrated by chemical as well as by biological procedures
is believed to account for the attachment and development of bacteria, although there
are other ways in which solid surfaces promote the growth of bacteria in dilute
nutrient solutions.
Barnacles, hydroids, bryozoa, tunicates, algae and other fouling organisms
attach to submerged surfaces coated with films of bacteria more readily than to
bacteria-free surfaces. Bacteria might promote the fouling of submerged surfaces
(a) by affording the larval forms of larger fouling organisms a foothold or otherwise
mechanically facilitating their attachment, (6) by serving as food, (c) by discoloring
bright or glazed surfaces, (d) by increasing the alkalinity of the film-surface interface
thereby favoring the deposition of calcareous cements, (e) by influencing the e.m.f.
potential of the surface or (/) by increasing the concentration of plant nutrients at
the expense of the organic matter which the bacteria decompose.
Cell division and differentiation in living plant meristems . E. W.
Sinnott.
A paper containing the material given in this talk is to be published by E. W.
Sinnott and Robert Bloch under the title: "Changes in intercellular relationships
during the growth and differentiation of the living plant tissues," in the Am. Jour.
Botany for October, 1939.
AUGUST 1
The ionic permeability of frog skin as determined with the aid of radioactive
indicators. Leonard I. Katzin.
The rate of penetration of ions through the excised skin of the frog was measured
using radioactive isotopes as markers for the salts which had passed through. Radio-
active sodium (as the chloride) was used alone and in mixtures with inactive potassium
chloride; radioactive rubidium, also in the form of the chloride, was used alone and in
mixture with inactive sodium chloride. Diffusion was measured for two-hour periods
between isotonic salt solutions bathing the inner side of the skin, and distilled water on
the outside. The results are expressed in terms of the permeability to the pure salt
in each case, and parentheses used to indicate extrapolated or estimated values.
Fraction NaCl 1.0 0.75 0.67 0.50 0.25 0.10 0.0
Permeability 1.0 1.0 1.1 1.15 1.6 1.8 (2.0)
Fraction RbCl 0.0 0.25 0.33 0.50 0.75 1.0
Permeability (0.5) (0.5) (0.65) 0.66 0.7 1.0
The absolute rates of passage for the pure salts are 4.7 X 10~12 mols cm."2 sec."1 for
sodium chloride and 125 X 10~12 mols cm.-2 sec."1 for rubidium chloride. Values for
the permeability ratio of RbCl and NaCl vary between 11 and 15, but are essentially
constant. These results indicate that KC1 (and probably RbCl) exert small influence
PRESENTED AT MARINE BIOLOGICAL LABORATORY 303
on the permeability level of the frog skin, and that the passage of individual salts is
essentially independent. Further, the large differences in rate of passage of rubidium
and sodium ions indicates what has long been postulated, that the mobility of ions
through tissues is not the same as in pure aqueous solution. An expression has
been derived for the electrical resistance of the skin, from the Nernst diffusion
coefficient and the Fick diffusion equation, on the assumption that the salts studied
are the principal carriers of the electrical current. Sample calculations show again
that there are differences in the ion mobility through the tissue.
Crystallization of myogen from skeletal muscle. Kenneth Bailey.
The main globulin component of skeletal muscle, myosin, is now well charac-
terized. The separation of the remaining components, Weber's globulin X and the
albumin myogen, has depended upon fractionation by dialysis, a procedure unsuited
to many tissue proteins which denature in an ion-free solution. An attempt has
been made to crystallize myogen by two methods: (1) The perfused, freshly excised
minced muscle from rabbit is extracted with 10 per cent KC1 at pH 7, and the solution
after nitration through pulp is treated with ammonium sulphate which is dialyzed
in through a collodian bag at room temperature. The precipitated myosin is filtered
off when the specific gravity of the solution reaches 1.14. The solution is now brought
to a pH of 6 by addition of sulphuric acid, and more ammonium sulphate is fed in
until a specific gravity of 1.18 is attained; the amorphous globulin precipitates are
separated, and the sulphate concentration again increased very slowly. Between a
specific gravity of 1.18 and 1.22 the myogen separates in crystalline form together
with some amorphous material which dissolves on cautious dilution, leaving the
crystals in suspension. (2) The minced muscle is mixed with an equal volume of ice
cold water and after standing for 20 minutes the juice is expressed. This is treated as
outlined above.
The crystallization of myogen has recently been reported ' by Baranowski; two
crystal types were obtained, the one, termed myogen A, crystallizing as hexagonal
bipyramids and the other, myogen B, as long thin plates. The A form is obtained by
fractionation of muscle press juice by a procedure which involves a heat treatment at
50°, and the B form, which appears to be identical with the author's preparation,
crystallizes fortuitously from the mother liquor. The physico-chemical properties of
myogen B (purity by classical solubility methods, molecular weight, dielectric
increment, titration curve) are now being investigated.
Chemical and histochemical observations on Macracanthorhynchus
hirudinaceus. Theodor von Brand.
The females of Macracanthorhynchus hirudinaceus contain 1.13 per cent glycogen
and 0.95 per cent ether extract. Phospholipids, unsaponifiable matter and unsatu-
rated fatty acids are the chief components of the ether extract. Relatively more
glycogen than fat is stored in the body-wall, whereas the contrary is true for the
reproductive cells. Differential staining showed that the chief places of glycogen
deposition are the hypodermis, the muscles and the mature eggs, those for the fat are
the hypodermis and the ovaries.
pH reactions during feeding in the ciliate Bresslaua (Accompanied by
photomicrographs taken on Kodachrome). C. Lloyd Claff and
G. W. Kidder.
The large ciliate Bresslaua, a carnivorous member of the family Colpodidae, was
used for experiments and observations on the pH condition during various phases
of feeding and digestion. The general method was as follows: Bresslaua were treated
1 Baranowski, Zeitschr.f. PhysioL, 260, 43 (1939).
304 PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS
with dilute indicator dyes and were later fed untreated Colpoda steini, the subsequent
color changes studied under the water immersion lens. When neutral red- or methyl
red-treated animals were used the color changes were very striking while other
indicators gave less satisfactory results.
In neutral red preparations the medium, being slightly alkaline, shows a pale
yellow coloration which extends into the oral cavity. As the first Colpoda is taken
into the mouth a secondary vacuole forms in the posterior protoplasm to receive the
prey. The contents of this vacuole appears faintly pink but as the prey is trapped by
a thin sheet of protoplasm cutting the secondary vacuole off from the mouth region
the periphery of the vacuole is outlined by a collection of brilliant red granules or
droplets from the cytoplasm of the carnivore. The prey is immediately killed.
Within a few seconds the granules disappear from view. The prey does not take on
any of the red coloration at this time but slowly becomes yellow, then, as digestion
proceeds, through orange to a cherry red.
The conclusions reached were that the actual killing of the prey is brought about
by some strong acid. A pressure system was considered but ruled out by direct
observation, in that the cilia of the prey stand out very straight from the body at the
moment of killing. Methylene blue-treated Colpoda showed no color reduction,
therefore lack of oxygen as a means of killing seems improbable. The color changes
in the food masses subsequent to killing indicate that digestion is alkaline in nature
and that the final residuum shifts back to an acid state.
AUGUST 8
The dielectric properties of insulin solutions. J. D. Ferry.
The material contained in this talk will appear shortly in Science under the title:
"Studies in the physical chemistry of Insulin. I. The solubility and dielectric
properties of insulin and its crystallization with radioactive zinc," by Edwin J. Cohn,
John D. Ferry, J. J. Livingood, and Muriel H. Blanchard.
The effects of lack of oxygen, and of low oxygen tensions, on the activities
of some Protozoa. J. A. Kitching.
A comparative investigation was undertaken of the effects of lack of oxygen, of
low oxygen tensions, and of some respiratory narcotics, on the activity of Protozoa.
The organisms were suspended in a thin hanging drop, and oxygen-free hydrogen or
nitrogen (purified over hot platinized asbestos or hot copper), or hydrogen mixed
with oxygen in known proportions by means of flow meters, were flushed continually
via lead tubing with seals of de Khotinsky cement through the observation chamber.
The purity of the oxygen-free gas was checked by the extinction of luminescence of
marine luminous bacteria and by mass spectrographic analysis — methods sensitive to
about 0.005 mm. of oxygen and to one part in 10s respectively.
In the peritrich ciliate Cothurnia kellicottiana pure hydrogen caused an almost
immediate stoppage of the contractile vacuole and cilia, and a swelling of the body;
very often the body blistered. Return to air led to an immediate recovery of the
vacuole to a rate of output at first much above the normal. The cilia resumed their
beat, and the body slowly shrank to its original size or less. A partial pressure of 1.1
mm. of oxygen was not enough to allow any vacuolar activity; at 1.6 mm. the con-
tractile vacuole stopped but recovered slightly after the body had swollen; and in 3
mm. there was full activity. In dilute cyanide there was little or no activity of the
contractile vacuole.
In fresh-water amoebae of the "proteus" type the contractile vacuole quickly
ceased all activity in absence of oxygen, or in cyanide; although amoeboid movement
continued for some hours. Recovery in air from lack of oxygen was rapid. Amoeboid
movement and the activity of the digestive vacuoles of the marine amoeba Flabellula
PRESENTED AT MARINE BIOLOGICAL LABORATORY 305
mira were rapidly and reversibly inhibited in absence of oxygen. Some measure of
recovery from lack of oxygen was obtained at 0.3 mm.
Paramecium spp. continued swimming for some time in the absence of oxygen,
but eventually stopped and cytolysed. The best survival, namely 12 hours, was
obtained in culture medium and with sufficient carbon dioxide added to the hydrogen
or nitrogen to maintain the pH at a reasonable value. Anaerobic survival was much
shorter in dilute phosphate buffer, or for starved animals. Some recovery from lack
of oxygen was obtained in 0.3 mm. oxygen.
No correlation could be found between the degree of sensitivity of Protozoa to
cyanide and the critical oxygen tensions at which these organisms were just able to
maintain some activity.
Nerve asphyxiation and aerobic recovery in relation to temperature.1
Herbert Shapiro.
The sciatic nerve of the Hungarian bullfrog, R. esculenta, was mounted on 3
platinum electrodes for stimulating, and 2 calomel recording electrodes in an all glass
chamber, which could be immersed in a Dewar flask and thus kept at constant
temperature over long periods. Moistened purified nitrogen or hydrogen was passed
through the chamber, and test stimuli from a commutator permitting condenser
charges and discharges through the nerve at any desired frequency, were applied at
regular intervals. The duration of the tetanus was controlled with a Lucas contact
breaker. The total action current was integrated ballistically by a Zernicke Zd
galvanometer of 4-second period. During anaerobiosis, the response of the nerve to a
standard stimulus gradually decreases, and finally is extinguished. At higher
temperatures "overshoot" occurs during early anoxia. After positivity disappears
earlier than action current, but injury potential was never completely abolished
during the experiment. Continuous tetanization shortens asphyxiation time. Time
for asphyxiation of action current is an exponential function of temperature, requiring
approximately an hour at 38° C., and about 1 150 minutes at 0° C. The form of the
function may be described by the Arrhenius equation, yielding a temperature charac-
teristic of 11,100 calories. Evidently the nerve utilizes energy from an anaerobic
reaction for setting up and conducting action currents, and this reaction runs to
completion at a rate determined by the temperature. Upon readmission of oxygen
to the nerve, action current, injury potential and after positivity showed recovery.
Rate of recovery of action current also conformed to the Arrhenius equation with a M
value of 28,000 calories. Examination of Amberson's data for temperature effect on
the absolute refractory period of nerve shows a ju value of 18,400. Underlying
chemical reactions for conduction, refractory period, and recovery in nerve proposed
by Gerard are such as to suggest three different types of reaction, and hence different
temperature characteristics. Though the latter by themselves do not identify the
reactants involved, it is of interest that the present study indicates chemical bases for
these several aspects of nerve activity, with different enzyme systems acting as the
controlling links.
Effects of hydrostatic pressure upon certain cellular processes. D. A.
Marsland.
The experiments demonstrate that pressure induces a solation of protoplasmic
gels in a number of different cells and in an inanimate gel prepared of the myosin of
rabbit muscle. In each of the cells studied, the solation is associated with a re-
tardation of movement — amoeboid movement, the pinching of the cleavage furrow,
and the streaming of plant cells.
1 This work was done during the tenure of a fellowship of the John Simon
Guggenheim Memorial Foundation.
306 PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS
In order to see cells while under compression, it was necessary to construct a
special chamber. This chamber permits objects within it to be viewed at a magnifica-
tion of 600 diameters at pressures up to 600 atmospheres.
The pressure effects upon the rigidity of the cellular gels were measured by the
centrifugal method. A centrifuge head was constructed which permits centrif ugation
during the period in which the cells are under compression.
When amoebae are suddenly exposed to a relatively high pressure (above 450
atmospheres) a peculiar reaction suddenly occurs. Each of the extended pseudopodia
collapses and tends to round up. This result would be expected if the plasmagel,
which supports the pseudopodia, were to undergo liquefaction. If the pressure is
maintained for a few minutes, the whole cell becomes spherical. However, when the
pressure is released, active amoeboid movement begins again within a minute or so.
Centrifuge experiments demonstrate that the form of the amoeba is related to
the structural properties of the plasmagel. When the amoebae are centrifuged under
pressure, the liquefaction is indicated by the rapidity with which the granular
components of the plasmagel undergo displacement. In the higher range of pressure,
the rigidity of the plasma gel is reduced to a small fraction of the normal value. In
this range of pressure, no pseudopodia may be sustained. In the lower range,
pseudopodia may be formed, but they display a graded diminution of diameter and
length as the pressure is increased.
Comparable results were obtained in studies of cleaving Arbacia eggs and the
streaming of the leaf cells of Elodea. The retardation of the furrowing parallels the
loss of rigidity of the gelated cortex of the egg, and the streaming of the Elodea cell is
slowed in proportion to the degree of liquefaction which occurs in the non-flowing
part of the protoplasm. Thus it appears that sol-gel reactions are providing a
machine by which the cell can transform chemical potential energy into mechanical
work.
AUGUST 15
On the nature of the material elaborated by fertilizable Nereis eggs inducing
spawning of the male. Grace Townsend.
The observations of Lillie on the spawning reaction of Nereis limbata led to the
formation of the "fertilizin theory" (Just, 1930). Lillie found evidence that the
material inducing spawning of the male came only from fertilizable eggs.
I have re-investigated the relation of the spawning inducing material to fertiliza-
tion and have found it to possess properties in common with material essential to egg
activation though not necessarily associated with the phenomenon of egg and sperm
union.
Egg-cell activation may plausibly involve processes common to all species and be
based on the same processes as may initiate cell division in any tissue. Corre-
spondingly, spawning was induced by extracts of many fresh tissues from many
species, all of which contain glutathione. Crystalline pure glutathione in one part in
a million in a single drop quantity, and the molecular constituent, cystine or cysteine,
in higher concentration, induced the natural spawning reaction.
Marine eggs are rich in glutathione (300-700 mg. per 100 gm. wet weight) and
it is especially concentrated in the germinal vesicle. A reducing substance passes
from Nereis eggs. The reaction of the male to glutathione, by a large variety of tests,
was indicated to be extremely specific.
All substances found to destroy the spawning inducing property of egg-water and
glutathione inhibit fertilization.
PRESENTED AT MARINE BIOLOGICAL LABORATORY 307
Properties of Egg- Water Spawning Inducing Material and Glutathione and
"Fertilizin" and Its Described Fractions
Analysis of properties:
Egg-
Water
Spawning-
Inducing
Material
Glutathione
Woodward's
Lime's
Fertilizin
Partheno-
genetic
Fraction
Agglutinin
Fraction
Elaboration from egg . .
+
+
+
+
+
+ + +
+ + +
+ + +
+ + +
+ + +
+
+
4-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+ + +
+ + +
+ + +
+
+
+
+
+
+
+
+
+
+
+
+
+
Xanthoproteic test ...
Stability in acid
Stability in alkali
Precipitation by acetone .
Berkfeld filter
Dialysis
Inactivation by aging
Inactivation by boiling ins. H2O.
Inactivation by irradiation
Inactivation by Au, Cu, Ni, etc. .
Inactivation by CH2ICOOH ....
Inactivation by IvCN
Inactivation by blood, coelomic
fluid
Inactivation by cytolyzed eggs. .
Inactivation by cytolyzed tissues
Induction of spawning
Ovum and spermatozoon age at the time of fertilization and the course of
gestation and development in the guinea pig. W. C. Young.
An abstract of this paper has already been published in the Anat. Rec. by Arnold
F. Soderwall, William C. Young and Richard J. Blandau, under the title : "Spermatozoa
vitality in the genital tract of the guinea pig" (Anat. Rec., vol. 73, Suppl. 2, p. 47,
1939).
Experiments on the production of haploid salamanders. Cornelius T.
Kaylor.
The eggs of many species of amphibians can be induced to begin their develop-
ment with only one set of chromosomes. This would then produce haploid embryos
and larvae.
In spite of the large number of experiments which have been performed on the
production of haploid amphibians (see review of Fankhauser, Jour. Hered., 28, 1937),
the results have varied with the species used, the degree of abnormality of embryos
produced, and with the extent of development. So far as demonstrated, only one
completely haploid amphibian has been reared to a stage approaching sexual maturity
(Baltzer and Fankhauser, 1922). This is in striking contrast to the fact that normal
haploid animals exist in nature, as well as to the fact that viable haploid plants have
been produced experimentally.
The present experiments were undertaken primarily to test, by new methods,
with the eggs of species which have not been used extensively before, the possibilities
of advanced haploid development in these species. Two species of newts were used:
Triturus viridescens and Triturus pyrrhogaster. The female chromosomes were
removed from the egg with a small pipette. All subsequent development then took
place by means of the male chromosomes.
308 PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS
In over 200 experiments with the eggs of T. viridescens, only about 15 per cent of
the embryos developed beyond the gastrula stage. In 76 experiments with
pyrrhogaster eggs, about 30 per cent of all embryos developed to stages ranging from
a neurula to a 120-day-old larva. This larva, however, was not completely haploid.
Another larva, which in external appearance resembles all haploid larvae which have
been produced in other experiments, was fixed at 47 days of age, at a time when
hind limb buds had appeared. It had a deformed jaw and could not take food.
It appears, then, that for the purpose of obtaining advanced haploid larvae in
these amphibians, the eggs of Triturus pyrrhogaster are much more adaptable than
eggs of Triturus viridescens.
Regulation in mosaic eggs. Alex B. Novikoff.
Transplantation experiments on the eggs of Sabellaria vulgaris by the author l
have demonstrated that the materials in the polar lobe which are involved in the
formation of the apical tuft and the post-trochal region do not diffuse from the
transplanted lobe into adjacent cells. But there were some indications that when the
polar lobe material was incorporated into a cell the course of development of the cell
was altered.
A 7.5 per cent solution of 2.5 Normal KCL in sea water inhibits cleavage in the
eggs of Sabellaria. Eggs are placed into the solution after the second maturation
division, and kept there until the controls have completed the first cleavage. Upon
returning them to sea water, as many as 90 per cent of the eggs develop into perfect
double embryos. These embryos possess two eye spots, two sets of posttrochal
bristles, two posterior cilia, two sets of dorsal cilia, two neurotrochs, two intestines,
one central stomach, probably one oesophagus, one mouth, and two mouth folds.
At least some of the cells in these larvae have developed into structures which
they do not form in normal development. Thus the prospective potency of these
cells is revealed to be wider than the prospective fate. This is a characteristic
generally associated with regulative eggs.
Eggs placed in the KCL solution after the completion of the first cleavage until
the controls have completed the second cleavage do not develop into double embryos.
Instead larvae which possess extra bristles, or extra eye spots, or both, are produced.
The formation of double embryos suggests that the material of the first polar
lobe has been distributed to each of the first two blastomeres where it 'organizes' in
each a new embryonic axis.
AUGUST 21
Micro ma nipulative studies. (Motion picture). Robert Chambers.
Living cells in action (motion pictures'). C. C. Speidel.
Cine-photomicrographs of the fast motion type have been taken of many kinds of
cells. The pictures are made directly from living frog tadpoles and they reveal
characteristic cellular movements and reactions under normal and experimental
conditions.
The pictures include examples of the growth, migration, mitosis, and differenti-
ation of connective tissue cells, epithelial cells, vacuolated sub-epidermal cells,
endothelial cells of blood and lymph capillaries, sheath cells, regenerating spinal cord
cells, pigment cells, and various kinds of leucocytes. A complete record of nerve
regeneration over a period of a month is given, including the stages featured by growth
cones, sheath cells, and myelin segments.
Case histories are also presented to show the changes in position from day to day
of the relatively stable cutaneous nerve endings which belong to myelinated fibers.
These include examples of extension, retraction, irritation, autotomy, and new growth
cone differentiation following loss by phagocytosis. Several cases are given which
., 1938, 74, p. 211.
PRESENTED AT MARINE BIOLOGICAL LABORATORY 309
reveal how red blood cells that have been extruded from blood vessels are engulfed by
macrophages.
Various types of behavior of localized contraction nodes in single muscle fibers
(from Palaemonetes leg and Limulus heart) are also presented. These include their
formation, progression, splitting, reflection, collision, and dissipation; also their
progression past thin clots resembling intercalated discs.
Other pictures (obtained with the cooperation of Dr. Ethel Harvey) show the
early developmental history of the sea urchin, Arbada, including the immature egg,
mature egg just before and just after fertilization, segmentation stages from 1 to 64
cells, free-swimming gastrula, and pluteus. Other pictures show abnormal cleavages
of centrifuged eggs and of the clear halves of centrifuged eggs.
Polariscopic pictures reveal the birefringent substances in pigment cells, epithelial
cells, muscle fibers during contraction and relaxation, and in the developing eggs and
larvae of Arbada.
AUGUST 22
Studies on the life history of Spelotrema Nicolli. R. M. Cable and
A. V. Hunninen.
Metacercariae occurring in the blue crab, Callinectes sapidus, were fed to six
young herring gulls which were killed and examined 12 hours to 18 days later. Each
bird yielded a large number of adult Spelotrema nicolli while six controls were negative
for this species. Since very young metacercariae indicated clearly that the cercaria
is of the Ubiquita type, an extensive search was made for such a larva in mollusks
occurring where infected crabs were abundant. After many unsuccessful attempts,
a cercaria of the type sought was found in the snail, Bittium alternatum. This cercaria
was found to enter the crab by way of the gills, passing with the circulation to
strands of muscle-like tissue where encystment occurred. The morphology of various
stages in the life history of S. nicolli has been studied in detail; the excretory
formula of the cercaria is 2[(1 + !) + (! + 1)], the metacercaria and adult,
2[(2 + 2) + (2 + 2)].
Stabilizing action of alkaline earths upon crab nerve membranes, as
manifested in resting potential measurements. Rita Guttman.
Resting potentials of the non-myelinated nerve of the proximal segment of the
claw or of the first walking leg of the spider crab, Libinia canaliculata, were measured
by means of a potentiometer and null point galvanometer. All solutions used were
approximately isotonic with sea water and pH was controlled.
It was found that the alkaline earths, in themselves, have no effect upon the
magnitude of the potential. Yet they are able to prevent the usual depressing
action of KC1 upon the potential when the alkaline earth and KC1 are simultaneously
applied to the nerve. Solutions containing two parts of BaCh to one part of KC1,
five parts of SrCU to one part of KC1, and eleven parts of CaCh to one part of KC1
are threshold values for the neutralization effect. The order of effectiveness of the
alkaline earths for counteracting the depression of the potential by KC1 is thus Ba,
Sr, Ca. This is the order in which these elements appear in the atomic table.
The alkaline earth, Ba, is also capable of preventing the depression of the resting
potential by various lipoid-soluble, surface active, highly polar substances, viz.,
veratrine sulphate, chloral hydrate, iso amyl urethane, sodium salicylate and saponin.
The neutralization effect of the alkaline earths may last over a period of many
hours. The alkaline earths are capable of preventing the action of depressants
strong enough to cause, when present alone, a decrease of potential of fifty per cent
or more.
310 PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS
A direct relationship was found between the amount of depression of the potential
per unit time caused by veratrine sulphate and the logarithm of the concentration of
veratrine sulphate acting. Although a concentration effect is obtained with both K
and veratrine sulphate, it is not necessary to assume that the action of these sub-
stances upon the nerve is identical. It should be noted that the relative concentra-
tion of KC1 (one part isotonic KC1 to eleven parts sea water, or 0.04 M) necessary to
depress the potential is of a much greater order of magnitude than that of the organic
substances (0.00004 M veratrine sulphate in sea water).
Two possible explanations for the neutralizing action of the alkaline earths are:
(1) change of effective pore size in a sieve-like membrane by the alkaline earths or
(2) alteration of the partition coefficients of the depressants by the alkaline earths.
There is at present no basis for deciding between these alternative concepts. The
phenomenon is certainly, however, not one of antagonism in the classical sense of
Jacques Loeb, inasmuch as, for one thing, the quantities of alkaline earth necessary
are much too large.
An experimental study of the pigment granules of the Arbacia egg. D. L.
Harris.
The pigment granules are isolated by breaking eggs in 0.35 m Nas citrate to
avoid the presence of Ca. Unbroken cells are removed by centrifuging. It is found
that the granules are actually vacuoles. They are easily deformed, and regain
spherical shape when external force is removed. In the presence of Ca, Mg, and Sr;
hypotonic solutions; and isotonic solutions of urea, acetamide, and ethylene glycol
they discharge pigment. Moreover, in Ca, Mg, and Sr solutions, contiguous granules
coalesce to form fluid vacuoles containing small granules in active Brownian move-
ment.
The discharge of pigment in hypotonic solutions was investigated quantitatively.
The number of granules present at various concentrations of Nas citrate was deter-
mined by counting. It was found that there was much more extensive breakdown
at low concentrations. Furthermore, there is a normal distribution of granules in
respect to ease of breakdown.
In the absence of Ca, Mg, or Sr, the pigment outside of the granule turns greenish
brown and finally green. A suspension of granules in Nas citrate treated with hypo-
tonic solutions will therefore change color from pink to green. The amount of
granule breakdown may be determined by comparing the color in a given hypotonic
suspension with standards prepared by making mixtures of pink suspension and green
suspension. This method gives results in agreement with those of the counting
method.
The rate of granule breakdown in various hypotonic concentrations was de-
termined by measuring the time to reach a given color, i.e., a given amount of
breakdown. Using eight end-points, it was found that as the concentration was
decreased the rate of reaction increased. This is clear evidence that the granules
behave as osmometers. It is concluded that the granules are vacuoles surrounded
by a semi-permeable membrane, and that they do not constitute osmotic dead space
within the cell.
The action of calcium on muscle protoplasm. L. V. Heilbrunn.
The complete paper is in press and will appear shortly in Physiol. Zool.
PRESENTED AT MARINE BIOLOGICAL LABORATORY 311
GENERAL SCIENTIFIC MEETINGS
AUGUST 29
The mechanism of membrane elevation in the egg of Nereis. D. P.
Costello and R. A. Young.
Certain agents may induce the formation of a very wide perivitelline space in
the Nereis egg. Alkaline NaCl (pH 10.5) was used by D. P. Costello, and X-rays
(8,800-41,300 r) were used independently by R. A. Young.
When placed in alkaline NaCl the uninseminated egg elevates the vitelline mem-
brane (occasionally double), and the width of the perivitelline space increases until
the membrane ruptures, setting free the ovum. It was demonstrated by means of
a Chinese ink suspension and by microneedles, that there is no jelly external to the
intact membrane, but that jelly fills the perivitelline space and escapes with the
ovum upon membrane rupture. Attempts to inseminate such eggs after return to
sea water have been unsuccessful. Alkaline NaCl is a parthenogenetic activating
agent. If normally inseminated eggs are subsequently treated with alkaline NaCl,
the passage of cortical jelly through the membrane ceases, and a perivitelline space
appears of width inversely proportional to the jelly already extruded. Protoplasmic
cone-shaped filaments, temporarily adhering to the elevating membrane, are gradually
retracted, except the one to which the activating spermatozoon is attached. The
spermatozoon passes through the membrane 7-10 minutes after the egg is introduced
into alkaline NaCl. The penetration of the sperm into the treated egg takes place
three times as rapidly as under normal conditions, and can be followed with un-
precedented clarity.
If Nereis eggs are treated with X-rays, and inseminated after 5-10 minutes,
a similar, but much slower, response is induced. The width of the perivitelline
space is directly proportional to the dosage, whereas the extrusion of jelly through
the membrane is inversely proportional to dosage. In these eggs the sperm frequently
does not complete the penetration process.
The colloidal nature of the perivitelline material is demonstrated by collapsing
the elevated membrane against the egg surface with a solution of gum arabic in
sea water.
It is suggested that exaggerated membrane elevation of the Nereis egg may be
obtained by agents which (1) initiate the outflow of the cortical jelly precursor
(if the egg has not been previously inseminated), and which (2) alter either the
vitelline membrane or the jelly in such a way that passage of the jelly through the
membrane is completely or partially prevented.
Determination and induction of the anuran olfactory organ. Edgar
Zwilling.
By means of large transplants of presumptive head ectoderm from gastrulae of
varied ages to the flank of older urodele embryos it was established that the olfactory
organ of Rana pipiens is determined (i.e. would self-differentiate) before the neural
folds are present. This determination probably occurs in the small yolk-plug stage.
The presumptive olfactory material of pre-neurula stages and of early neural plate
stages self-differentiated in the absence of brain tissue. Since it was possible that
the presumptive brain tissue might act laterally upon cells in the same layer and so
be responsible for the determination, the brain tissue was tested for its inductive
capacity. Forebrain material from various stages during neurulation was trans-
planted beneath the epidermis of the flank region of various host stages. In only one
case did olfactory material develop; and since this operation was an heteroplastic
one (to Rana palustris) it could be determined that the olfactory tissue was of donor
origin. The anterior portion of the roof of the archenteron was then implanted into
312 PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS
the blastocoeles of older gastrulae to test its inducing power for this structure. Of 19
surviving cases 5 developed small but perfect heads — which had olfactory organs.
One of the others developed an olfactory organ in the absence of brain. Of 9 controls
(where forebrain of the open neural plate stage was implanted into similar gastrulae)
one developed an olfactory canal. The origin of this tissue cannot be established with
certainty since this was an homoplastic graft.
A method of determining the sex of Arbacia, and a new method of producing
twins, triplets and quadruplets. Ethel Browne Harvey.
To determine the sex of Arbacia, inject a few drops of a saturated solution of
NaCl in sea water into the mouth; then with a fine pipette inject a little of the same
solution into one genital pore. A few eggs or a little sperm will exude only from the
opening injected, easily distinguishable by color. If thrown immediately into a jar
of still sea water, the animal will stop shedding, and the gonads remain normal and
intact. If the animal does not respond to NaCl, a molar solution of KC1 can be
used ; this is more drastic.
To obtain twins, place the eggs from which fertilization membranes have been
removed in a solution of 30 grams of NaCl per liter of sea water for 5 to 10 minutes,
immediately after first cleavage ; then remove to sea water. The first two blastomeres
are nicely separated, but connected by a thin strand, and they develop independently.
The blastulae at first swim in pairs and can easily be isolated. Some pairs gave
both normal plutei, some both abnormal, and some, one normal one abnormal.
In lots of 10 pairs, development was variable, but in one lot all but one became perfect
or almost perfect plutei. Quadruplets are obtained by similar treatment just after
second cleavage; four almost perfect plutei have developed from' the first four blasto-
meres in some cases. Triplets are obtained when two of tbr blastomeres of the
4-cell stage develop independently, and the other two togethe
y
An artificial nucleus in a non-nucleate half-egg. Etn'ci Browne Harvey.
The non-nucleate half -eggs of Arbacia punclulata, obtained by centrifugal force,
have been injected (by W. R. Duryee) with yeast nucleic acid and thymus nucleic
acid. The injected drop of fluid does not disperse through the cytoplasm, as does
sea water, but remains distinct in a small drop or vacuole surrounded .y a film or
membrane, resembling strikingly the appearance of the nucleus in th- living cell.
The possibility of supplying the non-nucleate eggs with chemical mate -\\s found in
the normal nucleus opens a new line of investigation in the effort to c use the par-
thenogenetic merogones (i.e. activated non-nucleate egg fractions) to df elop beyond
the blastula. Nucleic acid, nucleo-proteins, adenine, guanine, icil, auxins,
tobacco mozaic virus and many other substances, added to the mediu.i >efore, during
and after centrifugation have been found to have no effect.
Color responses of catfishes with single eyes. G. H. Parker.
As a rule fishes with only one eye respond by color changes to differences in their
environment as successfully as do those with two eyes. In this respect the trout has
long been known to be peculiar for on the loss of one eye it darkens contralaterally.
The common catfish, Ameiurus nebulosus, when deprived of one eye conforms neither
to the general rule for fishes nor to the special one for the trout. A one-eyed catfish
is at first very dark, after which it may change slowly in tint according to its environ-
ment though without ever becoming fully pale. Such fishes may finally assume in
the same environment somewhat different tints and retain these with considerable
individual persistence. This diversity appears not to be due to variations in the
irritability of the orbital wound which might influence to various degrees the stump
of the optic nerve. The cause of these more or less characteristic color differences is
unknown. In these respects one-eyed catfishes are unlike any other fishes thus far
PRESENTED AT MARINE BIOLOGICAL LABORATORY 313
described. The fact that intermedin, the secretion from the pituitary gland, plays a
very important part in the color changes in catfishes, and that in this fish chromato-
phoral nerves, both dispersing and concentrating, are less significant than the
pituitary gland, may be the occasion of the difference between Ameiurus and most
other teleosts whose chromatophores are often under almost exclusively nervous
control.
A vibration sense in a swarming annelid. Grace Townsend.
During sexual metamorphosis of Nereis limbata a vibration sense becomes highly
developed which equips the worms for orientation in rapid swimming. The sense
enables the worVns, typically, to avoid striking solid objects, to relate themselves to
smooth surfaces and to the opposite sex. Circling can be induced by the vibration
sense alone and the sense doubtless supplements the chemical sense (Lillie and Just,
1913) in the spawning integration of this species. The marked development of the
sense is associated with the metamorphosis of the dorsal and ventral cirri.
During metamorphosis the cirri become elongated beyond the surrounding
parapodial structures and equipped with budding elevations, or processes, along the
inner surfaces bordering the parapodial lobes. The elevations are filled with nerve
cells which connect with clusters of turgid cytoplasmic hairs.
If the cirri are clipped from the parapodia of the metamorphosed worms (with
use of iridectomy scissors), the worms lose their characteristic orientation to surfaces
and swim unrelated to surfaces as does the non-sexual phase.
The sensory sti ictures of the dorsal and ventral cirri are part of the lateral line
system of annelids < laborately described for the Capitellidae by Eisig (1879) and for
all groups by Jeenei (1928). Treadwell (1900) describes a striking sensory organ on
the dorsal cirrus of a swarming palolo (Eunice auriculata) . No work has been done
as to the general fi 'tional significance of the system. Stolte (1932) observed that
the dorsal cirri of ( 'era are not stimulated by chemicals or tactile stimuli but are
stimulated by slight i echanical oscillations of the structures.
The plausibility of a generally distributed vibration sense in annelids possessing
a pelagic phase is suggested.
Food habits of Endomoeba muris. D. H. Wenrich.
These studies were made on prepared smears of the caecal contents of rats and
mice, most ; • fixed with Schaudinn's fluid and stained with iron alum haematoxylin.
Endamaeba muris feeds on a great variety of food materials including various
kinds of ba'teria, Blastocystis, yeasts, filamentous organisms, starch grains, Tricho-
monas, Giai^jp,, Hexamitus, leukocytes and erythrocytes. On the same slide some
individuals i ; (,y be engorged with one kind of food while others will be engorged with
different kin" ' ; still others may contain a variety of food materials. There is a
tendency for the amoebae from one particular host to show food preferences different
from those from some other host. For example, on one set of slides about 60 per cent
of the amoebae contained a certain kind of colonial bacteria, while on another set
of slides about 75 per cent showed one or more individuals of Trichomonas muris in
their food vacuoles.
In ingesting different kinds of food objects, E. muris employs somewhat different
methods. Fairly large starch grains are apparently surrounded by slowly advancing
pseudopodia on all sides in close contact with the grain, but one pseudopodium may
be further advanced than the others. The edges of the advancing pseudopodia
often stain intensely with iron alum haematoxylin. Some food cups were surrounded
by a dense layer of cytoplasm which sometimes did and sometimes did not stain
intensely. Ingestion cones were extended along plant filaments, which became
coiled inside the cell. In most cases an ingestion cone included a definite pharynx-
like structure with deeply-stained walls and annular thickenings. Similar pharynx-
like tubes formed without protruding cones were employed in ingesting Trichomonas
314 PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS
muris. Comparable tubes were also found deep in the cytoplasm apparently being
used to break up food masses into smaller units.
A quantitative study of the hemopoietic organs of young albino rats. J. E.
Kindred.
A study has been made of the differential cell counts and mitosis rates in the
parotid lymph nodes, spleen, thymus and bone marrow of 15-day-old and 20-day-old
albino rats. These data have been used in conjunction with differential cell counts
of the blood and with the volumes of the several organs in an attempt to find out
what degree of relationship exists between cell production and differentiation in the
organs and the blood. Other investigators have suggested that in order to meet
the demands of granulocytopoiesis and erythrocytopoiesis, the bone marrow must
filter out small lymphocytes from the blood stream. The present data show that the
lymph nodes and spleen are deficient in the production of small lymphocytes needed
for the growth of these organs between the 15- and 20-day stages. On the other
hand, the bone marrow and thymus produce an excess of small lymphocytes during
this period. The excess small lymphocytes more than balance the needs of the
lymph nodes, spleen and blood. The principal reason for the excess production of
small lymphocytes (hemocytoblasts) in the bone marrow is an actual decrease in
numbers with time, whereas in the thymus, the excess is caused by a very high mitosis
rate in the peripheral zone of the cortex.
On the histology of the mammalian carotid sinus. William H. F.
Addison.
The carotid sinus is the dilated beginning of the internal carotid artery at the
bifurcation of the common carotid artery into the internal and external carotid
arteries. The wall of the carotid sinus has the three layers characteristic of arteries,
but its structure differs from that of adjoining regions of the arteries with which it is
continuous by the presence in the tunica media of a predominant amount of elastic
tissue and by the absence or the small amount of smooth muscle. In the sinus
of the dog the elastic tissue is arranged in 8-10 coarse lamellae with collagenous
tissue interspersed. The transition in structure from the sinus to the internal carotid
artery is abrupt and there is a conspicuous difference in the organization of the
walls. The diameter of the sinus is over twice as great as that of the adjoining
internal carotid artery in the living animal. Ordinarily the carotid sinus may pass
unnoticed in the dead animal because the lowering of blood pressure at death allows
the sinus to diminish in size until it is only slightly larger than the diameter of the
internal carotid artery. A type of carotid sinus similar to that in the dog has been
seen in other mammals, e.g., newborn child, rhesus monkey and cat.
On Clark's theory of muscular contraction. Alexander Sandow.
The theory of contraction for striated muscle proposed by Clark (Am. J. Physiol.,
V. 82, p. 181, 1927) depends on the following assumptions: (1) the muscle fibers
consist of alternately arranged isotropic and anisotropic layers which run unbroken
across each fiber; (2) upon stimulation chemical changes occur which transform the
liquid crystals of the anisotropic discs into a more solid crystalline state, while the
substance of the isotropic layers remains unaltered; (3) the new relation between the
substances of the layers results in the sudden production of a tension that can be
calculated by means of the formula F = 2A T/d where F = the force per fiber,
1 = the area of the fiber cross section, T = the surface tension of the material of the
isotropic layers, and d = the thickness of this layer. Using the values for the frog
sartorius: diameter of fiber = 50 M, T = 70 dynes/cm., d = 0.7 n, Clark finds
F = 39.25 dynes, in fair agreement with the experimental value for maximal isometric
PRESENTED AT MARINE BIOLOGICAL LABORATORY 315
tension of 55 dynes (Hill, 1926). Consideration of more recent work (Hill, 1938) and
allowance for Fenn's chloride space lead to the value for F of 37.5 dynes, in striking
agreement with Clark's calculated value. Although muscle physiologists have paid
little attention to this theory, it has received some notice in physiological literature.
(Evans, 1931 ; von Muralt and Edsall, 1930; Burns, 1929; Howell, 1936; Barnes, 1937.)
Clark's theory may be questioned on general grounds: the assumed value of T is
probably too high; there is a possibility of contradiction between the predicted and
observed changes in birefringence during contraction; the assumed mechanism for
tension production is itself open to criticism; and even if it be accepted it cannot be
applied to unstriated muscle. The theory is definitely at variance with observation
in indicating that during contraction the isotropic bands shorten relative to the
anisotropic bands. But difficulties of a decisive nature arise if, conforming to the
generally accepted view (Schmidt, 1926; Hiirthle, 1931; Chambers and Hale, 1932;
von Muralt, 1933) account is taken of the fibrillar structure of the muscle fiber.
Clark's method for calculating the tension must then be applied to the individual
fibrils, and the sum of their tensions taken as the tension developed by the whole
fiber. Now, however, the previously used formula is not valid, since the diameter of
the fibril is of the same order as the thickness of the isotropic segment of a sarcomere.
The correct formula for the tension per fiber is F = 2nAT(\fd — I ID), where
n = the number of fibrils per fiber, A = the cross-sectional area of a fibril, d = the
thickness of the isotropic segment, D = the diameter of the fibril, and T is as before.
Using Clark's values for T, d, and the diameter of the fiber, and taking D = 1 fj. and n
the value for closest packing of the cylindrical fibrils, it is found that F = 10.6 dynes.
This is clearly far too low in comparison with the experimental value to support the
theory. Moreover, the assumption that d = 0.7 n is open to question. The work
of Buchthal, Knappeis and Lindhard (1936) has shown that d in the frog sartorius
may vary from 0.81 n to 1.10 /x depending on the degree of stretch of the muscle. If
d = 0.9 ju, then F = 2.8 dynes. And if d > 1 ju, or if in general d > D, then F
becomes negative indicating instability of the system and the separation of the
fibrils into discrete sarcomeres. In view of the difficulties and inadmissable impli-
cations of Clark's theory we must conclude that it cannot be accepted as a valid
picture of the mechanism of contraction of striated muscle.
Conditions governing the frequency of contraction of the heart of Venus
mercenaria. Albert E. Navez and John D. Crawford.
Two characteristics: frequency and amplitude of the beat of the excised and
perfused ventricular portion of the heart have been studied. This note is concerned
only with the frequency.
If one plots against time, the frequency of the heart perfused with non-aerated
sea-water, one obtains a curve showing a rapid rise (in about 2 hours) to a maximum
frequency followed by a slow decline to zero in about 36 hours. Perfusion with
aerated sea-water determines a frequency curve which reaches a plateau, slightly
below the maximum value and which extends over 36 or more hours before final
decline sets in. If a solution of one part of dextrose into 250,000 parts of well
aerated sea-water is used as perfusion liquid, the plateau of constant frequency may
be extended to 72 to 96 hours. The rate of perfusion is sufficient when above 20
ml./min.
Frequency is a function of load (tension) on the heart. The load determines the
time elapsing between excision and maximum in frequency curve. In general less
time is required to reach maximum frequency under greater loads. The graph of
"time of excision to time of maximum frequency " against " load " is a hyperbola with
a short induction period for loads below 30 mg. The "excess frequency " (frequency
above that of the level plateau mentioned above) is also a function of the load. The
value of the "excess" frequency is greatest at about 80-95 mg. load and is of smaller
value for loads above or below this point.
316 PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS
For a heart beating at constant frequency under a given load, any change in load
below a critical tension of about 200 mg. effects a change in frequency that is com-
pletely reversible. When the load on a heart is changed, the frequency changes
correspondingly, reaching a new value through a series of " damped oscillations " over
a period of more than an hour at room temperature. Above the critical load, changes
in frequency are not exactly reversible. On passing from 100 mg. to 250 mg., the
frequency rises; on return to 100 mg. the frequency falls decidedly below its original
value.
In all experiments reported the temperature was kept constant to within
±0.01° C.
Small variations in temperature affect decidedly the rate of contraction. Four
(or five) temperature (characteristics) have been found for a large series of hearts
studied. Critical temperatures have been found around 10°, 15°, 20-21°, and 25°;
not all appear in every heart studied.
The refractory period in the non-conducted response of striated muscle.
F. J. M. Sichel.
In the isolated skeletal muscle fibre (frog), with cut ends, the contractile mecha-
nism can be excited to a normal type of response apparently without involving the
conductile mechanism (Brown & Sichel, J. C. C. P., 8, 315, 1936; Sichel & Prosser,
Biol. Bull., 73, 293, 1937). This non-propagated response has no absolute refractory
period and the size of the response is a function of the strength of the stimulus even
though the entire length of the fibre is involved in the contraction.
During the course of an extension of these experiments in collaboration with
D. E. S. Brown use was made of the fact that twitch-like contractions without
propagation can also be obtained in the intact entire sartorius provided the KC1
content of the medium is 70 mg. per cent. If the entire muscle is stimulated under
these conditions by means of massive electrodes so placed that the electrical field is at
right angles to the longitudinal axis of the muscle, the entire muscle is involved in the
contraction. Recorded isometrically these contractions have a normal form. They
resemble the contractions of the isolated fibre preparation and differ from those of the
normal muscle in that they are essentially local and non-propagated and also in that
no refractory period is involved in thel^excitation. The absence of the refractory
period was demonstrated as in the case of the isolated fibre preparation by stimulating
with two equal condenser discharges separated by a variable time interval. For the
whole muscle stimuli of rectangular form were also used. In the isolated fibre and in
the KCl-treated muscle the second stimulus will always contribute something to the
mechanical response; in the normal muscle there is an interval, related to the absolute
refractory period, during which the second stimulus can contribute nothing. Since
the response of the KCl-treated muscle is non-propagated, its grading presumably
does not necessarily involve the frequency distribution of the fibre thresholds.
Pigment inheritance in the Fundulus- Scomber hybrid. Alice M.
Russell.
Hybridizations between Fundulus heteroclitus 9 and Scomber scombrus o* were
made successfully during June and July, 1938 and 1939 at Woods Hole. The ab-
normality of the embryos, and the appearance of paternal pigmentation in the
hybrid, as previously described by H. H. Newman, were confirmed.
A systematic re-investigation of the inheritance of pigmentation, followed by a
cytological and morphological study of the hybrid seemed worthwhile.
Development of pigmentation in normal Scomber scombrus is described for the
first time. Tables comparing the number of melanophores on embryos, and yolk, in
parents and hybrids, seem to reveal a Scomber-effect in the hybrid.
PRESENTED AT MARINE BIOLOGICAL LABORATORY 317
As regards the chromatophores, the hybrid embryos appear to fall into three
categories: those with Scomber type, those with Fundulus type, and those with both
types of chromatophores. The melanophores and chromatophores in parents and
hybrids are distinguishable: those in the hybrid being identical in color and structure
with those of the parents. However, a close and systematic study of the hybrids
reveals an enormous variability of combinations of the parental pigment cells.
Actually, no two embryos are identical in their pigmentation pattern.
This enormous variation found in the Fi generation is unusual, and at present
inexplicable. The reciprocal cross was never successfully made.
The use of the swimbladder by fish in respiratory stress. Virginia Safford.
It is generally agreed that the chief function of the swimbladder is to maintain
buoyancy in the fish. The use of oxygen in the swimbladder for respiration has been
shown by Potter for physostomous fish (J. Exp. Zool., 49, 45, 1927). Physoclistous
fish when confined in a limited volume also show gaseous exchange in the bladder
without change in external pressure on the fish.
The scup, sea robin, cunner, tautog, fundulus and toadfish (all physoclisti) were
bottled by the method of Fry and Black (Am. J. Physiol., 126, p. 497, 1939) in water
with various pressures of CO2. The water was analyzed for CO2 and O2 at the death
of the fish and the results showed a characteristic curve for each species, i.e. the
ability to use oxygen decreases with the increase in pressure of CO2. At the same
time gas samples were taken from the swimbladders and analyzed for CO2 and O2 by
the use of Krogh's micro-gas-analyzer.
The analyses showed that for these species: (1) CO2 passes freely into the
swimbladder, the fish equilibrating with external CO2 up to the point where his
ability to use oxygen in the water decreases. (2) Oxygen in the bladder is used at
low CO2 pressures in the water but the ability to use oxygen in the bladder decreases
with the rise in CO2 in a manner parallel with the ability to use oxygen from the
water.
Species differences occur in the curves showing the ability of the fish to use
oxygen from water and swimbladder in the presence of CO2. The ratio of
CO2 in the water and swimbladder is practically constant for each species, i.e.
swimbladder CO2 ... r • ., ,
- = k, up to the point where utilization of oxygen in the water de-
water CO2
creases. The values of k in these six species were not very different.
It seems, therefore, that the same sort of mechanism effects gaseous exchange
between swimbladder and blood and between water and blood in the fish under the
conditions of the experiments described.
Water permeability of Chaetopterus eggs. Herbert Shapiro.
The permeability of the egg of the annelid worm, Chaetopterus pergamentaceus, to
water was determined by allowing the egg to swell in 60 per cent sea water. The
equilibrium volumes of about thirty eggs were measured at various dilutions of sea
water, and found to conform to Boyle's law. From the equation
Po(F0- 6) = PcX(Vcq-b)
(where F0 and P0 represent respectively the egg volume, and osmotic pressure in
normal sea water, and Veq the cell volume at equilibrium in diluted sea water of
osmotic pressure Pex), the "osmotically inert volume," b, was found to be about 34
per cent of the cell volume. Individual plots were made of the kinetics of osmosis of
27 unfertilized eggs and of 21 fertilized eggs (at room temperatures, 22 to 25° C.).
The permeability constant, K, was calculated for the first, third and fifth minute of
swelling from the relationship proposed by Lucke, Hartline and McCutcheon (Physiol.
Rev., 12, 68, 1932) viz.,
dV/dt = KS(P - Peq),
318 PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS
where dV/dt represents the rate of change of egg volume at any instant, S the surface
area of the cell, P — Peg the difference in osmotic pressure between the interior and
exterior of the cell, and K the cubic micra of water passing across a square micron of
cell surface per minute per atmosphere osmotic pressure difference. The "osmotically
inert volume" of each egg was taken into consideration. These values for the
unfertilized eggs obtained by direct microscopic measurement (K = 0.5) agree with
those previously reported by Lucke, Ricca and Hartline (Biol. Bull., 71: 397, 1936)
using a diffractometer method. It was also found that the permeability of the
Chaetopterus egg to water shows little change on fertilization, the data indicating a
slight increase (K = 0.6). Whitaker (Jour. Gen. Physiol., 16: 475, 1933) has demon-
strated a decline in respiration of the Chaetopterus egg when fertilized. Thus it is
evident that the parallelism between alterations in respiration and water perme-
ability, which appeared on fertilization of the eggs of Arbacia and Asterias, is not a
universal feature of the activity of marine eggs.
A mechanism of increased cell permeability resembling catalysis. M. H.
Jacobs and A. K. Parpart.
It was shown by 0rskov that the rate of entrance of NH4C1 into the mammalian
erythrocyte is greatly accelerated by low concentrations of bicarbonates. This
effect was attributed to an increased permeability of the cell to the NH4 ion. The
following is suggested as a more plausible explanation. NH4C1 enters the erythrocyte
by the penetration of NH3 followed by the exchange of OH for Cl ions at a rate that
depends on the value of [OH]t[Cl]0 — [OH]0[C1];. This rate is relatively slow
because of the low value of [OH];. On the addition of bicarbonate a second pene-
trating molecule, CO2, is formed, which for reasons discussed elsewhere (/. Cell.
Comp. Physiol., 7, 351, 1936) leads to the accumulation within the cell of NH4HCO3
at a higher concentration than outside. An exchange of HCO3 for Cl completes the
entrance of NH4C1, the bicarbonate again being available to repeat the cycle. Be-
cause of the more favorable value of [HCO3]i[Cl]o — [HCO3]0[C1],- the entrance
of Cl under these conditions is far more rapid than before. In agreement with the
theory, two parts of the swelling curve of the erythrocyte may be distinguished, the
first, which involves only undissociated molecules, being little affected by butyl
alcohol, the second, which depends on an ionic exchange, being strongly retarded.
Even without alcohol two parts of the curve are apparent if cyanide or sulphide be
substituted for bicarbonate. The ineffectiveness of acetates etc. seems to be due
both to a slower entrance of free acid and to a less ready ionic exchange. The
addition of bicarbonate to ammonium citrate gives only the first part of the swelling
curve, since there is no penetrating anion externally for which HCO3 can be ex-
changed. For reasons discussed previously (loc. cit.) even the initial swelling is
absent when borates are used.
Oxygen consumption and cell division of fertilized Arbacia eggs in the
presence of respiratory inhibitors. M. E. Krahl, A. K. Keltch, and
G. H. A. Clowes.
For fertilized eggs of Arbacia punctulata initially exposed to the reagents at 30
minutes after fertilization at 20° C., the levels of oxygen consumption prevailing in
the minimum concentrations of reagent producing complete cleavage block were:
In 0.4 per cent oxygen — 99.6 per cent nitrogen, 32 per cent of control oxygen con-
sumption; in 0.7 per cent oxygen — 99.3 per cent carbon monoxide, 32 per cent of
control oxygen consumption; in 1.6 X 10~4 M KCN, 34 per cent of control oxygen
consumption; in KCN at 24° C. the value was 16 per cent of the control oxygen
consumption.
The carbon monoxide inhibition of oxygen consumption and cleavage was
reversed by light from a powerful carbon arc lamp. The percentage of inhibition of
PRESENTED AT MARINE BIOLOGICAL LABORATORY 319
oxygen consumption by CO in the dark is described by the equation • - - = K
1 — n po-i
where n is the fraction of oxygen consumption not inhibited, pco and po-i the partial
pressures of CO and O2, respectively, and K = 60. A 20 per cent stimulation of
oxygen consumption occurred in 10 per cent oxygen — 90 per cent CO.
Spectroscopic examination of fertilized and unfertilized Arbacia eggs reduced by
hydrosulfite revealed no cytochrome bands, although a band at 600-605 m/u corre-
sponding to cytochrome a was found in Arbacia sperm. The thickness and density
of the egg suspension used was such as to indicate that, if cytochrome is present at all,
the amount in Arbacia eggs is extremely small as compared to that in other tissues
having a comparable rate of oxygen consumption.
Three reagents poisoning copper catalyses, potassium dithiooxalate (10~2 M),
diphenylthiocarbazone (10~4 M), and isonitrosoacetophenone (2 X 10~3 M) produced
no inhibition of division of fertilized Arbacia eggs.
These results indicate that the respiratory processes required to support division
in the Arbacia egg may be of a type not dependent on cytochrome for intermediate
hydrogen transport, not dependent on a copper containing catalyst, and perhaps
different in several essential steps from the principal respiratory processes of yeast or
mammalian muscle.
Some factors affecting the rate of hemolysis of the mammalian erythrocyte
by n-butyl alcohol. M. G. Netsky and M. H. Jacobs.
The hemolytic effect of solutions of w-butyl alcohol in isotonic NaCl varies greatly
with small changes in concentration of alcohol and temperature. In certain parts
of the range, concentration differences of 0.1 per cent or 0.0025 M butyl alcohol, and
temperature differences of 0.25° C. are readily detectable. For the erythrocytes of
eight species of mammals a maximum resistance to butyl alcohol hemolysis was
found at pH 6.7-6.8, with times of hemolysis lower in the acid than in the alkaline
range. The pH of the maximum lies near the isoelectric point of hemoglobin. If it
be assumed that the alcohol causes a condition of cation-permeability of the erythro-
cyte, an effect for which there is already some independent evidence, the Gibbs-
Donnan equilibrium demands a swelling of the cell on both sides of the isoelectric
point; this might be expected to hasten the rate of hemolysis. Optical studies of
volume changes made by the method of A. K. Parpart show that at a pH value of
8.60 hemolytic concentrations of butyl alcohol convert the usual shrinkage of the
cells in alkaline solutions into a rapid swelling as demanded by the theory; swelling
also occurs rapidly at pH 5.18, but very slowly at pH 6.70. Despite the similarity
of the pH effect in different species, marked and characteristic specific differences
were found in the absolute times of hemolysis. At the pH maximum, the order of
resistance was;
monkey > man > dog > cat > rat > rabbit > beef > pig.
In accordance with the Gibbs-Donnan principle even low concentrations of non-
penetrating non-electrolytes were found to cause a considerable retardation of alcohol
hemolysis, but the effect proved to be more complex than a simple osmotic phe-
nomenon, since with equimolecular concentrations of different non-electrolytes it
was generally greater the greater the molecular weight of the added substance.
This relation is seen in the following series for beef cells:
lactose > sucrose > mannitol > dextrose, levulose > xylose
> pentaerythritol, erythritol > malonamide.
I -Jk
; L I L R A R •-
\
r
320 PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS
»
Studies on the permeability-decreasing effect of alcohols and pharma-
cologically related compounds on the human erythrocyte. J. B. S.
Campbell and M. H. Jacobs.
In extending the work previously reported by Jacobs and Par part (Biol. Bull,,
73, 380, 1937), it was found that the hemolysis of human erythrocytes in 0.3 M solu-
tions of glycerol at pH 7.4 is characteristically retarded by methyl, ethyl, w-propyl,
w-butyl, w-amyl, n-hexyl and w-octyl alcohols, as well as by ethyl ether, chloroform,
ethyl and phenyl urethanes and several other substances in a manner that in general
runs parallel with their pharmacological activity. On reducing the pH below 6.0,
substances of this type were found to give an acceleration rather than a retardation
of hemolysis, as they do likewise with beef erythrocytes at all pH values studied.
While the absolute time of hemolysis in 0.3 M glycerol solutions is more than doubled
by lowering the temperature from 35° C. to 5° C., the minimal effective concentration
of alcohol remains approximately constant over this temperature range. At body
temperature, quantitatively measurable effects were obtained with ethyl ether,
chloroform, and ethyl alcohol at concentrations considerably lower than those re-
ported in the literature to exist in human blood in anesthesia and in alcoholic in-
toxication.
Quantitative studies of the rate of passage of protein and other nitrogenous
substances through the watts of growing and of differentiated mammalian
blood capillaries. Richard G. Abell.1
The rate of passage of nitrogenous substances through the walls of growing
blood capillaries was observed with a transparent chamber, inserted in the rabbit's
ear, called the "moat" chamber. In this chamber the growing capillaries can be
seen with the high powers of the microscope, their condition recorded, and their
area calculated. After passing through the walls of these capillaries, nitrogenous
substances diffuse into a moat, or reservoir, of known volume, from which they can
be removed and analyzed quantitatively.
(1) Analyses were made of the total nitrogen entering the moat during the first
24 hours following the introduction into the moat of a mammalian Ringer's solution.
(2) The total surface of the capillaries involved was obtained from measurements of
length and diameter. From these two sets of data the calculated amounts of total
nitrogen passing through per sq. mm. of endothelial surface per 24 hours were, in
6 different chambers, as follows: (1) 0.091 mg.; (2) 0.113 mg.; (3) 0.102 mg.; (4) 0.097
mg.; (5) 0.046 mg.; (6) 0.081 mg.
The slower rate of passage of these substances (through the walls of the capil-
laries) in chambers 5 and 6 was associated with a slower rate of circulation in these
two chambers, as observed with the microscope.
When the Ringer's solution was left in the moat for intervals of time longer than
24 hours, the total nitrogen content of the moat rose above the total non-protein
nitrogen level of rabbit's blood within 5 days, which would seem to indicate that
plasma protein passes through the walls of growing blood capillaries.
Analyses of the moat content for protein showed that this is the case. Using
calculations similar to those described above in the one chamber containing blood
capillaries in which such studies have so far been made (chamber 3), but basing the
estimation on a collection period 48 hours in length, protein nitrogen came through
the capillary wall at the rate of 0.039 mg. per sq. mm. of capillary surface per 24
hours. This compares with the figure for total nitrogen of 0.102 mg. Thus, of the
total nitrogen that came through the walls of the growing capillaries in this chamber
per 24 hours, approximately } was protein nitrogen.
The results secured with a new type of chamber, the "filter disc" chamber,
1 Department of Anatomy, University of Pennsylvania School of Medicine.
PRESENTED AT MARINE BIOLOGICAL LABORATORY 321
indicate that mature blood capillaries are permeable to protein, but less so than are
growing blood capillaries.
AUGUST 30
The occurrence of cytochrome and other hemochromogens in certain marine
forms. Eric G. Ball and Bettina Meyerhof.
The process of oxygen utilization in mammalian tissue appears to proceed
through a chain of iron porphyrin compounds composed of hemoglobin, myoglobin,
cytochrome oxidase and the three cytochromes a, b, and c. Certain marine animals,
however, possess instead of hemoglobin a copper blood pigment, hemocyanin, which
functions in a manner similar to hemoglobin. The question, therefore, arises as to
whether these organisms are also deficient in those other iron porphyrin compounds
that compose the respiratory chain in mammalian tissue. We have therefore
examined the following hemocyanin-containing animals for these compounds: Venus
mercenaria, Busycon canaliculatum, Limulus polyphemus, Homarus americanus, and
Loligo pealeii. The heart and some body muscles of all these organisms were found
to possess cytochrome oxidase and the three cytochrome components. In addition
we have tested extracts of these same tissues for succinic dehydrogenase and have
found that it is present in amounts which parallel roughly the concentration of
cytochrome in these same tissues. Myoglobin has been found only in Venus mercenaria
and Busycon canaliculatum. The radula muscles of the latter are extremely rich in
this iron compound.
Two additional hemochromogens have been observed in Limulus polyphemus.
One is present in the abundant clot obtained from the blood of the animal. Its
reduced form possesses an absorption band centered at X560 myu. The oxidized form
shows no characteristic band. The other is present in the eggs. Its reduced form
shows an absorption band centered at X625 m/Lt while the oxidized form has a band
centered at X570 m/i.
It would therefore appear that the process of oxygen utilization in these organ-
isms whose blood pigment is a copper compound is similar to that in mammals except
for the substitution of hemocyanin for hemoglobin. This substitution can therefore
not be ascribed to the inability of these animals to utilize iron or to synthesize the
porphyrin prosthetic group characteristic of the iron respiratory pigments.
The eggs and sperm of Arbacia punctulata were also examined. The sperm were
found to contain abundant cytochrome oxidase, cytochromes a, b, and c, and succinic
dehydrogenase. Tests for these same compounds in the eggs were negative. Upon
addition of pyridine and sodium hydrosulfite to a ground egg suspension a strong
hemochromogen band centered at X560 mju appeared indicating that a hemin is,
however, also a constituent of the eggs.
Some observations on cholinesterase in invertebrates. Carl C. Smith and
David Click.
A study of the distribution of cholinesterase in some invertebrate hearts and in
various tissues of Limulus polyphemus was made in an attempt to find some basis for
explaining certain observations previously made concerning their reaction to
cholinergic drugs.
The manometric method of Ammon utilizing the Warburg apparatus was used.
In the following table the activities found are expressed in cubic millimeters of carbon
dioxide produced per fifty milligrams of tissue per thirty minutes.
322 PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS
Invertebrate Hearts Tissues of Limulus polyphemus
Modiolus demissus 304 Cardiac nerve 446
Callinectes sapidus 51 Ventral nerve 216
Limulus polyphemus 50 Heart (segments 5-6) 50
Pagurus longicarpus 49 Heart (segments 1-2) 34
Libinia dubia 37 Blood serum 42
Venus mercenaria 5.5 Blood cells (clot) 33
Intestine 18
Skeletal muscle 4-5
The concentration of cholinesterase found seems to roughly follow the amount of
nervous tissue present. The enzyme was found in the blood and tissues of arthropods
and Crustacea in which it has previously been reported absent. The insensitivity of
the clam heart to eserinization can be explained on the basis of the low esterase
content found.
Crystalline myogen. Kenneth Bailey.
The albumin fraction of rabbit skeletal muscle is obtained in the form of long
thin needles in the following way: the perfused minced muscle is treated with an equal
volume of ice cold 1 per cent NaCl, and after standing for one hour is pressed dry.
The globulin fractions in the press juice are separated by addition of solid ammonium
sulphate until the specific gravity of the solution at 25° reaches 1.175, the pH being
maintained at 6.3. After filtration the liquid is acidified with dilute H2SO4 to a pH of
5.8 and after standing overnight is again filtered. More ammonium sulphate is fed in
with slow stirring through a collodion membrane, the crystals separating when the
specific gravity reaches 1.18; at a specific gravity of 1.21 crystallisation is complete.
Effect of increased intracellular pH on the physiological action of substi-
tuted phenols. J. O. Hutchens and M. E. Krahl.
1. The effects of five substituted phenols (2,4-dinitrophenol, 4,6-dinitro-o-cresol,
2,4,5-trichlorophenol, 2,4-dichlorophenol, and m-nitrophenol) on the respiration and
cell division of fertilized eggs of Arbacia punctulata have been determined at an
extracellular pH of 8.0 in the presence and absence of a concentration of ammonia
(0.004 M NH4C1) sufficient to increase the normal cytoplasmic pH from the normal
value of 6.8 ± 0.2 to approximately 7.2 ± 0.2.
2. The following results were obtained:
a. The relative and absolute stimulation of oxygen consumption produced by
suboptimum concentrations of each substituted phenol was greater in the presence
than in the absence of ammonia.
b. The relative inhibition of cell division by each concentration of each substituted
phenol was the same in the presence and absence of the ammonia.
c. The optimum respiratory stimulating concentration for each substituted
phenol was the same in the presence and absence of the ammonia.
3. These results confirm and extend the experiments of Krahl and Clowes
\_J. Cell, and Comp. Physiol., 11,1 (1938)] in which the cytoplasmic pH was decreased
by means of carbon dioxide. Both the present and previous series of experiments, so
far as the experimental and theoretical limitations of the method permit, indicate
that the substituted phenol anion is the intracellular active form for respiratory
stimulation and that the substituted phenol molecule is the intracellular active form
for inhibition of cell division. The experimental data are completely inconsistent
with the suggestion, advanced by Tyler and Horowitz \_Biol. Bull., 75: 209 (1938)],
that the substituted phenol anion is the intracellular active form for inhibition of cell
division.
PRESENTED AT MARINE BIOLOGICAL LABORATORY 323
Fatty acid compounds in the unfertilized egg of Arbacia punctulata.
Albert E. Navez.
In a note in this Bulletin (1938) * it was pointed out that some "oil " was reacting
with the Nadi reagent (used in experiments on indophenoloxidase) concurrently with
its reaction with the oxidase. Variable results in the saponification value and in the
iodine number of this "crude oil" raised doubts on the adequacy of ether as an
extraction fluid. Even carbon tetrachloride extractions showed differences unless the
solvent was specially purified.
In the present experiments cyclohexane and CCU were used as extraction
solvents; in some cases after treatment of eggs (packed solidly by centrifuging) with
alcohol, in others in an atmosphere of nitrogen.
In the "crude oil" sterols and phospholipids are present (cf. also Mathews,
1913;2 Page, 19273). This oil, dark red in color, heavy in consistency, with a strong
fish oil odor and slowly semi-drying in thin films, was fractionated after saponification.
The fatty acids separated are: saturated fatty acids, unsaturated fatty acids and
fatty oxyacids. Their relative quantities seem to be variable with successive batches
of eggs and moment in the season. The largest portion of fatty acids are unsaturated ;
we have isolated by Br derivatives small quantities of the diethylenic, larger amounts
of the triethylenic and definitely indications of tetraethylenic (clupanodonic?) in
small quantities.
The red color of the oil is interesting as it can be removed by adsorption on
norite but with concomittant removal of some fatty compound. By successive acid
and alkaline treatments the red color can be eluted from the adsorbing agent; it gives
the absorption spectrum of echinochrome in CCU. The possibility is seen of the
presence of a fatty acid derivative of echinochrome, playing a possible role on oxi-
dations in the egg, in view of the ease of oxidation of the unsaturated compounds.
The unsaponifiable as yielded — unfortunately in very small quantities — small
crystals on treatment by HC1 gas in anhydrous acetonic solution, which might point
to the presence of some unsaturated hydrocarbon (squalene). No detailed work done
due to small quantity available. From the present observations it appears that the
composition of the "crude oil " is not constant but varies with time, state of animals,
feeding, method and length of keeping, perhaps temperature of sea water. No
correlation has been found. Work on a larger scale is planned for the future.
Color changes in luciferin solutions. Aurin M. Chase.
During the spontaneous non-luminescent oxidation of Cypridina luciferin,
partially purified by Anderson's method, the visible absorption spectrum of the
solution, which has initially a slightly increasing absorption toward the shorter wave-
lengths, rises, producing a maximum at about 470 mju, and then subsequently falls.
This change is much more rapid and its magnitude greater in aqueous solutions of
luciferin than in butyl alcohol solutions and the loss of luciferin (as measured by
light emission) in aqueous solutions and butyl alcohol parallels the color change.
Hydrogen peroxide causes very rapid and almost complete loss of color in
luciferin solutions, together with very rapid oxidation of luciferin.
Measured at pH's from 5.1 to 10.2 the change in the absorption spectrum is
much faster at alkaline than at acid pH's and so is the decrease in concentration of
luciferin under these conditions, using the luminescent reaction as a measure of
luciferin concentration.
These facts indicate that the color changes observed represent changes in luciferin
itself.
1 Navez, A. E., 1938. Biol. Bull., 75: 357.
2 Mathews, A. P., 1913. Jour. Biol. Che
3 Page, I. H., 1927. Biol. Bull., 52: 164.
2 Mathews, A. P., 1913. Jour. Biol. Chem., 14: 465.
LIBRARY) =o|
324 PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS
Anderson has demonstrated that when luciferase is added to a partially oxidized
luciferin solution approximately the same amount of light is emitted, but much more
slowly, as is emitted when luciferase is added immediately upon dissolving the
luciferin. To explain this he has postulated that the non-luminescent oxidation of
luciferin is reversible and, in the presence of luciferase, the slowly emitted light in
the former case is controlled by the reduction of this reversibly oxidized luciferin.
This reversible oxidation is believed to be represented by the initial rise in the ab-
sorption spectrum of luciferin solutions during non-luminescent oxidation in air.
The subsequent decrease in absorption must indicate another reaction, possibly also
an oxidation; probably irreversible.
A change in the absorption spectrum of luciferin solutions also occurs as a
result of the luminescent reaction itself, and this color change may perhaps be used
in studying the luminescent reaction.
Photodynamic action in the eggs of Nereis limbata. Fred W. Alsup.
Photodynamic effects can be produced in the eggs of Nereis by exposing them
in solutions of rose bengal or eosin of the proper concentrations to sunlight or to
light from a 1000-watt electric bulb. The effects consist of: (1) elevation of wide
membranes, (2) germinal vesicle breakdown and (3) cytolysis of the eggs. The per-
centage of nuclear breakdown varies with exposure time and the concentration of dye.
Solutions of rose bengal stronger than 1 part to 20,000 parts of sea water produce
effects on the eggs in the dark. These dark reactions involve nuclear breakdown,
staining of the entire eggs and cytolysis. With eosin a much stronger concentration
is required to produce the same degree of reaction. Previously irradiated solutions
of rose bengal produce no observable effects on the eggs, but when eggs are put into
previously exposed weak solutions of the dye and then removed to sea water and
fertilized, many cleave irregularly or cytolyze, indicating that the previously exposed
solutions did have some effect on the eggs. Most eggs develop normally in these
weak solutions, if the solutions have not been exposed to light. Solutions of eosin
previously exposed to light produce nuclear breakdown in the eggs. No photo-
dynamic changes can be produced in the eggs in the absence of free oxygen. KCN
increases photodynamic action on the eggs as shown by increased percentages of
nuclear breakdown. Relatively concentrated solutions of rose bengal bleached by
sunlight have little or no effect on the eggs, since eggs can be fertilized and will
develop normally in such bleached solutions, whereas they can not develop in un-
bleached solutions of the same concentration.
The same general effects obtained with the eggs of Nereis limbata were obtained
with the eggs of Arbacia punctnlata.
Cleavage delay in A rbacia punctulata eggs irradiated while closely packed
in capillary tubes. Irving Cohen.
From the work of Henshaw and others it is known that X-rays administered to
Arbacia punctulata eggs prior to fertilization cause a delay in the occurrence of first
cleavage.
Doctor Failla suggested to the writer the problem of comparing the radio-
sensitivity of these eggs when irradiated in the ordinary way and closely packed in
capillary tubes.
The general technique worked out by Henshaw, (Am. Jour. Roentgenol. and
Rad. Ther., 27, No. 6, June, 1932) has been followed in these experiments. Individual
controls were set up for the two parallel series of experiments.
The results show that with equal dosages of radiation considerably less delay in
cleavage is produced in the eggs irradiated in the capillary tubes. Doctor Failla
predicted the result on the basis of his theory of the biological action of ionizing
radiations (Occ. Publ. Am. Ass. Adv. Sci. No. 4, June, 1937). He has suggested that
PRESENTED AT MARINE BIOLOGICAL LABORATORY 325
owing to the greater complexity of the molecules within the egg, the increase in ion
concentration resulting from the X-rays would be relatively greater in the cytoplasm
than in the sea water. Therefore the radiation would cause an initial ionic unbalance
across the cell boundary and this is assumed to enhance the radiation effect. On the
other hand, when the eggs are closely packed in capillary tubes there is practically no
sea water around the cells and the ionic unbalance should be much less. Accordingly
the radiation effect should be much less marked.
It should be noted that while the experimental results confirm Failla's pre-
diction it does not follow that the suggested explanation is necessarily correct. (This
point is fully appreciated by Failla who has followed the work with interest.) There
are, of course, other possible explanations.
The X-ray effect on the cleavage time of Arbacia eggs in the absence of
oxygen. Rubert S. Anderson.
From a chemical viewpoint it is possible to consider that one result of the
ionization produced by the absorption of X-rays is the formation of products having
oxidizing and reducing tendencies. Experimentally, a number of authors have
found that oxidation or reduction is one of the types of reactions produced by X-rays
in simple chemical systems. They are especially common in the dilute aqueous
solutions studied by Fricke and Clarke where most of the reactions occur indirectly
through the water molecules.
This same type of reaction almost certainly occurs within living cells during
irradiation and it is important to know if it plays any significant part in biological
effects. Skoog concluded that this was true for certain plants and Fricke has sug-
gested similar possibilities for some genie effects in Drosophila.
In the present preliminary experiments the primary object has been to see if
the X-ray effect could be modified experimentally, as a first step toward finding out
what reactions are important. Arbacia eggs have been used. Henshaw showed that
irradiation of the unfertilized egg increased the time from fertilization to the first
cleavage. If oxidations or reductions produced by the X-rays are an important
factor in this delay it seemed possible that irradiation in the absence of oxygen would
modify the effect.
Oxygen was removed from suspensions of eggs in Thunberg tubes by washing
out with purified hydrogen. These eggs and control eggs were irradiated with
15,000 r.
All of the eggs were fertilized in air within four minutes after the end of irradia-
tion and the time until 50 per cent of the eggs had cleaved was determined.
In all of fifteen lots of eggs the delay in cleavage was greater for the eggs irradi-
ated in air than for those irradiated in hydrogen and this increase in delay averaged
about 50 per cent. However, the variability was very large and ranged from a low
of 13 per cent, which is probably not outside the error, to over 100 per cent for eggs
from different females.
The absence of oxygen (or conceivably the presence of hydrogen) therefore
does modify this X-ray effect in most Arbacia eggs. This is not presented as an
argument for the importance of oxidations or reductions but these results do en-
courage investigation in that direction.
Fixation of X-ray effect by fertilization in Arbacia eggs. P. S. Henshaw.
We have shown previously that exposure of Arbacia punctulata eggs to X-rays
causes a delay in the occurrence of the first cleavage, and further, that if an interval
of time is allowed between the end of treatment and the moment of insemination,
the effect is reduced or lost as a function of time — the latter being a change referred
to as recovery.
While these findings were satisfactory in demonstrating that recovery takes
326 PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS
place so far as the first cleavage is concerned, they gave no information as to whether
it is significant for subsequent cleavages and later development or whether it con-
tinues after fertilization. It is the purpose of this report to deal with these points.
The procedure followed was to treat a collection of eggs giving all the same
exposure, fertilize samples of these at various times up to three hours after treatment,
allow to develop for 48 hours, and then to classify the embryos as to degree of develop-
ment. By the method used, it was possible to determine whether all samples had
developed to the same extent and to compare the development in samples which had
had different amounts of time for recovery to occur.
It was found first that development had proceeded farther in those samples
which had been allowed the most time for recovery before fertilization; and secondly,
that curves arranged to show the amount of development as a function of time, bore
characteristic similarities to those based on delay in the occurrence of the first
cleavage.
On the basis of these observations, therefore, it appears that the recovery from
X-ray effect, which takes place before fertilization and is manifest by the time of the
first cleavage, is significant in subsequent cleavage and later development; and
consequently, that fertilization acts to fix whatever X-ray effect is present at the
moment of fertilization.
Does the action of X-rays on the nucleus depend upon the cytoplasm?
William R. Duryee.
Germinal vesicles from small ovarian eggs of three local species of frogs were
irradiated both in intact eggs and in the isolated condition in Ca-free Ringer.
Dosages varied from 500 to 50,000 r.1 Chromosomes in nuclei isolated after previous
irradiation in situ showed progressive injuries starting from 1000 r, in contrast to
those in isolated irradiated nuclei, which even after 50,000 r showed no marked
differences from the controls. No appreciable latent period in any of the 28 experi-
ments was observed. In support of the conclusion that radiation damage to the
chromosomes results primarily from chemical products of the injured cytoplasm is the
fact that nuclei, having first been isolated and then placed in a concentrated egg brei
and exposed to 50,000 r, showed typical chromosome defects of nuclei irradiated
in situ.
Chromosome injuries were of three types: progressive loss of side branches or
chromomere loops (which I described here last summer), fragmentation of the
longitudinal chromonemata, and frequent separation of the members of synaptic
pairs. Contraction of the chromosomes occurred when they were exposed either to
irradiated or to non-irradiated injured cytoplasm as previously described under the
term Autofixation. These changes are distinct from simple displacements of the
chromosome pairs from their normal central positions in the nuclear matrix which
depend on other factors.
PAPERS READ BY TITLE
Moulting and viability after removal of the eyestalks in Uca pugilator.
R. K. Abramowitz and A. A. Abramowitz.
Operative mortality following eyestalk removal in Uca is about 8 per cent for the
first 24 hours. Eighty animals were isolated in paper cups (to abolish cannabalism)
on the second day following eyestalk extirpation, and 25 normal animals were kept as
controls under identical conditions. After one month, 12 per cent of the operated
animals died directly without moulting, whereas none of the normal animals died
without moulting. Moulting in the operated animals began on the tenth day, the
percentage increasing rapidly as a hyperbolic function of time. Fifty per cent of the
1 Irradiation experiments carried out by E. LP. ittle.
PRESENTED AT MARINE BIOLOGICAL LABORATORY 327
animals had moulted by the sixteenth day, and 96 per cent by the twenty-eighth day.
Only 5 of the normal animals had moulted by the end of a month beginning the
twenty-fifth day. The operated animals experienced considerable difficulty in
moulting, many of them dying during or shortly after moulting. The total mortality
was 74 per cent at the end of the month, 62 per cent being due to death in moulting.
Forty per cent of the normal animals that moulted died during moulting. Blinded
animals appear" to lose pigment, an effect which becomes especially evident after
moulting.
A new method for the assay of inter medin. A. A. Abramowitz.
The proposed method is based on previous observations that a pale denervated
caudal band cannot darken during black-adaptation in hypophysectomized Fundulus,
and that maximal sub-lethal doses of purified intermedin evoke a darkening in the
denervated band but nowhere else in the integument of white-adapted fishes. In the
caudal fins of 400 Fundulus, a 2 mm. band was made and the fishes white-adapted for
5 days, at which time both innervated and denervated regions of the tail were uni-
formly pale. In one series, graded doses of purified intermedin and in another,
weighed samples of commercial pituitary powder (sheep whole gland) emulsified in
distilled water were injected intraperitoneally into 20 fishes for each dose. The
percentage of animals which responded was determined after a half-hour. The
points fall on a smooth hyperbolic curve whose steepest part lies between 0 and
40 per cent. A unit of activity is defined as that amount of intermedin which
darkens the denervated band in 25 per cent of the animals, at least 20 animals being
injected. This test, in addition to being quantitative, seems to be quite specific.
Twenty drugs, mainly alkaloids, were tested in various dosages, and all were inef-
fective in producing this reaction. In fact, the drugs usually darken the entire
integument, leaving the pale band unaffected — an effect diammetrically opposite to
that of pituitary intermedin.
Analysis of the electrical discharge from the cardiac ganglion of Limulus.
Florence Armstrong, Mary Maxfield, C. Ladd Prosser, and Gordon
Schoepfle.
The median ganglion of the Limulus heart contains two types of nerve cell, large
unipolar ganglion cells found in segments 4 to 7 and small multipolar cells found in
the outer portion of the whole nerve cord (Heinbecker, A. J. P., 1933, 1936). In
action potential records of the activity of isolated portions of this ganglion we find no
spontaneous discharge from segments 1, 2, 3, and 8. Rhythmic cardiac discharges
occur in the intermediate segments. Occasionally, particularly in segments 5 to 7,
there is a continuous spontaneous background upon which the rhythmic cardiac
bursts are superimposed. Low potassium tends to favor the asynchronous back-
ground activity.
It has been postulated (Heinbecker et al.) that the small neurones are activated
by the large pacemaker cells. A 0.1 per cent solution of nicotine, which abolishes all
reflex activity in the central nervous system of the animal, was applied to the cardiac
ganglion. The duration of the bursts remained constant, the interval between bursts
diminished, thus increasing the cardiac frequency. This result indicates that
activation of small neurones by the large ones is not by way of synapses.
Analysis of the activity in ganglia dissected down to a very few fibers showed
that some neurones discharge only once per heart beat. Others discharge many times
at a declining frequency during the burst. The cells providing the spontaneous
background fire at a relatively constant frequency.
328 PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS
The intra-cellular distribution of reducing systems in the Arbacia egg.
Robert Ballentine.
A comparison of the distribution of reducing activity (the dehydrogenase
systems) with the cellular constituents has been studied by the manometric method of
Quastel and Wheatley (1938) in centrifugally fragmented and whole Arbacia eggs.
Allowing for considerable individual variation from urchin to urchin, it may be stated
that the sum of the activities of the two halves is greater than that of the unfertilized
egg, and approximately equivalent to that of the intact fertilized egg. Centrifugal
and osmotic stretching of the egg, provided it is sub-threshold for activation, has little
or no effect on the dehydrogenase systems. Since sedimentation of granules similar
to that obtained in the half eggs is present in the centrifugally stretched cells, one is
led to the conclusion that as far as the dehydrogenases are concerned, the process of
fragmentation is equivalent to activation, although the halves are not induced to
parthenogenetic development nor do they develop membranes. The exposure to
0.95 M sucrose, as employed in fragmentation, is without effect. On the basis of
equivalent volumes, the red half has a greater activity than the white half, thus
definitely indicating that the dehydrogenating systems are not exclusively limited to
the clear cytoplasm, as is the peptidase activity (Holter, 1936). Rather it leads to
the conclusion that a considerable portion of the substrate activation occurs at
heterogeneous phase boundaries between the granules and the clear cytoplasm, or
perhaps within the granular material itself.
Some effects of colchicine upon the first division of the eggs of Arbacia
punctulata. H. W. Beams and T. C. Evans.
At room temperature 0.0002 molar concentration of colchicine in sea water
inhibits cleavage of Arbacia eggs when applied at any time before (approximately) ten
minutes of the appearance of the first cleavage furrows. Eggs left in this solution for
one hour, washed and let stand in sea water show recovery as indicated by the
appearance of cleavage furrows, many of which are abnormal. Eggs which have
started to cleave when put into the colchicine solution continue the process until the
two blastomeres are formed. However, all further cleavages are suppressed unless
the colchicine is removed.
Eggs placed in the colchicine solution ten minutes after fertilization and cen-
trifuged ten minutes later show a more marked stratification than do controls
centrifuged for the same time and speed. Likewise, they show more stratification
than do controls of the same lot fertilized ten minutes later and which are in approxi-
mately the same stages of division as were the experimental eggs when placed in the
colchicine solution.
These results indicate that colchicine in concentrations sufficient to block
cleavage acts by lowering the viscosity; or by inhibiting the normal rise in viscosity
(gelation) that is associated with the appearance of the mitotic apparatus of the
cleavage process. This is further substantiated by the fact that a disintegration of
the asters may be observed when the eggs are placed in the colchicine approximately
ten minutes before the appearance of the cleavage furrows. The rays seem to fade
out, leaving only clear and often irregular areas in the position formerly occupied by
the asters.
Temporal summation in neuromuscular responses of the earthworm,
Lumbricus terrestris. E. Frances Botsford.
Temporal summation has been demonstrated in vertebrate smooth muscle and in
the muscle of crustaceans and coelenterates. The phenomenon in smooth muscle of
vertebrates has been attributed to the spreading of a chemical mediator through the
tissue so that with each additional stimulation more muscle fibers contract. This
PRESENTED AT MARINE BIOLOGICAL LABORATORY 329
study is to demonstrate temporal summation in the muscles of the body wall of the
earthworm and the dependence of this summation upon a chemical mediator.
The earthworm was arranged for recording the contractions of the longitudinal
muscles by a weak isometric lever. Stimulating the nerve cord at 8-second intervals
with a tetanizing current of constant intensity and brief duration produced a facili-
tation of the successive responses, in some cases for as many as sixteen times. The
dependence of the magnitude of the response upon the frequency of stimuli was
demonstrated conclusively by varying the frequency by means of a vacuum tube
stimulator. Furthermore, at constant frequency there was an increase in response
with increase in duration of the stimulation. Thus it is evident that the strength of
the response is dependent upon the number of stimuli and temporal summation is
shown to be characteristic of the muscle responses of the earthworm.
Since this summation was also true of a dorsal muscle strip, the phenomenon is
not dependent upon the nerve cord, but is produced either in the peripheral plexus or
at the neuromotor junction.
The dependence of temporal summation upon the chemical mediator acetylcholine
was demonstrated as follows:
(1) Eserine caused no response in an unstimulated dorsal muscle strip, but
electrical stimulation of an eserinized muscle strip caused an increase in tension
similar to that produced by acetylcholine.
(2) When the interval between 'stimulations was increased to 5 minutes there
was no summation of the successive responses. But when eserine was applied to the
muscle summation occurred in spite of the long interval between stimulations.
Since the muscle of the earthworm is very sensitive to acetylcholine, these
experiments indicate that temporal summation is brought about by the spread of
acetylcholine from the neuromotor junctions.
The source of chromatophorotropic hormones in crustacean eyes talks.
F. A. Brown, Jr.
Through a series of injection experiments in which extracts of whole eyestalks
and certain portions of eyestalk tissue were injected into Palaemonetes and Uca as
test animals it has been demonstrated that the active source of chromatophorotropic
hormones is a small, translucent, or bluish white mass located in the dorsal or dorso-
lateral region of the eyestalk. This tissue appears to be the sinusgland of Hanstrom
and constitutes a definite gland which can dissected out easily in the forms investi-
gated: Cambarus, Carcinus, Callinectes, Libinia, Uca, Pagurus, Crago, and Palaemo-
netes. In the last two named forms the gland is readily visible in the normal living
animal. Quantitative studies of the effects of extract of the gland show that better
than 80 per cent of the activity of the whole eyestalk extract is to be found in extracts
of the minute gland. The difference of about 20 per cent can be accounted for by
diffusion of substance from the gland into other eyestalk regions. Furthermore, the
effect of the sinusgland by itself is qualitatively indistinguishable from that of the
whole eyestalk of the same species judging by the relative effects of the sinusgland
and whole eyestalk extracts upon the red chromatophores of Palaemonetes and the
black ones of Uca. Implantation of the sinusgland of Carcinus into the abdomen of
eyestalkless Palaemonetes has given confirmation of its chromatophorotropic activity
as a single implant maintained the red pigment of Palaemonetes more or less concen-
trated over a five-day period at the end of which time the gland apparently became
functionless.
Comparative effects of sinusgland extracts of different crustaceans on two
chromatophore types. F. A. Brown, Jr. and H. H. Scudamore.
Extracts of the sinusglands of seven crustaceans (Callinectes, Carcinus, Libinia,
Pagurus, Uca, Crago, and Palaemonetes) were each tested simultaneously on the red
330 PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS
chromatophores of eyestalkless Palaemonetes and the black chromatophores of eye-
stalkless Uca. The order of effectiveness of the different sinusgland extracts upon
the two chromatophore types were not the same, some extracts having relatively
more effect upon the Uca black and others upon the Palaemonetes red chromatophores.
This was expressed in the form of a ratio:
Effect upon Uca black chromatophores
Effect upon Palaemonetes red chromatophores
The values of this ratio when sinusglands alone were used showed a definite sequence
which was substantially repeated when whole eyestalks were used. Of the crus-
taceans examined Crago showed the highest value for the ratio while Palaemonetes
and Callinectes showed the lowest. To support an hypothesis that two hormonal
substances are to be found in different proportions in the various sinusglands, the
discovery was made that dried sinusglands, or eyestalks, extracted with absolute
ethyl alcohol yielded a fraction with a very low value for the above-mentioned ratio
while a sea-water extract of the alcohol-insoluble residue yielded a fraction with a
very high value. Thus, there seems to have been effected a partial separation of
two chromatophorotropic principles from the crustacean sinusgland.
On the control of the dark chromatophores of Crago telson and uropods.
F. A. Brown, Jr. and H. E. Ederstrom.
Since Roller's work indicating the presence of a rostral organ secreting a pigment-
dispersing hormone there has been general lack of confirmation and even denial of
its actuality. An exhaustive series of injection experiments in which extracts of
various regions and tissues of the body of Crago were injected into eyestalkless Crago
has disclosed what appears to be the normal source of a dispersing humoral substance
for the dark pigment of the telson and uropods. The only tissue of the many tried
which yielded darkening of the tail in practically 100 per cent of the trials was the
central third of the circum-oesophageal connectives including the connective ganglion
and a short portion of the connectives immediately posterior to the ganglion. From
eyestalkless and black-adapted animals this middle third was usually the only effective
region but in the case of white-adapted animals frequently all three portions of the
connectives (anterior, middle, and posterior), and often even the posterior portion
of the brain were somewhat active. This last was probably due to the diffusion of
substance through the connectives. An extract of Palaemonetes connectives acts
similarly upon Crago, though extracts of connectives of Carcinus, Uca, Libinia,
Pagurus, and Callinectes fail to produce the response. The dispersing action of the
connective hormone is annulled by extracts of eyestalks of Crago or Palaemonetes
but is apparently uninfluenced by eyestalk extracts of Carcinus, Libinia, or Uca.
The latter extracts exert the interesting effect of blanching strongly the trunk and
leaving the black " tail " more conspicuous than ever, by contrast. The observations
thus indicate definitely that there is an extra-eyestalk origin of a chromatophorotropic
hormone and that Palaemonetes and Crago have a principle in their eyestalks not to
be found in the eyestalks of Carcinus, Libinia, and Uca.
Micromanipulation of salivary gland chromosomes. John B. Buck.
Micromanipulation of normal salivary gland chromosomes of Chironomus
phimosus, in vitro, proved infeasible because dissection of the cells causes immediate
and marked abnormalities in the chromosomes. However, immersion of the gland
in a hanging drop of isotonic Ringer's over the vapor of osmium tetroxide for 15 to
18 hours at 5° C. renders the cytoplasm and nuclear membrane sufficiently brittle so
that individual chromosomes can be isolated. These chromosomes resemble those
in vivo very closely in regard to minute visible structure and dimensions, and in
addition partly retain the power of living chromosomes of responding reversibly to
PRESENTED AT MARINE BIOLOGICAL LABORATORY 331
osmotic changes in their environment. The mechanism of action of the osmic vapor
is obscure, but apparently involves a surface reaction, rather than impregnation,
since the principal effect is loss of stickiness, and no osmium could be found inside
the chromosomes following reduction.
The following results were obtained from manipulation of these chromosomes:
(1) A photographic record was obtained of the stage-by-stage reversible trans-
formation of staggered transverse rows of achromatic droplets (honeycomb) into
longitudinal parallel thread-like striations, supporting Metz's view that the latter
are artifacts.
(2) Most of the longitudinal stretching occurs in the interband regions. Return
to original length in relaxation may occur after up to 300 per cent stretch, and at
least 500 per cent stretch may be sustained before breakage occurs. Breakage
always occurs in the interband regions and in a straight line at right angles to the
long axis of the chromosome.
(3) The somatically synapsed homologs are so intimately fused that forces
sufficient to break the chromosome cannot separate them.
Effects of Roentgen radiation on certain phenomena related to cleavage in
Arbacia eggs (Arbacia punctulata}. T. C. Evans and H. W. Beams.
Fertilized eggs irradiated at 7,400 r/m. (minute exposures with a maximum
dosage of six) showed subsequent delay in first cleavage which increased exponentially
with the dosage.
Clumping of the eggs was noticed in the irradiated lots and it was found (by
staining with Janus green) that the jelly, which surrounded the control eggs, was
missing in the irradiated lots. Absence of the jelly was also noted in irradiated lots
of unfertilized eggs. The effect was noticed immediately after irradiation and appears
to be a direct radiation action as eggs placed in irradiated sea water were found to
retain their enveloping jelly as long as did eggs in untreated water. Dosages below
10,000 r were not completely effective. Some fertilized eggs were supported in a
hanging drop over polonium and were examined at the time when the controls were
in the eight-cell stage. Some of the eggs in the drop were apparently not affected but
some of them showed unilateral delay in cleavage. Such eggs stained with Janus
green possessed the jelly only on the side of the unaffected blastomeres.
Irradiated sperm were found to lose their motility and fertility more rapidly
than did the controls. Irradiated sperm (radiation sufficient to delay subsequent
cleavage of eggs fertilized with treated sperm) were found to produce the initial rapid
increase in oxygen consumption, as noted for controls, upon fertilization. Dead
sperm, when added to the egg suspension in the respirometer, failed to produce the—
increased oxygen uptake. It appears that the rapid increase in oxygen consumption
noted in normal fertilization may be related to the entrance of the sperm (or other
related surface actions) regardless of the subsequent fate of the sperm nucleus.
Fertilized eggs irradiated as high as 37,000 r showed less stratification than
controls when centrifuged at the same time.
The above findings are apparently unrelated in the present preliminary state
of the investigation. It is evident, however, that radiation may produce several
quite different immediate biological effects in the same cell and that such effects must
be considered in attempting to formulate any possible fundamental biologic action
of radiation.
Chemical and mechanical properties of two animal jellies. John D.
Ferry.1
The jelly surrounding the eggs of Arenicola cristata, when dialyzed free of salts,
contained only 0.2 per cent solid matter. It could be reversibly shrunk by concen-
1 Society of Fellows, Harvard University.
332 PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS
trated salt solutions or by alcohol, acetone, or dioxane; it was not dissolved by zinc
chloride, urea, or dilute acid. Qualitative tests showed that the material was a
polysaccharide containing uronic acid. The jelly was precipitated by barium chloride
(thereby releasing the eggs) in the form of long fibers, which swelled only slightly in
cold dilute acid or salt solutions.
The relaxation of shearing stress in the dialyzed jelly, shrunk to different extents,
was examined in a concentric cylinder apparatus. Analysis of the decay curve
indicated two relaxation times of the order of 2 and 100 minutes respectively, which
decreased somewhat with increase of temperature, but showed little change in a
concentration range where the rigidity varied tenfold.
Fresh specimens of Mnemiopsis leidyi, drained of excess sea water, were treated
with borate buffer at pH 9.5 to dissolve the softer parts, and the residue discarded.
When the solution was brought to pH 3, a viscous gel formed, and separated as a
gelatinous, stringy clot. This was compressed by centrifuging and extracted with
alcohol, thereby removing a small quantity of yellow lipoid material. The clot was
resuspended in water and washed free of alcohol.
Qualitative tests on the material thus purified showed it to be a mucoprotein,
containing 12 per cent nitrogen, and small amounts of sulfate sulfur and cystine. It
swelled increasingly with increasing pH, dissolving alkaline to pH 8. In the neigh-
borhood of pH 8 it formed highly viscous gels; this property was, however, destroyed
by concentrated urea or by boiling.
The relaxation of shearing stress in a 0.1 per cent solution in M/6 borate buffer at
pH 8.8 showed a relaxation time of the order of 2 minutes, which decreased with
increasing temperature.
Response of frog striated muscle to CaCl2. Judith E. Graham and F. J.
M. Sichel.
The local application of CaCl2 to the surface of a length of isolated skeletal
muscle fibre causes a marked reversible shortening of the muscle substance, as pointed
out by Chambers and Hale and by Keil and Sichel. This behavior of the isolated
length of fibre is in marked contrast to that of the intact muscle, where no such
shortening occurs even with concentrations of CaCl2 as high as 400 mg. per cent.
Since KCl-treated muscles have been shown by one of us to resemble in some respects
the isolated fibre preparation, it was thought advisable to investigate the possibility
that such muscles might behave like the isolated fibre also with respect to CaCl2.
The isolated fibre shows no marked response to the injection or local application
of KC1. The intact muscle (frog) when placed in a modified Ringer's solution con-
taining 400 mg. per cent KC1 undergoes a rapid transient contracture which
disappears in 3 to 5 minutes. Subsequent to this treatment of the muscle with the
KC1, and its response, the muscle will shorten markedly if placed in a similar solution
containing 400 mg. per cent CaCl2. This shortening is reversibly maintained in the
presence of the excess CaCl2 if the excess KC1 is still present in the solution, but
subsides slowly if only the normal concentration of KC1 is present. This sensitization
of the muscle to CaCl2 by excess KC1 is reversible; that is, washing the muscle in
Ringer's solution after the transient KC1 contracture renders it insensitive again to
the action of CaCl2. No antagonism of the CaCl2 contracture by an equal KC1
concentration could be detected.
Curarized muscle in Ringer's solution, and muscle in isotonic sucrose behaves like
the KCl-treated muscle.
The permeability of the toadfish liver to inulin. Charlotte Haywood.
An earlier investigation (Haywood and Hober, Jour. Cell. Comp. Physiol., 10,
305, 1937) has indicated that the relatively large, lipoid-insoluble inulin molecule
penetrates the isolated bullfrog liver, from perfusion fluid to bile, as through a passive
PRESENTED AT MARINE BIOLOGICAL LABORATORY
filter. The present study demonstrates that in a living, unanaesthetized animal,
retaining its normal blood supply to the liver, administered inulin can also enter the
bile.
The toadfish was used because its aglomerular kidney fails to eliminate inulin.
(Shannon, Jour. Cell. Comp. Physiol., 5, 301, 1934) ; 1.5 to 2. grams inulin per kilogram
of body weight were injected intramuscularly a day or more before collection of bile
samples. During collection, fish were strapped down and kept alive by a stream of
water entering the mouth and directed over the gills. A ventral incision was made,
the bile duct ligated, the gall bladder drained and cleaned, and a cannula inserted,
after which the incision was closed, leaving the cannula protruding. Such prepara-
tions secreted up to 3 or 4 mg. bile per hour per gram of liver over a collecting period
of 1 1 to 21 hours, after which a blood sample was drawn, usually from a caudal vessel,
occasionally from the heart. Survival of fish following the operation was obtainable,
often for several days.
Twenty-fold dilutions of bile and of protein-free blood plasma were analyzed by
the Shaffer-Somogyi method for reducing substance. The difference in reducing
substance before and after hydrolysis with H2SO4 represents the amount of inulin
hydrolyzed to levulose.
A series of nine experiments showed the concentration of inulin in the bile to
range between 55 and 73 per cent of that in the blood plasma. These figures are
corrected for a small amount of reducing substance found in the bile in control
experiments, without inulin.
A further series to determine the effect of a choleretic is under investigation.
Activation of Cumingia and Arbacia eggs by bivalent cations. Josephine
Hollingsworth.
Eggs of Cumingia tellinoides are activated by isotonic solutions of SrCl2, CaCh
and BaCl2. The degree of effectiveness of the various salts follows the order named.
The various pHs from 6.1 to 8.6 are equally favorable for activation. The time of
polar body formation in eggs activated by bivalent cations is approximately the same
as the time of polar body formation in eggs activated by sperm. The addition of sea
water or the addition of isotonic solutions of NaCl or KC1 tends to inhibit activation
by bivalent cations. This inhibiting effect increases as the proportion of sea water or
the concentration of the monovalent cations increases.
Eggs of Arbacia are activated by isotonic solutions of CaCl2, SrCl2, MgCl2 and
BaCl2. Calcium ions are more effective than strontium, magnesium and barium
ions. The action of the last three cations is somewhat variable. Whereas the
bivalent cations act rapidly on Cumingia eggs, Arbacia eggs must be exposed to them
for hours before any effect is observed. In eggs exposed to calcium ions, there is a
much higher percentage of cleavage in ovary eggs which have been washed than in
ovary eggs which have not been washed; in shed eggs than in ovary eggs either
washed or unwashed; and there is a slightly higher percentage of cleavage in shed eggs
that have been washed than in shed eggs that have not been washed. The highest
percentage of cleavage takes place at pH 9.0. Below pH 8.8 the percentage of
cleavage is usually small and above pH 9.2 the percentage of cytolysis is large.
Whereas the addition to the calcium solution of isotonic solutions of NaCl or KC1
tends to inhibit the activation of Cumingia eggs, in certain proportions the addition of
these solutions to the calcium solution may increase the percentage of activation of
Arbacia eggs while in other proportions the addition of these solutions has the
opposite effect.
334 PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS
The vacuole systems of a fresh water limacine Amoeba. Dwight L.
Hopkins.
By means of high power apochromatic objectives and staining with Nile blue
sulfate, neutral red and Janus green, each type of vacuole and granule found in this
amoeba has been followed from its origin to its disappearance. In general there are
two systems of vacuoles. In active feeding amoebae the vacuoles arise from the
protoplasm. One set of vacuoles, by swelling and coalescence, form the "contractile
vacuoles" which are periodically evacuated to the outside. A second set of vacuoles
coalesce with engulfed food and form the food vacuoles which after digestion has
taken place are evacuated to the outside. Evacuation of the food vacuoles generally
is independent of the contractile vacuoles. Occasionally, however, a food vacuole
may coalesce entirely with a contractile vacuole and then this resulting food-contractile
vacuole is evacuated. Again, the fluid portion of the food may run into the contractile
vacuole leaving the food residue practically in contact with the protoplasm. Fol-
lowing this the food residue is soon evacuated but independently of the contractile
vacuole.
In feeding or slowly feeding amoebae granules stainable with neutral red, Janus
green B, and Nile blue sulfate arise in the protoplasm instead of food vacuoles.
Under certain conditions more abnormal than those which cause cessation of feeding
the contractile vacuole system is retarded and granular structures stainable with
Janus green B, but not with Nile blue or neutral red, replace the fluid vacuoles which
form the contractile vacuoles. Under favorable conditions the contractile vacuole
system is not conspicuously stained with Janus green B. At a certain intermediate
stage granulation of the contractile vacuole system becomes stainable with Janus
green, but still these Janus green stained vacuoles can be observed to coalesce, swell
and form the contractile vacuole in which a greenish-blue tinge definitely can be
detected.
Cytological studies on andro genetic embryos of Triturus viridescens which
have ceased development. Cornelius T. Kaylor.
Fankhauser (/. E. Z., 68, 1934) has shown that there is a high death rate in
developing egg fragments of the European newt, Triton palmatus, during blastula and
gastrula stages and that this is caused by the presence of irregular numbers of
chromosomes in the cells of the embryos. A correspondingly high mortality during
these same stages of development was observed in my experiments on androgenesis in
eggs of Triturus viridescens (J. E. Z., 76, 1937). It was, therefore, reasonable to
expect that the same abnormal chromosomal conditions as were found in Triton
palmatus egg fragments would be responsible for the death of these androgenetic
embryos of viridescens during the blastula and gastrula stages.
In a study of about 65 blastulae and 8 irregular gastrulae fixed at cessation of
development, it was found that the cells of all these embryos were equipped with
subhaploid to superhaploid numbers of chromosomes. The cessation of development
was substantiated by the onset of cytolysis in all these cases. In a preliminary study
of chromosome numbers in more advanced embryos which have ceased development,
that is, neurulae and tail bud stages, it was found that there were only four question-
able counts in over 100 which showed other than the haploid number of chromosomes.
It appears, then, that at least the complete haploid set of chromosomes is
necessary in androgenetic embryos of Triturus viridescens, the same as in merogonic
Triton palmatus embryos, if they are to develop beyond gastrulation.
Also a large number of mitoses were found in androgenetic embryos of T.
viridescens fixed while still developing, which had no chromosomes at all on the spindle.
Apparently cell division can proceed in the T. viridescens egg as well as in Triton
palmatus, in the absence of chromosomes.
PRESENTED AT MARINE BIOLOGICAL LABORATORY 335
Effect of leukotaxine on cellular permeability to water. Valy Menkin.
The writer has recently succeeded in demonstrating the presence of a crystalline
nitrogenous substance from inflammatory exudates capable per se of increasing
capillary permeability and of inducingthe migration of polymorphonuclear leukocytes.
The liberation of this substance offers a reasonable explanation for two of the basic
sequences in the development of the inflammatory reaction ( J. Exper. Med., 1936, 64,
485 and 1938, 67, 129, 145). This substance has been named leukotaxine.
In an endeavor to determine the mechanism of action on individual cells, the
effect of leukotaxine on the permeability of ova of Arbacia punctulata was studied.
Ova were exposed for intervals varying from 20 minutes to about an hour and a half to
concentrations of leukotaxine ranging from about 3 to 8 mg. per cc. The eggs were
subsequently immersed in hypotonic sea water (50 per cent concentration) and their
degree of swelling measured from minute to minute with an eyepiece micrometer. A
large number of ova were thus studied for a total period of 6 to 8 minutes. Their
mean diameter served as a basis for the calculation of cell volume as previously
described in the various contributions of Lucke and McCutcheon (cf. Physiol. Rev.,
1932, 12, 68). The permeability was also obtained from the equation utilized by these
workers: Permeability = dV/dtlS(P - Pex).
The duration of exposure to leukotaxine prior to immersion in the hypotonic
medium was found to be relatively inconsequential provided the pH had previously
been adjusted to approximate that of sea water. A failure to follow this precaution
might ultimately induce a change in ova exposed for long intervals to an acid pH that
tends to reduce their swelling capacity when placed in a hypotonic medium.
The results of several experiments on a considerable number of ova indicate that
leukotaxine appreciably increases the permeability of Arbacia ova to water. The
extent of augmented permeability over that found in the case of normal ova in a
similarly hypotonic medium is about twofold.
Effect of leukotaxine on cell cleavage. Valy Menkin.
Leukotaxine induces increased capillary permeability and migration of poly-
morphonuclear leukocytes in mammalian tissue (Menkin, Physiol. Rev., 1938, 18, 366).
Its effect on ova of Arbacia punctulata is to enhance further their permeability to
water when immersed in a hypotonic medium.
Does leukotaxine induce sufficient injury to ova to influence cleavage develop-
ment? A series of observations have yielded the following results, summarized in
brief:
1. Leukotaxine-treated ova manifest the usual fertilization reaction when
exposed to sperms. The fertilization membrane, however, appears as a distinctly
narrower zone than is seen under normal circumstances.
2. An appreciable number of fertilized ova fail to segment when exposed, for even
a few minutes prior to fertilization, to a solution of leukotaxine.
3. The rate of cleavage of leukotaxine-treated ova tends to be retarded.
4. In the leukotaxine-treated group a considerable number of dividing eggs
reveal atypical forms exemplified by unequal cleavage.
5. Sperms immersed for about an hour in sea water containing leukotaxine fail to
fertilize normal ova.
These results indicate that leukotaxine seems to be definitely injurious to isolated
cells as exemplified in the ova and sperms of Arbacia punctulata. In the case of ova
this is manifested by increased permeability to water and by an appreciable inhibition
to normal cleavage.
Response of the Arbacia egg cortex to chemical and physical agents in the
absence of oxygen. Floyd Moser and J. A. Kitching.
Previous attempts to determine whether membrane elevation can be initiated in
the Arbacia egg in the absence of oxygen have failed because of the fact that the
336 PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS
sperm is immobilized under these conditions. Thus there is no proof that the Arbacia
egg cortex could not respond in the complete absence of oxygen if it were given an
adequate stimulus. In the present experiments mechanical and chemical agents
have been used, to avoid the effect of oxygen lack upon the sperm.
Drops of Arbacia eggs and of the various agents required were suspended from a
cover slip sealed with vaseline over a modified Englemann gas chamber. A monolayer
of ferric stearate (see Ballentine, Science, 89, 1939) was laid down on the cover slip to
prevent coalescence when several drops were employed. A short, wide, bent,
mercury-filled, glass side-arm allowed gas-tight manipulation of a microneedle or fine
glass loop within the chamber. Oxygen-free hydrogen was passed through the
chamber at 50-75 cc. per minute, and adequate time was allowed for equilibration.
Mixing of the drop was achieved by slight shaking of the chamber, and transfer of the
eggs with little fluid from one reagent to another was accomplished by means of the
loop.
Eggs treated with saponin (1/4 of 1 per cent, in sea water), molar urea, and
molar sucrose solutions in the absence of oxygen exhibited the same characteristic
cortical response and membrane elevation as that obtained in air (see Moser, J. E. Z.,
80, 1939). Moreover, the time relationships were much the same, regardless of
whether the experiments were made in an atmosphere of hydrogen, carbon-dioxide-
free air, or air. Shortly after the response to the non-electrolyte solutions in the
absence of oxygen, the elevated membranes disappeared (see Moser, Biol. Bull., 73,
1937). Typically the response to saponin was followed some minutes later by
cytolysis. The response to pricking with a microneedle under anoxic conditions was
not unlike that obtained in air (see Moser, J. E. Z., 80, 1939).
Eggs transferred under anoxic conditions to hypertonic sea water exhibited
cortical alveolar swelling (see Hunter, J. C. C. P., 9, 1936), but no membrane ele-
vation. When, however, the eggs had previously been treated with urea, which
itself initiates the cortical response, no such cortical alveolar swelling took place.
When, by means of the loop, eggs were transferred in the absence of oxygen first
to urea, and then through several changes of sea water, they subsequently cleaved
when exposed to air, but did not cleave when kept under anoxic conditions. Eggs
left in urea undergo amoeboid movements in air (Moser, unpublished observations),
but in the absence of oxygen no such movement took place. Stoppage of this
movement in absence of oxygen was reversible.
Further studies on regeneration in Fundulus embryos. S. Milton Nabrit.
Due to the fact that time recorded in terms of days lapsed since fertilization is
not an adequate criterion of developmental time for Fundulus, the results obtained
from experimental development on this form cannot be readily compared. Some
of the differences in results obtained after the removal of the distal end of the tail
of the fish may be accounted for on that basis. Other differences, however, are not
so readily explained.
The Nicholas l technique was employed to remove Fundulus heteroclitus embryos
from their chorions six days after fertilization. At this time the embryos were
fitted to the Oppenheimer 2 developmental schedule for normal development at 25° C.
The embryos were between the stages 24-25, about 80 hours. The natatory fold
was elevated from the distal end of the tail up to the third segment from the distal
end. The rounding of the caudal fin had not begun. The operations for removal
of the chorions and for the distal two segments of the tail were performed in amphibian
Ringer's solution. After twelve hours some fish were transferred to sea water; after
twenty-four hours some fish were placed in 4/5 Ringer's and 1 J5 sea water by volume.
1 Nicholas, J. S., 1927, Proc. Nat. Acad. Sci., 13.
2 Oppenheimer, J., 1936, J. E. Z., 73.
PRESENTED AT MARINE BIOLOGICAL LABORATORY 337
About 20 per cent of the fish kept in the modified Ringer's solution and 10 per
cent of those transferred to sea water differentiated tail fins without replacing the
missing tail segments. New rays were first observed in those in the modified Ringer's
solution in 7 to 9 days. New rays were observed in those in sea water in 11 to
16 days.
Birnie 3 reported that five-day-old fish cut in sea water or in isotonic sodium
chloride solutions that afterwards were transferred to sea water did not differentiate
tail fins in 65 days. I previously reported that cauterized tail stumps would differ-
entiate new fins in fish that were 9 days old at the time of removal of the distal end.4
Therefore, to assume that some of the fish in these experiments had not arrived at
the critical period for setting the differentiation, and that most of them had, would
necessitate the supposition that cautery causes a reversal or recovery of the capacity
to differentiate the caudal fin. The other alternative appears to lie in the nature of
the healing process. If sloughing occurs, the chances that the natatory fold will
close over the cut stump in time to become infiltrated with mesenchyme are greatly
diminished. In such cases rays may not differentiate. It is rather striking that if
rays differentiate at all it is quite early. The fold is a regressive structure in Fundulus
and appears to depend upon fin differentiation for persistence. I have not obtained
caudal regeneration in the fry of Fundiilus, although it has been reported in several
other fishes.
The action of certain drugs on the intact heart of the compound ascidian,
Perophora viridis. A. J. Waterman.
Previous work has been done on Molgula, dona and certain salps (Hunter,
Schultze, Bacq, etc.). In Perophora the abvisceral beats (toward branchial basket)
greatly outnumber the advisceral; both are highly variable.
Adrenalin, mecholyl and acetylcholine excite dominance of the advisceral
center, but it is a question if they all act in the same way and on similar mechanisms.
Adrenalin increases the number of abvisceral beats and the length of rest periods,
decreases the advisceral beats and causes irregularity. In 1-125,000 dominance
lasts a few minutes. In 1-20,000 the advisceral beats are suppressed for many
hours when transferred during the abvisceral series; if during the advisceral, several
reversals occur before the abvisceral become continuous ('death sign' of Schultze).
After recovery in sea water or oxidation of adrenalin (Perophora accelerates the
latter) these results are reproducible. Occasionally 1-100,000 increases the number
proportionately in both directions. One to 15,000 inhibits dominance, and beating
from both ends occurs without coordinating rhythm. Mecholyl (1-5,000 to 1-30,000)
is less effective and abvisceral dominance lasts about 10-30 minutes. In acetylcholine
the time varies from 4-9 minutes. Certain other effects are also different.
Atropine (1-2,500 to 1-5,000) causes irregularity, stops the heart in 5-30
minutes, and tends to reduce or abolish the exciting action of mecholyl; but the
latter restores an atropine-poisoned heart to near normal. Acetylcholine and
mecholyl influence the action of each other. Strychnine and colchicine are depressant
without significant evidence of stimulation or of differential effect on the heart
centers. Reaction and recovery occur quickly. No prolonged contraction of the
animals occurred except with lethal concentrations of these drugs. Some of the
observed effects differ from those described for other ascidians. In certain respects
these results resemble those obtained with crustacean hearts.
3 Birnie, J. H., 1934, Biol. Bull., 66.
4 Nabrit, S. M., 1938, Jour. Exper. Zool., 79.
338 PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS
An effect of the injection of a solution of dihydroxyestrin into castrated
female frogs , Rana pipiens. Opal Wolf.
In 1928 Wolf reported that subcutaneous injections over a long period of time,
of a water-soluble extract prepared from mammalian ovaries prevented the involution
of the oviducts of female frogs castrated in the autumn. Wolf also demonstrated in
1929 that implants of the anterior lobe of frog pituitary evoked the reproductive
processes of both the male and female frogs, Rana pipiens, as far out of season as
September.
Further studies in 1938 showed that the ovary and the oviduct of the frog
during the summer months were enlarged following pituitary stimulation, the latter
presumably as a result of an increased output of ovarian hormone.
The present study was undertaken to show the effect of injections of a solution
in sesame oil of the pure crystals of dihydroxyestrin (a- estradiol benzoate).1 Female
frogs were castrated early in July when the oviducts are very small and were allowed
to recover fully from the effects of the operation. Approximately five grams of
lean beef were fed daily, the animals gained in weight and appeared in excellent con-
dition. From August 10 to August 17 inclusive, a total of 1000 rat units per frog
of the solution was injected into the thigh muscles. An average increase of 47.1
per cent in the weight of the oviducts in proportion to the body weight (more marked
in diameter than in the length of the oviducts) of the injected animals over the
castrate controls had occurred as a result of the eight days of injection.
1 The hormone was furnished through the courtesy of Dr. Max Gilbert of the
Schering Corporation.
I wish to thank Miss Naomi de Sola Pool for technical assistance.
Vol. LXXVII, No. 3 December, 1039
THE
BIOLOGICAL BULLETIN
PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY
THE EFFECTS OF A LACK OF OXYGEN AND OF LOW
OXYGEN TENSIONS ON PARAMECIUM
J. A. KITCHING i
(From the Department of Physiology, Princeton University, Princeton, N. J., and
the Department of Zoology, University of Bristol, England)
INTRODUCTION
The ability of Paramecium to survive without oxygen has been the
subject of many investigations, and the results previously published are
at variance with one another. Loeb and Hardesty (1895) confined
Paramecium in a special gas chamber which they freed of oxygen either
with a stream of hydrogen or by means of alkaline pyrogallol ; the
organisms died after twenty-four hours. Budgett (1898), using an
Engelmann gas chamber flushed with hydrogen, obtained a survival
time of several hours, after which the Paramecium blistered. Putter
(1905) found Paramecium caudatum to survive for five or six days in
a specially designed bottle which had been flushed thoroughly with
nitrogen. Faure-Fremiet et al. (1929) got a survival time of twenty-
four hours at 25° C. in sealed tubes containing leuco-methylene blue
(reduced by sodium hydrosulphite). Juday (1909) discovered Para-
mecium sp. in the deeper waters of Lake Mendota (Wisconsin) at a
time when he believed these waters to be devoid of free oxygen ; ac-
cording to him Paramecium is therefore able to live anaerobically for
several months. Fortner (1924) enclosed P. caudatum in an at-
mosphere of hydrogen together with aerobic bacteria to remove any
remaining oxygen ; they survived for several hours, and their con-
tractile vacuoles continued to function, although at a reduced frequency.
Gersch (1937) found that Paramecium died within 10 seconds in gas
purified of its oxygen by means of alkaline pyrogallol. In the work
which is described in this paper I have attempted to harmonize these
discrepancies.
In addition it has been claimed by several investigators that the
respiration of Paramecium is insensitive to cyanide (Lund, 1918;
Gerard and Hyman, 1931 ; Shoup and Boykin, 1931). I have therefore
1 Fellow of the Rockefeller Foundation.
339
340 J. A. KITCHING
made some observations to determine the capacity of Paratnecium to
carry on its normal activities, such as secretion by the contractile vacu-
oles and locomotion, in the presence of cyanide.
APPARATUS AND METHODS
Anaerobiosis in a Hanging Drop
In subjecting Protozoa 'to a lack of oxygen, it is necessary to ensure
the following conditions :
(1) A genuine lack of oxygen. It is not possible to assure com-
plete absence of oxygen molecules, but the oxygen content of the medium
surrounding the organisms must be so small that the organisms could
not possibly make any significant use of it for oxidative metabolism.
Oxygen must not be generated by accompanying plant cells or bacteria.
(2) No extraneous contamination. For instance, there must be no
harmful impurities in any gas used. For this reason the absorption of
oxygen by alkaline pyrogallol is perhaps to be avoided.
(3) No harmful secondary effects. For instance, the flushing of
the medium with an oxygen-free gas mixture must not result in a
harmful shift of the hydrogen ion concentration.
The general method used was to pass oxygen-free gas past a hanging
drop containing the organisms. For this purpose cylinder hydrogen,
or cylinder nitrogen (water-pumped), was first bubbled through con-
centrated sulphuric acid, dilute sulphuric acid, dilute potassium hy-
droxide and distilled water. Next it was purified of its oxygen. In
most experiments hydrogen was used, and was passed through an elec-
trically heated quartz tube containing platinized asbestos. The quartz
tube extended sufficiently far at each end beyond the heating coil to
avoid any significant warming of the deKhotinsky cement seals. In
some experiments, however, nitrogen was passed over hot copper in a
special internally heated furnace described by Kendall (1931). This
furnace consists of a Pyrex glass tube containing a cylinder of copper
gauze of large surface area, which is heated from within by means of
a coil of nichrome wire. In this furnace it is always possible to see
the condition of the copper, and in the time required for one experi-
ment only a small section of the copper, at the inflow end, became tar-
nished. Finally the gas was carried by pure lead tubing with seals of
deKhotinsky cement through a closed glass wash tube with distilled
water in it to the chamber containing the organisms.
The organisms were mounted in a hanging drop on a coverglass
which was sealed with vaseline or a mixture of vaseline and paraffin
EFFECTS OF OXYGEN LACK ON PARAMECIUM 341
wax to the chamber. The hanging drop was not allowed to touch the
vaseline. The chamber itself consisted of a glass ring about 2 cm. in
diameter and 1 cm. deep, closed underneath by a microscope slide to
which it was sealed with deKhotinsky cement, and with glass inlet and
outlet tubes. The upper edge of the ring was ground to support a
coverglass, and the microscope slide which formed the base of the
chamber fitted onto the mechanical stage of the microscope.
From the chamber the gas was carried by lead tubing to a light-
proof box, where it was bubbled either through a simple water trap or
through a suspension of marine luminous bacteria. These bacteria
(Achromobacter fischeri) luminesce in the presence of minute traces
of oxygen, and according to Harvey and Morrison (1923) about 0.005
mm. of oxygen can be detected in this way. After the purified hydrogen
or nitrogen had bubbled for five or ten minutes through the bacterial
suspension no luminescence could be detected with the dark-adapted
eye, but admission of air gave immediate recovery of luminescence.
An analysis of the purified gas made by mass spectrograph by Dr.
W. R. Guyer showed no trace of oxygen, although one part in 105 could
have been detected.
For control experiments another glass chamber, similar to the first,
was mounted on the stage of a second microscope, and CO,-free air
was drawn through gently with a suction pump.
Anaerobiosis in a Tube with Reduced Redo.v Indicator
The survival of organisms in the presence of a reduced redox in-
dicator low in the redox scale has been used as a demonstration of
anaerobic life. For instance, Clark (1924) found that certain bacteria
reduced indigo carmine until a high ratio of the reduced to the oxidized
substance was attained. He calculated that the oxygen tension in equi-
librium with this mixture was so low as to be physically meaningless.
For the present purpose, however, such a condition does not establish
anaerobiosis, since the bacteria were obtaining oxidative energy by re-
ducing the dye. The dye should therefore be reduced as completely as
possible to avoid this possibility. Furthermore, the use of methylene
blue, indophenols, or other indicators high in the series is to be avoided.
The reoxidation of indicators high in the series is relatively slow at
atmospheric oxygen tension (Barron, 1932), and might be very slow at
low oxygen tensions even at the experimental hydrogen ion concentra-
tion. Harvey (1929) has shown that indophenols in the presence of
luminous bacteria may remain reduced in the presence of a concentra-
tion of free oxygen sufficient to allow the bacteria to luminesce. Under
certain conditions equilibrium may never be attained, and the state of
342 J. A. KITCHING
the dye is then no indication of the oxygen tension in the solution. The
method described below to the best of my knowledge avoids these errors.
Paramecium in the requisite medium was placed in an internally
sealed glass wash tube with some platinized pumice and sufficient indigo-
trisulphonate to color the solution a clear blue. (Platinized pumice
was found more satisfactory than platinized asbestos, as the latter is
easily stirred up, and splits into fine sharp spikes which may damage the
organisms. Platinized pumice was prepared by boiling pumice chips in
chloroplatinic acid until most of the air had been driven out of the pores.
Then sodium formate was added until platinum was precipitated in the
pores and all over the surface of the pumice. The chips were then
washed very thoroughly in running water.) The wash tube was sealed
with deKhotinsky cement to the source of purified hydrogen, and the
gas escaping from it was carried by lead tubing to the anaerobic chamber
for a parallel experiment. The indicator dye bleached five or ten
minutes after the hydrogen was turned on. The wash tube was then
wrapped completely and thoroughly in black cloth so as to exclude all
light, and slow bubbling of the hydrogen was continued. This pre-
caution was taken although no photosynthetic organisms were ever
detected in the cultures.
Controlled Oxygen Tensions
In order to subject Paramecium to controlled and known oxygen
tensions, the apparatus used for anaerobiosis in a hanging drop was
modified. Hydrogen, purified of its oxygen as already described, and
oxygen were led through flow meters (see Harvey and Morrison, 1923)
to a T tap in which they were mixed. The resulting known gas mixture
was then conveyed through the wash tube with distilled water to the
observation chamber. Lead tubing and seals of deKhotinsky cement
were used throughout. According to a later refinement, condensation
of water in the capillaries of the flow meters was prevented by a tubular
show-case lamp placed alongside them. By manipulation of the T tap
the organism could be subjected rapidly either to pure hydrogen or to
any desired ratio of hydrogen and oxygen.
In a few experiments at very low oxygen tensions, oxygen was
mixed with hydrogen in proportions measured by flow meters, and a
small part of the resulting mixture was then mixed in the same way
with additional pure hydrogen. The unwanted part of the first mix-
ture was rejected through a mercury trap. In this way extremely low
tensions of oxygen could be provided with reasonable accuracy.
The tension of oxygen in the observation chamber was calculated
EFFECTS OF OXYGEN LACK ON PARAMECIUM 343
with clue regard for the barometric and water vapor pressures. The
total gas pressure in the observation chamber exceeded that of the air
by an insignificant amount.
The Diffusion of Oxygen in a Hanging Drop
I am indebted to Dr. H. P. Robertson of the Physics Department,
Princeton University, for a mathematical expression relating the thick-
ness of the hanging drop, the original concentration of oxygen in it,
the diffusion coefficient of oxygen in water, and the time required to
bring that drop to any given low oxygen tension after the drop has been
placed over an atmosphere devoid of oxygen. The drop has been re-
garded as a film parallel to the coverglass, which for my experiments is
reasonably true. Of the infinite series which was derived only the first
term is significant, viz.
07T2
I /">-' — - • I OP'
ATT2 ge 8
where t =the time required for the equilibration,
b = = the thickness of the drop,
6 = the ratio of concentration of oxygen attained at time / to
initial concentration,
A = the diffusion coefficient of oxygen in water.
For conditions approaching those of the experiments A is given by
Bruins (1929) as very nearly 2.0 X 10~5.
It will be seen that the time required for the drop to reach any given
oxygen tension varies as the square of its thickness. Let us choose
arbitrarily an oxygen tension of 10~4 X the oxygen tension of the at-
mosphere — less than one-tenth the minimal value necessary (as will be
shown later) to allow activity in Paramecium. For 0=lQr4, when
b = y± mm., t = 2 minutes ; and when b = i/£ mm., t = 8 minutes.
The films used were of this order of thickness, and in practice it is
probable that equilibration is accelerated by the water currents set up
by the swimming of the Paramecium, as well as by convection currents.
These theoretical results agree well with experiments on certain
marine amoebae, to be published later, in which oxygen tensions below
1/2 mm. are required for stoppage of movement, and which in a hanging
drop come to a standstill within five minutes of the time when pure
hydrogen is turned on. It may be concluded that in my experiments
adequately anaerobic conditions were attained within ten minutes.
Similarly the equilibration of a hanging drop with gas mixtures of
known low oxygen tension must be rapid, provided always that the drop
is a thin one.
344
J. A. KITCHING
MATERIAL
Paramecium was cultured in timothy hay infusion, and was obtained
from the following sources :
(1) P. multimicronucleatum collected from a backwater of the canal
near Princeton, N. J. Individuals of this race had three con-
tractile vacuoles.
(2) P. multimicronucleatum of a pure line (Clone I), with two con-
tractile vacuoles.
(3) P. caudatum of a pure line (Clone D).
TABLE I
Survival of Paramecium multimicronucleatum clone I under anoxic conditions in
a hanging drop of its own culture fluid. CO2, when used, was at a partial pressure of
12 mm., which was sufficient to maintain the culture fluid at pH 7.1 approximately.
Room temperature was 24-28°C.
Gas used
Time for first
Paramecium
to blister
Time for fifty
per cent to
blister
Time for last
to blister
Number of
animals used
Date (1939)
minutes
minutes
minutes
Pure H2
160
370
440
14
August 6
Pure N2
210
465
585
43
August 3
H2 + C02
360
650
690
19
July 29
H2 + CO2
595
665
710
28
August 1
N2 + CO2
500
660
720
12
August 5
N2 + C02*
500
660
725
9
August 7
CO2-free air
(control ex-
periment) ....
!
—
—
21
July 30-31
* Extra pure CO2: see text.
f All survived throughout experiment (31 hours) and remained normal in ap-
pearance and activity.
The last two were both kindly supplied to me through the courtesy
of Professor S. O. Mast of the Johns Hopkins University.
RESULTS
In all cases and in all media Paramecium multimicronucleatum and
P. caudatum, when mounted in a hanging drop in absence of oxygen,
continued to swim and to evacuate fluid by the contractile vacuoles for
a limited time. However, the speed of swimming and the rate of out-
put of the contractile vacuoles decreased, and eventually the organisms
stopped. Then the trichocysts were discharged ; the anterior end of the
body usually, but not always, became constricted ; blisters formed at the
surface of the body ; and cytolysis ensued. Paramecium which had
EFFECTS OF OXYGEN LACK ON PARAMECIUM
345
ceased all activity for lack of oxygen, and which had even begun to
blister, recovered rapidly on admission of sufficient oxygen (see Tables
III and IV).
The duration of anaerobic survival was very variable, and depended
partly on the media used. The longest survival times were got with
culture fluid. In hanging drops of culture fluid survival times in ab-
sence of oxygen ranged from one to twelve hours. It appeared (as
already shown by Putter (1905)) that Paramecium from well-fed,
flourishing cultures survived the longest. There were considerable dif-
ferences between cultures of the same clone. However, exposure of a
hanging drop of culture medium to pure hydrogen or nitrogen leads to
TABLE II
The effect of a lack of oxygen on Paramecium caudatum clone D, after segregation
in test tube in culture fluid without hay.
Medium
Culture fluid;
pure hydrogen
M/300 phosphate
buffer; pure Hz
M/300 phosphate
buffer; COs-free
air as control
Experiments on same day:
Time required for first one to stop
or blister, in minutes
88
23
no visible ad-
Same for 50 per cent
154
56
verse effects
Same for last one
235
76
Number of individuals used
28
8
12
Experiments five days later:
Time required for first one to stop
or blister
79
7
27
Same for 50 per cent
79
7
29
Same for last one
109
9
68
Number of individuals used
8
4
7
a loss of carbon dioxide, with a resulting shift of the hydrogen ion
concentration far into the alkaline range. Accordingly, pure hydrogen
was bubbled through some culture fluid and the resulting shift of pH,
according to measurements kindly made for me with the glass electrode
by Dr. Marshall E. Smith, was from about 6.2-6.4 to about 8.9-9.0.
Addition of about 12 mm. partial pressure of carbon dioxide to the hy-
drogen was sufficient to maintain the culture fluid at about pH 7.1.
When hydrogen or nitrogen together with this amount of carbon di-
oxide were passed through the observation chamber, the Paramecium
survived slightly but significantly longer than without the carbon di-
oxide (Table I) ; the series of changes leading to death was, however,
346
J. A. KITCHING
the same. In these experiments no purification of the carbon dioxide
was undertaken, but the connection bewteen the carbon dioxide cylinder
and the apparatus was entirely of glass, lead tubing, and deKhotinsky
cement, and the pressure was regulated by a double water trap of such
a nature as to prevent backward diffusion of oxygen. The manufac-
turers of the carbon dioxide stated that the oxygen content of their
cylinders varied between two and sixteen parts in ten thousand. The
oxygen content of the mixed carbon dioxide and nitrogen or hydrogen
must have been insignificantly low, and experiments with a cylinder
TABLE III
The effect of distilled water, as compared with culture fluid, on the ability of
Paramecium multimicronucleatum to withstand a lack of oxygen. For each gas mix-
ture two separate hanging drops, with the two media, were suspended from the same
coverglass.
Pure Hydrogen
CCh-free Air (Control)
Time
Culture fluid
Distilled water
Culture fluid
Distilled water
minutes
0
started
started
started
started
57
all very slow
1 blistered,
normal
normal
rest very slow
movement
movement
64
1 blistered,
* i
ii
ii
rest very slow
73
all stopped
all stopped
ii
ii
74
3.5 mm. oxygen admitted
80
3 swimming,
3 swimming.
1 1
it
rest appear
rest appear
dead
dead
395
2 swimming
2 swimming
ii
ii
normally,
normally,
rest dead
rest dead
Number of or-
ganisms used
15
12
12
8
which had been cleaned out and filled with special care by the manufac-
turers gave entirely similar results.
The results obtained in the presence of a reduced redox indicator
(indigo trisulphonate) in a wash tube in the dark agreed well with those
obtained by the hanging drop method. In several experiments the puri-
fied hydrogen was bubbled first through the wash tube with the platinized
pumice, and then through the anaerobic chamber. In all such experi-
ments a few individuals survived in the wash tube after all had cytolysed
in the anaerobic chamber. However, the wash tube contained many
thousands of individuals, and it is to be expected that out of so many
EFFECTS OF OXYGEN LACK ON PARAMECIUM
347
a few would be more hardy. At the time when the majority in the
anaerobic chamber cytolysed there was a marked decrease in the number
visible in the wash tube. The longest survival in culture fluid was about
twelve hours.
In other experiments the organisms were washed four times with
M/300 phosphate buffer (Na2HPO4 + KH2PO4) at pH 7.0-7.1, and
mounted over the anaerobic chamber in this medium. Under anoxic
conditions Paramecium underwent the same series of changes as in cul-
ture medium, although the constriction of the anterior end and the
blistering seemed more sudden and violent. Also it survived for much
less time in phosphate buffer than in culture fluid ; even though in air
i OCX?
a
z
o
<J
a
3«00
200
AIR
HYDROGEN
AIR
10
20
TIME
3O 40
IN MINUTES
8O
FIG. 1. The effect of pure hydrogen on the rate of output of the anterior
contractile vacuole of Paramecium multimicronuclcatum in a hanging drop of
dilute phosphate buffer.
it lived without apparent damage for many hours (in some cases ob-
servations were extended over two days) in the buffer solution. How-
ever, if some Paramecium were removed from the culture and placed
in a test tube with some of the culture fluid but without hay, their ability
to survive anaerobically in either medium (culture or buffer solution)
decreased progressively, and after a few days they became fatally sus-
ceptible to phosphate buffer even when in air. These results are illus-
trated in Table II.
In the absence of oxygen Paramecium was found (in a few experi-
ments) to survive equally well either in culture fluid or in distilled water
(Table III). However, in M/300 KC1 some individuals cytolysed
348
J. A. KITCHING
almost instantly in air, and in absence of oxygen the remainder survived
only a few minutes.
The contractile vacuoles, both in culture medium and in phosphate
buffer, continued to function under anaerobic conditions, but finally be-
15
05
Q
§ 00.
" 15
10
DC
111
O.
05
z 00
r IS
z 10
o
E 00
E 15
10
05
00
15
10
05
00
AIR
AIR
AIR.
AIR
AIR
7-0 MM. O,
N MM. O,
0-3 MM O.
0-2 MM. O.
0-1 MM.
AIR
Al R
A IR
AIR
50
100
260
TIME
IN
MINUTES
FIG. 2. The influence of low oxygen tensions on the rate of swimming of
Paramecium multimicronucleatum in dilute phosphate buffer.
came very slow. In two experiments Paramecium was slowed down by
means of an agar gel just sufficiently viscous to make observation pos-
sible. The result of one of these experiments is plotted in Fig. 1.
The stoppage of the contractile vacuoles after a period of lack of oxygen
was found to be reversible. Contrary to the findings of Frisch (1937),
EFFECTS OF OXYGEN LACK ON PARAMECIUM
349
the Paramecium which I used showed normal activity of the contractile
vacuoles whether they were swimming or stationary. This was true of
Paramecium in hanging drops of culture medium or dilute buffer in
contact with air, with or (usually) without agar. I attribute the dis-
crepancy to the fact that his organisms had been sealed in a vaseline
ring for many days and were not fully active.
A series of experiments was made to determine the survival and
activity of Paramecium multimicronucleatum at low oxygen tensions.
The rate of swimming of healthy Paramecium in phosphate buffer was
found, after an initial burst of high activity, to remain reasonably con-
stant for as long as observations were continued (up to 22 hours),
TABLE IV
Recovery of Paramecium caudatum at known oxygen tensions from lack of oxygen,
in dilute phosphate buffer solution.
Condition at end of
Duration of
anaerobic period:
number of animals
Duration
Condition after admission of oxygen
at this tension: number of animals
lack of
oxygen in
minutes
Stopped
but normal
in shape
Stopped
but pointed
and
blistered
admitted
in mm.
vations at
this tension
in minutes
Recovered
after
stoppage
Recovered
after
stoppage
and
blisters
Cytolysed
44
1
2
27
36
1
1
1
/46
6
2
7.0
38
6
1
1
131
5
2
0.28
21
3
1
3
36
1
7
1.4
38
1
6
1
31
1
5
0.85
41
1
1
4
57
3
2
0.57
22
3
0
2
53
11
4
0.23
24
1*
0
14
36
3
1
0.17
18
0
0
4
* Movement very slow.
Accordingly single organisms were acclimatized for 90 minutes in a
hanging drop of the buffer solution in a stream of moist carbon dioxide-
free air. After this observations were made of the rate of swimming
(a) in carbon dioxide-free air, (b) in a known mixture of oxygen and
hydrogen, and (c) in carbon dioxide-free air. The second period (&)
lasted usually about two hours. Readings of the time required for the
organisms to traverse the distance indicated by the ocular scale (2.06
mm.) were made in groups of twelve, and the mean for each group
determined. The standard error of the mean time usually lay between
3 and 5 per cent of the mean value. Results are shown in Fig. 2.
There was a slowing down of swimming in oxygen tensions below 1 mm.
Below 0.2 mm. approximately the organisms died, but above this value
350 J. A. KITCHING
they slowed down to a speed approximately constant within the duration
of the treatment ; and this effect was reversible. Individual variation
was such that it did not seem worthwhile to try to determine a detailed
relation between oxygen tension and rate of swimming.
In a further series of experiments a group of Paramecium was
mounted in a hanging drop of the dilute phosphate buffer in the an-
aerobic chamber, and subjected to anoxic conditions until all had stopped
and some of them had blistered. Then oxygen was admitted at a known
partial pressure, and the extent of recovery recorded. Results are
shown briefly in Table IV. Recovery could be obtained, on admission
of sufficient oxygen, even after the organisms had begun to blister, and
in such cases the blisters were gradually resorbed. In many of these
cases it is certain that if the organisms had been left for another one or
two minutes without oxygen irreversible cytolysis would have ensued.
The minimal oxygen tension needed for recovery was of the same order
as that which was found just to allow swimming in the previous series
of experiments.
The effect of cyanide on the secretory activity of the contractile
vacuoles and on the general activity of the animals was examined briefly
in three experiments. Single individuals of Paramecium multimicro-
nucleatum were mounted in a hanging film of river water just sufficiently
thin to prevent too rapid swimming. The cover glass was sealed with
vaseline over a small glass cell half filled with river water, so that
evaporation from the film was prevented. After a period of examina-
tion in river water the Paramecium was transferred with the usual four
washes to a dilute solution of sodium cyanide (M/200, M/ 1,000, M/
2,000, pH corrected to 7.1) in river water, and the fluid in the cell was
also replaced by the cyanide solution. This procedure was carried out
as quickly as possible in order to avoid loss of cyanide, and the examina-
tion of the organism was continued. After an initial depression of
activity the vacuoles continued to function regularly although slightly
less vigorously than in plain river water. The cilia also continued to
beat actively. Observations were continued in one case for over ten
hours after the cyanide treatment was begun.
DISCUSSION
The majority of workers are agreed that Paramecium at room tem-
peratures (20-25° C.) can survive for some hours, though not days,
without oxygen. In view of the indubitable demonstrations of this
fact, the results of Gersch (1937), who observed almost instantaneous
death, must be discounted. It seems somewhat questionable whether
Putter, who (1905) obtained a much longer supposedly anaerobic sur-
EFFECTS OF OXYGEN LACK ON PARAMECIUM 351
vival, really achieved strictly anoxic conditions; and it is clear that
Paramecium can make some use of oxygen at partial pressures below
1 mm. It also seems doubtful whether Juday (1909) would have de-
tected oxygen in such low concentrations in the bottom waters of Lake
Mendota (Wisconsin), where he claimed that Paramecium lived an-
aerobically. However, it remains possible that Paramecium might be
found to survive without oxygen for longer periods under experimental
conditions if it were supplied with suitable food. Slight discrepancies
between the results of other workers may be ascribed to variations in
the excellence of the oxygen " lack," possibly to the use of several
different species, and particularly to variations in the state of nutrition
of the organisms. This latter condition was stressed by Putter (1905),
and probably accounts for the beneficial effect of stirring the culture.
There is clearly some adverse influence in phosphate buffer which
a normal healthy Paramecium can withstand in air, but which success-
fully operates against a starved Paramecium in air or against a well-fed
one in absence of oxygen. The constriction of the anterior end of the
organism and the blistering just before death are probably the result
of a violent contraction of the myonemes. They were found to occur
even when the organism was in M/20 lactose solution, and it is there-
fore unlikely that they can be attributed to osmotic uptake of water by
the organism. A somewhat similar effect has been seen in Paramecium
subjected to an electric current (see Kalmus, 1931). The phosphate
buffer, either directly or indirectly, hastens the time for the myoneme
contraction. It seems probable that the harmful effect of this buffer is
due to a lack of balance of ionic concentrations, although this matter has
not yet been investigated in detail.
Whereas cyanide in very low concentration inhibits the action of the
contractile vacuoles of peritrich ciliates (Kitching, 1936), it has no very
marked effect on those of Paramecium. The prolonged anaerobic ac-
tivity of Paramecium might account for this. However, according to
the results of various workers, at least a considerable part of the respira-
tion of Paramecium must be insensitive to cyanide. Gerard and Hyman
(1931) found that substitution of phosphate buffer for a calcium-con-
taining water approximately halved the rate of oxygen consumption,
but addition of cyanide made no further difference. The persistence of
vacuolar activity in the presence of cyanide harmonises with the view
that the respiration of Paramecium is relatively insensitive to this
substance.
Paramecium is able to continue swimming, or to recover from a lack
of oxygen, at oxygen tensions down to 0.3 mm. However, it is possible
that higher tensions may be needed for growth and prolonged survival.
'QD
352 J. A. KITCHING
Amberson (1928), by a not very delicate method, obtained results which
suggest that the respiration of P. caudatum is depressed slightly at
tensions of oxygen below 50 mm.
A comparison of the results obtained with Parameciuin and with
other Protozoa will be made in another paper.
ACKNOWLEDGMENTS
I wish to thank Dr. E. N. Harvey for his guidance and criticism,
as well as for the facilities of the physiological laboratory, Princeton
University. The work was completed at the Marine Biological Lab-
oratory, Woods Hole.
SUMMARY
1. Parameciutn multimicronucleatum and P. caudatum were sub-
jected to pure hydrogen, pure nitrogen, and known mixtures of hydro-
gen and oxygen, while in a thin hanging drop under microscopical
observation.
2. In all cases there was a limited period of anaerobic survival, dur-
ing which activity of swimming and of contractile vacuoles was gradu-
ally diminished. Finally the organisms stopped, blistered, and cytolysed.
3. Admission of sufficient oxygen, even after blistering had begun,
gave recovery.
4. Survival under anoxic conditions was best in culture fluid main-
tained at a reasonable hydrogen ion concentration by the addition of
small quantities of carbon dioxide to the hydrogen or nitrogen. Under
these conditions some organisms survived as long as twelve hours.
5. Paramecium was extremely variable as regards the length of
anaerobic survival in any one medium. This variability is ascribed to
physiological condition and not to genetic factors.
6. Survival under anoxic conditions was much shorter in dilute phos-
phate buffer than in culture medium. This is tentatively ascribed to a
lack of balance of ionic concentrations.
7. In phosphate buffer the rate of swimming was reduced at ten-
sions of oxygen below 1 mm., and the organisms died within a short
time at tensions below 0.2 mm. At tensions above 0.3 mm. some
measure of recovery from lack of oxygen could be obtained.
LITERATURE CITED
AMBERSON, W. R., 1928. The influence of oxygen tension upon the respiration of
unicellular organisms. Biol. Bull, 55: 79.
BARRON, E. S. G., 1932. The rate of autoxidation of oxidation-reduction systems
and its relation to their free energy. Jour. Biol. Chem., 97 : 287.
EFFECTS OF OXYGEN LACK ON PARAMECIUM 353
BRUINS, H. R., 1929. Coefficients of diffusion in liquids. International Critical
Tables, 5 : 63.
BUDGETT, S. P., 1898. On the similarity of structural changes produced by lack
of oxygen and certain poisons. Am. Jour. Physiol., 1 : 210.
CLARK, W. M., 1924. Life without oxygen. Jour. Wash. Acad. Sci., 14: 123.
FAURE'-FREMIET, E., C. LEON, A. MAYER, AND L. PLANTEFOL, 1929. Recherches
sur le besoin d'oxygene libre. I. L'oxygene et les mouvements des Para-
mecies. Ann. de Physiol., 5 : 633.
FAURE-FREMIET, E., C. LEON, A. MAYER, AND L. PLANTEFOL, 1929. L'oxygene
libre et les mouvements des Paramecies. Compt. Rend. Soc. Biol., 101 :
627.
FORTNER, H., 1924. tiber die physiologisch differente Bedeutung der kontraktilen
Vakuolen bei Paramecium caudatum Ehrenb. Zool. Ans., 60 : 217.
FRISCH, J. A., 1937. The rate of pulsation and the function of the contractile
vacuole in Paramecium multimicronucleatum. Arch, filr Protist., 90: 123.
GERARD, R. W., AND L. H. HYMAN, 1931. The cyanide insensitivity of Para-
mecium. Am. Jour. Physiol., 97 : 524.
GERSCH, M., 1937. Vitalfarburg als Mittel zur Analyse physiologischer Prozesse
(Untersuchungen an Paramecium caudatum). Protoplasma, 27: 412.
HARVEY, E. N., 1929. A preliminary study of the reducing intensity of luminous
bacteria. Jour. Gen. Physiol., 13 : 13.
HARVEY, E. N., AND T. F. MORRISON, 1923. The minimum concentration of oxygen
for luminescence by luminous bacteria. Jour. Gen. Physiol., 6 : 13.
JUDAY, C., 1909. Some aquatic invertebrates that live under anaerobic conditions.
Trans. Wisconsin Acad. Sci., 16 : 10.
KALMUS, H., 1931. Paramecium. Jena, Verlag von Gustav Fischer, 188 pp.
KENDALL, E. C., 1931. The removal of traces of oxygen from nitrogen. Science,
N. S., 73 : 394.
KITCHING, J. A., 1936. The physiology of contractile vacuoles. II. The control
of body volume in marine Peritricha. Jour. Exper. Biol., 13: 11.
LOEB, J., AND I. HARDESTY, 1895. Ueber die Localisation der Athmung in der
Zelle. Pfliigers Arch. f. d. ges. Physiol., 61 : 583.
LUND, E. J., 1918. Quantitative studies on intracellular respiration. II. The rate
of oxidations in Paramecium caudatum and its independence of the toxic
action of KNC. Am. Jour. Physiol., 45 : 365.
PUTTER, A., 1905. Die Atmung der Protozoen. Zeitschr. f. allg. Physiol., 5: 566.
SHOUP, C. S., AND J. T. BOYKIN, 1931. The insensitivity of Paramecium to cya-
nide and effects of iron on respiration. Jour. Gen. Physiol., 15 : 107.
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Ciliata). Trans. Am. Micros. Soc., 47: 275.
DARK ADAPTATION AND REVERSAL OF PHOTOTROPIC
SIGN IN DINEUTES
J. E. G. RAYMONT
(From the Biological Laboratories, Harvard University)
INTRODUCTION
Animals which exhibit orientation and directed movement with re-
spect to a light source are said to be positively or negatively phototropic,
according as they move towards or away from the source. Under cer-
tain conditions, a change of phototropic sign may occur, e.g. an animal
which normally moves towards the source will move from it. Among
the more important factors which have been shown to produce such a
reversal of phototropic sign are: difference in absolute light intensity,
change of light intensity, temperature, pH, and some chemical sub-
stances. The possible effect on phototropic sign of the degree of dark
adaptation, however, has been largely neglected. Crozier and Wolf
(1928) demonstrated the effect of dark adaptation on the strength of
phototropism in Agriolimax and more recently Wolter (1936) has re-
ported that some specimens of Carcinus macnas show a change of photo-
tropic sign with dark adaptation. Since Clark (1931, 1933) demon-
strated the process of dark adaptation by means of phototropic reactions
for the "whirligig beetle" (Dineutes assimilis), it was decided to use
this beetle in testing for the possible effect of dark adaptation on the sign
of phototropism.
MATERIAL AND METHODS
The animals used in these experiments were of the species Dineutes
Iiorni,1 and were collected from Fresh Pond, Cambridge, Massachusetts.
They were kept in an aquarium in a lighted room, and were fed on pieces
of fresh meat and fish, floated on wood.
The eye in Dineutes is divided into dorsal and ventral parts on each
side, and the phototropic responses were compared when the whole, and
when only part of each eye was functional. Blinding was effected by
blackening the surface of part of the eye with asphaltum varnish.
In each experiment the beetle was first light-adapted and was then
left for the desired period in a covered vessel of water. Following this
1 1 am indebted to Mr. C. Parsons of Harvard University for the identification
of this species.
354
DARK ADAPTATION AND PHOTOTROPISM
period in darkness the phototropic reaction was observed by allowing the
animal to crawl on a dry, horizontal test-plate of ground glass. This
was illuminated from one side by a diverging beam of light emanating
from a slit (!%" X %") covered by a ground glass plate, behind which
a shielded 100-watt lamp was set. The test plate was level with the
bottom of the slit and rested on a dull black surface. A little light was
diffusely reflected up from the surface of the test plate but the greater
part of the light reached the insect directly from the horizontal beam.
The light intensities at the two ends of the test plate were 1.8 and 0.6 f .c.
respectively.2 The path taken by the insect was followed with a pencil,
and marked directly on the plate. This did not appear to disturb the
beetle.
Light adaptation was effected by placing the beetle, in water in a
glass cell (2%" X %") for 20 minutes, in the path of a beam of light
from a 500-watt projection lamp. A strongly-reflecting surface at the
" darker end " of the cell ensured fairly uniform lighting. Intensity of
adapting light was 4,800 f.c.2 Heating was avoided during the light
adaptation period by interposing heat-absorbing filters.
At the beginning of each experiment, the beetle was placed by means
of a piece of stiff paper at approximately the centre of the test plate.
It was not headed precisely in any special direction, as earlier experi-
ments had shown that the animal oriented immediately, irrespective of
the direction in which it was placed.
OBSERVATIONS
With all eyes functional, a strong, positively phototropic response
was invariably obtained, whether the animal was light- or dark-adapted.
When any two, or when only one of the four eyes 3 was functional,
a negative response was obtained if the beetle were dark-adapted. If
only one eye was blackened the animal was photopositive. In the great
majority of experiments, either the two upper, or the two lower eyes
were covered. This avoided any complications, such as possible circus
movements (cf. Clark).
It was often impossible to carry out all the experiments, with the
various combinations of eyes, on the same individual, but the behavior
was sufficiently constant to combine the results obtained from different
animals. (A very few individuals were found in which the photo-
tropic reactions were irregular.)
* 2 Light intensities were measured with a Macbeth Illuminometer, using dense
filters for the highest intensities.
3 To obviate needless repetition, the upper and lower halves of the eyes are
simply referred to in the results as upper and lower eyes.
356
J. E. G. RAYMONT
The results of experiments with (a) two upper eyes only functional
and (b) two lower eyes only functional were similar. The beetles were
always positively phototropic when they were light-adapted. They were
usually still positively phototropic after dark adaptation of less than
two hours duration, but became negatively phototropic after dark adap-
8
8
FIG. 1. Tracings of paths followed by Dinentes (Individual X) May 19,
1938, in 8 successive trials after dark adaptation for seven hours. The upper eyes
were functional. The numbers refer to the order of trials. L. indicates light
source.
tation of more than two hours duration. However, the actual period
of dark adaptation necessary to bring about the change in phototropic
sign showed considerable individual variation; in a very few cases,
even one hour was sufficient.
A beetle which had been dark-adapted for a period sufficient to be-
DARK ADAPTATION AND PHOTOTROPISM
357
come negatively phototropic would show a reversal to the original posi-
tive condition as it became partially light-adapted. This was shown as
follows. A beetle, after dark adaptation for many hours, was tested
repeatedly in the beam. The responses exhibited at first were all photo-
negative, but the sign of phototropism became reversed after a certain
11
10
FIG. 2. Tracings of paths followed by Dineutcs (Individual B) December 15,
1937, in 11 successive trials after dark adaptation for twelve hours. The upper
eyes were functional. No " run " was obtained in Trial 9.
amount of light adaptation had been brought about by the light from
the test beam itself. Subsequently, all further responses were con-
sistently photopositive.
This reversal of phototropic sign with light adaptation was also
observed, while avoiding the repetition of trials. A fully dark-adapted
beetle was tested once to demonstrate the negatively phototropic reaction.
358
J. E. G. RAYMONT
It was then left in the test beam, surrounded by a small glass cell, until
the light effected a sufficient degree of light adaptation. A single new
trial then showed the beetle to be positively phototropic.
FIG. 3. Tracings of paths followed by Dineutes (Individual V) May 20,
1938, in 7 successive trials after dark adaptation for seven hours. The lower eyes
were functional.
It was obvious that a considerable period of exposure to darkness
was necessary to elicit the negatively phototropic reaction. Experiments
were next carried out to test the possibility that with all eyes functional,
DARK ADAPTATION AND PHOTOTROPISM 359
a very prolonged period in darkness might cause a reversal of photo-
tropic sign. The results of these experiments, with seven individuals,
showed that reversal cannot be brought about by 24-65 hours of dark
adaptation, if all eyes are functional.
If the two upper or the two lower eyes were blackened but a very
few ommatidia of one of these eyes were left exposed, the beetle re-
mained photopositive when fully dark-adapted. On completely cover-
ing the eye, the beetle, when dark-adapted, became negatively phototropic.
A few experiments were carried out with any three eyes blackened,
and other experiments in which one upper eye and one lower eye of
the opposite side were blackened. Such experiments, with any two
or any three eyes covered, always showed that the dark-adapted beetles
were negatively phototropic, and that light adaptation caused a reversal
to positive phototropism.
TABLE I
Change in the direction of phototropic path followed by Dineutes (Individual V),
in 7 successive trials, consequent upon light adaptation. The animal was first dark-
adapted for 7 hours. The lower eyes were functional.
Angle of Deviation
Trial
Number
1
from Norn
Positive P;
degrees
180
2
150
3
135
4
110
5
no
6
50
7.
0
Exact time of each trial, since beginning of exposure to test light was not re-
corded.
On the orthodox Loebian view, it would be expected that on re-
peatedly testing an originally fully dark-adapted beetle, a number of
photonegative trials would be first obtained, and then, if a reversal
of sign occurs with light adaptation, a sudden and complete change to
positive phototropism would be observable. It was actually found,
however, that an incompletely light-adapted beetle pursued a path at an
angle to the light beam. If the direct negative response may be re-
garded as a deviation of 180° from the positive path, then with exposure
to light, the angle of deviation gradually diminished until it approached
0° (i.e. the beetle was again positively phototropic).
It was possible to obtain such records from several individuals, and
to repeat the observations on the same beetle. Although the actual
paths (Figs. 1-3) were not straight, and in spite of some irregularities
360 J. E. G. RAYMONT
TABLE II
Change in the direction of phototropic path followed by Dineutes (Individual B), in
6 successive trials, consequent upon light adaptation. The animal was first dark-
adapted for 12 hours. The upper eyes were functional.
Trial
Number
1
Total Time
of Exposure
to Test Beam
minutes
. 0
Angle of Deviation
from Normal
Positive Path
degrees
180
2
1
120
3 . . .
3
75
4
8
60
5
18
. . . . (no "run" obtained)
6.
.38..
20
in respect of angle, in general the results showed a surprisingly con-
sistent trend.
The time required for complete reversal of phototropic sign varied
from < 10 to > 30 minutes. It is believed that a rough estimate of
TABLE III
The effect of dark adaptation on the direction of phototropic path in Dineutes (Individual
V). The lower eyes were functional.
Result of Single
Trial in Test Beam:
Time in Dark
Angle of Deviation
Following Standard
from Normal
Light Adaptation
Positive Path
minutes
degrees
5
5
15
35
24
90
30
(140)
45
120
90
160
180.
.180
the course of light adaptation can be obtained by measuring the suc-
cessive deviation angles (Table I).
Table II shows an experiment conducted on a beetle which was dark-
adapted for a longer period. The total duration of exposure to light
from the test beam since the beginning of the experiment was also re-
corded for each trial.
If these results do really indicate the course of light adaptation, it
should be possible, using similar methods, to follow the course of dark
adaptation. To test this possibility, a number of experiments were
carried out using different periods of dark adaptation. In each experi-
ment the animal was first light-adapted by means of the usual adapting
light for 20 minutes. It was then dark-adapted for a given period, and
a single trial made in the test beam. By using various periods of dark
DARK ADAPTATION AND PHOTOTROPISM
361
adaptation a number of trials were obtained, and the results (e.g. Table
III) showed that with progressively longer periods of dark adaptation,
the paths pursued showed an increasingly greater deviation from the
positive path. An approximate dark-adaptation curve has been con-
structed from these data (Fig. 4) for one individual.
The possibility that the change in the paths pursued might be caused
by the repeated disturbance of the beetle when replacing it at the centre
of the test plate was investigated. The test plate was constantly moved
in such a way that a beetle which had been originally fully dark-adapted
was kept in the beam without being replaced. The animal at first
180
^160
o
£140
O
H 120
W
Q
fe
O
w
too
80
60
40
20
20 40 60 80 100 120 140 160
TIME IN DARK IN MINUTES
180
FIG. 4. Progressive deviation from the normal positively phototropic path
during reversal of phototropic sign with dark adaptation, in Dineutes horni (In-
dividual V) . (The lower eyes were functional.)
moved from the light source, but with continued exposure it turned
gradually towards the source until it reached the edge of the test plate.
The changes in path are therefore not the result of disturbance, but do
depend on the degree of adaptation.
DISCUSSION
Mclndoo (1929) states that Schmitt-Auracher believed there was
a relationship between the state of adaptation and pigment deposition
in insect ocelli and the sign of phototaxis. In the present observations
362 J. E. G. RAYMONT
on Dineutes, a reversal of phototropic sign could never be obtained when
all four eyes were functional, but if any two, or any three, eyes were
blackened, the fully dark-adapted beetles were always negatively photo-
tropic. The difference in behavior may depend upon the area of photo-
sensitive surface stimulated. Although one cannot compare human,
subjective sensations with animal tropisms, it is interesting that experi-
ments on the intensity discrimination of the human eye have shown
that the use of a small test field of high intensity may cause uncom-
fortable glare and even pain, while with a larger test field of the same
average intensity, vision is normal.
When only partially light- (or dark-) adapted, Dineutes, with only
two eyes functional, moves at an angle to the light beam. Radl, Car-
penter, and especially Dolley (1916) and Clark (1931 and 1933) have
discussed movement of phototropic insects at an angle to a beam of
light, but in all such cases only one of a pair of symmetrical eyes was
functional, and deviation might be then expected. Clark supposed that
light from the direct beam, and light from the background acted on
the functional eye. But in the experiments described, with both upper,
or both lower eyes functional, light from both background and beam
should act equally on the two sides, and therefore, according to Loeb,
the insect should move directly to or from the source. Indeed, Clark
states that if in Dineutes both upper or both lower eyes are blackened,
the beetle moves straight towards the light. But in Clark's experiments
the beetles were consistently photopositive, and provided D. horni is
strongly positively phototropic, it moves straight to the source also.
Mast (1938) has shown that the phototropic reflexes in insects vary
according to the region of the eye stimulated. In Dineutes, according to
Clark, the posterior ommatidia are much more sensitive than the anterior
ommatidia. Possibly then, during reversal of phototropic sign with
light adaptation in D. horni, some of the ommatidia become light-adapted
more rapidly and give rise to reflexes which are opposed by the less
sensitive ommatidia. Morphologically symmetrical ommatidia also may
not adapt at exactly similar rates, and therefore photochemical reactions
will proceed at different rates on the two sides during partial adaptation,
and a deviation would result. When all ommatidia are fully adapted,
the beetle will move straight to the source.4
Although an exact explanation must therefore await further work,
it is obvious that the simple Loebian theory will not account for the facts
here presented. Light must act in a more complex manner, and among
Some recent experiments on the related genus, Gyrinus, have shown that even
with all eyes functional, a positively phototropic beetle, when it is fully dark-
adapted, may pursue a path deviating widely from the normal straight photopositive
path.
DARK ADAPTATION AND PHOTOTROPISM 363
other factors, the phototropic responses must depend to a considerable
extent upon the region of eye stimulated, as Mast has repeatedly
emphasized.
SUMMARY
1. When all eyes are functional, Dineutes horni is positively photo-
tropic after dark or light adaptation.
2. With one or with two eyes functional, Dineutes is positively
phototropic when light-adapted, but is negatively phototropic when fully
dark-adapted.
3. At intermediate stages of dark and light adaptation, the beetle
moves at an angle to the light rays. The courses of dark and of light
adaptation were followed by a study of these " angles of deviation "
from the incident rays.
4. Possible theories are discussed to account for these results.
ACKNOWLEDGMENTS
This work was carried out at Harvard University during the tenure
of a Henry Fund Fellowship. The author wishes to thank Dr. G. L.
Clarke, of Harvard University, for his constant advice and criticism
throughout the investigation, and Mr. J. Armstrong, of Harvard, for
his contribution to the discussion.
BIBLIOGRAPHY
CLARK, L. B., 1931. Some factors involved in the reaction of insects to changes in
luminous intensity. Shock reactions in Dineutes assimilis. Jour. Expcr.
Zool, 58: 31.
CLARK, L. B., 1933. Modification of circus movements in insects. Jour. Expcr.
Zool, 66: 311.
CROZIER, W. J., AND E. WOLF, 1928. Dark adaptation in Agriolimax. Jour. Gen.
Physio!., 12: 83.
DOLLEY, W. L., 1916. Reactions to light in Vanessa antiopa with special reference
to circus movements. Jour. Expcr. Zool., 20 : 357.
MAST, S. O., 1938. Factors involved in the process of orientation of lower or-
ganisms in light. Biol. Rev., 13 : 186.
MclNooo, N. E., 1929. Tropisms and sense organs of Lepidoptera. Smiihson.
Misc. Coll., 81 : 10.
WOLTER, H., 1936. Beitrage zum Lichtsinn von Carcinus maenas. Zool. Jahrb.
Abt. Zool. und Physiol, 56 : 581.
MODIFIED SEXUAL PHOTOPERIODICITY IN COTTON-
TAIL RABBITS *
THOMAS HUME BISSONNETTE AND ALBERT GEORGE CSECH
(From the Shade Sivawf* Sanctuary, Farmington, Connecticut)
INTRODUCTION
Studies too numerous to cite here show that the sexual cycles of many
birds and mammals and of some fish and reptiles can be modified and
their breeding seasons changed by manipulating daily cycles of exposure
to light. Not all are susceptible in the same way nor to the same degree
(Bissonnette, 1936, 1938; Marshall, 1936, 1937).
The development of proper methods of conservation and wild life
management require knowledge as to what wild animals have photo-
periodic breeding cycles and how they react to management of light-
cycles. Breeding seasons of some animals have been prolonged to per-
mit two litters per year in place of one, with better than normal growth
of early-induced litters (Bissonnette and Csech, 1937, 1938, 1939).
The cotton-tail or gray rabbit of New England (Sylvilagas transi-
t'wnalis (Bangs)) is shot for sport and food, furnishes food for fur-
bearing carnivores, and may injure fruit trees in some places and sea-
sons. It exhibits a limited breeding season with three or four litters,
beginning about mid-April. It is, therefore, more polyoestrous than
most of the animals so far investigated and intermediate between strictly
monoestrous or dioestrous and completely polyoestrous forms. It,
therefore, has been tested by " night-lighting " in autumn and winter.
MATERIAL AND METHODS
Three pairs of cotton-tails were placed, each in a wire enclosure
raised from the ground, with wooden " den " at one end. In each den
a few inches of earth were covered with loose dry grass about
two inches deep for bedding. Twenty-five-watt bulbs were so placed
as to shine into both den and enclosure and controlled by a time switch
so that lights were lit for one hour each night for the first week, begin-
ning on October 10. " Night-lighting " was increased one hour each
1 Aided by grants 'from the National Research Council, Committee for Re-
search in Problems of Sex, 1935-8, and from the Penrose Fund, American Phil-
osophical Society, 1938-9, and by cooperation and animals from the State
Department of Fish and Game, Connecticut.
364
PHOTOPERIODICITY IN COTTON-TAIL RABBITS 365
ten days thereafter to eight hours on December 17 and maintained into
February, and, in one case, into April. Lights came on each evening
at six o'clock. All pens were outside, without heating, except from the
bulb, throughout the experiment. Feeding and care were similar for all
rabbits at the sanctuary.
Replacements were made without altering schedule when animals
killed one another and the exact lighting history of each animal recorded.
The gonads of killed animals were secured for histological study. But
none were sacrificed expressly for such material, because our experience
with raccoons and the behavior of these rabbits suggested that matings
would lead to litters out of season. Sex-organs were obtained also from
unlighted males on December 8 and January 25 for comparison with
those of a male killed by his mate, January 12, after night-lighting eight
hours each night from December 20.
OBSERVATIONS AND RESULTS
After varying periods of lighting and after matings, two experi-
mental females killed their original mates by biting them through the
back. One male killed his mate by grasping her anal region with his
teeth and pulling out her abdominal organs which became useless for
study. Replacement of males may account for failure of matings to
induce pregnancy in December and again beginning on January 5. Con-
trols also mated somewhat later in December and about January 10, all
without pregnancies. Bissonnette's studies on male ferrets indicate that
willingness to mate antedates potency and fertility by a considerable
time. This was probably true here also.
After matings in January, two experimental females made nests and
one lined hers with fur to receive young that failed to come. No con-
trol did so then, nor until near littering time in April.
The first " experimental " litter (of two) was born on April 4 and
died April 10, from exposure. The nest was not heated and the mother
left it for long periods. On May 31 and June 30 she produced second
and third litters (of one and six) which survived. The first " control "
litter came on April 18 and all six died of exposure. None of the other
experimental or control females had litters before June 8, although the
above-mentioned control female mated again the day her litter was born
and others probably did so too. In that season no controls had litters
live through. They normally should have had litters every thirty days
after mid-April. It was a poor rabbit-breeding season, for reasons as
yet unknown. Experimentally lighted animals succeeded slightly better
than controls on normal light.
A male, used for replacement on December 20, lighted eight hours
366 T. H. BISSONNETTE AND ALBERT G. CSECH
each night until killed by his mate on January 12, had mated about Jan-
uary 5-7 with her. His testes showed sperms just metamorphosed in
small numbers in some tubules but none had yet reached the middle part
of the epididymis. The apparent breeding condition of this epididymis,
with tall columnar epithelium and well developed ciliary processes, in-
dicates functional activity of the interstitial cells of the testis and ac-
counts for sexual libido and matings.
Much smaller testes from the control of December 8 showed smaller
tubules with no stages of germ-cells beyond synizesis. Its epididymis,
in a partly activated, partly regressed condition, suggested some activity
of interstitial cells, which may account for December and January mat-
ings of controls. Its epithelium was short columnar with some ciliary
processes, less developed than those of the lighted male.
Testes from the January twenty-fifth control were slightly more
advanced in spermatogenesis than those of December 8. Its most ad-
vanced stages were synizesis and a few growing primary spermatocytes.
No germ-cell debris was found in the middle part of the epididymis
which was more regressed than that of December 8. Its lining epi-
thelium was more nearly cuboidal, with no ciliary processes evident.
CONCLUSIONS
Increasing night-lighting induces cotton-tail rabbits to undergo sexual
activation in winter. In males, it leads to complete libido and sperma-
togenesis in twenty-three days at January 12, on eight hours of added
light from December 20. It would probably induce complete breeding
effectiveness in little longer time. In females, it induces repeated re-
ceptivity followed by nest-making and even lining of nests with fur.
December and January matings do not alone indicate increased sexual
activation, for controls on normal light reacted similarly. Killing of
mates is not attributable to added light ; but rather it was permitted by
the close confinement's preventing escape from an aggressor. Making
and lining of nests in January, not done by controls at that time, sig-
nify activation above the normal for that time, and suggest pseudo-preg-
nancy after winter copulations.
Even if litters can be born under the conditions of temperature pre-
vailing in these experiments in winter, they cannot be raised by their
mothers, even in their fur-lined nests. The mothers do not keep the
nests warm, as do raccoons, but leave them closed for long periods, re-
turning only at intervals to suckle the young, born naked and defenseless
against cold. It is suggested, however, that, with warmed nesting places,
the long absences of the mother may not permit the young to die nor
prevent the raising of litters in winter. In addition, by arranging run-
PHOTOPERIODICITY IN COTTON-TAIL RABBITS 367
ways so that males and females can have separate dens and can be sepa-
rated after matings by a wire screen which permits them to remain ac-
quainted without being able to kill each other, the same pairs may be
kept on the lighting schedule and make winter breeding successful.
Further experiments along these lines seem to be indicated.
SUMMARY
1. Three pairs of cotton-tail rabbits were confined in dens and run-
ways and subjected to increased lighting at night from October 10 on-
ward. Controls were not lighted but fed similarly.
2. The original objective, induction of winter litters, was not at-
tained because, after varying times of lighting, one member of the pair
killed the other and replacements were made on schedule.
3. Sex organs of males were modified to complete sperm formation
in twenty-three days in December and January and mating libido
reached, accompanied by breeding conditions of the epididymis. Con-
trols showed mating libido but no spermatogenesis nor epididymal ac-
tivation.
4. Lighted females mated and made nests; and one lined its nest
with fur. Controls mated but made no nests. No pregnancies resulted
with any female until April.
5. The induced changes indicate that these rabbits can be brought
into breeding condition in winter by increased lighting, but modification
of the method used and the provision of warmed nest-boxes are neces-
sary for successful winter breeding and rearing of these animals.
LITERATURE CITED
BISSONNETTE, T. H.. 1936. Sexual photoperiodicity. Quart. Rev. BioL, 11: 371-
386.
BISSONNETTE, T. H., 1938. Experimental control of sexual photoperiodicity in
animals and possible applications to wild life management. Jour. Wild
Life Management, 2: 104-118.
BISSONNETTE, T. H., AND A. G. CSECH, 1937. Modification of mammalian sexual
cycles. VII. Fertile matings of raccoons in December instead of Febru-
ary induced by increasing daily periods of light. Proc. Roy. Soc. Ser. B,
122 : 246-254.
BISSONNETTE, T. H., AND A. G. CSECH, 1938. Sexual photoperiodicity of raccoons
on low protein diet and second litters in the same breeding season. Jour.
Mammal, 19: 342-348.
BISSONNETTE, T. H., AND A. G. CSECH, 1939. A third year of modified breeding
behavior with raccoons. Ecology, 20 : in press.
MARSHALL, F. H. A., 1936. Sexual periodicity and the causes which determine it.
The Croonian Lecture. Phil. Trans. Roy. Soc., Ser. B,226: 423-456.
MARSHALL, F. H. A., 1937. On the change over in the oestrous cycle in animals
after transference across the equator, with further observations on the in-
cidence of the breeding seasons and the factors controlling sexual
periodicity. Proc. Roy. Soc., Ser. B, 122 : 413-428.
THE LIFE CYCLE OF DACTYLOMETRA QUINQUECIRRHA,
L. AGASSIZ IN THE CHESAPEAKE BAY 1
ROBERT A. LITTLEFORD
(From the Chesapeake Biological Laboratory, Solomon's Island, Maryland)
INTRODUCTION
The common jellyfish or sea nettle of the Chesapeake Bay is the
large scyphozoan medusa Dactyloinetra quinquecirrha, L. Agassiz. This
species is a coastal form occurring in tropical and temperate seas
throughout the world. In the Chesapeake Bay, as in other brackish
water areas (Mayer, 1910; Menon, 1930), it is accompanied by a color-
less, milky-white medusa which lacks the pigmented areas of Dacty-
loinetra.
The primary object of this study has been to elucidate the problems
presented by the white medusa, which becomes sexually mature in a
growth stage having twenty-four marginal tentacles, thus answering the
taxonomic description of the genus Chrysaora of Eschscholtz. Certain
workers, including Bigelow (1880), have considered the white medusa
a member of the genus Chrysaora, on a basis of the sexually mature
form, possessing twenty-four marginal tentacles. Mayer (1910) con-
siders this medusa to be a growth stage of Dactylometra quinquecirrha,
the so-called " Chrysaora-stage," believing that the premature develop-
ment of the gonads is the result of the brackish water conditions. More
recently Papenfuss (1936) has considered this white medusa to be a
variety of D. quinquecirrha and named it chcsapeakcii.
The literature on the life cycles of these two forms is limited to
brief and scattered observations by Mayer (1910), Stiasny (1919-
1921), Papenfuss (1934), and Truitt (1934). For this reason an in-
vestigation of the complete life cycles of the red and the white medusae
was undertaken. The results obtained from this investigation are
presented at this time.
PROCEDURE
The organism was reared from the egg stage to the medusa under
controlled conditions, and the data obtained in this way were supple-
1 Contribution number 31 from the Chesapeake Biological Laboratory. Part
of a thesis_ presented to the Faculty of the Graduate School of the University of
Maryland in partial fulfillment of the requirements for the degree of Doctor of
Philosophy.
368
DACTLYOMETRA IN THE CHESAPEAKE BAY 369
merited by observations in nature. Fertilization was obtained in the
laboratory (1) by crowding sexually mature males and females to-
gether in a small container, and (2) by placing segments of the ripe
gonads in fingerbowls 4 by 10 cm. in size and partly filled with brackish
water.
The data reported in this paper are based on observations on be-
tween two and three thousand individual scyphostomae, about five hun-
dred of which have been reared through four successive years. About
one thousand additional embryos were followed to the scyphostoma
stage.
The embryonic material was reared in a basement room of the lab-
oratory, where changes in temperature and light were comparatively
slight, though not controlled. The temperature of the water in the
fingerbowls varied between 20 and 24° C. The lighting was indirect,
light entering the room through a small window just below the ceiling on
the eastern wall.
Scyphostomae were reared in the laboratory by placing a finger-
bowl in a battery jar of three-liter capacity partly filled with bay water.
Others were reared in fingerbowls placed overboard under natural con-
ditions in nearby waters and protected by wire cages measuring 24 X 9
X 9". The wire was of hardware screen measuring eight strands to
the inch. Each cage, containing five bowls held in a wooden rack, was
lowered to the bottom in eight or ten feet of water in a protected cove
near the laboratory.
The water containing the free developing embryos was changed daily
by pipetting out a part of it and refilling the jars with fresh brackish
water, whereas the water on the attached forms was changed weekly.
The larvae were fed daily on material gathered by means of plankton
tows, and on small pieces of oyster.
The embryonic material was studied by using hanging drops and
depression slides, which made it possible to follow development of a
single egg or group of eggs up to and through the planula stage. Ma-
terial intended for cytological or histological examination was fixed in
standard solutions of Kaiser's., Bouin's, Flemming's, or Zenker's fixa-
tives and stained according to standard methods. Iron hematoxylin and
Mallory's triple stain as given by Galigher proved to be particularly
effective. Chloral-hydrate-menthol was most satisfactory for anes-
thetizing the scyphostomae.
OBSERVATIONS
Mature males and females of D. quinquecirrha may be recognized
on a basis of the color of the gonads, which in the males are bright pink
370
ROBERT A. LITTLEFORD
and in the females grayish brown or yellowish brown in appearance.
The egg is a colorless cell with a prominent nucleus. The mature egg
(Fig. 2), which is yellow in color, resulting from the rilling of the cyto-
plasm with yolk material, varies in diameter from 0.07 to 0.19 mm.,
with an average diameter of 0.15 mm. The spermatozoa are developed
in sacs (Fig. 1), many of which are found in a single gonad. These
sacs show great variation in size and shape.
The eggs are released from the gonads into the stomach cavity and
fertilization takes place either there or externally. Numerous laboratory
observations strongly suggest that fertilization occurs in the stomach
cavity. This is substantiated by the collection of fifty-one females
bearing embryos in the gastric cavity.
The relationship between the initiation of development and the time
of day is shown in Table I, which is based on 710 cases. These results
are supported (1) by the collection, between eight and nine o'clock in
the evening, of females having eggs in the first and second cleavage
stages, and (2) by the collection before ten o'clock the next morning of
females bearing gastrula and planula stages in the gastric cavity.
TABLE I
Time of Fertilization
Time
Number
Time
Number
3:00-6:00 p.m.
0
9:00-10:00 p.m.
206
6:00-7:00 p.m.
2
10:00-11:00 p.m.
24
7:00-8:00 p.m.
124
11:00-12:00 p.m.
16
8:00-9:00 p.m.
329
after 12:00 p.m.
9
The fertilized egg (Fig 3), which is distinguished by the presence
of a distinct membrane, may divide immediately or it may remain quies-
cent for six or seven hours after fertilization. The first sign of de-
velopment is the elongation of one end of the egg (Fig. 4), producing
a prominent, knob-like protrusion (Fig. 5). This knob, the function
of which is unknown, is resorbed prior to the first cleavage, which oc-
EXPLANATION OF PLATE I
Photomicrographs from living material.
FIG. 1. Section of gonad of male medusa showing sacs containing sper-
matozoa. X 146.
FIG. 2. Section of gonad of female medusa showing mature and immature
eggs. X 146.
FIG. 3. The fertilized egg. X 182.
FIG. 4. Elongation of the fertilized egg. X 182.
FIG. 5. Knob-like protrusion of fertilized egg. X 95.
FIG. 6. The two-celled embryo. X 146.
ROBERT A. LITTLEFORD
curs a little less than one hour after fertilization. The two-celled
embryo (Fig 6) divides almost immediately to form four equal blasto-
meres (Fig. 7). At the end of three or four hours, under laboratory
conditions, a third cleavage results in an embryo of eight equal cells.
Succeeding divisions result in the formation of a blastula in about ten
or twelve hours. During these cleavage stages the fertilization mem-
brane, which surrounded the egg and embryo during early segmentation,
is lost. The cells on one end of the blastula now invaginate and a
free-swimming planula develops within sixteen to twenty hours after
fertilization.
No provision is made for the protection of the developing embryo,
such as the pockets on the oral arms of Aurcllla. The rate of develop-
ment of the embryonic stages shows great variation among individuals
as well as with the external factors of the environment. A more de-
tailed discussion of the stages of cleavage and gastrulation is to be
presented in a subsequent paper.
The planula (Fig. 8) is at first round or oval in shape, but within
two or three hours it adopts a definite pyriform outline. The first sign
of movement in the planula is a regular rotation that is observed at the
end of twenty or twenty-four hours. This is produced by the action
of cilia which are developed by certain cells of the ectoderm. After
adopting the pyriform outline, the planula moves through the water by
means of a fringe of cilia confined to the cells of the periphery. Move-
ment is rapid and the direction is changed continuously. The broad end
is always directed anteriorly.
THE SCVPHOSTOMA
Completing its free-swimming existence after a period varying from
three to five days, the planula becomes attached to some object, and is
then known as the scyphostoma or hydra-tuba. The larva thus formed
lias a " ninepin " shape. In the process of attachment, the cells of the
anterior region secrete an adhesive substance which forms a disc sur-
rounding the stalk of the polyp.
The scyphostoma stage of D. quinquecirrha follows the general pat-
tern of the group and lends support to the statement of Fowler (1900)
EXPLANATION OF PLATE II
Photomicrographs from living material.
The four-celled embryo. X 165.
The planula previous to adopting a pyriform outline. X 165.
Scyphostomae having three to seven tentacles. X 26.
10. Schyphostoma having eight tentacles. X 66.
DACTLYOMETRA IN THE CHESAPEAKE BAY
8
u
»
'A
" ' 'A \ ,
10
• \
PLATE II
374 ROBERT A. LITTLEFORD
that the scyphozoan polyp is insignificant in size and monotonous in
structure. The adult scyphostoma, which has sixteen or, rarely, twenty
tentacles, shows three clearly defined stages of intermediate development,
having four, eight, and twelve tentacles respectively. This fact prob-
ably explains the observations of Mayer (1910) that reproduction was
through a scyphostoma stage having normally four tentacles, and of
Stiasny (1919) that the number of tentacles was eight.
Development
Immediately after attachment, the oral region shows a great pro-
liferation of endodermal cells resulting in the formation of the oral cone.
This development, in turn, stretches the ectodermal layer and changes
the mouth from a small almost invisible slit into a wide, yawning open-
ing. This process is completed in from one to three hours after at-
tachment. The sides of the oral cone become secondarily cleft to form
a cruciform mouth.
The tentacles are produced singly and arise as wart-like evaginations
from the region of the body surrounding the mouth. At the end of five
days, the scyphostoma has four primary tentacles (Fig. 9) developed in
the perradii of the body. Alternating with these tentacles, there arise
four taeniolae, or ridges of the ectoderm, projecting into the stomach
cavity in the interradial axes.
A circular depression appears on the upper part of the larva on the
sixth day, marking off the oral cone from the bases of the tentacles.
Several days later four deep depressions, the septal funnels, appear in
this furrow just above the taeniolae. At the end of ten days a scyphos-
toma with eight tentacles (Fig. 10) is produced as the result of the
formation of four secondary tentacles in the four remaining interradii.
The scyphostoma continues to increase in size, and at the end of fifteen
days, eight more tentacles have been developed in the adradii of the
polyp (Fig. 11). Although the typical number of tentacles is sixteen,
occasionally an individual which has developed twenty tentacles will lie
noted. This, however, is a rare exception.
EXPLANATION OF PLATE III
Photomicrographs from living material.
Oral view of scyphostoma having sixteen tentacles. X 26.
Lateral view of scyphostoma showing the pedal discs. X 26.
I'll,. Colony of scyphostomae. X 10.
Apical view of the strobila. X 26.
The ephyra immediately after being released. X 46.
FlG. 16. Contracting ephyra showing the cruciform mouth, the heavy nema-
tocyst aggregations, and the beginning of the gastric cirrha. X 119.
DACTLYOMETRA IN THE CHESAPEAKE BAY
375
16*
PLATE III
376 ROBERT A. LITTLEFORD
In typical specimens, the scyphostoma has reached complete develop-
ment by the middle of August, although longer periods are required for
certain individuals. The only observed change taking place from Sep-
tember until the following April or May is a slight increase in size. The
scyphostoma then becomes bright pink in color and begins to undergo a
process of transverse fission known as strobilization.
After strobilization is completed, the remaining scyphostoma, which
is about one millimeter in height, has a small cruciform oral slit, sur-
rounded by sixteen normal-size tentacles produced before the ephyral
discs were released. It may be distinguished from those scyphostomae
which have not undergone strobilization by ( 1 ) the small mouth opening,
(2) the short and broad appearance of the body, and (3) the crater-like
mouth area in contrast to the normal oral cone. This old scyphostoma
regains its normal appearance in from five to seven clays. The following
spring it again undergoes strobilization, producing the typical number of
ephyrae.
Associated groups or colonies of scyphostomae (Fig. 13) are a
regular occurrence in D. quinquecirrha. Such colonies, consisting of
seven or eight individuals ranging in height from one to four milli-
meters, are produced by means of (1) stolons, (2) buds from the stalk
of the parent scyphostoma, and (3) development of scyphostomae from
the pedal discs. The pedal discs (Fig. 12) are formed as a result of
movement of the scyphostoma from place to place, in the course of
which it leaves behind on the substrate a protoplasmic disc. The
groups of polyps developed from these discs may be either linear or
irregular in formation, depending on the movement of the " parent '
scyphostoma. The colonies formed either by means of buds from the
stalk or by means of stolons are irregular in arrangement because of the
irregular development of buds and stolons.
During the course of this study, it was observed that certain culture
dishes in open water no longer contained scyphostomae. Instead, the
bottom surface was covered with small, brownish, wart-like cysts. In
one case (December, 1935) 51 cysts were counted and their positions
marked on the culture dishes. The following February, 21 cysts had
disintegrated, while normal scyphostomae had developed in the remain-
ing 30 cases. In November, 1937, several culture dishes containing
cysts were brought into the laboratory. The cysts in these dishes were
kept under constant observation from that time. A count of 27 cysts
was made in one dish, and two weeks later it was observed that the
number was reduced to eighteen. Scyphostomae were produced from
these cysts, and development took place in a normal manner.
It is to be noted that the cysts occurred in both cases in material
DACTLYOMETRA TN THE CHESAPEAKE BAY 377
that was being held in cages under conditions as nearly natural as pos-
sible. Scyphostomae which were retained in the laboratory for long-
periods without food or change in water resorhed themselves until they
diminished to the size of one-week-old forms, but at no time did they
encyst. At present, therefore, we can offer no valid explanation for the
production of cysts. The same phenomenon has been observed in
C/irysaora by Chuin (1930), who also has been unable to offer an ade-
quate explanation. In Chrysaora these cysts produce a ciliated larva
which swims about and then attaches itself to develop into a polyp. In
D. quinquecirrha a larva was not produced, but a large number of these
cysts produced new polyps in the same manner as has been reported
for pedal discs.
Morphology
The scyphostoma of D. quinquecirrha is a colorless, goblet-shaped
polyp averaging 3.5 mm. in height, with a diameter of 0.6 mm. The
mouth is cruciform and occupies the entire oral surface of the organism.
The body of the scyphostoma is divided into two distinct regions : ( 1 )
the long, stalk-like, tapering basal region, and (2) the cup-like body, or
apical region. The relative size of the two parts is dependent upon the
degree of contraction of the polyp. The tentacles are solid, averaging
6 mm. in length, and bear many nematocysts, which are regularly ar-
ranged, forming a series of successive rings around the tentacle. The
aboral end of the stalk is fixed to a sub-strate and is surrounded at the
point of attachment by a pedal disc. In older specimens the pedal disc
may be one of a group, each of which marks a previous place of attach-
ment. The body wall is divided into the three layers, ectoderm, enclo-
derm, and mesoglea. The mesoglea is a thin, almost invisible lamella
separating the two germ layers in the early development of the scyphos-
toma. This gelatinous layer increases after the eight-tentacle stage
until it becomes a prominent structure of the mature polyp, containing
muscle fibers and other cellular structures produced by the germ layers.
Reproduction
Reproduction in the scyphostoma stage is of two types: (1) budding,
resulting in the production of additional polyps, and (2) transverse
fission, or stabilization, resulting in the production of ephyrae.
Additional scyphostomae are produced as the result of three types
of budding: (1) somatic buds from the stalk region of the polyp, (2)
production of stolons, which may be considered a form of somati
budding, and (3) development from pedal discs.
A
ROBERT A. LITTLEFORD
Stolun formation and somatic budding, the common methods of
asexual reproduction in the polyp stage of the Scyphomedusae, have
been found to be rare in this species. Likewise the statement of Truitt
(1934), that the polyp buds profusely, has not been borne out by this
study. The common method of asexual budding in D. quinquecirrha
was found to be by means of pedal discs, the formation of which has
been reported by Herouard (1907) as occurring in TacnioUiydra ros-
coffcnsis. Other investigators (Mayer, 1910) have claimed that
Herouard was dealing with an abnormality of development of Aur cilia
This study has shown, however, that the formation of pedal discs is a
normal occurrence in D. quinquecirrha.
Strobilization occurs in April or early May (June or July under
controlled conditions). At this time the scyphostomae become bright
pink in color and develop a series of circular furrows in the wall of the
flask-like part of the body. As these furrows become deeper, the oral
tentacles of the polyp are resorbed. This process may be completed
before further development of the furrows occurs, or the tentacles may
remain until the furrows mark off a series of well-defined discs. The
furrows eventually divide the polyp into a series of saucer-like segments
connected by a central cord and borne on a slender stem. Each of these
saucers develops eight deep clefts, which in turn separate the periphery
into eight lobes. Each lobe becomes cleft to form a bifurcation. At
the apex of each bifurcation a deeply pigmented club, the future ten-
taculocyst of the medusa, is produced. The scyphostoma reaches the
stage of a strobila (Fig. 14) in a period of four or five days and the
saucers are then released into the water.
The number of discs produced remains remarkably constant at either
five or six. The process of separation of the discs from the base or
stalk region of the scyphostoma is completed in from ten to sixteen
hours. Shortly before being released, the discs begin to pulsate and con-
tinue a characteristic movement of short, rapid pulsations until release
is gained.
THE EPHYRA
The general structure of the ephyra was observed by Stiasny ( \(M\ ),
and A layer (1910) had earlier published figures of the ephyra drawn
by Brooks.
The newly liberated ephyra (Fig. 15) is about 0.84 mm. in diameter,
measured from the distal end of one arm to the distal end of the oppo-
site arm. The most prominent structure of its anatomy is the rhophalia.
or tentaculocyst, of which there are eight, one in the bifurcation of each
ot the ephyral arms. Tentacles are lacking. The manubrium measures
DACTLYOMETRA IN THE CHESAPEAKE BAY 379
approximately 0.23 mm. in length and is flared in the radii, forming a
cruciform structure. The nematocysts are grouped in capsules of three
different sizes and also appear singly covering the exumbrellar surface.
The ends of the ephyral arms appear knobbed as a result of nematocyst
aggregations. The middle of each of the arms is marked by the pres-
ence of a pair of large nematocyst capsules, and the region where the
arms join the disc is heavily covered with nematocysts.
The ephyra spends the first three or four days of its existence close
to the bottom. During this time the subumbrellar surface is outward
and the manubrium is carried in an upright position. A single tentacle
is developed in each of the deep clefts that separate the ephyral arms.
These tentacles, which appear four or five days after the ephyra has
become separated from the scyphostoma, are the eight primary tentacles
of the medusa, and the lobes of the arms are the primary lappets of the
bell margin. Following the appearance of the eight primary tentacles,
the ephyral disc grows outward, filling the clefts which separate the
arms until it reaches the radius of the rhophalia. The margin then
becomes cleft into a series of lappets, the number of which varies with
the age, bell diameter, and stage of development of the individual.
When the eight tentacles have formed and the manubrium has in-
creased in length until it is slightly longer than the bell diameter, the
bell becomes inverted and the manubrium hangs downward from the
center of the subumbrella. Inversion occurs between the sixth and the
eighth day in typical specimens. The oral lips of the ephyra, which are
heavily covered with nematocysts, are simple folds of the body wall,
produced from the connecting tube of the strobila. As the ephyra de-
velops into the medusa, these lips become folded and develop curtain-like
margins on their internal edges.
Four hollow, tentacle-like outgrowths, which are the first gastric
cirrha (Fig. 16), appear on the subumbrellar surface at the interradii
of the manubrium three days after separation. They increase rapidly
until as many as thirty-five or forty appear in each interradius. An
ephyra 5 mm. in diameter has eight gastric cirrha in each interradius,
while the 8-mm. one has ten, and the ephyra of 15 mm. has twenty-five.
These cirrha increase in number as development in the medusa continues.
Development in the ephyra is completed in from six days to two weeks.
Upon obtaining the bell shape, it is considered as the early " post-ephyral
stage " of the medusa.
SUMMARY AND CONCLUSIONS
Investigation of the life cycle of Dactylometra quinque cirrha, L.
Agassiz in the Chesapeake Bay has been in progress since 1935. The
380 ROBERT A. LITTLEFORD
results have been obtained ( 1 ) from observations made on the organism
reared under controlled laboratory conditions and (2) from specimens
reared under natural conditions. These observations have been supple-
mented by continuous investigation of the life cycle as it occurs under
normal conditions.
This study has shown that the metagenetic cycle, previously reported
by Agassiz and Mayer (1898), requires a period of from ten to twelve
months for development from the egg to the medusa. The collection
of scyphostomae and ephyrae from the waters of the Bay, as well as the
successful rearing of the polyp stage under natural conditions over a
period of four years substantiates the opinion of Cowles (1930) that the
sea nettle breeds in the Bay. There is no evidence to support the con-
tention that it breeds in the salter ocean water and migrates into the
Bay. As was pointed out by Littleford and Truitt (1937), ephyrae
have been collected in large numbers up deep creeks and inlets at times
when they were not found in open Bay waters.
The common method of asexual reproduction in the scyphostoma
stage is by means of pedal discs, budding being of rare occurrence. In
the course of the study, it was noted that the scyphostoma had the capa-
city to encyst under certain environmental conditions. These cysts later
produced polyps that continued development in the normal manner.
Strobilization differs markedly from that known to occur in other species,
in that the number of ephyral discs produced is constant at either five
or six, at no time approaching the condition where a large and variable
number is produced, as in Aurellia aurita.
The fact that the scyphostoma can live for very long periods of time
was pointed out over one hundred years ago by Dalyell (1836), the dis-
coverer of the polyp of Aurellia. It is of interest to note that this in-
vestigation established the fact that in D. quinquecirrha the scyphostoma
can live for rather long periods of time. Certain individual polyps have
been reared through four successive years and have undergone strobili-
zation each summer during that time.
The ephyra, when small, lives close to the bottom and swims with
the subumbrellar surface upward. After development of the inanu-
brium and the eight primary marginal tentacles, the normal position is
attained. Growth of the ephyra is a rapid process ; the organism may
increase ten times in size within a few days.
The life cycles of the red and white medusae are identical as regards
rate of development and actual size of the morphological stages. The
two " varieties " readily interbreed and the resulting cross shows no
deviation from normal in its developmental history.
DACTLYOMETRA IN THE CHESAPEAKE BAY 381
ACKNOWLEDGMENT
The writer wishes to express his appreciation to Professor R. V.
Truitt, Director of the Chesapeake Biological Laboratory, for suggesting
the problem and for his continuous advice and assistance during its
prosecution; to Dr. C. L. Newcombe and Professor N. E. Phillips of
the University of Maryland for helpful advice and suggestions ; and to
Professor W. L. Threlkeld, of the Virginia Polytechnic Institution, for
his association with the investigation during the summer of 1935.
Particular thanks are due Professor E. A. Andrews, of the Johns
Hopkins University, for a critical reading of the manuscript and for
offering valuable suggestions.
LITERATURE CITED
AGASSIZ, A., AND A. G. MAYER, 1898. On Dactylometra. Bull. Mus. Compai:
Zool, 32: 1.
AGASSIZ, L., 1862. Contributions to the Natural History of The United States,
Vol. 4, pp. 125-166.
BIGELOW, R. P., 1880. A New Chrysaoran Medusa. Johns Hopkins Univ. Cir-
culars, 9: (No. 8) 66.
CHUIN, T. T., 1930. Le cycle evolutif du Scyphostome de Chrysaora etude his-
tophysiologique. Trav. Stat. Biol. Roscoff., Vol. 8, 1930.
COWLES, R. P., 1930. A biological study of the offshore waters of Chesapeake
Bay. Bull. U. S. Bur. Fish., 46: 331-332.
DALYELL, J. G., 1836. Further illustrations of the propagation of Scottish zo-
ophytes. Edinburgh New Philos. Jour., 21 : 88.
FOWLER, G. H., 1900. The Scyphomedusae, Chapter V, Part II, Treatise on
Zoology, Edited by E. Ray Lankester, London, 1900.
HEROUARD, E., 1907. Taeniolhydra roscoffensis. Compt. Rend. Paris, 145: 601.
HEROUARD, E., 1907. Sur un acraspede sans meduse : Taeniolhydra Roscoffensis.
Compt. Rend. Paris, 147: 1336.
LITTLEFORD, R. A., AND R. V. TRUITT, 1937. Variation of Dactylometra quinque-
cirrha. Science, 86 : (No. 2236) : 426-427.
MAYER, A. G., 1910. Medusae of the world. Carnegie Institution, Publ. No. 109,
Vol. III. The Scyphomedusae. Pp. 585-588.
MENON, M. G., 1930. The Scyphomedusae of Madras and the Neighboring Coast.
Bulletin, Madras Government Museum, Nciv Series, Natural History
Section, Vol. 3, No. 1, pp. 7-8.
PAPENFUSS, E. J., 1934. The sea nettle of the Chesapeake, Maryland fisheries.
Published by the Conservation Department of Maryland, No. 28, pp. 14-17.
PAPENFUSS, E. J., 1936. The utility of nematocysts in the classification of certain
Scyphomedusae. Lunds Universitets Arsskrift, N. F. Avd. 2, Bd. 31, Nr.
11, Kungl. Fysiografiska Sallakapets Handlinger, N. F. Bd. 46, nr. 11,
pp. 14-19.
STIASNY, G., 1919. Zoologische Mededeelingen, Rijks Museum van Natuurlijke
Historic, Leiden, Vol. V, pp. 75-85.
STIASNY, G., 1921. Zoologische Mededeelingen, Rijks Museum van Natuurlijke
Historic, Leiden, Vol. VI, pp. 112-113.
TRUITT, R. V., 1934. Eleventh Annual Report, Conservation Department of the
State of Maryland, Report of the Chesapeake Biological Laboratory, p. 45.
THE BLOCKING OF EXCYSTMENT REACTIONS OF
COLPODA DUODENARIA BY ABSENCE
OF OXYGEN J
MORDEN G. BROWN
(Frotn the School of Biological Sciences, Stanford University 2)
The encysted state of a protozoon may be considered one of high
stability in that little or no energy is required for its maintenance. Cells
in this condition are not in any dynamic equilibrium of diverse reactions,
but in a static state. This investigation is concerned with the funda-
mental problems of the nature of the changes from the dynamic to the
static and from the static to the dynamic state as found in the encyst-
ment and excystment of protozoa.
The excystment process of the holotrichous ciliate, Colpoda duo-
denaria, is more than a reactivation of metabolic enzyme systems. The
process involves a redifferentiation of protoplasmic structures, cilia, etc.,
along with special physical-chemical systems such as the contractile
vacuole system and in addition involves the processes for escape from the
cyst membranes.
Though like all ontogenetic processes, the excystment process is thus
complex, it may prove amenable to analysis since the encysted organisms
may be made very nearly uniform and will remain in a resting state with
little or no change until reception of an excystment-inducing substance
from their environment. The uniformity of the cyst preparation is
obtainable since encystment as well as excystment depends on environ-
mental conditions which may be controlled (Taylor and Strickland,
1938). A standardized, constant biological material may thus be made
available for an extended series of experiments, and quantitative as well
as qualitative results compared throughout the series.
The investigation into the nature of the physiological processes in-
volved in excystment has been (1) by chemical analysis of substances
which will induce the process (see Haagen-Smit and Thimann, 1938)
1 This study comprises part of the Ph.D. dissertation (Brown, 1938a) and has
been briefly reported at the Richmond meetings of the A. A. A. S. (Brown, 19386).
Equipment used throughout this research was made available directly through
the courtesy of Dr. C. V. Taylor and indirectly through a grant from the Rocke-
feller Foundation to Stanford University for Dr. Taylor's research in chemo-
physical biology.
Now in the Department of Zoology, Washington University, St. Louis.
382
EXCYSTMENT REACTIONS AND ABSENCE OF OXYGEN 383
and (2) by determination of the relations between the excystment time
(the time elapsed between substitution of the excystment solution for
the salt solution in which the organisms are kept and emergence from
the cyst membranes) and controlled environmental factors such as con-
centration of the excystment solution, temperature, oxygen tension, and
x-ray irradiation (see Taylor, Brown, and Strickland, 1936; Brown
and Taylor, 1938; and Brown, 1938a). This report presents the ex-
perimental data obtained in the study of oxygen tension as a limiting
factor in excystment together with a further analysis of the physical-
chemical processes of excystment of Colpoda.
EXPERIMENTAL
The Colpoda used in these experiments were carefully cultured and
selected as to interfission age, then encysted in grooves in cellophane.
The cyst-cellophane preparation was then thoroughly washed and then
kept in a continuously flowing, dilute, balanced salt solution. The tech-
nique of making this preparation was developed by Mr. Strickland
(Taylor and Strickland, 1935) and the cysts used throughout this study
were prepared by him.
The time for excystment was determined by counting the number
of still encysted organisms (100 to 150 at start of each test) at intervals
throughout the period of emergence from the cyst membranes. The
geometric mean time was then evaluated by graphical methods as de-
scribed by Brown and Taylor (1938). This geometric mean time which
is equal to the median excystment time is referred to throughout this
paper as excystment time.
In each experiment a series of concentrations of excystment solution,
Difco yeast extract, was used. This enables one to separate the excyst-
ment processes into two periods : ( 1 ) that inversely proportional to the
concentration of the excystment solution, and (2) that independent of
the concentration of the excystment solution (Brown and Taylor, 1938).
The control of oxygen tension necessitated the design and construc-
tion of a special airtight excystment chamber through which gases of
various composition could be passed. This chamber must be mounted
on a mechanical microscope stage and fitted to a microscope of approxi-
mately 150 X magnification. The final design (Fig. 1) was the result
of a long series of improvements of chambers and mechanical stages.
The chamber will contain a set of six Columbia dishes which can be suc-
cessively observed by rotation of the glass plate forming the floor of the
chamber. The upper, stationary part is made from a large Petri dish
cover which is ground into the plate. This Petri dish cover is drilled
at four points, a large hole, shown in the figure through which the
384
MORDEN G. BROWN
microscope objective projects, and three small ones, one for the inflow
of the gas mixture, one for its outflow into a %-inch tube about a foot
long, and one, normally sealed and close to the gas outlet tube, through
which twice concentrated excystment solutions, previously brought to
equilibrium with the oxygen tension being tested, could be added to equal
amounts of salt solution in the excystment dishes with a negligible ad-
mixture of air. The joint between the cover and floor and all joints
about the objective have been sealed with paraffin oil throughout each
experiment.
In the first series of experiments, the partial pressure of oxygen was
reduced to %0 that of air (from 150 mm. Hg to that of tank nitrogen,
FIG. 1. Chamber and mechanical stage for studies of excystment of Colpoda.
(1) Columbia dish containing cyst-cellophane preparation. (2) Glass cover of
excystment chamber made from large, 150 mm., Petri dish. (3) Circular glass
plate forming floor of chamber. (4) Moveable part of stage attached to a standard
mechanical stage. (S) Microscope stage (specially constructed). (6) Micro-
scope objective (10 X Zeiss, small size). (7) Microscope condenser. (8) One
of three ball bearings in groove in moveable stage which support plate 3 and
permit easy rotation of dishes. (9) One of three ball bearings between moveable
stage and fixed stage.
approximately 15 mm. Hg for the tank used). The control experiments
were identical in all respects with those with reduced oxygen tension
except that air was flowing through the excystment chamber instead of
the tank nitrogen. No differences of any kind were found when the
excystment under an oxygen tension %0 that of air was compared with
the controls. A typical experimental run and two controls are shown
in Figure 2. These results are in agreement with studies of respiratory
rate under low oxygen tension for the free-swimming stages of the
ciliates Paramecium (Lund, 1918, and Amberson, 1928) and Colpoda
(Adolph, 1929) ; and further indicate that in none of the preceding
work involving measurement of excystment time in solutions in contact
with air was oxygen ever a limiting factor.
To obtain lower oxygen tensions, the nitrogen was purified by
EXCYSTMENT REACTIONS AND ABSENCE OF OXYGEN 385
bubbling through an acid chromous sulphate solution (M/10
pH = 2). The reduced state of the solution was maintained by the
presence of amalgamated zinc prepared according to the methods of
Stone and Beeson (1936). Gas exchange with the solution was facili-
tated by use of a sintered glass bubbler which broke up the gas stream
into very small bubbles. After the air was washed out of the excyst-
ment chamber with this nitrogen and sufficient time elapsed so that equi-
librium between the gaseous and liquid phase was approached, then ex-
cystment solution which had been de-oxygenated was added. There
350
300
c/)
Ld
h-
z
250
ill
200
150
100
LITERV'GRAM
5 10 15 20
RECIPROCAL CONCENTRATION
FIG. 2. Excystment of Colpoda at 20° C. O, two control series; A, oxygen
tension = 15 mm. Hg.
were no signs of excystment for periods of as long as 25 hours, though
at 15 mm. Hg partial oxygen pressure, excystment at 20° C. would have
been completed in two to three hours.
The data demonstrate much more than just a prevention of excyst-
ment by absence of oxygen, for upon admittance of air, normal excyst-
ment ensued ; and further, the excystment time following the block was
found to be independent of the duration of the block, independent of the
concentration of the excystment solution, and equal in length to the
period that was found from studies on relation between concentration
386
MORDEN G. BROWN
and excystment time to be independent of the concentration of the ex-
cystment solution. The experimental data for two of the series of
experiments at 20° C. are shown in Figure 3. Detailed tables of these
data can be found in Brown ( 1938a) .
Apparently the reactions of the period in excystment depending on
concentration of the excystment solution go to completion and the reac-
tions of the subsequent periods are completely blocked in the absence
of oxygen. These results are in agreement with the hypotheses previ-
ously made that the first period is controlled by a diffusion phenomenon
and that the following periods at 20° C. are controlled by a reaction of
1900
1700-
1500
LJ
200
150
100
e
e
LITERS/GRAM
500
450
400
250
150
100
to
u
H
Z
-5
e
-t-
LITERS/GRAM
10
15
20
10
15
20
RECIPROCAL CONCENTRATION
FIG. 3. Excystment of Colpoda at 20° C. after being blocked by very low
oxygen tension. •, time between admittance of air and emergence; ©, time be-
tween addition of excystment solution and emergence.
oxidative metabolism (Brown and Taylor, 1938). The fact that the
reactions do not proceed in the second period in the absence of oxygen
does not in itself prove that the normal limiting reaction is the oxidative
metabolism — more refined experiments in which tests are made over a
temperature range and in which excystment proceeds, but at a reduced
rate due to oxygen tension being a limiting factor, are required.
Though the period dependent on extract concentration changes with
temperature in the range 12° to 32° C. as though it were controlled by
the time required for diffusion of a substance from the excystment
solution, below 12° C. this period changes with temperature according
EXCYSTMENT REACTIONS AND ABSENCE OF OXYGEN 387
to the Arrhenius equation with a very high ^ value (Brown and Taylor,
1938). From this, one might expect that a different process limits
this period in the low temperature range. However, this other process,
if it exists, is also independent of oxygen, for when the experiments
were repeated at 11° C. it was found that the time after admittance of
air for completion of excystment is at this temperature also independent
of concentration and equal in duration to the period which is found by
study of the relation between excystment time and concentration to be
2000
1500
LJ
1000
500
e
e
2000
1500
1000
500
LITERS/GRAM
e
D
Z
LITERS /GRAM
RECIPROCAL CONCENTRATION
FIG. 4. Excystment of Colpoda at 11° C. after being blocked by very low
oxygen tension. •, time between admittance of air and emergence; ©, time be-
tween addition of excystment solution and emergence ; O, control-time between
addition of excystment solution under aerobic conditions and emergence.
independent of concentration. Two of the low temperature series are
shown in Figure 4. In the second of these series shown, the set of
six dishes included four which were blocked by absence of oxygen and
two to which the excystment solution was added at the time of admission
of air ; the conditions in the experimental and control dishes seem much
more comparable in this case for it is seen that the extrapolated value
for duration of the period independent of concentration coincides much
more closely with the time for excystment after the block than in the
cases in which the controls were run separately.
MORDEN G. BROWN
DISCUSSION
At present four physiological periods in excystment of Colpoda have
been sorted out by a quantitative study of excystment time under a
variety of environmental conditions. Separation and characterization
of the first and subsequent periods is by Brown and Taylor (1938) and
this report and separation of the later periods is through the work of
Taylor, Brown, and Strickland (1936) on the effects of x-ray irradia-
tion at different stages of excystment.
The experimental characterization and physiological interpretation
of these periods is briefly as follows :
I. A period whose duration is inversely proportional to the concen-
tration of the organic constituents of the excystment solution (Brown
and Taylor, 1938), and independent of oxygen tension (this report).
Its duration changes with temperature as does the viscosity of the
cytoplasm for a considerable temperature range (Brown and Taylor,
1938). This period is considered to be one during which diffusion of
essential substances from the excystment solution takes place and pos-
sibly also an anaerobic reaction with high activation energy (Brown and
Taylor, 1938, and this report).3
II -\- III -f IV. A period whose duration is independent of the
concentration of the excystment solution, and which is dependent upon
oxygen (Brown and Taylor, 1938, and this report). The change in
duration with change in temperature follows the Arrhenius equation
with /A = 44,000 calories/ mole below 15° C., 18,000 calories/mole be-
tween 15° and 25° C, and zero above 25° C. (Brown and Taylor, 1938).
It is suggested that the value of //. = 18,000 is associated with oxidative
metabolism and /u, = 44,000 with an anabolic reaction of excystment
(Brown and Taylor, 1938).
II. A period during which x-ray irradiation increases excystment
time to the same extent as does irradiation at any time in Period I
(Taylor, Brown, and Strickland, 1936).
III. A short period during which emergence from the cyst is pre-
vented by the same x-ray dose that at other periods only delays excyst-
3 A recent abstract of a paper by Danielli (1939) not yet published indicates
that one might expect the rate of diffusion through living cell membranes to change
with temperature according to the Arrhenius equation. This suggests that below
12° C. diffusion is the limiting factor in this first period in encystment rather than
some postulated chemical reaction (Brown and Taylor, 1938) but that the
mechanism limiting diffusion above 12° C. and below is different, i.e., a barrier of
the type suggested by Danielli which requires diffusing molecules to possess
greater than a certain kinetic energy in order to penetrate into the cell is limiting
below 12° C., whereas cell structures that control diffusion rate according to their
viscosity are limiting above 12° C.
EXCYSTMENT REACTIONS AND ABSENCE OF OXYGEN 389
ment or has no effect (Taylor, Brown, and Strickland, 1936). This
period seems to be critical to the later building up of hydrostatic pres-
sure which results in rupturing of the ectocyst membrane, for the ir-
radiation prevents emergence but does not prevent the completion of
differentiation of cilia or their functioning (unpublished observations
of Taylor, Brown, and Strickland; see also Brown, 1938a).
IV. A period throughout which administration of an x-ray dose
which caused a three-fold increase in excystment time if given during
Periods I or II and prevented emergence if given in Period III has
almost no influence on excystment time (Taylor, Brown, and Strickland,
1936). This period is considered separated from preceding ones by
completion of a developmental reaction involving a substance which may
be inactivated by x-ray irradiation during any preceding period.
That Colpoda blocked from excystment by absence of oxygen do not
die or show any adverse effects for a block of at least 25 hours at 20° C.
is opposite to the interpretation of some experiments with free-swim-
ming Colpoda (Taylor and Strickland, 1938). In these experiments it
was observed that free-swimming organisms die within a short time (97
per cent in two hours) in an unaerated dense bacterial suspension but
do not die in a similar suspension which is aerated. From this it was
concluded that low oxygen tensions cause death of free-swimming
Colpoda within a few hours. This may indicate that certain enzyme
systems which may be thrown out of balance by removal of oxygen and
which then destroy the free-swimming organism are not activated until
the second or later periods of excystment.
SUMMARY
1. Excystment time is independent of oxygen tension down to 15
mm. Hg.
2. Excystment is blocked by very low oxygen tensions. This block
is at a developmental stage between the excystment period dependent on
concentration of the excystment solution and the periods independent of
concentration.
3. The excystment process may be divided into four physiological
periods characterized by the influence of temperature, concentration of
the excystment solution, oxygen tension, and x-ray irradiation on the
excystment time.
LITERATURE CITED
ADOLPH, E. F., 1929. The regulation of adult body size in the protozoan Colpoda.
Jour. Ex per. Zool., 53 : 269.
AMBERSON, W. R., 1928. The influence of oxygen tension upon the respiration of
unicellular organisms. Biol. Bull., 55 : 79.
390 MORDEN G. BROWN
BROWN, M. G., 1938a. A chemophysical investigation of the excystment process
of Colpoda duodenaria. Dissertation, Stanford University Library.
BROWN, M. G., 19386. The blocking of excystment reactions of Colpoda duo-
denaria by absence of oxygen. Anat. Rec. (Suppl.), 72: 51.
BROWN, M. G., AND C. V. TAYLOR, 1938. The kinetics of excystment in Colpoda
duodenaria. Jour. Gen. Physiol., 21 : 475.
DANIELLI, J. F., 1939. A contribution to the theory of diffusion in non-ideal liquids
and membranes. Proc. Roy. Soc., Ser B, 127 : S71.
HAAGEN-SMIT, A. J., AND K. V. THIMANN, 1938. The excystment of Colpoda
cucullus. I. The chemical nature of the excysting factors in hay infusion.
Jour. Cell, and Camp. Physiol. , 11 : 389.
LUND, E. J., 1918. Quantitative studies on intracellular respiration. I. Relation
of oxygen concentration and the rate of intracellular oxidation in Para-
mecium caudatum. Am. Jour. Physiol., 45 : 351.
STONE, H. W., AND C. BEESON, 1936. Preparation and storage of standard chro-
mous sulphate solutions, hid. and Eng. Chem., Anal. Ed., 8: 188.
TAYLOR, C. V., M. G. BROWN, AND A. G. R. STRICKLAND, 1936. Effects of a given
x-ray dose on cysts of Colpoda steini at successive stages of their induced
excystment. Jour. Cell, and Comp. Physiol., 9 : 105.
TAYLOR, C. V., AND A. G. R. STRICKLAND, 1935. Some factors in the excystment
of dried cysts of Colpoda cucullus. Arch. f. Protist., 86: 181.
TAYLOR, C. V., AND A. G. R. STRICKLAND, 1938. Reactions of Colpoda duodenaria
to environmental factors. I. Some factors influencing growth and en-
cystment. Arch. f. Protist., 90: 396.
N
THE RELATION BETWEEN KIND OF FOOD, GROWTH,
AND STRUCTURE IN AMOEBA1
S. O. MAST
(From the Zoological Laboratory of the Johns Hopkins University and the Marine
Biological Laboratory, Woods Hole, Mass.)
It is well known that amoebae usually feed on living organisms and
that they ordinarily ingest several different kinds. It has, however,
been demonstrated that for some species, one kind suffices for growth
(Oehler, 1916, 1924; Rice, 1935; Hopkins, 1937). No observations
have been made on the relation between the kind of food and the struc-
ture of amoebae. This is the main problem involved in the following
experiments.
Amoeba proteus and Amoeba dubla grown in Hahnert solution -
containing rice grains were fed on Chilomonas paramecium raised on
sterile acetate-ammonium 3 and glucose-peptone 4 solutions respectively
and Colpidium striatwn raised on sterile tryptone-phosphate 5 solution.
The experiments were made as follows :
Numerous amoebae were taken from vigorous cultures, passed
through several separate portions of distilled water so as to remove the
food, and then left in distilled water several hours. In this, many of
them became stellate in form. The largest of these were selected and
five of them put into each of four 6 cc. glass salt dishes containing 3 cc.
Hahnert solution each. Then numerous chilomonads or colpidia which
by means of the centrifuge had been passed successively through 4
separate portions of fresh Hahnert solutions were added to the solution
in each, and left two hours, i.e. until the amoebae had ingested many
chilomonads or colpidia, then the amoebae with as little solution as pos-
sible were transferred to clean salt dishes containing Hahnert solution.
This was repeated until the solution was free of chilomonads or colpidia,
after which the process of feeding and transferring was repeated and
the number of amoebae in each dish recorded daily for 9 days, then 5
1 1 am much indebted to Drs. R. A. Fennell and William J. Bowen for very
efficient assistance in the experimental part of this work.
* Hahnert solution— KC1, 4 mg. ; CaCL, 4 mg. ; CaH4(PO4),, 2 mg. ; Mgs(PO«)2,
2 mg.; Cas(PO*)2> 2 mg.; water, 1000 cc.
3 Acetate-ammonium solution— NaQH3O2, 150 mg. ; NEUCl, 46 mg.; (NH4>2-
SO*, 10 mg.; K.HPO4, 20 mg.; MgCU, 1 mg.; CaCl2, 1.16 mg.; water, 100 cc.
4 Glucose-peptone solution — peptone, 8 g. ; glucose, 2 g. ; water, 1000 cc.
5 Tryptone-phosphate solution— tryptone, 15 g. ; KK.PO,, 2 g. ; water, 1000 cc.
1000 cc.
391
392
S. O. MAST
of the specimens in each dish were transferred to clean dishes containing
Hahnert solution and the rest discarded or used for the study of struc-
ture, after which the process of feeding, transferring, and recording
TABLE I
Growth of Amoeba fed on chilomonads and colpidia respectively. Temperature,
21°-25°C.; x, all but five discarded; * Several specimens removed for study of
structure.
Amoeba proteus
Food
Number of Specimens
August
September
Chilomonas in glucose-peptone
7
10
12
14
16
16
20
23
26
29
1
4
solution
5
8
8
9
6*
5
6
13
12
11
12*
5
9
12
15
11*
5
6
7
7
10
9*
5
7
6
7
6*
5
5
7
7
10
9*
5
6
7
4
3*
3
0
Chilomonas in acetate-am-
5
3
0
monium solution
5
7
0
5
7
1
Colpidium
5
16
24
34
35*
5
12*
17
26
29
42*
7
3
6
16
23
16*
5
8
13
14
20
23*
24
4
12
24
50
90*
5
8
9
5
11
15
20
5
8
17
27
45*
. 5
7
7
11
18
29
40
Amoeba dubia
Chilomonas in glucose-peptone
5
6
4
2
5
5
6
solution
5
4
3
5
5
5
0
5
7
5
4
5
6
3
5
4
5
5
5
5
0
Chilomonas in acetate-am-
5
2
0
monium solution
5
3
0
5
5
2
Colpidium
5
10
13
17
22*
5
5
5
2
0
5
13
24
26
44*
5
3
6
2
1
5
9
11
13
21*
5
6
3
3
0
5
12
13
12
20*
5
1
0
was again repeated daily for 16 days. The results obtained are pre-
sented in Table I.
This table shows that both Amoeba proteus and Amoeba dubia fed
exclusively either on chilomonads or colpidia increased in number, but
FOOD, GROWTH AND STRUCTURE IN AMOEBA 393
that the increase continued thruout the experiment only in Amoeba
proteus fed on colpidia. It shows that some of the specimens of Amoeba
pro tens fed on chilomonads were still alive at the close of the experi-
ment, but that the number had decreased ; and microscopic examination
showed that they were in very poor condition. The table shows that
the specimens of Amoeba dubia fed on colpidia increased in number
much more rapidly and lived much longer than those fed on chilomonads
and it shows that for several days those fed on colpidia increased in
number as rapidly as Amoeba proteus fed on these organisms, but that
they then decreased rapidly in number and soon died. The table shows
that no increase in number occurred in the specimens of either of the
two species of Amoeba fed on chilomonads grown in acetate-ammonium
solution and that they did not live so long as those fed on chilomonads
grown in glucose-peptone solution.
This experiment was repeated in part several times. In some of
the tests made, the colpidia used were taken from a culture which con-
tained an unidentified mold, but no bacteria. In some of these tests,
the amoebae were left with the food 2 hours, i.e. the same length of time
as in the preceding experiments, but in others they were left only 15
minutes and in still others they were left 24 hours.
In the tests in which the amoebae were left with the food only 15
minutes there was no increase in number, either in those fed on chilo-
monads or in those fed on colpidia. The time was obviously not long
enough for the amoebae to ingest sufficient food for growth. The re-
sults obtained in the tests in which the amoebae were left with the food
2 and 24 hours respectively are essentially the same as those presented
in Table I. That is, in the tests in which chilomonads were used as
food, the amoebae usually increased in number fairly rapidly for several
days and then decreased, and in those in which colpidia were used, the
increase in number continued longer and, under some conditions, doubt-
less would have continued indefinitely if the tests had not been closed.
For example, in one test with Amoeba proteus fed on chilomonads, the
number increased from 5 to 330 in ten days after which there was a
slight increase for a few days, then a gradual decrease to zero, and in
another with Amoeba proteus fed on colpidia there was a slow, but con-
sistent increase in number for 34 days, i.e. thruout the entire experi-
ment, with no indication of deterioration whatever, altho the increase
during this entire time was only from 10 to 106.
The results obtained seem to demonstrate therefore that Amoeba
proteus can grow and live indefinitely on sterile colpidia as food, but
that Amoeba dubia cannot, and that neither can live indefinitely on chilo-
monads as food, but that chilomonads grown in glucose-peptone solution
394
S. O. MAST
are more nearly adequate as food than those grown in acetate-ammonium
solution.
The chilomonads grown in acetate-ammonium solution contained
much starch and little fat, while those grown in glucose-peptone solution
contained considerable starch but no fat and they were much smaller
than the former (Fig. 1). The difference in their food value is, there-
fore, doubtless due to difference in their chemical structure and content
correlated with the chemical composition of the medium in which they
grow. Growth in Amoeba is consequently not only correlated with the
kind and the quantity of organism they ingest, but also with the physio-
logical condition of the organism ingested.
FIG. 1. Camera outlines of Chihmonas paramecium showing the effect of the
kind of food in the culture medium on size and content. A, specimens taken at
random from a vigorous culture in sterile glucose-peptone solution; B, specimens
taken at random from a vigorous culture in acetate-ammonium solution ; O, starch ;
•, fat. The flagella are not represented.
Note that the chilomonads grown in glucose-peptone solution were much
smaller and contained much less starch and fat than those grown in acetate-am-
monium solution. Growth is more rapid in the former solution than in the latter.
In the experiments on growth in Amoeba fed exclusively on sterile
chilomonads and colpidia respectively, specimens were taken from the
cultures at different times and studied in reference to behavior and
structure. The results obtained are summarized in the following pages.
The specimens of Amoeba proteus. which had fed exclusively on
colpidia for several days were extraordinarily large (Fig. 2) and liter-
ally packed full of globules of fat, especially those which had fed on
colpidia from the culture which contained mold.6 They had only a few
6 This mold contained much fatty acid but no neutral fat and the colpidia
contained enormous quantities of neutral fat but no fatty acid. In fresh cultures
the colpidia multiplied rapidly and became abundant in 24 hours. At this time the
solution was perfectly clear and the colpidia in it contained but little or no fat.
Then the solution gradually became turbid and in 4 or 5 days, mold was clearly
visible and at this time the colpidia were well filled with globules of fat and each
one usually contained 3 or 4 fragments of mold hyphae or spores which contained
liberal quantities of fatty acid which was doubtless changed to neutral fat in the
cytoplasm of the colpidia.
FOOD, GROWTH AND STRUCTURE IN AMOEBA
395
pseudopods and these were very short, thick, and blunt without a hy-
aline cap. They were only slightly attached to the substratum and
moved about very slowly and irregularly, now in one direction, then in
another, giving the impression of very sluggish, aimless, rolling about.
Many had two nuclei. The alpha and beta granules were normal in
number and structure, but the bipyramidal crystals were scarce and much
FIG. 2. Camera outlines showing the size, form, and structure of Amoeba
protens fed exclusively on colpidia and chilomonads respectively.
A, optical section of Amoeba protcus fed on colpidia; n, nucleus; c.v., contrac-
tile vacuole ; /, fat globules in one focal plane ; F ', same, enlarged ; C, bipyramidal
crystals in A; R, largest refractive bodies in A (substance in them not differen-
tiated) ; r, one of these drawn out in the form of a fiber; C\, bipyramidal crystals
in a specimen fed on chilomonads; s.v., side view; e.v., end view; R\, refractive
bodies in a small area in an optical plane in a specimen fed on chilomonads (sub-
stance in these highly differentiated) ; o, outer layer; s, shell; c, central substance;
mm, projected scale.
shorter and thicker and more truncated than usual and there were usually
only a few spherical bodies and some specimens had none at all. The
spherical bodies were with few exceptions very small and the substance
in them undifferentiated. There was nothing in them similar to the
fragile shell usually found and all the substances in them usually stained
crimson with neutral red, but the central portion often appeared lighter
396 S. O. MAST
in color and somewhat more granular than the rest, and did not stain so
readily. This substance was usually so elastic that if the bodies were
released after they had been flattened by means of pressure on the cover-
glass they soon assumed their original shape and it was so adhesive and
viscous that if the cover-glass was pushed sidewise on the slide after
the bodies had been flattened by pressure on it, the substance in them,
owing to adhesions to the glass, was often drawn out in the form of a
long slender fiber (Fig. 2).
It is consequently obvious that if the food of specimens of Amoeba
proteus is restricted to colpidia great changes occur in them in reference
to size, form, behavior, and structure; in fact, changes so great that if
such specimens were examined without information as to their origin
they would certainly be designated as a new species and probably as a
new genus.
Specimens of Amoeba proteus which for several days had fed ex-
clusively on chilomonads were normal in size, form, and activity; but
they contained an extraordinarily large number of spherical bodies
(often a thousand or more) and numerous bipyramidal crystals and very
little or no fat. The spherical bodies were relatively very large and the
substances in them well differentiated into a central mass surrounded
with a prominent fragile shell which was covered with a thin layer of
oily substance (Fig. 2). In solutions containing neutral red, the outer
layer became deep red (crimson) in color, but the central portion and
the shell did not stain. The spherical bodies in these amoebae were,
therefore, similar to some described by Mast and Doyle (1935, p. 167)
but differed radically in number, size, and structure from those found
in the amoebae fed exclusively on colpidia.
The bipyramidal crystals were relatively long and but little trun-
cated and in some specimens as many as 2 percent of them were not
truncated at all (Fig. 2).
The facts that there were many more refractive bodies in the amoebae
which had fed on chilomonads than in those which had fed on colpidia
and that they were much larger and the substance in them much more
differentiated, show that these structures are closely correlated with the
kind of food ingested. They therefore support the contention of Mast
and Doyle (1935, p. 291) and others that they are cytoplasmic inclusions
and not cytoplasmic structures, i.e. secondary nuclei (Calkins, 1905),
cysts (Taylor, 1924), Golgi bodies (Brown, 1930), mitochondria,
(Horning, 1925, 1928), vacuome (Volkonsky, 1933).
Amoeba dubia usually contains relatively few crystals (some irregu-
lar or roughly bipyramidal in form with the edges and corners rounded,
and some thin rectangular plate-like in form) and not much fat.
FOOD, GROWTH AND STRUCTURE IN AMOEBA 397
In the specimens fed on colpidia the irregular crystals decreased
greatly in number and often disappeared entirely and the plate-like
crystals increased considerably and there was marked accumulation of
fat, altho not nearly so much as in Amoeba proteus. In those fed on
chilomonads the irregular crystals increased greatly in number and the
plate-like crystals decreased considerably and the fat usually disappeared.
There was no significant change in size, form, or activity in those
fed on colpidia or those fed on chilomonads.
SUMMARY
1. If specimens of Amoeba proteus are fed exclusively on colpidia,
they become very large and extremely fat and sluggish and grow and
multiply slowly, but indefinitely. The refractive bodies in them decrease
greatly in number and size and their content becomes homogeneous and
very adhesive, elastic and viscous. The crystals decrease in number and
become shorter and more truncated.
2. If they are fed exclusively on chilomonads, they grow and mul-
tiply for several days, then decrease in number and soon die, but they live
longer if the chilomonads have grown in glucose-peptone solution than
if they have grown in acetate-ammonium solution. The refractive
bodies increase greatly in size and number and the content of these
bodies becomes sharply differentiated ; the bipyramidal crystals increase
in number and become less truncated, and the fat decreases in quantity.
3. If specimens of Amoeba dubia feed exclusively on chilomonads,
they multiply for a few days, then cease and soon die. The irregular-
shaped crystals increase and the plate-like crystals decrease considerably
in number and the fat disappears.
4. If they feed exclusively on colpidia, they multiply more and live
longer than if they feed exclusively on chilomonads, but they do not
live indefinitely. The plate-like crystals increase in number and the
irregularly shaped crystals usually disappear entirely and the fat in-
creases in quantity, but not so much as it does in Amoeba proteus.
5. Amoeba is in reference to form, size, behavior, and structure
closely correlated with the kind of organisms it eats and their physio-
logical condition.
LITERATURE CITED
BROWN, V. E., 1930. The Golgi apparatus of Amoeba proteus Pallas. BioL Bull.,
59 : 240-246.
CALKINS, G. N., 1905. Evidences of a sexual-cycle in the life-history of Amoeba
proteus. Arch. f. Protist., 5 : 1-16.
HOPKINS, D. L., 1937. The relation between food, the rate of locomotion and
reproduction in the marine amoeba, Flabellula mira. Biol. Bull., 72 :
334-343.
398 S. O. MAST
HORNING, E. S., 1925. The mitochondria of a protozoan (Opalina) and their
behavior during the life cycle. Austral. Jour. Expcr. Biol. and Med., 2 :
167-171.
HORNING, E. S., 1928. Studies on the behavior of mitochondria within the living
cell. Ibid., 5 : 143-148.
MAST, S. O., AND W. L. DOYLE, 1935. Structure, origin and function of cyto-
plasmic constituents in Amoeba proteus (with special reference to mito-
chondria and Golgi substance). I. Structure. Arch. f. Protist., 86:
155-180. II. Origin and function based on experimental evidence; effect
of centrif uging on Amoeba proteus. Ibid., 86 : 278-306.
OEHLER, R., 1916. Amobenzucht auf reinem Boden. Arch. f. Protist., 37 : 175-190.
OEHLER, R., 1924. Gereinigte Zucht von freilebenden Amoben, Flagellaten und
Ciliaten. Arch. f. Protist., 49 : 287-296.
RICE, N. E., 1935. The nutrition of Flabellula mira Schaeffer. Arch. f. Protist.,
85: 350-368.
TAYLOR, MONICA, 1924. Amoeba proteus : some new observations on its nucleus,
life history and culture. Quart. Jour. Micros. Sci., 69: 119-143.
VOLKONSKY, M., 1933. Digestion intracellulaire et accumulation des colorants
acides. Bull. Biol. France et Belg., 67 : 135-275.
THE EFFECT OF ELECTRIC CURRENT ON THE RELATIVE
VISCOSITY OF SEA-URCHIN EGG PROTOPLASM *
C. A. ANGERER 2
(From the Zoological Laboratory, University of Pennsylvania,3 and the Marine
Biological Laboratory, Woods Hole, Mass.)
Becquerel (1837) was the first investigator to study the action of
electricity on protoplasm of single cells. [The greater part of the lit-
erature treating of the effect of electric current on cyclosis may be ob-
tained from Ewart (1903).] The conclusion to be drawn from the
literature is that electric current, depending on the current density em-
ployed, produces a progressive decrease in, followed ultimately by cessa-
tion of, cyclosis providing the current flows for a sufficient interval of
time. However, Velten (1876), Ewart (1903) and Koketsu (1923)
observed an initial increase prior to the characteristic progressive slow-
ing of cyclosis. The results obtained by Briicke (1862) on human
leucocytes, Chifflot and Gautier (1905) on Cosmarium, Bayliss (1920)
on Tradescantia and Amoeba by the Brownian movement method and
Bersa and Weber (1922) on Phaseolus by the centrifuge method are
in essential agreement, i.e., electric current produces an increase in
protoplasmic viscosity.
This investigation was undertaken to continue and extend the study
of electric current as a stimulating agent to some type of protoplasm
other than that of the protozoan cells Amoeba dubia and A, proteus
already studied (1937).* It is of interest to know whether the proto-
plasm of such distantly related biological groups, e.g., Amoeba and the
unfertilized eggs of Arbacia, responds to this stimulating agent in a
comparable manner.
MATERIAL AND METHODS
The experiments were performed on the unfertilized eggs of the
sea-urchin, Arbacia punctulata. The eggs were treated according to
method "3" as described by Just (1928). Eggs were shed in about
1 This investigation was aided by a grant-in-aid from the Society of the
Sigma Xi.
- Now of the Dept. of Physiology, The Ohio State University, Columbus.
3 I wish to express my profound gratitude to Professor C. E. McClung for
his many kindnesses during the present investigations.
4 Our own studies on amoebae are now nearing completion and a detailed
report will appear shortly.
399
400 C. A. ANGERER
250 cc. of sea water and washed once in an approximately equal volume
of the medium.
The eggs were subjected to either direct or alternating electric cur-
rent, according to the experiment in question, in a celluloid trough, the
sides of which were perfectly milled and thus parallel to the lines of
current flow. The current was applied through a Zn/ZnSO4/ sea-
water-in-agar system, which, in turn, was in circuit with a reversing
switch, rheostat and milliammeter. The agar bridges were cut to fill
the ends of the trough completely and were finally sealed in place by
means of hot agar. The available electrode surface, i.e., the cross-sec-
tional area of the available medium in the trough, was 40 mm2. The
source of the electric current was the regular service line (110 volts)
running into the laboratory.
The protoplasmic viscosity was determined by the centrifuge method.
The handle of an Emerson hand centrifuge, when turned at the rate of
one revolution per two seconds, developed a centrifugal force of 2,531
times gravity, after allowance was made for the depth to which the eggs
settled in the centrifuge tube when cushioned on an isosmotic (0.73 m.)
sucrose solution. The eggs were centrifuged until 80 per cent, or
more, showed a fine hyaline band 2/15 the diameter of the egg appearing
between the oil cap and the yolk granules. (This fraction was equal
to one division of the arbitrary scale of the ocular micrometer employed
in these experiments.) The time in seconds necessary to move the
yolk granules the specified distance, and thus show the requisite width
of the hyaline band at the centripetal pole of the egg, is designated the
"centrifuging value." This is the end-point to which all experimental
centrifugalizations are referred.
The experiments were conducted in the following manner. Each
batch, i.e., eggs from one female, was tested for ' normalcy.' A batch
of eggs was declared ' normal ' if, on sampling, 95 per cent or more
showed membrane elevation after insemination and 80 per cent or more,
showed the desired width of the hyaline area when centrifuged for 60
seconds at room temperature (19°-24° C.). These conditions prevail-
ing, approximately uniform quantities of eggs were placed in the stimu-
lating trough and while the eggs were more or less suspended, an
electric current of known intensity and duration of flow was admitted.
At the cessation of the current the eggs were immediately pipetted into
the centrifuge tube, which contained a known depth of isosmotic sucrose
solution, and were centrifuged respectively for various known periods
of time. The lowest value to which the interval of time elapsing be-
tween the cessation of the application of the stimulating agent and in-
cipient centrifugalization could be reduced was 7 seconds. There was no
ELECTRIC CURRENT ON PROTOPLASM
401
apparent difference between this and the 10-second interval which was
used throughout these experiments unless otherwise stated.
The points of curves A and B (Fig. 1) which represent centrifuging
values plotted as functions of the time of exposure to the electric cur-
rent in question were obtained in the following manner. After pre-
liminary tests in which various constant current densities were studied,
when applied for varying intervals of time, it was apparent that a cur-
rent density 5 of 0.005 amperes/mm.2 served best to illustrate the results
and this density was employed throughout these experiments.
On the basis of the above procedure the curves were developed as
140
g!20
yioo
80
LD
Z
LD
L_
CC
60
•10
20
_CL
ZD
m
n
10'
2 4 6 8 10 12 14
TIME DF EXPOSURE 5EEDND5
FIG. 1. Centrifuging time in seconds (viscosity) of Arbacia egg protoplasm
vs. time of exposure in seconds to alternating (curve A) and direct (curve B) elec-
tric current. Curve C represents the rate of thermal increase to which the stimu-
lating trough is subjected vs. time of exposure to either type of current. Current
density = 0.005 amperes/mm.2; pH 8.2; room temperature = 21.5° C.
follows. In the early experiments several batches of eggs were neces-
sary in order to work out a complete curve. With more experience
one batch of eggs sufficed for the determination of the points for any
one curve. Twelve curves for direct (B) and ten curves for alternating
(A} current were thus worked out. Finally, with these data as a basis,
all points for both alternating and direct current curves were obtained
by employing one batch of eggs. This procedure was repeated for three
different batches of eggs and thus assured relatively constant conditions
of material, temperature (21.5° C.), etc. Hence fewer experiments were
performed in the latter instance but these data were substantiated by the
5 In a preliminary note (1938) amperes/cm.2 should have read amperes/mm.2.
402 C. A. ANGERER
more detailed experiments performed during the early part of the work.
Under the conditions of the experiments no point on the curves deviated
more than ten arbitrary units in centrifuging value in repetitive tests.
Eggs were not used for experimental purposes after being shed
longer than three hours. The viscosity of the main protoplasmic mass,
as determined by the method here employed, does not undergo any ap-
preciable change during this three-hour period (Goldforb, 1935; An-
gerer, 1937).
There was no observable difference when experimentally treated eggs
were compared with their controls for membrane elevation and cleavage.
RESULTS
Direct Current
When sea-urchin eggs are subjected to a current density of 0.005
amperes/mm.2 flowing for varying known intervals of time, there is, in
the majority of experiments, no observable change in the centrifuging
value (curve B) after one second of continuous exposure.6 On con-
tinuous application of the current for two seconds, there is, in all ex-
periments, a decrease in the centrifuging value; while with three seconds
of continuous exposure the viscosity decreases from the control centri-
fuging value of 60 to a minimum value of 40 arbitrary units, i.e., a
decrement of 33 per cent in three seconds. There is no further change
in viscosity for the next four or five seconds, respectively, of continuous
exposure to the current.7 However, if the current is allowed to flow for
six seconds, the centrifuging value increases from a transient minimum
value until after seven seconds of constant exposure the centrifuging
value is identical with that of the control. There is a progressive in-
crease on further exposure, so that after fifteen seconds, when these
experiments were discontinued, the centrifuging value had increased
225 per cent above the previous minimum value.
Alternating Current
When the centrifuging values are plotted as functions of the time of
exposure in seconds (curve A) to alternating current of 0.005 amperes/
6 In a few experiments the viscosity at the end of this period of time was
found to show a slight decrease which was never greater than a centrifuging value
of ten seconds.
Occasionally eggs, after exposure to electric current, showed a tendency for
the intracellular granules to stick in the cortical area. This condition, though in-
frequent, was confined, more particularly, to eggs from certain females. Though
these eggs were discarded in the final count, since the behavior of the main mass
of the protoplasm was of chief interest, their number was not of such magnitude
as to affect appreciably the results.
ELECTRIC CURRENT ON PROTOPLASM 403
mm.2 there is, in all experiments, after one second of constant current
flow a decrease in the viscosity of the protoplasm as measured by the
centrifuging value. This value is decreased further when the eggs are
exposed to the current for two seconds, while after three seconds the
centrifuging value undergoes no further decrease but levels at the new
minimum value which is 67 per cent of the control. This minimum
value is maintained after continuous exposure for three, four and five
seconds, respectively.7 Continuing the exposure to the stimulating agent
further, there is, after five seconds, a perceptible increase in the vis-
cosity value. Thereafter, with each successive second of exposure to
the electric current a progressive increase is noted in the centrifuging
value. The increment is of the same value as that recorded for direct
current (curve £), namely 225 per cent in ten seconds. The results
recorded (curve A} were discontinued in this experiment after ten
seconds exposure to alternating current because of the expiration of the
time limit set upon the use of shed eggs.
It was of interest, in view of the high resistance at the sea water-
agar interface, to determine the thermal change occurring within the
stimulating trough as a result of the passage of an electric current of
specified density. The main coordinates of the broken-line curve C
(labeled at the right and lower sides of Fig. 1) represent temperature as
a function of time during which the eggs contained in the trough are
exposed to the thermal effect induced by passage of the electric current.
The temperature data were obtained by a specially constructed, direct
reading thermometer, the bulb of which was of such size as to be sub-
merged completely when immersed in the stimulating trough.
That thermal effects of the magnitude present during the course of
these experiments have no observable effect on the centrifuging value
is shown on immersing eggs in sea water which has been warmed pre-
viously to 32° C. When eggs are exposed to this temperature for
greater intervals of time (e.g., 20 seconds) than that to which they are
subjected during the course of these experiments and are simultaneously
centrifuged with eggs serving as controls, it is found that the centrifug-
ing values are identical. Heilbrunn (1924), though not primarily in-
terested in this phase of the question, states in the protocol (p. 192) of
his experiments on heat coagulation in sea-urchin eggs that after five
minutes exposure to 32.9° C. the heat-treated eggs were found to show
the same width of the hyaline area as the controls.
Experiments were conducted to test whether varying the quantity
of eggs suspended in unit volume of sea water in the specified electric
field had any tendency to alter the shape of the electric current- viscosity
curves. Batches of eggs were allowed to settle under the influence of
404 C. A. ANGERER
gravity in a four-inch finger bowl and minimum amounts of sea water
plus the relatively concentrated eggs were picked up by means of a regu-
lar medicine dropper. Various points on the electric current-viscosity
curves were investigated using one, ten and twenty drops respectively
of the egg suspension in unit volume of bathing medium. In the vari-
ous concentrations of eggs employed there is no observable difference
in the centrifuging values other than that which is within the range of
experimental error.
DISCUSSION
When either direct or alternating electric current of the intensity
employed in these experiments is used as a stimulating agent, there is
initially a transient decrease followed by a progressive increase in the
viscosity of the protoplasm of sea-urchin eggs. An ultimate increase in
the centrifuging value is in line with the literature (see introduction) ;
though no lucid evidence is to be found in favor of a transitory decrease
in viscosity prior to the ultimate increase.
In view of Heilbronn's (1914) results on the attempt to correlate
cyclosis in terms of viscosity data, it is justifiable to consider only, at
the present time, data as obtained from the methods of Brownian move-
ment and centrifugalization. Briicke (1862), Kuhne (1864), Chifflot
and Gautier (1905), and Bayliss (1920) have observed a decrease or
stoppage of Brownian movement on passage of an electric current
through the cell. These data, insofar as a definite statement as to the
experimental procedure is given, were obtained during the actual passage
of electric current through the material in question and not immediately
thereafter as in the experiments here reported. There may be some
criticism of studying Brownian movement during the actual passage of
the current since cytoplasmic granules undergoing electrophoresis lose
their characteristic trembling movements (unpublished results). Mast
(1931) should be consulted in this connection. Bersa and Weber
(1922), using the centrifuge method, observed an increase in the vis-
cosity of the protoplasm of PJiaseolus on the passage of electric current
for relatively long periods of time. It would be of interest to ascer-
tain data for shorter intervals of time.
There is no difference in the results whether one employs alternating
or direct current providing identical current densities are employed
(compare curves A and B). Alternating current tends to be more ef-
fective initially owing, apparently, to the greater shearing effect pro-
duced by the protoplasmic granules suspended in an oscillating electric
field which would tend to break down the protoplasmic structure. This
effect may be reenforced by the apparently thixotropic character of
ELECTRIC CURRENT ON PROTOPLASM 405
protoplasm. For a review of the literature on thixotropy in living
cells see Angerer (1936).
The question arises as to the congruity of applying a stimulating
agent for the duration of a few seconds while a minimum of 50 seconds
is required for obtaining the viscosity determination. When varying
intervals of time are permitted to elapse from the cessation of the elec-
tric current to incipient centrifugalization, the results obtained are found
not to vary for at least two minutes.
The data presented here are in accord with the known facts con-
cerning the action of certain stimulating agents on sea-urchin egg proto-
plasm. Heilbrunn and Young (1930) and Angerer (1937),8 employing
respectively ultra-violet radiations and mechanical agitation, found a
transitory liquefaction prior to an ultimate increase in viscosity. Sim-
ilar results were obtained for ultra-violet radiations (Heilbrunn and
Daugherty, 1933), mechanical agitation, electric current and suddenly
applied thermal increments (Angerer, 1936, 1938, 1940) on Amoeba
protoplasm. To explain their results, Heilbrunn and Daugherty (1933)
proposed a theory in terms of colloid chemical changes in protoplasm ;
for a detailed review of this theory one is referred to Chapter 37 of
Heilbrunn's book (1937).
SUMMARY
1. The centrifuge method was used to determine the viscosity of
sea-urchin egg protoplasm after exposure to either direct or alternating
electric current to a current density of 0.005 amperes/mm.2 for various
known intervals of time.
2. There is, on exposing eggs to either direct (curve B) or al-
ternating (curve A) current, a transient decrease followed ultimately
by a progressive increase in the centrifuging value (Fig. 1).
3. Since the data for the action of electric current, as employed in
these experiments, show a striking similarity to those results as ob-
tained by the use of certain other stimulating agents on Amoeba and
Arbacia egg protoplasm, it is suggested that the mechanism offered by
Heilbrunn (1937) may be applicable here.
LITERATURE CITED
ANGERER, C. A., 1936. The effects of mechanical agitation on the relative vis-
cosity of Amoeba protoplasm. Jour. Cell, and Compar. Physiol., 8: 329.
ANGERER, C. A., 1937. The effect of salts of heavy metals on protoplasm. I.
Jour. Cell, and Compar. Physiol., 10 : 183.
ANGERER, C. A., 1937. The effect of thermal and electrical stimulation on the vis-
cosity of ameba protoplasm (abstract). Anat. Rec. (suppl.), 70: 52.
8 Footnote p. 340.
406 C. A. ANGERER
ANGERER, C. A., 1938. The effect of electric current on the physical consistency
of sea urchin eggs (abstract). Biol. Bull., 75: 366.
ANGERER, C. A., 1940. The effect of thermal increments on and the subsequent
adjustment of the protoplasmic viscosity of Amoeba proteus. Physiol.
Zool., vol. 13.
BAYLISS, W. M., 1920. The properties of colloidal systems. IV. Proc. Roy. Soc.
(London) B, 91 : 196.
BERSA, E., AND F. WEBER, 1922. Reversible Viskositatserhohung des Cytoplasmas
unter der Einwirkung des elektrischen Stromes. Ber. d. dcutsch. Bot. Gcs.,
40: 254.
BRUCKE, E., 1862. Uber die sogenannte Molecularbewegung in thierischen Zellen,
insonderheit in den Speichelkorperchen. Sitsungber. Math.-Naturwiss.
Classe d, K. Akad. d. Wiss. Wien, 45: 629, Pt. II.
CHIFFLOT, J., AND C. GAUTIER, 1905. Sur le mouvement intraprotoplasmique a
forme brownienne des granulations cytoplasmiques. Jour, de Bot., 19: 40.
EWART, A. J., 1903. On the Physics and Physiology of Protoplasmic Streaming
in Plants. Oxford.
GOLDFORB, A. J., 1935. Viscosity changes in ageing unfertilized eggs of Arbacia
punctulata. Biol. Bull, 68: 191.
HEILBRONN, A., 1914. Zustand des Plasmas und Reizbarkeit. Ein Beitrag zur
Physiologic der lebenden Substanz. Jahrb. f. wiss. Bot., 54 : 357.
HEILBRUNN, L. V., 1924. The colloid chemistry of protoplasm. IV. Am. Jour.
Physiol., 69 : 190.
HEILBRUNN, L. V., 1937. An Outline of General Physiology. Philadelphia.
HEILBRUNN, L. V., AND K. DAUGHERTY, 1933. The action of ultra-violet rays on
Amoeba protoplasm. Protoplasma, 18 : 596.
HEILBRUNN, L. V., AND R. A. YOUNG, 1930. The action of ultra-violet rays on
Arbacia egg protoplasm. Physiol. Zool., 3 : 330.
JUST, E. E., 1928. Methods for experimental embryology. The Collecting Net, 3 :
7.
KOKETSU, R., 1923. Uber die Wirkungen der elektrischen Reizung an den
pflanzlichen Zellgebilden. Jour. Dept. of Agric., Kyushu Imperial Univ.,
1: 1.
KUHNE, W., 1864. Untersuchungen iiber das Protoplasma und die Contractilitat.
Leipzig.
MAST, S. O., 1931. The nature of the action of electricity in producing response
and injury in Amoeba proteus (Leidy) and the effect of electricity on the
viscosity of protoplasm. Zeitschr. f. vcrgl. Physiol., 15 : 309.
VELTEN, W., 1876. Einwirkung stromender Elektricitat auf die Bewegung des
Protoplasma, auf den lebendigen und todten Zelleninhalt, sowie auf ma-
terielle Theilchen iiberhaupt. Sitzungsbcr. Math.-Natunviss. Classe d. K.
Akad. d. Wiss. Wien, 73 : 343, Pt. 1.
DEVELOPMENT OF EYE COLORS IN DROSOPHILA: PRO-
DUCTION OF v+ HORMONE BY FAT BODIES *
G. W. BEADLE, E. L. TATUM AND C. W. CLANCY
(From the School of Biological Sciences, Stanford University, California)
Of the two diffusible substances known to be concerned in the pro-
duction of eye pigments in Drosophila, only z/+ hormone is produced by
fat bodies (Beadle, 1937). This was demonstrated by transplantation
experiments. Attempts to extract this hormone from larval fat bodies
were unsuccessful and it was therefore concluded that the hormone is
produced after the time of puparium formation. It is the purpose of
this paper to summarize additional experiments designed to determine
when and under what conditions fat bodies produce this hormone.
Unless otherwise indicated, fat bodies were taken from wild-type larvae
or prepupae. All tests for v+ hormone were made by using vermilion
brown flies as described by Tatum and Beadle (1938). The few tests
made for cn+ hormone were made in a similar way using cinnabar
brown flies.
LARVAL FAT BODIES
Although v* hormone could not be extracted from larval fat bodies
with Ringer's solution at 100° C., it was felt that the hormone as such
might nevertheless be present but in such a state that it was not ex-
tracted by the method used. Accordingly, several additional methods of
extraction have been used.
Fifty sets of dissected fat bodies were heated in distilled water,
oven-dried, and extracted with chloroform. The chloroform-insoluble
material was taken up in hot Ringer's solution and injected into ver-
milion brown test larvae. The results were negative (8 flies). Since
the hormone is known to be chloroform-insoluble and water-soluble,
these tests confirm those previously made. Other tests in which the
dissected fat bodies were ground with powdered silica were likewise
negative (3 flies). Alternate freezing of fat bodies (in an acetone and
solid CO2 mixture) and thawing for six successive times failed to yield
any hormone in subsequent extracts made with hot Ringer's solution
(6 flies).
Digestion of larval fat bodies with trypsin failed to liberate any
1 Work supported by funds granted by the Rockefeller Foundation.
407
408 BEADLE, TATUM AND CLANCY
hormone. In one experiment 20 sets of fat bodies from mature larvae
were incubated for 24 hours at 37° C. in 0.03 cc. of a solution of 0.5
per cent of NaHCCX and 0.025 per cent of trypsin made up in Ringer's.
The clear solution obtained after heating and centrifuging through a
microfilter gave negative results (20 flies). Appropriate controls
showed that under these conditions the trypsin used was active in di-
gesting casein and that it did not alter the activity of concentrated ex-
tracts of the hormone. Observations showed that the trypsin-treated
fat bodies were visibly broken down.
Further attempts to determine whether any v+ hormone is present
in larval fat bodies were made by freezing such tissues with solid CO,
and then transplanting them to test animals. This was done by taking
up the fat body tissue in a regular transplantation pipette (Ephrussi and
Beadle, 1936) and then placing the shaft of the pipette in contact with
a small piece of solid CO2. The temperature actually attained by the
tissue itself was not determined ; it was without question well above
that of the CO2. Ten flies to each of which a section of fat body (at-
tached along one margin to the salivary gland) had been transplanted
after being frozen three times, showed little or no eye-color modifica-
tion. Other experiments using a more or less similar technique were
made with fat bodies immersed in boiling water before transplantation.
Considerable difficulty was encountered in doing this, but by coating the
inside of the pipettes with a thin film of agar, drawing up the fat-body
tissue, and then immersing the pipette in boiling water, a number of
successful transplants were made. Of seven test animals to which such
heated fat bodies were transplanted, six were quite negative. The
seventh showed a color modification of 2.5 (medium response — see
Tatum and Beadle, 1938, for significance of color values). This ex-
ceptional animal was undoubtedly one to which by mistake an unheated
fat body had been transplanted. Because of the technical difficulty of
making such transplants such an error could easily have been made.
Living fat bodies were transplanted as controls for both the frozen and
heated series. Eight such control transplants gave a mean color value
of 2.4 (1.8 to 3.0). A single control transplant gave a negative test,
presumably due to failure of the operation.
These experiments agree with those previously made and indicate
that little or no v* hormone is present in larval fat bodies prior to
puparium formation, and consequently that the major portion of the
hormone produced by such tissues is elaborated after puparium forma-
tion. It is possible that a small amount of hormone is produced before
this time but in too small an amount to be detected by the methods used.
PRODUCTION OF V+ HORMONE BY FAT BODIES 409
FAT BODIES OF PREPUPAE
Preliminary experiments indicated that v+ hormone is present in
prepupal fat bodies and can be extracted from them during this stage.
Several series of extractions of prepupal fat bodies taken from animals
of various ages were therefore made. In each case 20 sets of fat bodies
were heated in 0.03 cc. of Ringer's solution and the solution removed by
centrifuging through a microfilter. One series, using prepupae from
the Oregon-r wild-type stock gave the results shown in Table I.
TABLE I
Age in Hours Number Eye Color,
after Puparium of Test Mean and
Formation Animals Range
0-1 11 0.1 (0.0-0.4)
3-4.5 14 0.0
6-8 10 2.2 (1.5-3.0)
7-9 15 2.3 (0.0-3.2)
10-11.8 10 0.8(0.0-2.7)
There is no apparent reason why the 3^1. 5 hour prepupal fat bodies
gave negative results. A number of other tests indicate that the results
are generally erratic for young prepupae. Thus a separate set gave
a mean color value of 1.3 for an extract of 0-1 hour prepupal fat bodies.
A series of tests of prepupae from the Canton-S wild-type stock gave the
results shown in Table II.
TABLE II
Age in hours Number Eye Color
larval 10 0.0
0-1 10 0.3 (0.1-0.6)
1.8-3.5 8 0.7 (0.0-1.6)
8-9.5 8 1.4(0.7-2.0)
An additional experiment using 10-12-hour Oregon-r prepupae gave
a test of 2.3 (6 animals 1.3-3.0). It should be pointed out that the
tests of older prepupae are unreliable because of the impossibility of
being sure of getting all of the fat body tissue. At this time the fat
bodies are undergoing the breakdown process characteristic of meta-
morphosis.
These results suffice to show that the hormone is present in fat body
tissue and may be extracted over most of the prepupal period. Because
of the several difficulties involved in such tests as these, the results are
only roughly indicative of quantitative relations.
V ^^
410 BEADLE, TATUM AND CLANCY
CORRELATION OF HORMONE PRODUCTION AND PUPARIUM FORMATION
Various attempts have been made to alter the conditions so that
larval fat bodies would produce v+ hormone. Unheated wild-type larval
fat bodies were allowed to stand in Ringer's solution for 5 to 6 hours
at room temperature. Extracts of these gave negative results. Several
series of 48-hour-old wild-type larvae were subjected to semi-starvation
conditions by transferring them to 0.25 per cent dry brewers' yeast in
I per cent agar as described by Beadle, Tatum and Clancy (1938).
This reduced food supply prolongs larval life. Extracts of the fat
bodies of such delayed larvae made just prior to puparium formation
failed to show the presence of hormone. On the assumption that en-
zymes might be involved in the production of v* hormone by the fat
bodies, pupal fluid from vermilion brown animals selected from 0 to 30
hours after puparium formation was injected into wild-type larvae 117-
124 hours after egg-laying. Three to 7 hours after these injections
were made the fat bodies of the hosts were removed and extracted with
hot Ringer's. These extracts were negative in tests for v+ hormone. A
similar experiment in which vermilion brown pupal fluid was injected
into 92-97-hour wild-type larvae gave negative results in tests of fat
body extracts made 23-25 hours later.
A marked delay in puparium formation brought about by subjecting
mature larvae to low temperature apparently does not break down the
synchronism between hormone production and puparium formation.
An experiment in which wild-type mature larvae were kept at 8-10° C.
for 18.5 hours showed that a Ringer extract of fat bodies of 0-1-hour-
old prepupae taken at the end of this time gave a mean color value of
0.3 when tested in 11 vermilion brown animals. A comparable ex-
tract made from prepupal fat bodies from mature larvae kept con-
tinuously at 25° C. gave an average color value of 0.6 (11 flies).
Considering the low values obtained from these two extracts and the
variation (0.1-0.7 and 0.1 to 0.8 respectively), this difference cannot be
regarded as significant.
Prepupal fat bodies 0-1 hours after puparium formation apparently
do not continue hormone production when explanted to Ringer's solu-
tion. In one experiment 20 sets of such fat bodies were placed in 0.03
cc. of Ringer's solution and allowed to stand at 22° C. for 27-28 hours.
At the end of this time a hot-Ringer extract gave a color value of 0.4
(range 0.0-0.7, 14 animals). A control series extracted in a similar
way immediately on dissection gave a color value of 0.6 (range 0.1-0.8,
II animals). The explanted fat bodies did not undergo the breakdown
processes characteristic of metamorphosis.
PRODUCTION OF V+ HORMONE BY FAT BODIES 411
Superfemales of Drosophila (individuals with 3 X chromosomes and
2 sets of autosomes) are known to show a delay of one to three days in
puparium formation as compared with their normal sisters (Brehme,
1937). During this period, suhsequent to puparium formation by their
sibs, there is relatively little growth of the superfemale larvae. Extracts
of fat bodies of such superfemale larvae taken shortly before puparium
formation show that v+ hormone is present at this time. Thus an ex-
tract of 20 sets of fat bodies from mature superfemale larvae in 0.03
cc. of Ringer's solution gave a mean eye-color modification of 0.7 (range
0.0-1.0, 10 animals). Extracts of prepupal fat bodies of superfemales
are likewise positive. It is clear, then, that under the particular set of
developmental conditions of superfemale larvae the synchronization of
fat-body hormone production with puparium formation characteristic
of normal larvae is broken down. This shows that the two processes
are not inseparably associated at least as regards their time sequence.
The mechanism by which the two processes are normally related, how-
ever, is entirely a matter of conjecture at the present time.
While under none of the environmental and experimental conditions
to which normal larvae were subjected was there any appreciable pro-
duction of v+ hormone by the fat-body cells prior to puparium formation,
the fact that the sequence of these two processes is modified by the genie
imbalance characteristic of superfemales suggests that it might be pos-
sible to induce the formation of hormone by cells of this tissue before
puparium formation in normal larvae if the proper conditions were
brought about. Certainly this possibility is not excluded by any of
the work reported in this paper.
In order to determine whether or not fat bodies might have any
effect on the eye-color hormones in vitro, an experiment was made in
which fat bodies were explanted to a Ringer's solution containing par-
tially purified v+ and cn+ hormones. As a control, fat bodies heated
for several seconds at 100° C. were allowed to stand in a similar solu-
tion of the hormones. In both cases the fat bodies were kept in the
solution for 4 hours at room temperature. The results are shown in
Table III.
Living fat bodies appear to have no significant effect on the hormones
in solution. Since the hormones may be inactivated through oxidation
in the presence of certain enzymes present in the organism (Thimann
and Beadle, 1937), it may be concluded that the fat body either does not
contain or does not liberate such enzymes under the conditions of this
experiment.
412
BEADLE, TATUM AND CLANCY
RELATION OF THE FAT BODY TO THE STARVATION EFFECT
It has been shown that low food level at a certain period of develop-
ment modifies vermilion flies in some manner such that they produce v*
hormone (Khouvine, Ephrussi and Chevais, 1938; Beadle, Tatum and
Clancy, 1938). Normally such flies produce little or no v+ eye-color
hormone. Since this modification evidently must be due to some altera-
tion in metabolism, attempts have been made to determine what tissues
or organs might be involved. It has been found that the fat body is
modified by subjection of larvae to low food.
Larvae were transferred from full food to low food at about 48
hours after egg-laying and allowed to complete larval development under
these conditions. The methods of inducing an eye color modification
in this way are described in the papers referred to above. Fat bodies
taken from mature larvae which had been subjected to such semi-star-
vation conditions were transplanted to vermilion brown larval hosts
TABLE III
Test for v
K hormone
Test for en
+ hormone
Unheated
Heated
Unheated
Heated
Number of tests .
11
11
8
9
Mean eye color
3.0
3.1
2.5
2.4
Range
2-3 5
3.0-3.5
2 5
2.0-2.5
grown under standard full-food conditions. In one experiment in
which fat bodies from vermilion brown larvae grown on low food were
transplanted, 21 host animals eclosed. Of these, 16 showed an eye-
color modification (mean 0.8, range 0.1-2.0). The remaining 5 were
negative, possibly because of unsuccessful operations. Since the fat
body normally breaks down during metamorphosis there is no easy way
of checking for the presence of implanted tissue. In another series fat
bodies from vermilion larvae subjected to a low food level were trans-
planted to vermilion brown test larvae. Ten animals developed and all
showed a positive effect of the implant (mean eye color 1.3, range
0.8-1.9).
Since it is well established that fat bodies of fully fed vermilion (or
vermilion brown) larvae give negative results when transplanted to ver-
milion brown hosts, it is evident that low food of the kind used so modi-
fies the fat body that it subsequently produces v+ hormone. These
results have been checked by direct extraction of the hormone from pre-
pupal fat bodies. Extraction of fat bodies of mature vermilion brown
PRODUCTION OF V+ HORMONE BY FAT BODIES 413
larvae that had been subjected to low food conditions yielded solutions
that were negative in tests for v* hormone. Fat bodies from prepupae
(4.5-6.5 hours after puparium formation) were extracted with hot
Ringer's solution. This extract gave a slight but definitely positive
modification of the eyes of vermilion brown test animals (11 flies, eye
color 0.1-0.2). It appears that in such larvae, as in normal wild-type
larvae, the fat body produces v+ hormone subsequent to the time of
puparium formation.
Preliminary studies have indicated that subjection of larvae to low
food conditions brings about changes in the cytoplasmic inclusions of
the fat body cells. These changes may possibly be correlated with the
production of hormone by vermilion larvae which have been grown
under semi-starvation conditions. Since these investigations are as yet
incomplete, discussion of them will be deferred.
Malpighian tubes of wild-type larvae are known to contain z/+ hor-
mone and there is evidence that they produce this substance. In order
to determine whether the low food level might also have an effect on
these organs, Malpighian tubes from semi-starved vermilion brown (or
vermilion) larvae were transplanted to normal vermilion brown test
larvae. It was discovered that tubes from larvae subjected to a low
food level tend to kill the hosts to which they are transplanted. Pre-
sumably the tubes accumulate toxic substances under such conditions.
In a preliminary series four mature recipients showed no eye color
modification. In this series, however, no dissections were made to de-
termine whether the implant was present. A second series in which
sets of four Malpighian tubes from mature semi-starved vermilion were
transplanted to vermilion brown test larvae, nine adult recipients were
obtained which dissections showed to contain implanted tubes. Eight
of these showed a relatively weak eye color response (0.5) indicating
that hormone was present or was produced — the ninth was negative.
It appears that the Malpighian tubes of vermilion larvae contain or
produce some v+ hormone under the semi-starvation conditions to which
these larvae were subjected. The effect, however, seems to be less
strong than that on the fat bodies. It is possible that the hormone re-
leased from larval Malpighian tube transplants represents accumulation
and is not produced by the tubes themselves. It does not seem prob-
able that the hormone is produced by the fat body, although we have not
entirely excluded the possibility that the larval fat body produces hor-
mone at a low rate. The fact that no hormone (or very little) ac-
cumulates in the larval fat body argues that if it is produced in this
tissue during larval life, it must diffuse out approximately as fast as it is
414 BEADLE, TATUM AND CLANCY
SUMMARY
Under normal genetic and environmental conditions fat-body cells
produce v+ hormone after the time of puparium formation but not before.
Attempts to induce hormone production by fat-body tissue before
puparium formation were unsuccessful. Since it is shown that larval
fat bodies of mature superfemale larvae contain v+ hormone, however,
it is clear that the normal sequence of puparium formation and hormone
production is not a necessary and invariable one.
Active solutions of v* hormone are readily obtained by extracting
prepupal fat bodies over practically the entire period of prepupal de-
velopment.
It is shown that the so-called " starvation effect " on eye pigmenta-
tion involves a modification of genetically vermilion fat body cells such
that they produce v* hormone, whereas normally they are unable to do
so. It is possible but not definitely established that a somewhat similar
modification is brought about in cells of the Malpighian tubes by semi-
starvation of larvae.
LITERATURE CITED
BEADLE, G. W., 1937. Development of eye colors in Drosophila : fat bodies and
Malpighian tubes in relation to diffusible substances. Genetics, 22 : 587-
611.
BEADLE, G. W., E. L. TATUM, AND C. W. CLANCY, 1938. Food level in relation to
rate of development and eye pigmentation in Drosophila melanogaster.
Biol. Bull, 75 : 447-462.
BREHME, K. S., 1937. Effects of the triplo-X condition on development in Dro-
sophila melanogaster. Proc. Soc. Exper. Biol. and Med., 37 : 578-580.
EPHRUSSI, B., AND G. W. BEADLE, 1936. A technique of transplantation for Dro-
sophila. Am. Nat., 70: 218-225.
KHOUVINE, Y., B. EPHRUSSI, AND S. CHEVAIS, 1938. Development of eye colors
in Drosophila : nature of the diffusible substances ; effects of yeast, peptones
and starvation on their production. Biol. Bull., 75 : 425-446.
TATUM, E. L., AND G. W. BEADLE, 1938. Development of eye colors in Drosophila:
some properties of the hormones concerned. Jour. Gen. PhysioL, 22 :
239-253.
THIMANN, K. V., AND G. W. BEADLE, 1937. Development of eye colors in Dro-
sophila : extraction of the diffusible substances concerned. Proc. Nat.
Ac ad. Sci., 23 : 143-146.
EFFECT OF DIET ON EYE-COLOR DEVELOPMENT IN
DROSOPHILA MELANOGASTER x
E. L. TATUM AND G. W. BEADLE
(From the School of Biological Sciences, Stanford University)
Vermilion brown (v bw) larvae of D. melanogaster placed on a low
food level diet produce v+ eye-color hormone, and therefore, as flies, de-
velop pigmented eyes (Khouvine, Ephrussi and Chevais, 1938). Beadle,
Tatum and Clancy (1938) showed that larvae are affected in this way
by low food level during a certain sensitive period lying between 60
and 70 hours from egg laying. Khouvine, Ephrussi and Chevais re-
ported that sugar added to the starvation diet inhibits the starvation
effect. Their work, however, did not eliminate the possibility that the
sugar effect was associated only indirectly with hormone production in
the flies, possibly through the intermediation of growing yeast or other
micro-organisms. We have investigated the effects under aseptic con-
ditions of various supplements to a low yeast diet on the growth and
eye-color development of vermilion brown animals. Under these con-
ditions carbohydrates and related substances inhibit the starvation effect,
while proteins and amino acids do not. The present paper summarizes
these results.
EXPERIMENTAL
Culture and Methods
The aseptic cultures of vermilion brown larvae used throughout this
work were obtained by a slight modification of Baumberger's (1919)
alcohol sterilization method. Eggs were collected over a 2- to 3-hour
period on freshly autoclaved standard corn-meal molasses agar, without
added yeast. Shortly after collection 20 to 30 eggs were picked up on
a single small sterilized glass rod flattened at the end. The rods with
the eggs were then placed individually in small sterile vials containing
85 per cent alcohol. After 10 minutes the rod was removed and the
eggs were pushed off onto the sterile test medium, using ordinary bac-
teriological methods to insure sterility. All cultures were incubated at
25° C. unless otherwise stated.
The standard starvation food contained 1.5 per cent agar and 0.5
per cent Fleischmann's dry brewers' yeast made up with distilled water.
1 Work supported by funds granted by the Rockefeller Foundation.
415
416
E. L. TATUM AND G. W. BEADLE
Ten cc. of this mixture and the desired amounts of the various supple-
ments were placed in 35 cc. vials which were stoppered with cloth-
covered cotton plugs. After sterilization in the autoclave the vials were
cooled and agitated, and finally slanted so that the solid yeast remained
suspended throughout the medium. Routine checks of sterility were
made after pupation of the larvae by streaking a loopful of the medium
onto yeast extract -glucose agar. Any vials which were not bacteriologi-
cally sterile at this time were discarded.
Cultures were observed every 24 hours, so that the time to emergence
of the flies was accurate only within this period. The observed pro-
longation of larval and pupal life as compared with the normal 215
hours on full food is given in days from egg-laying. The delay actually
represents prolongation of larval life, since Beadle et al. (1938) showed
TABLE I
Influence of yeast concentration and temperature on the starvation effect. Basic
medium: 1.5 per cent agar.
Yeast concentration (per cent)
0.5
1.0
3.0
5.0
Temp-
erature
Days
No.
Days
No.
Days
No.
Days
No.
°C.
to
of
Eye
to
of
Eye
to
of
Eye
to
of
Eye
emer-
adult
color
emer-
adult
color
emer-
adult
color
emer-
adult
color
gence
flies
gence
flies
gence
flies
gence
flies
17°
22-25*
7
3.5-4.5
27-29
22
3.5-5.0
24-26
39
1.0-3.0
24-26
35
0.5-2.5
25°
11-13
33
2.5-3.5
10-11
15
0.5-1.5
10
20
0.0-0.2
9-10
42
0.0
28°
10-11
26
0.5-1.2
9-10
19
0.0
8-9
29
0.0
8-9
34
0.0
* Normal developmental time on full food is 9 days (215 hours) from egg-laying.
that duration of pupal life is practically constant under all conditions.
After emergence of the flies the intensity of pigmentation of the eyes
was graded according to the scale of eye-color values described by
Tatum and Beadle (1938). These values have a definite relation to
the amount of hormone available to the fly, but for simplification all
results are given only as color values. It should be remembered that
the increased intensity of eye pigmentation resulting from starvation
involves the actual production of v+ hormone (Beadle et al., 1938).
Effect of Yeast Concentration and Temperature
In order to determine the most suitable conditions for the starvation
effect, series with varying yeast concentrations were incubated at dif-
ferent temperatures, 18°, 25° and 28° C. The results are given in
Table I. It was found that 0.5 per cent dry yeast at 25° C. was most
EFFECT OF DIET ON EYE-COLOR DEVELOPMENT 417
TABLE II
Influence of carbohydrates on the starvation effect. Basic medium: 0.5 per cent
brewers' yeast in 1.5 per cent agar.
Carbohydrates added (2 per cent concentration)
None
Starch
Sucrose
Glucose
Delay in days
2-3
9
2.0-3.5
2-5
62
0.0-0.5
1-4
67
0.0-0.3
1-4
45
0.1-1.0
Number of flies
Eye color
suitable, both for the intensity of the effect and for the developmental
time required. Lower concentrations of yeast at this temperature gave
somewhat stronger effects, but mortality was higher. The higher tem-
perature, 28° C., speeded up development and greatly decreased the in-
TABLE III
Influence of sucrose concentration on eye-color (starvation effect) and length of
larval life. Basic medium : 0.5 per cent brewers' yeast in 1 .5 per cent agar. (Figures
in parenthesis indicate number of flies.)
Prolongation of
Sucrose concentration (per cent)
larval life
0
0.05
0.1
0.3
0.5
1.0
2.0
4.0
days
1
4.0-4.5
(4)
1.5-2.0
(21)
1.0-2.5
(60)
0.3-1.5
(22)
2
2.0-3.5
(2)
4.0
(2)
3.5
(17)
3.5
(19)
2.5-3.0
(18)
0.5-1.5
(21)
0.0-0.2
(11)
3
2.0-3.5
(7)
3.5
(20)
3.0
(10)
3.0
(7)
2.0-3.0
(6)
0.0-1.5
(8)
0.0-0.6
(19)
4
2.0-4.0
(17)
2.0-3.0
(5)
3.0
(5)
2.5
(2)
0.0-0.5
(5)
0.0
(2)
5
2.5-3.5
(2)
1.5
(1)
0.0-0.8
(5)
0.0
(20)
Total
2.0-4.0
(28)
1.5-4.0
(28)
3.0-4.5
(36)
1.5-3.5
(49)
1.0-3.0
(84)
0.0-1.5
(51)
0.0-0.8
(40)
0.0
(22)
tensity of the starvation effect; i.e., pigment production did not take
place on yeast concentrations over 0.5 per cent. At 25° C., 3 per cent
yeast or more prevented the starvation effect, while pigment appeared
on all concentrations up to and including 5 per cent yeast at 17° C.
This effect of temperature may be due to a differential influence on
418
E. L. TATUA1 AND G. W. BEADLE
larval activity (food intake) and on the rate of metabolic processes.
The medium containing 0.5 per cent yeast was selected for standard
starvation and used throughout further work. At 25° C. it consis-
tently delayed larval development from 2 to 4 days and gave eye-color
values of from 2.5 to 3.5. This is equivalent to a v+ hormone produc-
tion of 3.5 to 8.0 units per individual (Tatum and Beadle, 1938). Con-
trols were made for each series of experiments, with similar results.
These control starvation values are omitted from the tables in most
cases.
DRY YEAST, 0.5 7°
-DRY YEAST, 0.5 %, WITH
HYDROLYZED CASEIN,
O
•J
O
Uj
PERC EN T
SUCROSE
FIG. 1. Influence of temperature and of hydrolyzed casein on sucrose inhibi-
tion of the starvation effect (maximum color values for each sugar concentration
used in plotting curves). Basic medium: 0.5 per cent brewers' yeast in 1.5 per
cent agar.
Effect of Carbohydrates
Table II shows the influence of added carbohydrates on the starva-
tion effect. Starch, sucrose, and glucose almost completely inhibited
the production of pigment, although larval life was prolonged as much
as or more than in the controls without carbohydrate. Several series of
experiments were made to establish the relation of sugar concentration
to prolongation of larval life and intensity of the starvation effect.
Table III gives the combined results of these series. Concentrations of
sucrose up to 0.5 per cent shortened larval life as compared to the con-
trol, without very marked effect on eye pigmentation. Higher sucrose
concentrations progressively prolonged larval life and inhibited pig-
EFFECT OF DIET ON EYE-COLOR DEVELOPMENT
419
mentation. Two per cent sucrose caused about the same delay as in
the control, but almost completely prevented v+ hormone production.
Four per cent sucrose seemed to be toxic and prolonged larval life even
more than in the control, but completely suppressed the starvation effect.
The inhibiting effect of varying concentrations of sugar at 28° C.
was also determined. The influence of temperature on the sucrose
effect is shown in Fig. 1. It required almost the same concentration of
sugar (2 per cent) to inhibit completely pigmentation at the higher
temperature as at 25° C., although the production of pigment on the
control starvation food without sugar was much less at 28° C.
Influence of other substances on the starvation effect. Basic medium: 0.5 per cent
brewers' yeast in 1.5 per cent agar.
Substance added
Prolongation of
larval life in days
Number of
adult flies
Eye color
Sodium benzoate, 1 per cent* ....
Sodium benzoate, 1 per centf ....
Calcium acetate, 2 per cent
1-3
2-3
6-9
20
5
30
0.1-1.5
0.5-0.8J
0.0-0.2
Calcium lactate, 3 per cent
5-9
30
1.0-3.0
Calcium carbonate, 2 per cent . . .
6-9
20
2.0-3.5
Ethyl alcohol, 5 per cent§
1-5
22
0.0-0.1
Glycerol, 2 per cent
3-7
38
2.0-3.0
Butter fat. 4 per cent .
3-8
11
0.0-0.2
* Sterile 60-hour-old fully fed larvae transferred aseptically to test medium.
t Eggs not sterilized.
t Control with no benzoate; color = 3.0.
§ Alcohol added after cooling medium to 35°C.
The results of these experiments with carbohydrates show that pro-
longation of larval life is not necessarily accompanied by v+ hormone
and eye pigment production. However, the starvation effect is ob-
served only when larval life is prolonged.
Influence of Other Substances on the Starvation Effect
It seemed possible that some indication of the nature of the starva-
tion effect might be obtained by similarly testing substances other than
carbohydrates. Table IV summarizes the results of these experiments.
Calcium lactate, calcium carbonate, and glycerol had only very slight
inhibiting effects on pigmentation. On the other hand, ethyl alcohol,
butter fat, and calcium acetate prevented the starvation effect almost
completely. Sodium benzoate was quite toxic, but under non-lethal
conditions it prevented pigment production to a considerable degree.
420
E. L. TATUM AND G. W. BEADLE
Each of these various additions to the starvation diet considerably pro-
longed larval life, but production of v+ hormone was suppressed only
by certain specific substances, all of which, with the exception of sodium
benzoate, may be assumed to be metabolized in a manner similar to
carbohydrates. No explanation can be suggested for the inability of
glycerol and calcium lactate to function in this way. Concentrations of
calcium lactate and glycerol from 0.5 to 3.0 per cent have been used with
similar results in every concentration.
Effect of Proteins and Ammo-acids
In contrast to carbohydrates, which definitely inhibit the starvation
effect, whole and hydrolyzed proteins and mixtures of amino acids, in-
TABLE V
Influence of protein and amino acids on starvation effect. Basic medium: 0.5 per
cent brewers' yeast in 1.5 per cent agar.
Substance added
Prolongation of
larval life in days
Number of
adult flies
Eye color
Gelatine, 3 per cent
5-6
2
1.5-2.0
Gelatine, 3 per cent; Trypto-
phane, 1 per cent
5-9
7
1.5-2.5
Mixture of amino acids* ....
Hydrolyzed casein, 0.5 per
cent
11
2-4
2
13
3.5
2.5-3.5
Hydrolyzed casein, 1 per
cent
2-4
18
2.0-3.5
Hydrolyzed casein, 2 per
cent ....
2-4
11
1.0-3.0
Hydrolyzed casein, 4 per
cent .
5-6
9
0.2-3.0
* Tryptophane, tyrosine, cystine, leucine, asparagine, glycine, alanine; 0.1 per
cent each.
eluding tryptophane, have no significant effect in reducing either dura-
tion of larval life or pigment production. These results are given in
Table V.
Although hydrolyzed casein alone had very little effect on pigmen-
tation, it greatly intensified the sucrose effect. The result of a series
containing 2 per cent hydrolyzed casein and increasing amounts of
sugar is graphically represented in Fig. 1. In the presence of hydro-
lyzed casein, a sucrose concentration of 0.3 per cent almost completely
inhibited hormone production. The other curves in Fig. 1 give for
comparison the effect of sucrose without hydrolyzed casein. In the
presence of an excess of amino acids, the sugar concentration effective
in pigment inhibition was about that optimal for growth (see Table III).
EFFECT OF DIET ON EYE-COLOR DEVELOPMENT 421
DISCUSSION
Khouvine et al. (1938) suggested that sugar may have a protein-
sparing action, and that the starvation effect and production of v+ hor-
mone involves an abnormal protein degradation in the larva. It seems
possible, from our results, that the action of carbohydrates and similar
substances may be due to their protein-sparing action. However, it is
probable that other factors are also involved since the sugar concentra-
tion optimal for growth does not inhibit the starvation effect and pig-
ment production. This concentration (0. 5 per cent) should have the
same protein-sparing action as higher concentrations. In the presence
of an adequate supply of amino acids (hydrolyzed casein), however,
sugar completely inhibits the starvation effect at the 0.5 per cent con-
centration optimal for growth.
Carbohydrates seem to inhibit pigment production in starvation by
altering the starvation metabolism in such a way that v+ hormone is not
produced, and not by affecting the utilization of the hormone. Khou-
vine et al. showed that a diet containing sugar did not affect the utiliza-
tion of ingested v+ hormone supplied as a Calliphora extract. In addi-
tion, we have injected mixtures of glucose with extracts containing v+
substance into v biv larvae with no decrease in the effectiveness of the
hormone.
Substances other than carbohydrates which also prevent the starva-
tion effect probably act in the same way, since theoretically they ma}- be
metabolized in a similar manner. The action of sodium benzoate, since
it has no relationship to carbohydrates metabolically, may have a dif-
ferent basis. Sodium benzoate acts similarly to sugar in that it inhibits
the production of v+ hormone by v bw larvae on a starvation diet. How-
ever, it has no effect on the normal hormone production by sif-v, v bit'
larvae (normal eye-color ^= = 1.0;? = = 2.0) . Nor does sodium benzoate
influence the utilization of ingested v+ hormone by v bw larvae.
Beadle et al. (1938) showed that starvation is effective only during
a certain sensitive period in larval development. This period was found
to lie between 60 and 70 hours of normal development. The starva-
tion effect may be assumed to be a result of prolonging this specific
developmental period. Preliminary experiments designed to determine
the effect of sucrose on this sensitive period were carried out under aseptic
conditions by placing fully fed 54-hour-old larvae on low food with and
without sugar. At intervals thereafter larvae were removed from the
starvation food to plain agar. The ability to pupate served as the cri-
terion of the end of the 60-70-hour sensitive period (see Beadle et al.,
1938). The results seemed to indicate that this period is significantly
shortened by sugar in the starvation food.
422 E. L. TATUM AND G. W. BEADLE
Whether the action of carbohydrates in inhibiting the starvation
effect is due to a direct influence (possibly through a protein-sparing
action) on specific processes which during the starvation period lead to
the production of z/+ hormone, or whether it is due to a differential ac-
celeration of development during the 60-70-hour sensitive period, thereby
shortening the effective time of starvation, cannot be definitely decided
at present.
SUMMARY
The production of z>+ eye-color hormone and development of pigment
in the double recessive vermilion brown of D. melanogaster may be
brought about by feeding the larvae on sub-optimal levels of dead yeast
under aseptic conditions.
With a given concentration of yeast, culture of larvae at low tem-
perature (17° C.) greatly increases the intensity of the starvation effect.
High temperature (28° C.), on the other hand, decreases the intensity
of the starvation effect.
Carbohydrates and related substances (acetate, fat, and ethyl al-
cohol) added to the low yeast diet, under aseptic conditions, completely
inhibit the starvation effect by their direct action on larval metabolism
and development.
Proteins and amino-acids have very little influence on the starvation
effect, but greatly lower the carbohydrate level required to completely
inhibit pigment production.
The starvation effect is always associated with prolongation of larval
life, but great prolongation of life is possible under certain conditions
without any modification of eye color.
The inhibition by carbohydrates may be due to a direct influence on
processes proceeding during starvation or to a specific acceleration of
development during the period sensitive to starvation, or to both.
LITERATURE CITED
BAUMBERGER, J. P., 1919. A nutritional study of insects, with special reference to
microorganisms and their substrata. Jour. Expcr. Zool., 28: 1-81.
BEADLE, G. W., E. L. TATUM, AND C. W. CLANCY, 1938. Food level in relation to
rate of development and eye pigmentation in Drosophila melanogaster.
Biol. Bull., 75: 447-462.
KHOUVINE, Y., B. EPHRUSSI, AND SIMON CHEVAIS, 1938. Development of eye
colors in Drosophila: nature of the diffusible substances; effects of yeast,
peptones and starvation on their production. Biol. Bull., 75 : 425-446.
TATUM, E. L., AND G. W. BEADLE, 1938. Development of eye colors in Dro-
sophila: some properties of the hormones concerned. Jour. Gen. Physiol..
22: 239-253.
PIGMENT INHERITANCE IN THE FUNDULUS-SCOMBER
HYBRID
ALICE RUSSELL
(From the Marine Biological Laboratory, Woods Hole, Mass.}
The hybrid between Fundulus hcteroclitus (L.) 9 and Scomber
scombrus (L.) 3 is first mentioned by H. H. Newman in 1915. J. Loeb
had stated his belief that development in intergenic hybrids is partheno-
genetic. Newman cites the inheritance of Scomber pigmentation in the
Fundulus-S comber cross as a proof that fertilization had taken place.
In a later paper, 1918, he continued the discussion of this hybrid.
His account of the abnormalities found in the embryos made it seem
worthwhile to obtain the cross again, to make a cytological and mor-
phological study of early stages, and to make a detailed study of the
chromatophores in parent and hybrid embryos.
Preliminary hybridizations were made successfully late in June,
1937, at the Marine Biological Laboratory. During the summers of
1938 and 1939 numerous hybridizations were made from June 10- July 5.
After July 5 it is usually impossible to procure spawning mackerel,
before June 10 it is difficult to obtain spawning Fundulus. For best
results, with a large percentage of hybrid embryos, both parents must
be at the height of sexual activity.
Ripe F. heteroclitus females were selected and isolated in tanks of
running sea water for at least 18 hours before they were to be used : this
assures the absence of Fundulus sperm. As mackerel do not live long
after being caught, hybridizations were carried out at the traps. Fun-
dulus females were carried to the fish traps in clean buckets or bowls of
sea water. Scomber scombrus males were stripped into finger bowls
containing a small quantity of sea water. The F. heteroclitus females
were stripped into the sperm suspension, the eggs from each female
being kept in separate bowls. After 10-15 minutes the sperm suspen-
sion was washed off and fresh sea water was added. In the laboratory
the eggs were placed a few in each bowl, and allowed to develop. Con-
trols were carried as follows: (1) Unfertilized F. heteroclitus eggs from
some of the females in each set of hybridizations were observed as a
check on the possible presence of F. heteroclitus sperm in sea water, in
the tanks, or on the fish. (2) F. heteroclitus eggs were fertilized with
F. hcteroclitus sperm to check on the fertilizability and rate of develop-
423
424 ALICE RUSSELL
ment of normal Fundulus heteroclitus. (3) Scomber scombrus eggs
were fertilized with Scomber scombrus sperm to check on the normal
Scomber scombrus development. (4) The reciprocal cross with Scom-
ber scombrus eggs and F. heteroclitus sperm was tried many times,
always unsuccessfully.
The egg of Fundulus heteroclitus is 2-2.5 mm. in diameter, well
yolked, demersal, developing slowly and hatching out in 12-16 days.
The egg of Scomber scombrus is smaller, 1 mm. or less in diameter,
transparent, pelagic, developing quickly and hatching in 60-72 hours.
The hybrid develops more slowly than normal F. heteroclitus, forming
defective embryos which, in our experience, never hatch, even though
kept for 30-35 days.
Normal stages of Scomber scombrus have not been described, al-
though Worley (1933) mentions that they resemble closely those of
sea bass as described by Wilson (1889). As there was no published
account of pigment development in Scomber scombrus, this had to be
studied. Scomber scombrus eggs were obtained and fertilized at the
fish traps. As the eggs are pelagic, it is difficult to wash off the excess
milt while in transit from traps to laboratory; sea water can, however,
be added from time to time. Scomber eggs are very sensitive to tem-
peratures above 17° C, and will not develop at all above 21° C. (Worley,
1933), therefore care must be taken that the water in the shallow bowls
is not warmed by the sunshine, or by heat from the decks or engine.
In the laboratory the fertilized and developing eggs soon float to the
top of the water, and can be skimmed off and transferred to fresh sea
water. If the bowls are placed in baths of running sea water the eggs
develop quite normally, and hatch out in 60-72 hours. The first pig-
ment cells to appear are the slender branching melanophores on the
dorsal surface of the embryo at 27 hours. Later more melanophores
appear and form the characteristic pattern ; a band across the dorsal
surface of the head at the level of the optic vesicles, and a row along
the lateral line region. A few migrate to the yolk sac and to the oil
drop. At 36 hours there appears just behind the optic vesicles a group
of cells containing yellowish green pigment granules. Soon the granules
increase in number, the pigment cells fuse, forming two large brilliantly
green chromatophores persisting at least as long as the fry live in the
laboratory. Other green chromatophores may appear behind the otic
vesicles, on the oil drop or near Kupfer's vesicle. Upon hatching the
young fry drop to the bottom of the vessel and lie there until they are
able to swim about easily.
Normal stages of Fundulus heteroclitus have been described (Op-
penheimer, 1937). Typical pigment formation has been described, also
PIGMENT INHERITANCE-FUNDULUS-SCOMBER HYBRID 425
(Bancroft, Stockard, Newman). Four days after fertilization a first
head crop of melanophores appears. Another crop appears on the fifth
day. The pigment cells are of three types : those on the yolk are large
polygonal melanophores with but few processes, those on the embryo
are smaller and more branched: the reddish-orange much-branched
chromatophores found on embryo- and yolk sac. On the sixth day,
when circulation begins, the melanophores of the yolk sac migrate to
the blood vessels and fuse. The reddish-orange chromatophores also
arrange themselves along the course of the blood vessels, but do not seem
to fuse. A number migrate to the lateral line. Melanophores are
rarely seen in this region, the absence of a visible lateral line being one
of the species characteristics of the adult F. heteroclitus. After hatch-
ing few reddish chromatophores are to be found on the exterior of F.
heteroclitus.
The melanophores of F. heteroclitus and S. scombrus, the green
chromatophores of Scomber, and the reddish chromatophores of Fun-
dulus all contain a granular pigment. No green chromatophores are
ever present in Fundulus heteroclitus, and no red chromatophores in
Scomber scombrus.
Fimdulus-S comber hybrids cleave at the same rate, or more slowly,
than normal Fundulus heteroclitus. In a series of 15 hybridizations,
3,097 eggs were fertilized : 3,084, or 99 per cent of these cleaved.
Many died at gastrulation and during early embryonic life, but 1,205 or
39 per cent formed advanced embryos. Development in the hybrid is
slower than in normal F. heteroclitus: pigmentation develops later, the
heart does not begin to pulsate as early, circulation is feeble, or not
established in most of the hybrids. As a result, the chromatophores
remain scattered for a longer period, eventually migrating to the heart,
or to the site of its attachment to the yolk. As has been noted by Ban-
croft, Newman and others, the hybrid embryos show various combina-
tions of the parental types of chromatophores.
Figure 1 a shows the average melanophore counts for parent and
hybrid embryos. As their rate of development differs widely stages
which are equivalent were arbitrarily chosen. Normal F. heteroclitus
five days after fertilization shows both first and second crops of mel-
anophores not as yet fused on the blood vessels. Scomber scombrus at
30 hours shows first and second crops of melanophores also, not yet
migrated to head and lateral line regions ; the hybrids at 7 days showed
the melanophores well developed, not yet fused. F. heteroclitus em-
bryos show from 8-18 melanophores, or an average of 13 — on the dorsal
surface; S. scombrus has from 34-51, averaging 40. The Fundulus-
S comber hybrid shows a great variation, from 2-44. The majority of
426
ALICE RUSSELL
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PIGMENT INHERITANCE-FUNDULUS-SCOMBER HYBRID 427
hybrid embryos seem to show a tendency to conform to the F. hetero-
clitits type of distribution, having 8-18 melanophores on the dorsal sur-
face of the embryos. However, we have seen no normal F. heteroditiis
embryos of 5 days with over 20 melanophores — the increase in number
of melanophores present in a large number of hybrid embryos may be
assumed to be the influence of Scomber scombrus.
600-
500-
400-
300-
200-
100-
0-
M^BV
1
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A JL £
RED MIXED RED GREEN
CHROMATCPHORES AND OREEN CHROMATOPHOftES
ONLY CHROMATOPHORES ONLY
CHROMATOPHORE INHERITANCE IN THE HYBRID EMBRYO
FIG. 2. Chromatophore inheritance in the hybrid embryos.
Figure 1 b shows the distribution of yolk melanophores, counted
on the embryonic hemisphere of the same individuals used above. In
F. heteroclitus the yolk melanophores are large polygonal cells, varying
from 26-55 in 5-day-old embryos. Scomber scombrus at 30 hours
rarely shows any yolk melanophores. In the 7-day-old hybrid embryos
the number varies from 6-80, with scarcely two embryos having identi-
cal numbers of melanophores.
428 ALICE RUSSELL
The hybrids can be grouped roughly into three categories : those
showing green chromatophores of the Scomber type, but no red F.
heteroclitus type chromatophores : those showing the red Fundulus type,
but no green Scomber type chromatophores : those showing both red and
green chromatophores. Figure 2 will show the distribution of 1,205
ten-day-old hybrid embryos in these three categories. There seems to
be a very significant relation at first glance, since about equal numbers
of individuals show only maternal or paternal type chromatophores, and
about twice as many show both types combined. As a matter of fact,
examination shows that no two embryos are identical. In the group
showing green chromatophores only, the individuals range from those
brilliantly green laterally to some with only a few green head chromato-
phores: some show green chromatophores on the embryo only, some
show them on the yolk also. In the group showing only red chromato-
phores of Fundulus type, the individuals are equally variable, some
embryos being brilliantly reddish-orange, others grading to some re-
sembling closely F. heteroclitus embryos. In the large group showing
both red and green chromatophores there is every conceivable type of
combination, no two individuals are identical. In addition, the Scomber
and Fundulus types of melanophores are present in all possible combina-
tions with the red and green chromatophores, in all three categories.
No consistency of pigment distribution is found in sets of Fundulus
eggs from various females, fertilized at the same time by sperm from
the same mackerel": some may show a preponderance of green, of red,
or of mixed green and red chromatophores as will be seen in Table I.
TABLE I
Green Chromatophores Only Mixed Red and Green Red Only
84 57 43
27 41 3
27 107 35
2 29 21
2 12 29
These counts were made on eggs from various F. heteroclitus ferti-
lized by sperm from the same mackerel on June 17, 1939, and counted
June 27, 1939.
Unlike Newman, who reports that green chromatophores can be
found only in hybrid embryos obtained before mid June, we have found
green chromatophores in the hybrids whenever we got successful
crosses.
DISCUSSION
Many intergenic and interspecific crosses have been described.
Some of these " hybrids " do not develop beyond late embryological
PIGMENT INHERITANCE-FUNDULUS-SCOMBER HYBRID 429
stages, as is true of the Fundulus-S comber hybrid we are discussing.
Here we are dealing with a much wider cross, for the two genera be-
long to different sub-orders. They differ widely in ecological relations,
in habitat, and in structure. Morphological studies of hybrid and parent
embryos may show significant combinations of structural peculiarities,
for example : F. heteroclitus has an air bladder, while this structure is
absent in Scomber scombrus. Cytological studies may show successive
elimination of chromosomes during early cleavages. It is probable that
some of the embryos showing only Fundulus type chromatophores, and
typical Fundulus distribution of melanophores, may be haploid indi-
viduals. These individuals are rare, however, for even in this group
one generally finds some Scomber type melanophores, or some Scomber
effect on the number or distribution of melanophores. (Figure 1, a
and b.)
It is probable that in the teleosts, as in the amphibians, pigment dif-
ferentiation depends on neural crest development. If so, it is reason-
able to suppose that wherever Scomber type chromatophores are present,
Scomber chromosomes may have been retained throughout cleavage,
gastrulation and differentiation.
We have purposely refrained from a discussion of the size of mel-
anophores and chromatophores in parents and hybrids, for any meas-
urements would be open to the criticism that metabolic processes in the
embryo are abnormal, or at least, much disturbed. In shape, the mel-
anophores of Fundulus are quite distinct and distinguishable (New-
man and others). In color the reddish chromatophores in the hybrids
are identical with those of Fundulus heteroclitus, the green chromato-
phores identical with those of the Scomber parent.
In the literature on interspecific and intergenic crosses, the F1 gen-
eration is generally reported as intermediate. A closer scrutiny of the
hybrids may reveal a much wider variation than hitherto suspected, as,
for instance, in the case of the melanotic hybrids between Platypoc-
cilius ? and Xiphophorus c? described by Gordon. Apparently a case of
Mendelian dominance is revealed in the universally melanotic F1 genera-
tion, but there is reported a variation in degree of melanosis in the
progeny.
Earlier reports (Newman, Bancroft) have attempted to explain the
results of the Fundulus-S comber hybridizations on the basis of Men-
delian dominants and recessives, or of " blending " inheritance. How-
ever, as no detailed study of pigmentation was attempted, the enormous
variability actually present in the hybrids escaped attention.
Pinney, reporting on other inter-sub-order hybrids, reports chromo-
some elimination during early cleavages.
430 ALICE RUSSELL
We have at this time no satisfactory explanation for the phenomenon
presented by the pigment inheritance in this cross, although we may
assume that there is a complicated random assortment, combination and
elimination of chromosomes.
We are indebted to Mr. Robert Coffin of the U. S. Bureau of Fish-
eries, at Woods Hole, and to the crew of the " Sagitta " of the Marine
Biological Laboratory for their assistance in getting to the fish traps
and obtaining mackerel. We wish also to thank Dr. H. B. Goodrich,
for his interest, and also Rev. F. W. Ludwig, Ph.D., for help with the
microphotographs.
SUMMARY
1. Methods for hybridizing Fundulus hcteroclitus and Scomber
scombrus are described.
2. The pigment development in hybrid and parents is described.
3. Comparison of the inheritance of embryo and yolk melanophores
reveals a Scomber-effect in the embryo.
4. The hybrid embryo shows chromatophore inheritance from both
parents.
5. Actually, as regards inheritance of melanophores and chromato-
phores, there is enormous variability, no two embryos being identical
as to pigment distribution.
6. This variation in the Fx generations is unusual and at present
inexplicable.
BIBLIOGRAPHY
BANCROFT, F. W., 1912. Heredity of pigmentation in Fundulus hybrids. Jour.
Exper. Zoo/., 12 : 153-178.
DOBZHANSKY, TH., 1937. Genetics and the Origin of Species, page 63.
Du SHANE, G. P., 1935. An experimental study of the origin of pigment cells in
Amphibia. Jour. Exper. Zool., 72: 1-31.
GOODRICH, H. B., 1929. Mendelian inheritance in fish. Quart. Rev. Biol., 4 : 83-99.
GOODRICH, H. B., 1933. One step in the development of hereditary pigmentation
in the fish Oryzias latipes. Biol. Bull., 65 : 249-252.
GOODRICH, H. B., 1935. The development of hereditary color patterns in fish.
Am. Nat., 69 : 267-277.
GOODRICH, H. B., AND NICHOLS, R., 1933. Scale transplantation in the gold fish,
Carassius auratus. Biol. Bull., 65: 253-265.
GORDON, MYRON, 1931. Hereditary basis of melanosis in hybrid fishes. Am.
Jour. Cancer, 15: 1495-1519.
MORRIS, MARGARET, 1914. The behavior of the chromatin in hybrids between
Fundulus and Ctenolabrus. Jour. Exper. Zool., 16: 501-521.
NEWMAN, H. H., 1908. The process of heredity as exhibited by the development
of Fundulus hybrids. Jour. Exper. Zool, 5: 503-561.
NEWMAN, H. H., 1910. Further studies of the process of heredity in Fundulus
hybrids. Jour. Exper. Zool., 8: 143-161.
NEWMAN, H. H., 1914. Modes of inheritance in teleost hybrids. Jour. Exper.
Zool., 16 : 447-499.
PIGMENT INHERITANCE-FUNDULUS-SCOMBER HYBRID 431
NEWMAN, H. H., 1915. Development and heredity in heterogenic teleost hybrids.
Jour. Ex per. Zool, 18: 511-576.
NEWMAN, H. H., 1917. On the production of monsters by hybridization. Biol.
Bull, 32 : 306-321.
NEWMAN, 1918. Hybrids between Fundulus and mackerel. Jour. Exper. Zool..
26 : 391-421.
OPPENHEIMER, J. M., 1937. The normal stages of Fundulus heteroclitus. Anal.
Rec., 68: 1-15.
PINNEY, EDITH, 1918. A study of the relation of the behavior of the chromatin to
development and heredity in teleost hybrids. Jour. Morph., 31 : 225-261.
PINNEY, EDITH, 1922. The initial block to normal development in cross-fertilized
eggs. Jour. Morph., 36: 401-415.
PINNEY,, EDITH, 1928. Developmental factors in teleost hybrids. Jour. Morph.,
45: 579-598.
REAGAN, F. P., AND J. M. THORINGTON, 1915. The vascularization of the em-
bryonic body of hybrid teleosts without circulation. Anat. Rec., 10 : 79-98.
STOCKARD, C. R., 1915. Development of wandering mesenchyme cells, etc. Am.
Jour. Anat., pp. 525-594.
WERBER, E. I., 1916. Experimental studies on the origin of monsters. Jour.
Ex per. Zool., 21 : 485-584.
WILSON, H. V., 1889. The embryology of the sea bass (Serranus atrarius).
Bull. U. S. Fish Com., 9 : 209-277.
WORLEY, L. G., 1933. Development of the egg of the mackerel at different con-
stant temperatures. Jour. Gen. Physiol., 16 : 841-857.
THE EFFECT OF INCREASING TIME OF DEVELOPMENT
AT CONSTANT TEMPERATURE ON THE WING SIZE
OF VESTIGIAL OF DROSOPHILA MELANOGASTER
GEORGE CHILD
(From the Department of Biology, Amherst College, Amherst, Massachusetts)
INTRODUCTION
This paper deals with the effect of nipagin (methyl parahydroxy
benzoate) and poor food conditions on the wing size of the mutant
vestigial in D. melanogaster. Nipagin is being used in many laboratories
as an antiseptic for mold control in Drosophila culture media. It was
found at this laboratory that the time of development of an isogenic
stock was increased when the larvae were raised on nipagin-treated food.
This suggested a method for increasing the time of development at
constant temperature, a new tool in phenogenetic research.
The effect of temperature on the wing size of vestigial has been
studied by a number of investigators (Harnly, 1930, 1932; Stanley,
1928, 1931, 1935; Hersh and Ward, 1932; Li and Tsui, 1936). They
find that the wing size of vestigial increases with increasing tempera-
ture. Furthermore, the temperature-effective period occurs during a
relatively short portion of the larval life. This present work represents
a different approach to the problem in that it concerns the effect of pro-
longing the duration of the larval period at constant temperature.
METHODS
The culture medium consisted of 850 cc. water, 100 grams corn-
meal, 150 cc. molasses, 13 grams agar-agar, and 6 grams brewer's yeast,
made up in the usual manner. The cultures were seeded with dry yeast.
In the starvation experiments the dry yeast was not added. The
nipagin was weighed on a standard quantitative balance and thoroughly
mixed with the food before pouring. Half-pint milk bottles containing
60 cc. food were used.
The isogenic vestigial stock was obtained from Dr. A. Hersh of
Western Reserve. About 20 pairs were used for egg-laying. It was
found that the flies did not lay well on the food containing the higher
concentrations of nipagin. It was necessary to use long egg-laying
periods of twelve hours and for this reason the time of development
was determined only approximately for these concentrations.
432
TIME OF DEVELOPMENT AND WING SIZE VESTIGIAL 433
The egg-laying and total development was carried out in an incubator
held constant at 28° ± 0.1°. The incubator was kept in a constant
temperature (16° ± 1.°), constant humidity (60 per cent ± 5 per cent
relative humidity) room. The apparatus is fully described in Droso-
phila Information Service 6, April 1936.
As the flies hatched they were examined, the sexes were separated
and they were placed in vials containing 70 per cent alcohol. An un-
selected sample of control flies and 0.2 per cent nipagin flies were bred
for a second generation to determine any " carry-over " effect. The
wings of the flies wrere removed under a binocular microscope and
TABLE I
Effect of nipagin on wing size of vestigial. Temperature, 28° ±0.1°.
Cone, of
Xipagin in
per cent
Time of
Pupation
(hours)
9 9
cfcf
No.
Length
in mm.
Area
in mm.2
No.
Length
in mm.
Area
in mm.2
±s.e.
±s.e.
±s.e.
±s.e.
0.0
98
40
0.85
0.178
14
0.76
0.132
±0.017
±0.0047
±0.023
±0.0033
0.05
104
44
0.90
0.186
25
0.91
0.177
±0.007
±0.0043
±0.035
±0.0078
0.1
112
37
0.90
0.171
41
0.95
0.189
±0.015
±0.0044
±0.024
±0.0072
0.2
146
50
1.02
0.213
45
1.06
0.221
±0.003
±0.0094
±0.035
±0.0094
Carry-over effect
from random sample of .2 per cent nipagin-treated flies
0.00
103
31
0.95
±0.023
0.206
±0.0055
38
0.85
±0.0075
0.167
±0.0038
mounted on slides with a drop of cedar oil. The right wing was used
unless it was torn or mutilated.
The wings were projected with a Proni projection apparatus. The
magnification set at 75 X was checked periodically with a stage microm-
eter. The periphery of the wings were traced and from these tracings
the maximum lengths and areas were measured. A Glogau vernier
caliper and a Keuffel and Esser planimeter were used.
In the later experiments 0.1 per cent nipagin was used. It was sus-
pected that nipagin produced its effect by slowing down the growth of
yeast and thereby decreasing the food supply. To check this a number
of cultures were prepared with no addition of live yeast. In these non-
434 GEORGE CHILD
seeded bottles a number of old larvae were added after the egg-laying.
This was done to remove any yeast carried in on the bodies of the adult
flies.
The time of development was determined by removing pupae at in-
tervals of four hours and placing them on agar slants. Many of these
flies were used for a second generation test. The matings were control
X control, control <3<3 X nipagin-treated ??, control ?? X nipagin-treated
<$d etc. to determine how much of the carry-over effect was maternal or
paternal.
FIG. 1. A normal 28° vestigial wing compared with wings from 28° nipagin
treated flies.
EXPERIMENTAL
The Effect of Varying Concentrations of Nipagin
In these experiments the larvae were raised on nipagin-treated food
to determine the effect of nipagin on the time of development and wing
area. The concentrations of nipagin used were 0.05 per cent, 0.1 per
cent, 0.2 per cent, 0.4 per cent, and 0.8 per cent of the food weight. A
few larvae in the 0.4 per cent and 0.8 per cent developed to pupation
but failed to hatch. It was found that the time of development in-
creased with increasing concentrations of nipagin, the 0.2 per cent
pupating two days later than the controls.
Table I shows the wing length and area as affected by nipagin. With
one exception, the 0.1 per cent for females, the lengths and areas of the
vestigial wings increase with increasing concentrations of nipagin. The
wing size of the control males is smaller than that of the females. With
increasing concentrations of nipagin, however, the male wing size in-
creases faster than the female. At 0.1 per cent and 0.2 per cent the
TIME OF DEVELOPMENT AND WING SIZE VESTIGIAL 435
male wings are larger than the female. A similar result is obtained with
temperature, the male wing size exceeding that of the females at high
temperature (Harnly, 1930; Stanley, 1931).
It was found when preserving the flies at hatching, that with in-
creasing time of development there was an apparent increase in wing
size within each nipagin-treated population. The change in wing size
due to nipagin is, therefore, greater than the means given in Table I ;
these means having been obtained by including all the flies in a given
population irrespective of their time of development. This general re-
sult has been recently reported by Braun (1939) on notch.
TABLE II
Relation between time of pupation and wing area. Control series.
rfc?
9 9
Time
of Pupation
in hours
No.
Area in mm.
No.
Area in mm.
1.
82
8
0.130
3
0.164
2.
86
6
0.138
6
0.137
3.
90
15
0.137
6
0.159
4.
94
25
0.136
10
0.143
5.
98
7
0.154
11
0.173
6.
. 102
3
0.158
4
0.147
7.
106
3
0.133
2
0.187
8.
110
0
2
0.167
9.
114
3
0.107
2
0.154
mean time, 93. 2 ±0.87 hours
mean area, 0.139±0.004 sq. mm.
mean time, 96.1 ±1.24 hours
mean area, 0.156±0.0035 sq. mm.
Figure 1 illustrates the appearance of the larger wings obtained from
a 0.2 per cent nipagin population as compared with a " normal " ves-
tigial wing. The larger wings simulate the expression of other vg.
alleles when raised under normal environmental conditions.
The results obtained by raising a random sample of 0.2 per cent
nipagin-treated flies for another generation but in untreated food are
shown in Table I. Both the mean length and area show a significant
carry-over effect. Some of these data had been reported previously
(Child and Albertowicz, 1936).
Effect of Time of Development
In the second series of experiments the relation between time of
development and wing area was determined. Starvation and 0.1 per
cent nipagin were used. The larvae were removed from the culture as
436
GEORGE CHILD
they pupated and the areas of the wings were determined separately for
each pupating group. The results (Table II) indicate that in the con-
trol series there is no apparent effect of time of development (from egg-
laying to pupation) on the size of the wings. The larvae pupate be-
tween 82 hours and 114 hours and the wing areas among the different
groups do not differ significantly from one another.
The time of development is very markedly increased in the nipagin
and starvation series (Tables III and IV). The wing areas of the
various groups show greater differences than in the control series.
There is an apparent relation between the time of pupation and wing
TABLE III
Relation between time of pupation and wing area after treatment with
0.1 per cent nipagin.
Time of Pupation
in hours
cfcf
9 9
No.
Area in mm.
No.
Area in mm.
1.
93
10
0.158
12
0.167
2.
97
8
0.231
5
0.181
3.
101
2
0.437
4
0.140
4.
107
23
0.299
12
0.172
5.
118
9
0.368
8
0.181
6.
129
5
0.592
5
0.236
7.
141
6
0.517
1
0.268
8.
153
1
0.505
3
0.233
9.
165
25
0.268
8
0.151
10.
179
23
0.267
23
0.165
11.
191
7
0.318
14
0.208
12.
203
12
0.379
14
0.190
13.
215
14
0.354
27
0.201
mean time, 152.4±3.39 hours
mean area, 0.315±0.0113 sq. mm.
mean time, 163.5±3.81 hours
mean area, 0.185 ±0.0044 sq. mm.
area. This relation is more easily observed on the imagoes as they
hatch. With increasing time of development the larvae (and the flies)
become smaller and smaller so that the relative difference between wing
area and body size is very great in the delayed flies although the absolute
area increases and then decreases. Unfortunately the body size of the
adults was not measured and we are unable to show this difference
quantitatively.
The Carry-over Effect
Lhe carry-over effect was studied using normal food. To determine
whether both sperm and eggs from treated parents transmitted the fac-
TIME OF DEVELOPMENT AND WING SIZE VESTIGIAL 437
TABLE IV
Relation between time of pupation and wing area after starvation.
cTc?
0 Q
Time of Pupation
¥ f
in hours
No.
Area in mm.
No.
Area in mm.
90
7
.204
5
0.190
94
3
.164
3
.310
98
2
.197
12
.185
102
5
.194
2
.238
106
1
.224
4
.188
110
2
.169
7
.169
116
17
.277
16
.201
122
4
.258
8
.196
126
4
.283
4
.215
130
3
.212
4
.100
138
6
.242
9
.300
150
0
3
.231
162
1
.345
1
.212
174
2
.642
mean time, 116.4±2.53 hours
mean area, 0.254±0.0174 sq. mm.
mean time, 115.0±2.13 hours
mean area, 0.213 ±0.0102 sq. mm.
tors for increased wing size, treated males and females were mated with
control females and males respectively. Treated males were also mated
with treated females. The results of these various reciprocal crosses
are shown in Table V which also includes the control areas and the
means of Tables III and IV. The carry-over effects are more apparent
TABLE V
Effect of .1 per cent nipagin and starvation.
cfcf
9 9
Experiment
Time of
Pupation
No.
Area±s.e.
Time of
Pupation
No.
Area±s.e.
hours
m m .2
hours
mm?
Control
93 2±0 87
70
0.139 ±.0040
96 1±1 24
48
0 156 ± 003 S
.1% Nipagin ....
152 4 ±3.39
145
0.3 15 ±.01 13
163. 5 ±3. 81
136
0.185± 0044
Starved. . . .
116 4±2 53
57
0 254± 0174
115 0±2 13
78
0 213± 010'
Previous Treatment
Carry-over effect
Fi from control and treated flies
Control 9 X Nipagin <?
110
94
0.203 ±.005 8
110
81
0.181±.0019
Control 9 X Starved d"
110
33
0.203 ±.0163
110
49
0.183 ±.0025
Nipagin 9 X Control c?
116
25
0.229 ±.0270
116
36
0.201±.0t67
Starved 9 X Control cf
116
45
0.281 ±.0203
116
56
0.192±.0060
Nipagin 9 X Nipagin cf
116
40
0.279 ±.02 15
116
44
0.174±.0042
Starved 9 X Nipagin cf
116
55
0.356 ±.0259
116
31
0.224±.0141
Control 9 X Control cT
95
22
0.143 ±.0052
95
19
0.161 ±.0037
GEORGE CHILD
in the male offspring than in the female offspring, since in the latter the
total effect is smaller. The time of development is only approximate,
not having been measured by pupa removal but by simply noting the
time when about half the larvae had pupated. It is quite evident that
in all of the matings the wing areas are greater when affected flies of
either sex are used as parents. When treated females are used as
parents the difference is greater than when males are used. Treated
males and females as parents have offspring with greater wing areas than
those obtained when only one treated parent is used. Starvation of the
parents seems to produce a greater effect in the offspring than nipagin
treatment.
DISCUSSION
It is well known that with increasing temperature there is an increase
in the wing size of vestigial. A sharp increase is not obtained, however,
until very high temperatures are reached. It is generally accepted that
temperature produces its effect by affecting differentially the rate or
duration of the " vestigial reaction " as compared with the rate or dura-
tion of other developmental processes. By vestigial reaction we mean
the developmental reaction or reactions in the vestigial fly which differ
in rate or duration from the reactions in their isogenic wild type.
In the experiments with nipagin there is little reason to suspect that
the change in wing size is due to a direct effect of nipagin on the ves-
tigial reaction. The evidence, moreover, indicates that nipagin produces
its effect by increasing the time of development. The temperature-
effective period of the vestigial reaction is known to occur during a
portion of the larval development. Thus, by increasing the larval period
at constant temperature an effect on the vestigial wings will be produced
if the duration of the vestigial reaction as compared with the rest of
development is differentially affected. It appears from these results
that such is the case.
In the first experiments, using varying concentrations of nipagin, it
appeared that this chemical increased the time of development by de-
creasing the food supply. The yeast did not grow very well in the
treated food although all bottles started with equal amounts of dead
brewer's yeast and live yeast. The starvation experiments showed that
this was the case. There was a definite increase in the time of develop-
ment in wing area under both types of environmental conditions.
As stated previously, the exact relation between time of develop-
ment and wing area is obscured because of the decrease in the size of
the fly with increasing time of development. Under normal conditions
the larvae begin to pupate at 82 hours and the last larvae pupate at 114
TIME OF DEVELOPMENT AND WING SIZE VESTIGIAL 439
hours in these experiments. This variation is great because of the four-
hour egg-laying periods but with even shorter egg-laying periods a
spread of 18-24 hours is obtained (Powsner, 1935; Child, 1935).
This variation is a direct corollary to the nature of development
which as Wright (1934) points out is the result of a large number of
physical and chemical reactions, the rates and durations of which are
determined by the history of the organism prior to the stage in question,
correlative reactions within the organism, external environmental fac-
tors, actions of the genes within each cell, etc. In the highly hetero-
geneous systems of a developing larva these reactions will not go on
at exactly the same rates in all organisms and there will of necessity
be a spread in time of development as well as wing area under normal
conditions but no correlation betiveen these measurements. However,
when an additional factor, lack of food, is superimposed upon this nor-
mal variation a new set of conditions prevails. The duration of the
larval (feeding stage) period is lengthened, various reactions may pro-
duce minimal or even subminimal concentrations for further develop-
ment and development will become somewhat disorganized. In other
words, there will be a differential effect on the rates and durations of
many embryological processes resulting in a modified phenotype. Un-
der such conditions there will be a definite correlation between time of
development and wing area.
With this general hypothesis in mind it is possible to postulate a
number of mechanisms to account for the increased wing size. A
simple scheme would allow the wing formation reactions to proceed at
their normal rate but the developmental reactions which normally paral-
lel them are slowed down, especially those reactions which determine
the time at which the wing development stops. This would permit of
an increased wing area. With further starvation even the wing reac-
tions are slowed down or produce subminimal concentrations and the
size of the wing decreases. This outline is, of course, very general and
is not the only one which can be postulated. It merely illustrates how
the general theory can be utilized.
Carry-over Effect
The carry-over effect experiments were unfortunately not extended
beyond the first generation. They show, however, that there is a defi-
nite effect on the offspring of parents raised under poor food condi-
tions — a sort of dauermodification (Jollos, 1934). It is well known
that starved flies lay smaller eggs than normal ones. Powsner (1935)
found that eggs laid by flies raised on poor food had a longer develop-
mental period than eggs laid by flies raised on good food. If this delay
440 GEORGE CHILD
in development were the only factor involved a definite carry-over effect
should be expected on the maternal side. In these experiments, how-
ever, there was also a paternal effect. To account for this result one
must assume an effect of starvation on developing sperm. This may
concern the small amount of cytoplasm carried by the sperm or perhaps
a direct effect on its genie material.
The recent series of investigations at Columbia University by Ritten-
berg, Schoenheimer, Clarke and others in which deuterium, isotopes of
nitrogen, and other elements were used to follow intermediary metabo-
lism may bear on this problem. These workers have shown that many
of the organic substances in protoplasm, even proteins, are not in a
static condition. The " living proteins " are constantly interacting with
their environment and may exchange their hydrogen for deuterium,
and even nitrogen for one of its isotopes. Thus the composition and
behavior of protoplasm is directly modified by the composition of its
environment. Should the chromosomes or the genes behave in this
kinetic manner of extracting substrates from the cytoplasm and releas-
ing equivalent substances in exchange, we would have a mechanism for
the production of these starvation effects and other dauermodifications,
production of immunity, even differentiation during ontogeny. It is
necessary, of course, to assume that the cytoplasm of the treated flies
differs from that of normal cytoplasm. In this manner a modified cyto-
plasm may produce a change in the chromosomes. It is also of interest
to note that if this is the case, we have a mechanism for an " inheritance
of acquired characters," not in the old sense of the phrase but on a
molecular level. This would allow the environment to produce " ge-
netic changes " which need not be permanent. These " mutations " could
return to normal in one or more generations. Plough and Ives (1935)
found that variations continued to appear in generations later than those
actually treated with a high temperature of 36.5° for 24 hours. These
variations decreased in number in subsequent generations.
These experiments are to be continued for a number of generations.
The original vestigial stock used has been discontinued in this laboratory
and another isogenic stock is being prepared.
SUMMARY
The time of development of an isogenic vestigial stock of D. mclano-
gastcr was increased by two methods: (1) by adding nipagin (ethyl
parahydroxy benzoate) to the food, and (2) by adding only very little
yeast to the food. Both methods are essentially the same in that the
developing larvae are under starvation conditions. With increasing
TIME OF DEVELOPMENT AND WING SIZE VESTIGIAL 441
concentration 0.05, 0.1, 0.2 and 0.4 per cent, there was an increase in
the time of development and increase in the size of the wings, males
showing a greater effect than females. The large wings resembled those
of other vestigial alleles raised under normal conditions.
In another series of experiments 0.1 per cent nipagin and starvation
were used. The larvae were removed from the culture as they pupated,
to determine the relation between time of pupation and wing size. The
first flies to pupate did not differ significantly in wing size from con-
trols at that temperature. With increasing time of development there
was an increase in wing size. Larvae which were very much delayed,
however, developed into small flies with small wings. These wings,
although small, were more differentiated and larger than the control
wings.
The " carry-over " effect was studied using normal food. The
treated females and males were mated with control males and females
respectively. Treated males were also mated with treated females. The
wings of flies from the latter mating showed the greatest carry-over
effect. Treated females by control males resulted in flies having a
significantly larger wing size than flies from the reciprocal cross. These
results indicate that there is a definite effect on the germ cells of flies
raised under starvation conditions, which effect shows itself in the sub-
sequent development of the zygote.
LITERATURE CITED
BRAUN, W., 1939. The role of developmental rates in the production of notched
wing character in D. melanogaster. Proc. Nat. Acad. Sci., 25 : 238-242.
CHILD, G. P., 1935. Phenogenetic studies on scute-1 of Drosophila melanogaster.
II. Genetics, 20 : 127-155.
CHILD, G. P., AND T. ALBERTOWICZ, 1936. The effect of Nipagin on the wing size
of vestigial of D. melanogaster. Rec. Genet. Soc. Amer., 5; and Genetics,
22 : 188, 1937.
GOLDSCHMIDT, R., 1935. Gen und Aussencharakter. III. Biol. Zentralbl, 55:
535-554.
HARNLY, MORRIS H., 1930. A critical temperature for lengthening of the vestigial
wings of D. melanogaster with sexually dimorphic effects. Jour. Expcr.
Zool., 56: 363-368.
HARNLY, MORRIS H., 1932. The temperature-effective period for the lengthening
of the vestigial wings of Drosophila. Proc. Sixth Inter. Congress of
Genetics, 2 : 224-230.
HERSH, A. H., AND ESTHER WARD, 1932. The effect of temperature on wing size
in reciprocal heterozygotes of vestigial in Drosophila melanogaster. Jour.
Expcr. Zool., 61 : 223-244.
JOLLOS, V., 1934. Dauermodifikationen und Mutationen bei Protozoen. Arch. f.
Protist., 83 : 197.
Li, Ju-Cni, AND Yu LIN Tsui, 1936. The development of vestigial wings under
high temperature in Drosophila melanogaster. Genetics, 21 : 248-263.
PLOUGH, H. H., AND P. T. IVES, 1935. Induction of mutations by high temperature
'in Drosophila. Genetics, 20 : 42-69.
442 GEORGE CHILD
POWSNER, L., 1935. The effects of temperature on the durations of the develop-
mental stages of Drosophila melanogaster. Physiol. Zool., 8 : 474—520.
RlTTENBERG, D., A. S. KESTON, R. SCHOENHEIMER, AND G. L. FOSTER, 1938.
Deuterium as an indicator in the study of intermediary metabolism. Jour.
Biol. Chem., 125 (1) : 1-12.
STANLEY, WILLARD F., 1928. The temperature coefficient and temperature-effec-
tive period for wing size in Drosophila. Anat. Rec., 41 : 114.
STANLEY, WILLARD F., 1931. The effect of temperature on vestigial wing in Droso-
phila melanogaster, with temperature-effective periods. Physiol. Zool., 4 :
394-408.
STANLEY, WILLARD F., 1935. The effect of temperature upon wing size in Droso-
phila. Jour. Exper. Zool., 69: 459^195.
WRIGHT, SEWALL, 1934. Physiological and evolutionary theories of dominance.
Am. Nat., 68 : 24-53.
THE METHOD OF FEEDING OF TUNICATES
G. E. MAcGINITIE
(From the William G. Kerckhoff Marine Laboratory of the
California Institute of Technology)
INTRODUCTION
This, the second of a series of papers (MacGinitie, 1939) on the
feeding mechanisms of marine invertebrates, deals with the method of
feeding in three tunicates, namely, Ciona intestinalis (Linn.), Ascidia
calif arnica Ritter and Forsyth, and Diplosoma pisoni Ritter and Forsyth.
The first two are simple ascidians and the latter is a colonial form.
Since both simple and colonial forms have been investigated, I feel that
it is fairly safe to state that the method described below is typical of all
ascidians.
Young specimens of Ciona intestinalis and of Ascidia calif ornica,
especially those which have been reared in the laboratory, are quite trans-
parent, and the observations here recorded were made upon animals
which were in no way disturbed while they were carrying on their
natural feeding activities. The same can be said of Diplosoma [>izoni,
as the matrix of the colony is perfectly clear and transparent. All
observations were made upon undisturbed individuals of the colony.
MECHANISM FOR FEEDING
The structures which are strictly connected with the feeding activi-
ties of tunicates are the endostyle with its mucous glands, the peri-
pharyngeal grooves, the dorsal groove, the stigmata, the esophagus, and
the cilia lining all grooves, bars and the inner edges of the stigmata.
The pharynx or branchial basket has been too well described in text-
books of zoology to make it necessary to redescribe it here. A current
of water is maintained through the branchial cavity almost continually,
whether the animal is feeding or not. The only time that the current
is stopped is when the animals are left exposed by the tide or when they
have been disturbed by some outside stimulus, and at such times the oral
aperture and atriopore are usually closed.
The cilia lining the stigmata and the branchial basket may be divided
into two groups, each group having a particular function. Those lining
the stigmata have the function of maintaining the current of water,
while those on the inner surface of the branchial bars and in the endo-
443
444 G. E. MACGINITIE
style, the peripharyngeal grooves and dorsal groove have the function
of moving mucus.
FOOD AND THE METHOD OF FEEDING
The endostyle is richly supplied with mucous glands, and when a
tunicate starts to feed it begins to secrete mucus throughout the length
of the endostyle. This mucus is moved by the cilia of the branchial
bars around the branchial basket in two sheets, one on either side.
When the edges of the mucous sheets arrive at the dorsal groove, they
are taken up by it and formed into a thread, and this string is passed
posteriorly along the dorsal groove to the esophagus. The function of
the peripharyngeal grooves is to hold and move the oral ends of the two
mucous sheets.
The water entering the branchial basket through the oral funnel
passes into the atrial cavity through the stigmata in all directions with
the exception of the region of the endostyle and dorsal groove, and when
the animal is feeding such water must also pass through the sheet of
mucus which covers the interior of the basket. This mucus intercepts
and entangles all solid material entering with the v/ater, and such
material comprises the food of tunicates. On rocky shores it consists
almost entirely of plankton, often greatly enriched by algal spores from
seaweeds. Within the estuaries it consists largely of material in sus-
pension, mainly stirred-up detritus from the shores and bottom. Dur-
ing the summer season in Southern California this detritus in suspension
is usually enriched by one or more species of dinoflagellates.
While a tunicate is feeding mucus is constantly being secreted, and
the mucous sheets covering the inner walls of the branchial basket move
continuously from the endostyle toward the dorsal groove. Hence,
while the tunicate is feeding, the food-laden thread of mucus enters the
esophagus in an unbroken string. As it enters the stomach this mucous
string is folded back and forth and remains intact for some time. It is
only that portion near the pyloric valve that coalesces and becomes semi-
liquid as it passes into the intestine.
Although the cilia of the stigmata and branchial basket beat almost
continuously, the mucous sheets are formed discontinuously. Upon the
least disturbance the animals will cut off the secretion at the endostyle,
and the remnants of the mucous sheets will continue to pass around to
the dorsal side until the ends reach the dorsal groove. From then on
until the animal begins to feed again the branchial basket is practically
free of mucus. When a tunicate is not feeding, small particles may be
seen to pass readily through the stigmata into the atrium and out with
the atrial current.
FEEDING METHOD OF TUNICATES 445
If material which is foreign to the usual run of food material is
introduced into the current of water entering the oral funnel, feeding
will cease at once, and the undesirable material will be quickly forced
from the branchial basket by a quick contraction of the body wall. The
current will be renewed immediately, and, if no further undesirable ma-
terial is taken in, feeding will soon be resumed. If the stimulus from
the introduced material is rather strong the animal will cease feeding
and will forcibly eject what water is in the branchial basket and atrium,
and will remain closed for a considerable length of time, depending
upon the strength of the stimulus.
In tunicates there is a ring of tentacles which interlace across the
oral funnel which prevents the entrance of large particles. Such large
particles as do find their way into the branchial basket are not incor-
porated in the mucus, but are in some way dropped from it into the
branchial basket, and at intervals are forcibly ejected from the oral
funnel by a sudden contraction of the body wall of the tunicate.
It is characteristic of animals which use mucus to entrap their food
that they are able to drop from such mucus at least a portion of the
undesirable material which is entrapped. Just how this is accomplished
is not at present clear. It may be that the cilia which move the mucus
can, by pressing outward through the mucus cause such particles to drop
out. Many animals have specialized regions where the cilia perform
this function. In the tunicates it is the cilia bordering the dorsal groove,
in the pelecypods (future paper) it is the cilia of the lower edge of the
gills and those of the labial palps, and in the echiuroid Urechis it is the
outer cilia bordering the proboscis. In such regions the cilia are usually
considerably larger than elsewhere.
When large particles strike the tentacles of the oral funnel they are
usually blown away by a quick contraction of the mantle wall with little
cessation of the feeding current. As most single tunicates hang verti-
cally with the osteum downward one ejection movement serves to re-
move the large object. But, because of the separate action of indi-
viduals of a colony, a particle upon the surface of a colonial form which
is fairly level may be bounced over the surface for some time before it is
carried away by currents or is rolled over the edge. Since there is a
constant current out of the atriopore (which in colonial forms may be
common to several individuals), no particles find their way into the
atrium and no tentacles are necessary, for if the current is stopped the
atriopore closes.
As has been stated above, the cilia beat almost continually, and nor-
mally when the tunicate is not feeding most of the solid particles pass
through the stigmata and out the atriopore. However, even when the
'
446 G. E. MACGINITIE
branchial basket is not lined with the mucous sheets, some of the solid
particles may find lodgment upon the ciliary tracts lining the branchial
basket (particularly those of the endostyle, peripharyngeal grooves and
dorsal groove), and will follow more or less the definite tracts. This is
especially true of specimens that are handled or cut open, since they may
secrete mucus along these grooves, whereas normally they would not do
so. The mucus which carries such particles as are transported along
these ciliary tracts may enter the esophagus or may be dropped into the
branchial basket and be ejected through the oral funnel. The more or
less abnormal performance just described has led to the erroneous ideas
found in textbooks about the feeding of tunicates.
CILIARY ACTION
The ciliary action of Ascidia calif ornica was studied in detail. The
oral aperture, the atriopore, the cilia of the basket, and the cilia of the
stigmata may all function independently of each other or they may all
function together. The cilia of the stigmata may be stopped without
stopping those of the basket. When the cilia of the stigmata cease
vibrating they lie down against the edges of the openings, leaving the
stigmata wide open. However, when the animal is contracted the edges
of the stigmata are approximated and the openings closed. At such
times, of course, the cilia are still and lie flat against the sides of the
openings.
After the cilia have been stopped they resume their beating by start-
ing to vibrate in a small circle at the center of the stigmatal opening, and
this ring spreads towards either end of the opening until all are again
beating. The beating cilia surrounding a stigmata remind one of an
elongated wheel organ of a rotifer or a veliger larva. In the ascidian
investigated the apparent movement was in an anti-clockwise direction
as viewed from the outside.
There is no doubt that the cilia of the branchial bars, ridges and
grooves actually hold and move mucus. The cilia seem partially to
enter the sheet of mucus and force it forward. During part of the beat
the cilia are more or less hooked into the mucus and this serves to hold
it so that the cilia following are able to penetrate and in turn do their
share of pushing and holding. This action of the cilia is further evi-
denced by the fact that the mucous sheet which is present on the inside
of the basket when the animal is feeding has in it waves which cor-
respond to the wave motion of the cilia. These waves in the mucus
appear when water heavily laden with food is introduced into the oral
funnel. As the food material collects in the mucous sheet it sometimes
FEEDING METHOD OF TUNICATES 447
appears in streaks which are more accentuated as the mucus nears the
dorsal groove.
SUMMARY
1. The feeding method of dona intestinalis and Ascidia calif 'arnica
(simple ascidians), and of Diplosoma pizonl (a colonial form) was
investigated.
2. Tunicates feed by straining the solid material from a current of
water as it passes through a thin film of mucus lining the branchial
basket.
3. The mucus is constantly secreted at the endostyle and is con-
tinually moved to the dorsal groove in two sheets which line the interior
of the basket. The dorsal groove forms the edges of the food-laden
sheets into a thread which is passed posteriorly to the esophagus and
enters it in an unbroken string. The peripharyngeal grooves serve
to hold the anterior ends of the mucous sheets and move them around
to the dorsal groove.
4. When a tunicate is not feeding, the inside of the branchial basket
is not lined with mucus, and the solid materials pass out with the atrial
current.
5. Some sorting is carried out by the cilia of the dorsal ridges. The
cilia which line the openings of the stigmata, and whose vibration
creates the current of water passing through the basket, may be stopped
without stopping the cilia lining the basket or without closure of the
oral aperture and atriopore.
6. After the cilia lining the stigmata have been stopped they com-
mence to beat in what appears to be a ring at the center of the opening.
The cilia of one side of a stigmata are in perfect synchronism with those
of the opposite side of the opening, and, by the continual inclusion of
other cilia, all finally vibrate and resemble somewhat an elongated wheel
organ of a rotifer.
LITERATURE CITED
MAC&NITIE, G. E., 1939. The method of feeding of Chaetopterus. Biol. Bull.,
77: 115.
TEST SECRETION IN TWO SPECIES OF FOLLICULINA
VIRGINIA C. DEWEY
{From the Arnold Biological Laboratory, Brozm University and The Marine
Biological Laboratory, Woods Hole, Massachusetts)
INTRODUCTION
The fact that Folliculina passes through a free-swimming stage at
some point in its life cycle was recognized by Wright (1859) and by
Claparede and Lachmann (1858-61). The former author observed
the transformation of the free-swimmer of F. producta into the adult
form, but did not discover the mode of origin of the " larva " nor any
details of the metamorphosis. For this reason these observations were
later questioned by Stein (1867), but they are completely confirmed by
more recent work. That the larvae are formed as the result of cell
division was pointed out by Mobius (1887), but the presence of a larva
and an adult lying side by side in a single test he interpreted as a case
of longitudinal division. In his figures he even shows an " umbilical
cord " projecting laterally from the larva. Folliculinas do not, however,
depart from the general rule of transverse division in ciliates as was
pointed out by Sahrhage (1917). Andrews (1920) has shown that
free-swimming larvae may also result from the dedifferentiation of an
adult form.
Test secretion and metamorphosis are described by Penard (1919)
for F. boltoni, a freshwater species. Too few details are given to de-
cide whether or not this species passes through the same stages as are
described for F. product a by Andrews (1923) or for F. simplex (pre-
viously called F. ampulla) by Faure-Fremiet (1932). Observations on
F. aculeata and F. elegans indicate that the process of test formation is
somewhat different in these species. During the summer of 1936 at
Woods Hole, while examining fresh preparations for specimens of
Folliculina, a form was discovered of which no description could be
found. The posterior part of the body resembled that of a Folliculina
seen on the slide, but the anterior portion was drawn out into one long,
slender, flexible process bearing membranelles only at the end. Circum-
stantial evidence pointed to the conclusion that these forms represented
a stage in the life history of Folliculina. Definite proof of this theory
was lacking, since the larvae did not remain in good condition long
enough for complete transformation to take place. This stage has evi-
448
TEST SECRETION IN FOLLICULINA 449
dently been noted by Faure-Fremiet (1936), but his descriptions are not
detailed. In order to obtain more complete evidence concerning the
significance of this stage in the life cycle, observations were resumed in
1939. A complete life history can now be given placing this stage in
its proper sequence.
The classification of the members of the family Folliculinidae is still
in a somewhat unsatisfactory state in spite of the fact that a number of
investigators have given the matter a great deal of attention. The sep-
aration of the genus Folliculina into a large number of species on the
basis of characteristics which are subject to considerable variation seems
to be the rule. For this reason the classification given by Faure-Fremiet
(1936), in which several species which have a number of common
characteristics are combined into a single species, is to be preferred.
Two species, as described by this author, F. aculeata and F. elegans,
seem to prevail at Woods Hole. The two are alike in many ways ; the
chief differences are in size, the presence or absence of pointed tips on
the peristomeal lobes and in the pigmentation of the animal and of the
test. Since all of these traits are subject to variation, so that one species
may resemble the other very closely, it was difficult to determine which
species was under observation. Certain organisms, however, presented
all the criteria of one species or the other. Since the greater number
of individuals observed were of a paler color and possessed the pointed
peristomeal lobes characteristic of F. aculeata, this species is figured.
The only point in which they differed from the descriptions of Faure-
Fremiet was in the color of the test which was often colorless or faintly
blue. Both species have an ovoid nucleus, but, as in F. bottom, it is
often notched or bi-lobed.
METHODS
The organisms were collected by placing glass slides in crystallizing
dishes containing quantities of the hydroid Tubularia and leaving them
for several days in running sea water. The slides were then removed
and placed in Petri dishes containing sea water. Observations were
made with a binocular dissecting microscope, with the 16 mm. objective
of a compound microscope or with a water immersion lens (40 X)- In
order to maintain the organisms in good condition over periods of sev-
eral hours, a stream of fresh sea water was run into the dish while on
the stage of the microscope. The overflow was carried off by means of
an inverted siphon. Temperature readings were regularly recorded and
during the time that the observations were being made the temperature
of the sea water varied from 21° C. to 24° C.
450 VIRGINIA C. DEWEY
OBSERVATIONS
The complete life history of a larva resulting from cell division, from
the time of departure from the test containing the sister cell until the
adult form was attained, was followed for a number of individuals. In
some cases free-swimming larvae were found and their subsequent his-
tory observed. In a few cases it was found that adult forms dedifferen-
tiated into the larval form, left the test and settled down to secrete a
new test.
After cell division (Fig. 1) the anterior individual, which will even-
tually leave the test as a free-swimming larva, remains for from 30 to
60 minutes in the test contracting and extending beside its sister cell,
which remains attached and which has already begun the metamorphosis
into the adult form. At each extension of the larva the anterior end is
projected farther and farther out of the neck of the test until finally the
whole organism is free. The swimming stage (Fig. 2) may last for
from 15 to 90 minutes, during which time the larva swims slowly along
the substratum or more rapidly near the surface of the water in the dish.
At intervals it pauses to contract and extend itself at one spot and then
swims on. Just before the larva settles down to secrete its test it may
be seen to repeat this process of contraction and elongation a number of
times in a single spot, changing direction each time it contracts until it
has described a complete circle at least once and sometimes several times.
It then flattens itself out on the substratum and its outlines become very
irregular (Fig. 3). Occasionally larvae were seen secreting tests at-
tached to the surface film of the water as described by Wright in 1859.
This flattened stage lasts for several minutes, after which time it is dif-
ficult to dislodge the organism from the slide even with a fairly strong
stream of water. It is at this time that a broad layer of cement sub-
stance is being secreted which will serve to attach the test to the sub-
stratum. During this stage and for the entire time that the test is being
secreted the anterior end of the animal is raised above the rest of the
body.
Having attached itself firmly, the animal now assumes a more regular
ovoid form, becoming thicker and rounder (Fig. 4). Soon granules
may be seen around the body among the cilia. These collect all around
the periphery and harden to form the bottle-shaped part of the test.
FIGS. 1-4. Folliculina aculeata. X 300.
FIG. Late division stage. Macronucleus not yet completely divided.
FIG. 2. Free-swimming larva.
FIG. 3. Larva flattened out on the substratum during the process of cement
secretion.
IMC. 4. Beginning of the secretion of the test.
m .
;
FIGS. 5-8. Folliculina aculeata. X 300.
FIG. 5. Side view of larva with long proboscis at the beginning of secretion
of the neck of the test.
FIG. 6. Same as Fig. 5. Neck secretion nearly completed.
FIG. 7. Same as Fig. 5. Top view.
FIG. 8. Beginning of secretion of the collar of the test.
TEST SECRETION IN FOLLICULINA
453
The upraised anterior portion of the body secretes the base of the neck
of the test. As the formation of the body of the test is completed, the
anterior end of the animal elongates to form a proboscis-like projection
&>*&>Yti I ; / '
;..:.>,AM^''
FIG. 9. Adult Folliculina aculeata. X 300.
of about the same length as the body. Upon the end of this proboscis
the membranelles are borne (Figs. 5, 7). The proboscis is seen to
sweep about in circles and while it is present the neck of the test becomes
454 VIRGINIA C. DEWEY
longer and the spiral rings are laid down (Fig. 6). This stage is ob-
served about 50 to 75 minutes after the larva has become attached and
lasts about 70 to 100 minutes.
It is difficult to state whether or not this stage has previously been
described. Penard (1919) states that Lachmann observed the fixation
of a larva of F. elegans " after which ... at the anterior end a mem-
branous extension appeared, which I should be disposed to consider as
moribund phenomena" (p. 312). Upon referring to the original paper
this stage is described as presenting an " epanouissement membraneux "
(p. 219). This seems clearly to refer to the next stage to be described
in this cycle. It is improbable that this is an abnormality in the develop-
ment, since this stage was observed in every larva whose development
was followed. In the metamorphosis of F. producta and F. simplex
this stage is apparently lacking (Andrews, 1923; Faure-Fremiet, 1932).
The figures of Faure-Fremiet (1936) cannot be definitely identified
with this stage.
Upon completion of the tubular part of the neck of the test, the
proboscis is retracted and a double fold of cytoplasm is extended around
the opening to form a collar (Fig. 8). During the 55 to 115 minutes
that this stage lasts the collar of the test is secreted. When this has been
finished the cytoplasm frees itself from the rim of the collar and the
ragged edges are withdrawn into the test. In this contracted state the
animal remains for from 3 to 5 hours. The changes occurring during
this time are described in detail by both Andrews and Faure-Fremiet
and seem to be essentially similar in all forms. At the end of this
period the animal protrudes from the test the long peristomeal lobes
characteristic of the adult form (Fig. 9) The entire process of test
secretion and morphogenesis may take from 4% to 8% hours.
A single small larva of a yellow color, probably F. viridis, was dis-
covered on the slide in the early stages of test secretion and was fol-
lowed through to the adult stage. The development followed that of
F. aculcata and F. elegans, although the proboscis was relatively shorter
than in these species and the peristomeal lobes of the adult were smaller
and more rounded.
I acknowledge gratefully the advice and encouragement of Dr.
George W. Kidder.
•
SUMMARY
Test secretion and metamorphosis in Folliculina aculeata and
Folliculina elegans are described.
2. These processes may be divided into the following six stages :
TEST SECRETION IN FOLLICULINA 455
a. The free-swimming stage.
b. The stage of cement secretion.
c. The secretion of the body of the test.
d. The secretion of the neck of the test, during which process the animal
puts forth a long, proboscis-like projection. This stage is, as far
as is known, peculiar to F. aculeata, F. elegans and F. viridis and
has not been described before in detail.
e. The secretion of the collar of the test.
/. Formation of the peristomeal lobes characteristic of the adult.
LITERATURE CITED
ANDREWS, E. A., 1920. Alternate phases in Folliculina. Biol. Bull., 39: 67-87.
ANDREWS, E. A., 1923. Folliculina : Case making, anatomy and transformation.
Jour. Morph., 38 : 207-278.
CLAPAREDE, E., AND J. LACHMANN, 1858. fetudes sur les Infusoires et les Rhizo-
podes.. Imprimerie Vaney, Geneve.
FAURE-FREMIET, E., 1932. Division et morphogenese chez Folliculina ampulla
O. F. Miiller. Bull. Biol. France et Bclg., 66: 77-110.
FAURE-FREMIET, E., 1936. La famille des Folliculinidae (Infusoria Heterotricha).
Mem. Mus. Roy. d'Hist. Nat. Belg., Ser. 2, Fasc. 3, pp. 1129-1175.
MOBIUS, K., 1887. Das Flaschentierchen, Folliculina ampulla. Abhandl. d.
naturwiss. Vereins in Hamburg, 10 : 3-14.
PENARD, E., 1919. On Folliculina boltoni (S. Kent). Jour. Roy. Mic. Soc., pp.
305-319.
SAHRHAGE, H., 1917. Uber die Organisation und den Teilungsvorgang des Flas-
chentierchens (Folliculina ampulla). Arch. f. Protist., 37: 139-174.
STEIN, F., 1867. Der Organismus der Infusionsthiere. II Abth., Wilhelm Engel-
mann, Leipzig.
WRIGHT, S., 1859. Description of new protozoa. Edin. New Phil. Jour., N. S.,
10 : 97-100.
INDEX
A BELL, RICHARD G. Quantitative
studies of the rate of passage of pro-
tein and other nitrogenous sub-
stances through the walls of growing
and of differentiated mammalian
blood capillaries (abstract), 320.
ABRAMOWITZ, A. A. A new method for
the assay of intermedin (abstract),
327.
— , R. K. AND A. A. Moulting and
viability after removal of the eye-
stalks in Uca pugilator (abstract),
326.
Activation, Cumingia and Arbacia eggs,
by bivalent cations (abstract), 333.
ADDISON, WILLIAM H. F. On the his-
tology of the mammalian carotid
sinus (abstract), 314.
"Agglutination" with spermatozoa of
Chiton tuberculatus, 157.
Alcohols, permeability-decreasing effect,
on human erythrocyte (abstract),
320.
Alkaline earths, stabilizing action on
crab nerve membranes (abstract),
309.
ALSUP, FRED W. Photodynamic action
in the eggs of Nereis limbata (ab-
stract), 324.
Amblystoma punctatum embryo, differ-
entiation of isolated rudiments (ab-
stract), 299.
Amoeba, growth and structure, as af-
fected by kind of food, 391.
— — , vacuole systems (abstract), 334.
ANDERSON, PRISCILLA L. See Goodrich
and Anderson, 184.
ANDERSON, RUBERT S. The x-ray effect
on the cleavage time of Arbacia
eggs in the absence of oxygen (ab-
stract), 325.
Androgenetic development, egg of Rana
pipiens, 233.
ANGERER, C. A. The effect of electric
current on the relative viscosity of
sea-urchin egg protoplasm, 399.
Animal jellies, chemical and mechanical
properties (abstract), 331.
Aphids, female, germaria in ovariole
differentiation, 135.
Arbacia egg, activation by bivalent ca-
tions (abstract), 333.
— , cleavage, effects of Roentgen
radiation on related phenomena,
331.
- cortex, response to chemical
and physical agents, in absence of
oxygen (abstract), 335.
fertilized, oxygen consump-
tion and cell division, in presence of
respiratory inhibitors (abstract),
318.
, intra-cellular distribution of
reducing systems (abstract), 328.
pigment granules (abstract),
310.
— , hermaphroditic, 74.
, method of determining sex and
producing twins, triplets and quad-
ruplets (abstract), 312.
punctulata egg, cleavage delay
after irradiation while closely packed
in capillary tubes (abstract), 324.
Arbacia punctulata egg, first division,
effects of colchicine (abstract), 328.
— , unfertilized, fatty acid
compounds in (abstract), 323.
ARMSTRONG, FLORENCE, MARY MAX-
FIELD, C. LADD PROSSER AND GOR-
DON SCHOEPFLE. Analysis of the
electrical discharge from the cardiac
ganglion of Limulus (abstract),
327.
Ascidia, embryonic induction, 216.
T3ACTERIA, in fouling of submerged
surfaces (abstract), 302.
BAILEY, KENNETH. Crystallization of
myogen from skeletal muscle (ab-
stract), 303.
, . Crystalline myogen (ab-
stract), 322.
BALL, ERIC G., AND BETTINA MEYERHOF.
The occurrence of cytochrome and
other hemochromogens in certain
marine forms (abstract), 321.
457
458
INDEX
BALLENTINE, ROBERT. The intracellu-
lar distribution of reducing systems
in the Arbacia egg (abstract), 328.
EARTH, L. G. Neural differentiation
without organizer (abstract), 299.
BEADLE, G. W., EDWARD L. TATUM AND
C. W. CLANCY. Development of
eye colors in Drosophila: production
of v+ hormone by fat bodies, 407.
BEADLE, G. W. See Tatum and Beadle,
415.
BEAMS, H. W. See Evans and Beams
(abstract), 331.
— , — . — ., AND T. C. EVANS. Some
effects of colchicine upon the first
division of the eggs of Arbacia
punctulata (abstract), 328.
BlSSONNETTE, THOMAS H., AND ALBERT
CSECH. Modified sexual photoperi-
odicity in cotton-tail rabbits, 364.
Blood capillaries, mammalian, passage of
protein and other nitrogenous sub-
stances through walls (abstract),
320.
— , and respiratory ability, freshwater
fish (abstract), 300.
BOTSFORD, E. FRANCES. Temporal sum-
mation in neuromuscular responses
of the earthworm, Lumbricus ter-
restris (abstract), 328.
Brachydanio rerio, karyokinesis during
cleavage, 79.
BRAGG, ARTHUR N. Observations upon
amphibian deutoplasm and its rela-
tion to embryonic and early larval
development, 268.
VON BRAND, THEODOR. Chemical and
histochemical observations on Ma-
cracanthorhynchus hirudinaceus
(abstract), 303.
— , - — , NORRIS W. RAKESTRAW
AND CHARLES E. RENN. Further
experiments on the decomposition
and regeneration of nitrogenous or-
ganic matter in sea water, 285.
Bresslaua, pH reactions during feeding
(abstract), 303.
BROWN, F. A., JR. The source of chro-
matophorotropic hormones in crus-
tacean eyestalks (abstract), 329.
— , — . — ., — ., AND ONA CUNNING-
HAM. Influence of the sinusgland of
crustaceans on normal viability and
ecdysis, 104.
— , — . — ., — ., AND H. E. EDERSTROM.
On the control of the dark chromato-
phores of Crago telson and uropods
(abstract), 330.
, — . — ., — ., AND H. H. SCUDAMORE.
Comparative effects of sinusgland
extracts of different crustaceans on
two chromatophore types (abstract),
329.
BROWN, MORDEN G. The blocking of
excystment reactions of Colpoda
duodenaria by absence of oxygen,
382.
BUCK, JOHN B. Micromanipulation of
salivary gland chromosomes (ab-
stract), 330.
BURGER, J. WENDELL. Some experi-
ments on the relation of the external
environment to the spermatogenetic
cycle of Fundulus heteroclitus (L.),
96.
BUTCHER, EARL O. The illumination of
the eye necessary for different
melanophoric responses of Fundulus
heteroclitus, 258.
, R. M., AND A. V. HUNNINEN.
Studies on the life history of Spelo-
trema Nicolli (abstract), 309.
Calcium chloride, response of frog stri-
ated muscle (abstract), 332.
CAMPBELL, J. B. S., AND M. H. JACOBS.
Studies on the permeability-decreas-
ing effect of alcohols and pharma-
cologically related compounds on
the human erythrocyte (abstract),
320.
Carassius auratus, differential effect of
radiations on Mendelian pheno-
types, 192.
— , variations of color pattern in
hybrids, 184.
Cell cleavage, as affected by leukotaxine
(abstract), 335.
, living, in action (abstract), 308.
permeability, increased, mechanism
(abstract), 318.
to water, effect of leukotaxine
(abstract), 335.
processes, effects of hydrostatic
pressure (abstract), 305.
Centropages typicus, reactions to light
and gravity, 200.
Chaetopterus eggs, water permeability
(abstract), 317.
— , method of feeding, 115.
CHASE, AURIN M. Color changes in
luciferin solutions (abstract), 323.
INDEX
459
Chemical and mechanical properties of
two animal jellies (abstract), 331.
CHILD, GEORGE P. The effect of in-
creasing time of development at
constant temperature on the wing
size of vestigial of Drosophila
melanogaster, 432.
Chilomonas paramecium, respiration (ab-
stract), 298.
— , temperature and starch and
fat (abstract), 298.
Cholinesterase, in invertebrates (ab-
stract), 321.
Chromatophores, dark, control of, in
Crago telson and uropods (abstract),
330.
Chromatophore types, comparative ef-
fects of sinusgland extracts of differ-
ent crustaceans (abstract), 329.
Chromatophoric hormones, source in
crustacean eyestalks (abstract), 329.
CLAFF, C. LLOYD AND G. W. KIDDER.
pH reactions during feeding in the
ciliate Bresslaua (abstract), 303.
CLANCY, C. W. See Beadle, Tatum and
Clancy, 407.
Cleavage delay in Arbacia eggs after
irradiation while closely packed in
capillary tubes (abstract), 324.
Cleavage, effect of leukotaxine (ab-
stract), 335.
— , effects of Roentgen radiation on
related phenomena, Arbacia eggs
(abstract), 331.
, first, Arbacia eggs, effects of col-
chicine (abstract), 328.
, karyokinesis during, zebra fish, 79.
- time of Arbacia eggs in absence
of oxygen, x-ray effect (abstract),
325.
CLOWES, G. H. A. See Krahl, Keltch
and Clowes (abstract), 318.
COHEN, IRVING. Cleavage delay in
Arbacia punctulata eggs irradiated
while closely packed in capillary
tubes (abstract), 324.
Colchicine, effects on first division of
Arbacia eggs (abstract), 328.
Color patterns, variations in goldfish
hybrids, 184.
responses of catfishes with single
eyes (abstract), 312.
Colpidium campylum, growth as affected
by biologically conditioned medium
(abstract), 297.
Colpoda duodenaria, oxygen lack and
blocking of excystment reactions,
382.
Contraction, frequency, conditions gov-
erning, heart of Venus mercenaria
(abstract), 315.
— , muscular, Clark's theory (ab-
stract), 314.
COSTELLO, D. P., AND R. A. YOUNG.
The mechanism of membrane eleva-
tion in the egg of Nereis (abstract),
311.
Crago telson and uropods, control of
dark chromatophores (abstract),
330.
CRAWFORD, JOHN D. See Navez and
Crawford (abstract), 315.
Crayfish, retinal pigment and theory of
asymmetry of flicker-response con-
tour, 126.
, retinal pigment migration, effect of
eye-stalk extracts, 119.
CROZIER, W. J., AND ERNST WOLF. The
flicker-response contour for the cray-
fish. II. Retinal pigment and the
theory of the asymmetry of the
curve, 126.
CSECH, ALBERT. See Bissonnette and
Csech, 364.
Cumingia eggs, activation by bivalent
cations (abstract), 333.
CUNNINGHAM, ONA. See Brown and
Cunningham, 104.
Cytochrome, occurrence of, and other
hemochromogens in certain marine
forms (abstract), 321.
Cytoplasm, does action of x-rays on
nucleus depend on? (abstract), 326.
•QACTYLOMETRA quinquecirrha, L.
Agassiz, life cycle, in Chesapeake
Bay, 368.
Dark-adaptation and reversal of photo-
tropic sign in Dineutes, 354.
Decomposition, nitrogenous organic mat-
ter in sea water, 285.
Deutoplasm, amphibian, and embryonic
and early larval development, 268.
DEWEY, VIRGINIA C. Test secretion in
two species of Folliculina, 448.
Diet, effect on eye color development,
Drosophila, 415.
Differentiation, isolated rudiments of
Amblystoma punctatum embryo
(abstract), 299.
460
INDEX
— , neural, without organizer (ab-
stract), 299.
Dihydroxyestrin, effect of injection into
castrated female frogs (abstract), 338.
Dineutes, dark adaptation and reversal
of phototropic sign, 354.
Dogfish, fetal, absence of epithelial hy-
pophysis, and head and pigmenta-
tion abnormalities, 174.
Drosophila, eye color development, v+
production by fat bodies, 407.
— , eye color, effect of diet, 415.
- melanogaster, wing size of vestigial,
effect of increasing development
time at constant temperature, 432.
DURYEE, WILLIAM R. Does the action
of x-rays on the nucleus depend upon
the cytoplasm? (abstract), 326.
EARTHWORM, temporal summation
in neuromuscular responses (ab-
stract), 328.
Ecdysis, influence of sinusgland of crus-
taceans, 104.
EDERSTROM, H. E. See Brown and
Ederstrom (abstract), 330.
Egg-sea-water-neutralizing substances
from spermatozoa, Echinometra sub-
angularis, 147.
Eggs, mosaic, regulation in (abstract),
308.
Electric current, effect on viscosity of
protoplasm sea urchin egg, 399.
Embryo, androgenetic, Triturus viri-
descens, after cessation of develop-
ment (abstract), 334.
— , development, related to amphibian
deutoplasm, 268.
— , induction in, Ascidia, 216.
Emplectonema kandai, Kato, lumines-
cence, 166.
Endamoeba muris, food habits (ab-
stract), 313.
Erythrocyte, human, permeability-de-
creasing effect of alcohols (abstract),
320.
— , mammalian, factors affecting he-
molysis by rc-butyl alcohol (ab-
stract), 319.
EVANS, T. C. See Beams and Evans
(abstract), 328.
EVANS, T. C. AND H. W. BEAMS. Ef-
fects of Roentgen radiation on cer-
tain phenomena related to cleavage
in Arbacia eggs (Arbacia punctulata)
(abstract), 331.
Excystment reactions, blocked by oxygen
lack in Colpoda duodenaria, 382.
Eye color development, effect of diet on,
Drosophila, 415.
in Drosophila, 407.
illumination, necessary for different
melanophoric responses, Fundulus,
258.
Eye-stalk extracts, action on retinal pig-
ment migration in crayfish, 119.
Eyestalks, removal, moulting and via-
bility after, Uca pugilator (abstract),
326.
pAWCETT, DON WAYNE. Absence
of the epithelial hypophysis in a
fetal dogfish associated with ab-
normalities of the head and of pig-
mentation, 174.
Feeding, method of, in Chaetopterus,
115.
— — , — — , — tunicates, 443.
— , pH reactions during, in ciliate
Bresslaua (abstract), 303.
Fermentation and respiration in higher
plants (abstract), 301.
FERRY, JOHN D. Chemical and me-
chanical properties of two animal
jellies (abstract), 331.
Fertilization, activity-preventing and
egg-sea-water neutralizing sub-
stances from spermatozoa, Echinom-
etra subangularis, 147.
— , "agglutination" phenomenon with
spermatozoa of Chiton tuberculatus,
157.
, fixation of x-ray effect in Arbacia
eggs (abstract), 325.
Flicker-response contour, asymmetry of,
and retinal pigment, crayfish, 126.
Folliculina, test secretion in two species,
448.
Food, growth, and structure in Amoeba,
391.
habits of Endamoeba muris (ab-
stract), 313.
Forty-first report of the Marine Biolog-
ical Laboratory, 1.
Fouling, r&le of bacteria, submerged
surfaces (abstract), 302.
Fundulus embryos, regeneration (ab-
stract), 336.
, eye illumination for different mel-
anophoric responses, 258.
- heteroclitus (L.), spermatogenetic
cycle and external environment, 96.
INDEX
461
-, male sexual cycle and effects of
light and temperature, 92.
—Scomber hybrid, pigment inherit-
ance (abstract), 316.
- scomber hybrid, pigment inheri-
tance, 423.
QANGLION, cardiac, electrical dis-
charge from, Limulus (abstract),
327.
Germaria, in differentiation of ovarioles,
female aphids, 135.
CLICK, DAVID. See Smith and Click
(abstract), 321.
GODDARD, DAVID R. The relation be-
tween fermentation and respiration
in higher plants (The Pasteur Effect)
(abstract), 301.
GOODRICH, H. B., AND PRISCILLA L.
ANDERSON. Variations of color pat-
tern in hybrids of the goldfish,
Carassius auratus, 184.
— , — . — ., AND J. P. TRINKAUS. The
differential effect of radiations on
Mendelian phenotypes of the gold-
fish, Carassius auratus, 192.
GRAHAM, JUDITH E., AND F. J. M.
SICHEL. Response of frog striated
muscle to CaCU (abstract), 332.
Gravity, reactions to, Centropages typ-
icus, 200.
Growth and structure, Amoeba, effect of
kind of food, 391.
, Colpidium, effect of biologically
conditioned medium (abstract), 297.
GUTTMAN, RITA. Stabilizing action of
alkaline earths upon crab nerve
membranes, as manifested in resting
potential measurements (abstract),
309.
IJ ARRIS, D. L. An experimental
study of the pigment granules of the
Arbacia egg (abstract), 310.
HARVEY, ETHEL BROWNE. An her-
maphrodite Arbacia, 74.
, . A method of deter-
mining the sex of Arbacia and a new
method of producing twins, triplets
and quadruplets (abstract), 312.
An artificial nucleus
r
in a non-nucleate half-egg (abstract),
312.
HAYWOOD, CHARLOTTE. The permea-
bility of the toadfish liver to inulin
(abstract), 332.
Heart, as affected by certain drugs, in
ascidian Perophora viridis (ab-
stract), 337.
— , contraction, conditions governing
frequency, Venus mercenaria (ab-
stract), 315.
Hemolysis, rate of, by n-butyl alcohol,
factors affecting, in mammalian
erythrocyte (abstract), 319.
Hemopoietic organs, young albino rats,
quantitative study (abstract), 314.
HENSHAW, P. S. Fixation of x-ray effect
by fertilization in Arbacia eggs
(abstract), 325.
Hermaphrodite Arbacia, 74.
HOLLINGSWORTH, JOSEPHINE. Activa-
tion of Cumingia and Arbacia eggs
by bivalent cations (abstract), 333.
HOPKINS, DWIGHT L. The vacuole sys-
tems of a fresh water limacine
Amoeba (abstract), 334.
Hormones, chromatophorotropic, source
in crustacean eyestalks (abstract),
329.
HUNNINEN, A. V. See Cable and Hun-
ninen (abstract), 309.
HUTCHENS, JOHN. Respiration in Chilo-
monas paramecium (abstract), 298.
, - — , AND M. E. KRAHL. Effect
of increased intracellular pH on the
physiological action of substituted
phenols (abstract), 322.
Hydrostatic pressure, effects on certain
cellular processes (abstract), 305.
Hypophysis, epithelial, absence of, and
abnormalities of head and pigmenta-
tion in fetal dogfish, 174.
INHERITANCE, pigment, Fundulus-
Scomber hybrid, 423.
Intermedin, method for assay (abstract),
327.
Inulin, permeability to, of toadfish liver
(abstract), 332.
Invertebrates, cholinesterase in (ab-
stract), 321.
IRVING, LAURENCE. The relation of
blood to the respiratory ability of
fresh water fish (abstract), 300.
JACOBS, M. H. See Campbell and
Jacobs (abstract), 320.
— , — . — . See Netsky and Jacobs
(abstract), 319.
— , — . — ., AND A. K. PARPART. A
mechanism of increased cell per-
462
INDEX
meability resembling catalysis (ab-
stract), 318.
Jellies, animal, chemical and mechanical
properties (abstract), 331.
JOHNSON, W. H., AND J. E. G. RAYMONT.
The reactions of the planktonic
copepod, Centropages typicus, to
light and gravity, 200.
17" ANDA, SAKYO. The luminescence
of a nemertean, Emplectonema
kandai, Kato, 166.
Karyokinesis during cleavage of zebra
fish, 79.
KATZIN, LEONARD I. The ionic permea-
bility of frog skin as determined
with the aid of radioactive indicators
(abstract), 302.
KAYLOR, CORNELIUS T. Cytological
studies on androgenetic embryos of
Triturus viridescens which have
ceased development (abstract), 334.
— , - — . Experiments on the pro-
duction of haploid salamanders (ab-
stract), 307.
KELTCH, A. K. See Krahl, Keltch and
Clowes (abstract), 318.
KIDDER, G. W. The effect of biolog-
ically conditioned medium upon the
growth of Colpidium campylum
(abstract), 297.
KINDRED, J. E. A quantitative study
of the hemopoietic organs of young
albino rats (abstract), 314.
KITCHING, J. A. The effects of a lack
of oxygen, and of low oxygen ten-
sions, on Paramecium, 339.
- , • — . — . See Moser and Kitching
(abstract), 335.
— , — . — . The effects of lack of
oxygen and of low oxygen tensions,
on the activities of some Protozoa
(abstract), 304.
KRAHL, M. E. See Hutchens and Krahl
(abstract), 322.
KRAHL, M. E., A. K. KELTCH AND G. H.
A. CLOWES. Oxygen consumption
and cell division of fertilized Arbacia
eggs in the presence of respiratory
inhibitors (abstract), 318.
— , effect on cell permeability to water
(abstract), 335.
Light, effects on male sexual cycle,
Fundulus, 92.
, reactions, Centropages typicus,
200.
Limulus, analysis of electrical discharge
from cardiac ganglion (abstract),
327.
LITTLEFORD, ROBERT A. The life cycle
of Dactylometra quinquecirrha, L.
Agassiz in the Chesapeake Bay, 368.
Luciferin solutions, color changes (ab-
stract), 323.
Luminescence of a nemertean, Emplec-
tonema kandai, Kato, 166.
^JACGINITIE, G. E. The method
of feeding of Chaetopterus, 115.
-, — . — . The method of feeding of
, CHESTER A. The signifi-
cance of germaria in differentiation
of ovarioles in female aphids, 135.
Leukotaxine, effect on cell cleavage
(abstract), 335.
tunicates, 443.
Macracanthorhynchus hirudinaceus,
chemical and histochemical observa-
tions (abstract), 303.
Marine Biological Laboratory, forty-first
annual report, 1.
MARSLAND, D. A. Effects of hydro-
static pressure upon certain cellular
processes (abstract), 305.
MARTIN, W. E. Studies on the trema-
todes of Woods Hole. II. The life
cycle of Stephanostomum tenue
(Linton), 65.
MAST, S. O. The relation between kind
of food, growth and structure in
Amoeba, 391.
MATTHEWS, SAMUEL A. The effects of
light and temperature on the male
sexual cycle in Fundulus, 92.
MAXFIELD, MARY. See Armstrong,
Maxfield, Prosser and Schoepfle
(abstract), 327.
Melanophore responses, and eye illu-
mination, Fundulus, 258.
Membrane elevation, mechanism in
Nereis egg (abstract), 311.
MENKIN, VALY. Effect of leukotaxine
on cell cleavage (abstract), 335.
? . Effect of leukotaxine on
cellular permeability to water (ab-
stract), 335.
MEYERHOF, BETTINA. See Ball and
Meyerhof (abstract), 321.
Micromanipulation of salivary gland
chromosomes (abstract), 330.
INDEX
463
MOSER, FLOYD. The differentiation of
isolated rudiments of the Ambly-
stoma punctatum embryo (ab-
stract), 299.
, AND J. A. KlTCHING. Re-
sponse of the Arbacia egg cortex to
chemical and physical agents in the
absence of oxygen (abstract), 335.
Moulting and viability after removal of
eyestalks in Uca pugilator (ab-
stract), 326.
Muscle contraction, Clark's theory (ab-
stract), 314.
, skeletal, crystallization of myogen
(abstract), 303.
— , striated, refractory period in non-
conducted response (abstract), 316.
— , , response to CaCl2 in frog
(abstract), 332.
Myogen, crystalline (abstract), 322.
— , crystallization from skeletal muscle
(abstract), 303.
N^ ABRIT, S. MILTON. Further studies
on regeneration in Fundulus em-
bryos (abstract), 336.
NAVEZ, ALBERT E. Fatty acid com-
pounds in the unfertilized egg of
Arbacia punctulata (abstract), 323.
— , — — ., AND JOHN D. CRAWFORD.
Conditions governing the frequency
of contraction of the heart of Venus
mercenaria (abstract), 315.
Nereis egg, membrane elevation, mechan-
ism of (abstract), 311.
- limbata eggs, photodynamic action
(abstract), 324.
— — spawning, induced by material
elaborated by fertilizable Nereis
eggs (abstract), 306.
Nerve asphyxiation and aerobic recovery
in relation to temperature (abstract),
305.
, differentiation without organizer
(abstract), 299.
• — — membranes, crab, stabilizing action
of alkaline earths (abstract), 309.
NETSKY, M. G., AND M. H. JACOBS.
Some factors affecting the rate of
hemolysis of the mammalian ery-
throcyte by w-butyl alcohol (ab-
stract), 319.
Neuromuscular responses, temporal sum-
mation, in earthworm (abstract),
328.
Nitrogenous organic matter, in sea water,
decomposition and regeneration,
285.
NOVIKOFF, ALEX B. Regulation in
mosaic eggs (abstract), 308.
Nucleus, artificial, in non-nucleate half-
egg (abstract), 312.
(~\LFACTORY organ, anuran, deter-
mination and induction (abstract),
311.
Ovariole differentiation, germaria in,
female aphids, 135.
Oxygen consumption and cell division,
fertilized Arbacia eggs in presence of
respiratory inhibitors (abstract),
318.
- lack, and blocking of excystment
reactions, Colpoda, 382.
— and low oxygen tension, effect
on Paramecium, 339.
and response of Arbacia egg
cortex to chemical and physical
agents (abstract), 335.
Oxygen lack, and x-ray effect on cleavage
time of Arbacia eggs (abstract),
325.
— , effects on activities of some
Protozoa (abstract), 304.
pARAMECIUM, effect of oxygen lack
and low oxygen tension, 339.
PARKER, G. H. Color responses of cat-
fishes with single eyes (abstract),
312.
PARPART, A. K. See Jacobs and Parpart
(abstract), 318.
Permeability, cell, effect of leukotaxine
(abstract), 335.
— , - — , increased, mechanism of, re-
sembling catalysis (abstract), 318.
— , ionic, of frog skin, determined by
radioactive indicators (abstract),
302.
— , to inulin, of toadfish liver (ab-
stract), 332.
— , water, of Chaetopterus eggs (ab-
stract), 317.
Perophora viridis, action of certain drugs
on intact heart (abstract), 337.
Phenols, substituted, effect of increased
intracellular pH on physiological
action (abstract), 322.
pH reactions during feeding in ciliate
Bresslaua (abstract), 303.
464
INDEX
Photodynamic action in eggs of Nereis
limbata (abstract), 324.
Photoperiodicity, sexual, modified, in
cotton-tail rabbits, 364.
Pigmentation abnormalities, and absence
of epithelial hypophysis in fetal dog-
fish, 174.
Pigment granules of Arbacia egg (ab-
stract), 310.
- inheritance in Fundulus-Scomber
hybrid (abstract), 316.
— , in Fundulus Scomber hybrid,
423.
— , retinal, and asymmetry of flicker-
response contour, crayfish, 126.
PORTER, K. R. Androgenetic develop-
ment of the egg of Rana pipiens, 233.
Program and abstracts of scientific papers
presented at the Marine Biological
Laboratory, summer of 1939, 297.
PROSSER, C. LADD. See Armstrong,
Maxfield, Prosser and Schoepfle
(abstract), 327.
Protein, passage of, and other nitrog-
enous substances through walls of
growing and differentiated mam-
malian blood capillaries (abstract),
320.
Protoplasm, viscosity, as affected by
electric current, sea urchin egg, 399.
Protozoa, oxygen lack and low oxygen
tensions, effects on activities (ab-
stract), 304.
"D ABBITS, cotton-tail, modified sexual
photoperiodicity, 364.
Radiations, differential effect, on Men-
delian phenotypes of goldfish, 192.
— , Roentgen, effects on phenomena
related to cleavage in Arbacia eggs
(abstract), 331.
RAKESTRAW, NORRIS W. See von Brand,
Rakestraw and Renn, 285.
Rana pipiens, androgenetic development
of egg, 233.
— , effect of injection of solution
of dihydroxyestrin into castrated
females (abstract), 338.
RAYMONT, J. E. G. Dark adaptation
and reversal of phototropic sign in
Dineutes, 354.
— , — . — . See Johnson and Ray-
mont, 200.
Reducing systems, intracellular distri-
bution, in Arbacia egg (abstract),
328.
Regeneration, Fundulus embryos (ab-
stract), 336.
— , nitrogenous organic matter, in sea
water, 285.
RENN, CHARLES E. See von Brand,
Rakestraw and Renn, 285.
Respiration and fermentation in higher
plants (abstract), 301.
— , Chilomonas paramecium (ab-
stract), 298.
, freshwater fish, relation of blood to
(abstract), 300.
Retinal pigment migration, crayfish,
action of eyestalk extracts, 119.
Roentgen radiation, effect on phenomena
related to cleavage in Arbacia eggs
(abstract), 331.
RoosEN-RuNGE, EDWARD C. Karyo-
kinesis during cleavage of the zebra
fish Brachydanio rerio, 79.
ROSE, S. MERYL. Embryonic induction
in the Ascidia, 216.
RUSSELL, ALICE M. Pigment inherit-
ance in the Fundulus-Scomber hy-
brid (abstract), 316.
— , . Pigment inheritance in
the Fundulus scomber hybrid,
423.
CAFFORD, VIRGINIA. The use of the
swimbladder by fish in respiratory
stress (abstract), 317.
Salamanders, haploid, production (ab-
stract), 307.
Salivary gland chromosomes, micro-
manipulation (abstract), 330.
SANDOW, ALEXANDER. On Clark's the-
ory of muscular contraction (ab-
stract), 314.
SCHOEPFLE, GORDON. See Armstrong,
Maxfield, Prosser and Schoepfle
(abstract), 327.
SCUDAMORE, H. H. See F. A. Brown,
Jr. and H. H. Scudamore (abstract),
329.
Sex cycle, light and temperature effects,
Fundulus, 92.
— , determination, in Arbacia (ab-
stract), 312.
— , photoperiodicity, modified, in cot-
ton-tail rabbits, 364.
SHAPIRO, HERBERT. Nerve asphyxia-
tion and aerobic recovery in relation
to temperature (abstract), 305.
INDEX
465
, — . Water permeability of
Chaetopterus eggs (abstract), 317.
SICHEL, F. J. M. The refractory period
in the non-conducted response of
striated muscle (abstract), 316.
Sinusgland, crustacean, influence on
normal viability and ecdysis, 104.
extracts, different crustaceans, com-
parative effects on two chromato-
phore types (abstract), 329.
Sinus, mammalian carotid, histology of
(abstract), 314.
SMITH, CARL C., AND DAVID CLICK.
Some observations on cholinesterase
in invertebrates (abstract), 321.
— , JAY A. Temperature and starch
and fat in Chilomonas paramecium
(abstract), 298.
SOUTHWICK, WALTER E. Activity-pre-
venting and egg-sea-water neutral-
izing substances from spermatozoa
of Echinometra subangularis, 147.
— , — . The "agglutination"
phenomenon with spermatozoa of
Chiton tuberculatus, 157.
Spawning, of male, induction by material
elaborated by fertilizable Nereis eggs
(abstract), 306.
SPEIDEL, C. C. Living cells in action
(motion picture) (abstract), 308.
Spelotrema nicolli, life history (ab-
stract), 309.
Spermatogenetic cycle, Fundulus, and
external environment, 96.
Spermatozoa, "agglutination" phenom-
enon with, Chiton tuberculatus,
157.
Stephanostomum tenue (Linton), life
cycle, 65.
Swarming annelid, vibration sense (ab-
stract), 313.
Swimbladder, use by fish, in respiratory
stress (abstract), 317.
'pATUM, E. L., AND G. W. BEADLE.
Effect of diet on eye-color develop-
ment in Drosophila melanogaster,
415.
— , — . — . See Beadle, Tatum and
Clancy, 407.
Temperature, constant, and increased
time of development, effect on wing
size vestigial Drosophila, 432.
, effects on male sexual cycle, Fun-
dulus, 92.
— , nerve asphyxiation and aerobic
recovery (abstract), 305.
— , starch and fat in Chilomonas para-
mecium (abstract), 298.
Test secretion, two species of Folliculina,
448.
Toadfish liver, permeability to inulin
(abstract), 332.
TOWNSEND, GRACE. A vibration sense in
a swarming annelid (abstract),
313.
— , . On the nature of the
material elaborated by fertilizable
Nereis eggs inducing spawning of the
male (abstract), 306.
Trematode, Stephanostomum tenue
(Linton), life cycle, 65.
TRINKAUS, J. P. See Goodrich and
Trinkaus, 192.
Triturus viridescens, androgenetic em-
bryos which have ceased develop-
ment (abstract), 334.
Tunicates, feeding method, 443.
TTCA pugilator, moulting and viability
after eyestalk removal (abstract),
326.
\7+ hormone, production by fat bodies,
Drosophila, 407.
Vacuole systems of fresh water limacine
Amoeba (abstract), 334.
Vibration sense in a swarming annelid
(abstract), 313.
Viscosity of protoplasm, sea urchin egg,
effect of electric current, 399.
Vision, retinal pigment, and theory
of asymmetry of flicker-response
contour, crayfish, 126.
TyATERMAN, A. J. The action of
certain drugs on the intact heart of
the compound ascidian, Perophora
viridis (abstract), 337.
WELSH, JOHN H. The action of eye-
stalk extracts on retinal pigment
migration in the crayfish, Cambarus
bartoni, 119.
WENRICH, D. H. Food habits of Enda-
moeba muris (abstract), 313.
Wing size, vestigial, Drosophila, effect
of increasing development time at
constant temperature, 432.
WOLF, ERNST. See Crozier and Wolf,
126.
466
INDEX
WOLF, OPAL. An effect of the injection
of a solution of dihydroxyestrin into
castrated female frogs, Rana pipiens
(abstract), 338.
V-RAY effect, fixation, by fertilization,
Arbacia eggs (abstract), 325.
, on cleavage time of Arbacia
eggs in absence of oxygen (abstract),
325.
nucleus, dependent on
on
7
cytoplasm? (abstract), 326.
•yOUNG, R. A. See Costello and
Young (abstract), 311.
rr EBRA fish, karyokinesis during cleav-
age, 79.
ZoBELL, CLAUDE E. The role of bac-
teria in the fouling of submerged
surfaces (abstract), 302.
ZWILLING, EDGAR. Determination and
induction of the anuran olfactory
organ (abstract), 311.
Volume LXXVII
THE
Number 1
BIOLOGICAL BULLETIN
PUBLISHED BY
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Zoology, Botany, and Genetics
including Algae, Protozoan
cultures, Drosophila cultures,
and animals for experimental
and laboratory use.
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Zoology, Botany, Embryology,
Histology, Bacteriology, and
Parasitology.
All our materials are fully guaranteed to give complete satisfaction and
we will gladly replace at our expense any shipment proving unsatisfactory.
Supply Department
MARINE
BIOLOGICAL LABORATORY
Woods Hole, Mass.
CONTENTS
Page
FORTY-FIRST REPORT OF THE MARINE BIOLOGICAL LABORATORY i
MARTIN, W. E.
Studies on the Trematodes of Woods Hole. II. The life
cycle of Stephanostomum tenue (Linton) 65
HARVEY, ETHEL BROWNE
An Hermaphrodite Arbacia 74
ROOSEN-RUNGE, EDWARD C.
Karyokinesis during Cleavage of the Zebra fish Brachydanio
rerio 79
MATTHEWS, SAMUEL A.
The Effects of Light and Temperature on the Male Sexual
Cycle in Fundulus 92
BURGER, J. WENDELL
Some Experiments on the Relation of the External Environ-
ment to the Spermatogenetic Cycle of Fundulus heteroclitus
(L.) 96
BROWN, F. A., JR., AND ONA CUNNINGHAM
Influence of the Sinusgland of Crustaceans on Normal
Viability and Ecdysis 104
MACGINITIE, G. E.
The Method of Feeding of Chaetopterus 115
WELSH, JOHN H.
The Action of Eye-stalk Extracts on Retinal Pigment Migra-
tion in the Crayfish, Cambarus bartoni 119
CROZIER, W. J., AND ERNST WOLF
The Flicker-response Contour for the Crayfish. II. Retinal
pigment and the theory of the asymmetry of the curve 126
LAWSON, CHESTER A.
The Significance of Germaria in Differentiation of Ovarioles
in Female Aphids 135
Volume LXXVII
Number 2
THE
BIOLOGICAL BULLETIN
PUBLISHED BY
THE MARINE BIOLOGICAL LABORATORY
Editorial Board
GARY N. CALKINS, Columbia University
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
E. E. JUST, Howard University
FRANK R. LELLIE, University of Chicago
CARL R. MOORE, University of Chicago
GEORGE T. MOORE, Missouri Botanical Garden
T. H. MORGAN, California Institute of Technology
G. H. PARKER, Harvard University
F. SCHRADER, Columbia University
ALFRED C. REDFIELD, Harvard University
Managing Editor
OCTOBER, 1939
Printed and Issued by
LANCASTER PRESS, Inc.
PRINCE & LEMON STS.
LANCASTER, PA.
NEW BOOKS
An Introduction to
Modern Genetics
By C. H. WADDINGTON
The theories and experimental results of modern genetics are thor-
oughly discussed here, not as a separate and specialized science, but as
an essential part of the larger science of biology. The author shows
especially the importance of genetics to cytology, embryology, the study
of evolution and of the biochemical nature of cell constituents. Full
bibliographies are included. Illustrated. $4.00.
The Genetics of
Garden Plants
SECOND EDITION
By M. B. CRANE and W. J. C. LAWRENCE
The new revised edition of this standard work is an event of im-
portance to all plant scientists. Both authors have made substantial
contributions to our present knowledge of plant genetics and speak
with authority on the practical as well as the theoretical aspects of
this science. Now brought thoroughly up to date, the book will be
invaluable to the horticulturist and practical plant breeder as well as
in the botanist. Illustrated. $3.25.
\
MACMILLAN60""^"
New York
C-980
C-990
c-n&i
C-1981
Micro-Dissecting
Instruments
C-980 Forceps, straight with slightly rounded
points, finely milled serrations, slender shank.
Easy working spring, 4" long. Chrome plated
each $1.25
C-990 Forceps. As above, but with curved
ends. Chrome plated, 4" length each $1.50
C-1221 McCLURE Scissors, improved. Blade
length Vz". Chrome plated. Total length
43/4" each $8.00
C-1981 Iridectomy Scissors. Blade length J/2n.
Chrome plated. Total length 53A". In metal
case, , each $10.80
*C-975 Swiss Watchmakers' Forceps, 4%" long,
very fine points each $1.50
if Not Illustrated
"ADAMS"
and
"GOLD SEAL"
INSTRUMENTS
SUPPLIES
MODELS - CHARTS
SKELETONS
CLAY- ADAMS CO,
INC.
44 EAST 23RD STREET, NEW YORK
A Perfect Illustration
Or the lack of it, may make
or mar a scientific paper.
For 65 years we have specialized in
making reproductions by the Helio-
type process of the most delicate
pencil and wash drawings and photo-
graphs; and by the Heliochrome proc-
ess, of paintings and drawings in
color.
Ask the editor to whom you submit
your next paper to secure our esti-
mates for the reproduction of your
illustrations.
The Heliotype Corporation
Est. 1872
172 Green St., Jamaica Plain,
Boston, Mass.
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.
THE BIOLOGICAL BULLETIN
THE BIOLOGICAL BULLETIN is issued six times a year. Single
numbers, $1.75. Subscription per volume (3 numbers), $4.50.
Subscriptions and other matter should be addressed to the
Biological Bulletin, Prince and Lemon Streets, Lancaster, Pa.
Agent for Great Britain: Wheldon & Wesley, Limited, 2, 3 and
4 Arthur Street, New Oxford Street, London, W.C. 2.
Communications relative to manuscripts should be sent to the
Managing Editor, Marine Biological Laboratory, Woods Hole,
Mass., between June 1 and October 1 and to the Biological Lab-
oratories, Divinity Avenue, Cambridge, Mass., during the re-
mainder of the year.
INSTRUCTIONS TO AUTHORS
Preparation of Manuscript. In addition to the text matter, manuscripts
should include a running page head of not more than thirty-five letters.
Footnotes, tables, and legends for figures should be typed on separate sheets.
Preparation of Figures. The dimensions of the printed page (4^x7
inches) should be borne in mind in preparing figures for publication. Draw-
ings and photographs, as well as any lettering upon them, should be large
enough to remain clear and legible upon reduction to page size. Illustrations
should be planned for sufficient reduction to permit legends to be set below
them. In so far as possible, explanatory matter should be included in the
legends, not lettered on the figures. Statements of magnification should take
into account the amount of reduction necessary. Figures will be reproduced
as line cuts or halftones. Figures intended for reproduction as line cuts
should be drawn in India ink on white paper or blue-lined coordinate paper.
Blue ink will not show in reproduction, so that all guide lines, letters, etc.
must be in India ink. Figures intended for reproduction as halftone plates
should be grouped with as little waste space as possible. Drawings and
lettering for halftone plates should be made directly on heavy Bristol board,
not pasted on, as the outlines of pasted letters or drawings appear in the
reproduction unless removed by an expensive process. Methods of repro-
duction not regularly employed by the Biological Bulletin will be used only
at the author's expense. The originals of illustrations will not be returned
except by special request.
Directions for Mailing. Manuscripts and illustrations should be packed
flat between stiff cardboards. Large charts and graphs may be rolled and
sent in a mailing tube.
Reprints. Authors will be furnished, free of charge, one hundred re-
prints without covers. Additional copies may be obtained at cost.
Proof. Page proof will be furnished only upon special request. When
cross-references are made in the text, the material referred to should be
marked clearly on the galley proof in order that the proper page numbers
may be supplied. Manuscripts should be returned with galley proof.
Entered October 10, 1902, at Lancaster, Pa., as second-class matter under
Act of Congress of July 16, 1894.
Hormones and Diets
In the new catalog No. 40 of Turtox Biological
Supplies we offer many new listings of plant hor-
mones, desiccated animal glands, gland extracts,
and fresh frozen glands. We also offer and carry
in stock for immediate delivery carefully com-
pounded diets for experiments in nutrition.
Information on these products, as well as price
quotations on living experimental animals of
known age and pedigree, will be forwarded at
your request.
GENERAL BIOLOGICAL SUPPLY HOUSE, INC.
761-763 East Sixty-ninth Place
CHICAGO
OUTLINE OF
PHYSIOLOGY
By WILLIAM R. AMBERSON
and DIETRICH C. SMITH
University of Maryland
This essentially mammalian physi-
ology lays a ground-work of basic
biochemical and biophysical concep-
tions as preparation for the later
more strictly physiological discus-
sions. Historical material is intro-
duced to give desirable perspective
and to increase student interest. The
authors treat modern physical and
chemical concepts briefly and simply.
Most of the 177 figures have been
especially prepared for this book.
412 pages, illustrated, $4.00
Published jointly by the
Williams and Wilkins Company and
F. S. CROFTS & CO.
41 UNION SQUARE NEW YORK
NOTICE
TO
SUBSCRIBERS
/LIBRARIES and individuals
^-* desiring to complete sets
or runs of the BIOLOGICAL
BULLETIN will be able to
obtain certain numbers and
volumes at reduced prices.
Please address communi-
cations to :
Secretary,
THE BIOLOGICAL BULLETIN
Marine Biological Laboratory
Woods Hole, Massachusetts
Biology Materials
We have a complete stock of the following freshly-collected
material for the opening of schools in September, and shall
be very glad to send you a copy of our current catalogue, or
quote prices on specimens needed for the fall term.
PRESERVED SPECIMENS
for
Zoology, Botany, Embryology,
and Comparative Anatomy
LIVING SPECIMENS
for
Zoology, Botany, and Genetics
including Algae, Protozoan
cultures, Drosophila cultures,
and animals for experimental
and laboratory use.
MICROSCOPE SLIDES
for
Zoology, Botany, Embryology,
Histology, Bacteriology, and
Parasitology.
All our materials are fully guaranteed to give complete satisfaction and
we will gladly replace at our expense any shipment proving unsatisfactory.
Supply Department
MARINE
BIOLOGICAL LABORATORY
Woods Hole, Mass.
CONTENTS
Page
SOUTHWICK, WALTER E.
Activity-preventing and Egg-Sea-Water Neutralizing Sub-
stances from Spermatozoa of Echinometra subangularis .... 147
SOUTHWICK, WALTER E.
The " Agglutination " Phenomenon with Spermatozoa of
Chiton tuberculatus 157
KAN DA, SAKYO
The Luminescence of a Nemertean, Emplectonema kandai,
Kato 166
FAWCETT, DON WAYNE
Absence of the Epithelial Hypophysis in a Fetal Dogfish
Associated with Abnormalities of the Head and of Pigmenta-
tion 174
GOODRICH, H. B., AND PRISCILLA L. ANDERSON
Variations of Color Pattern in Hybrids of the Goldfish,
Carassius auratus 184
GOODRICH, H. B., AND J. P. TRINKAUS
The Differential Effect of Radiations on Mendelian Pheno-
types of the Goldfish, Carassius auratus 192
JOHNSON, W. H., AND J. E. G. RAYMONT
The Reactions of the Planktonic Copepod, Centropages
typicus, to Light and Gravity 200
ROSE, S. MERYL
Embryonic Induction in the Ascidia 216
PORTER, K. R.
Androgenetic Development of the Egg of Rana pipiens 233
BUTCHER, EARL O.
The Illumination of the Eye Necessary for Different Melano-
phoric Responses of Fundulus heteroclitus 258
BRAGG, ARTHUR N.
Observations upon Amphibian Deutoplasm and its Relation
to Embryonic and Early Larval Development 268
VON BRAND, THEODOR, NORRIS W. RAKESTRAW AND CHARLES
E. RENN
Further Experiments on the Decomposition and Regenera-
tion of Nitrogenous Organic Matter in Sea Water 285
PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS PRESENTED AT
THE MARINE BIOLOGICAL LABORATORY, SUMMER OF 1939 297
Volume LXXVII
Number 3
THE
BIOLOGICAL BULLETIN
PUBLISHED BY
THE MARINE BIOLOGICAL LABORATORY
Editorial Board
GARY N. CALKINS, Columbia University
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
ALFRED C. REDFIELD, Harvard University
Managing Editor
E. E. JUST, Howard University
FRANK R. LILLIE, University of Chicago
CARL R. MOORE, University of Chicago
GEORGE T. MOORE, Missouri Botanical Garden
T. H. MORGAN, California Institute of Technology
G. H. PARKER, Harvard University
F. SCHRADER, Columbia University
DECEMBER, 1939
Printed and Issued by
LANCASTER PRESS, Inc.
PRINCE & LEMON STS.
LANCASTER, PA.
Biology Materials
LIVING MARINE MATERIALS
AND AQUARIA SETS
For several years we have been furnishing living marine ma-
terials and live aquaria sets, information or prices on which
will be gladly given on request. From November 1st until
March 1st we guarantee delivery on all these materials to
points indicated in our catalogue.
PRESERVED SPECIMENS
for
Zoology, Botany, Embryology,
and Comparative Anatomy
LIVING SPECIMENS
for
Zoology, Botany, and Genetics
including Algae, Protozoan
cultures, Drosophila cultures,
and animals for experimental
and laboratory use.
MICROSCOPE SLIDES
for
Zoology, Botany, Embryology,
Histology, Bacteriology, and
Parasitology.
All our materials are fully guaranteed to give complete satisfaction
Supply Department
MARINE
BIOLOGICAL LABORATORY
Woods Hole, Mass.
C-980
C-990
C-1231
C-1981
Micro-Dissecting
Instruments
C-980 Forceps, straight with slightly rounded
points, finely milled serrations, slender shank.
Easy working spring, 4" long. Chrome plated
each $1.25
C-990 Forceps. As above, but with curved
ends. Chrome plated, 4" length each $1.50
C-1221 McCLURE Scissors, improved. Blade
length Vz". Chrome plated. Total length
43/4" each $8.00
C-1981 Iridectomy Scissors. Blade length W.
Chrome plated. Total length 5%". In metal
case, each $10.80
*C-975 Swiss Watchmakers' Forceps, 4%" long,
very fine points each $1.50
if Not fllHttr ateri
"ADAMS"
and
'GOLD SEAL'
INSTRUMENTS
SUPPLIES
MODELS - CHARTS
SKELETONS
CLAY-ADAMS CO.
INC.
44 EAST 23RD STREET, NEW YORK
A Perfect Illustration
Or the lack of it, may make
or mar a scientific paper.
For 65 years we have specialized in
making reproductions by the Helio-
type process of the most delicate
pencil and wash drawings and photo-
graphs; and by the Heliochrome proc-
ess, of paintings and drawings in
color.
Ask the editor to whom you submit
your next paper to secure our esti-
mates for the reproduction of your
illustrations.
The Heliotype Corporation
Est. 1872
172 Green St., Jamaica Plain,
Boston, Mass.
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.
THE BIOLOGICAL BULLETIN
THE BIOLOGICAL BULLETIN is issued six times a year. Single
numbers, $1.75. Subscription per volume (3 numbers), $4.50.
Subscriptions and other matter should be addressed to the
Biological Bulletin, Prince and Lemon Streets, Lancaster, Pa.
Agent for Great Britain: Wheldon & Wesley, Limited, 2, 3 and
4 Arthur Street, New Oxford Street, London, W.C. 2.
Communications relative to manuscripts should- be sent to the
Managing Editor, Marine Biological Laboratory, Woods Hole,
Mass., between June 1 and October 1 and to the Biological Lab-
oratories, Divinity Avenue, Cambridge, Mass., during the re-
mainder of the year.
INSTRUCTIONS TO AUTHORS
Preparation of Manuscript. In addition to the text matter, manuscripts
should include a running page head of not more than thirty-five letters.
Footnotes, tables, and legends for figures should be typed on separate sheets.
Preparation of Figures. The dimensions of the printed page (4*4x7
inches) should be borne in mind in preparing figures for publication. Draw-
ings and photographs, as well as any lettering upon them, should be large
enough to remain clear and legible upon reduction to page size. Illustrations
should be planned for sufficient reduction to permit legends to be set below
them. In so far as possible, explanatory matter should be included in the
legends, not lettered on the figures. Statements of magnification should take
into account the amount of reduction necessary. Figures will be reproduced
as line cuts or halftones. Figures intended for reproduction as line cuts
should be drawn in India ink on white paper or blue-lined coordinate paper.
Blue ink will not show in reproduction, so that all guide lines, letters, etc.
must be in India ink. Figures intended for reproduction as halftone plates
should be grouped with as little waste space as possible. Drawings and
lettering for halftone plates should be made directly on heavy Bristol board,
not pasted on, as the outlines of pasted letters or drawings appear in the
reproduction unless removed by an expensive process. Methods of repro-
duction not regularly employed by the Biological Bulletin will be used only
at the author's expense. The originals of illustrations will not be returned
except by special request.
Directions for Mailing. Manuscripts and illustrations should be packed
flat between stiff cardboards. Large charts and graphs may be rolled and
sent in a mailing tube.
Reprints. Authors will be furnished, free of charge, one hundred re-
prints without covers. Additional copies may be obtained at cost.
Proof. Page proof will be furnished only upon special request. When
cross-references are made in the text, the material referred to should be
marked clearly on the galley proof in order that the proper page numbers
may be supplied. Manuscripts should be returned with galley proof.
Entered October 10, 1902, at Lancaster, Pa., as second-class matter under
Act of Congress of July 16, 1894.
CONTENTS
Page
KlTCHING, J. A.
The Effects of a Lack of Oxygen and of Low Oxygen Tensions
on Paramecium 339
RAYMONT, J. E. G.
Dark Adaptation and Reversal of Phototropic Sign in Dineutes 354
BISSONNETTE, THOMAS HUME AND ALBERT GEORGE CSECH
Modified Sexual Photoperiodicity in Cotton-tail Rabbits .... 364
LlTTLEFORD, ROBERT A.
The Life Cycle of Dactylometra quinquecirrha, L. Agassiz in
the Chesapeake Bay 368
BROWN, MORDEN G.
The Blocking of Excystment Reactions of Colpoda duodenaria
by Absence of Oxygen 382
MAST, S. O.
The Relation between Kind of Food, Growth, and Structure
in Amoeba 391
ANGERER, C. A.
The Effect of Electric Current on the Relative Viscosity of Sea-
Urchin Egg Protoplasm 399
BEADLE, G. W., E. L. TATUM AND C. W. CLANCY
Development of Eye Colors in Drosophila: Production of
v+ Hormone by Fat Bodies 407
TATUM, E. L., AND G. W. BEADLE
Effect of Diet on Eye-Color Development in Drosophila me-
lanogaster 415
RUSSELL, ALICE
Pigment Inheritance in the Fundulus-Scomber Hybrid 423
CHILD, GEORGE
The Effect of Increasing Time of Development at Constant
Temperature on the Whig Size of Vestigial of Drosophila
melanogaster 432
MACGINITIE, G. E.
The Method of Feeding of Tunicates 443
DEWEY, VIRGINIA C.
Test Secretion in Two Species of Folliculina 448
INDEX FOR VOLUME 77. 456
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