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
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
ALFRED C. REDFIELD, Harvard University
Managing Editor
VOLUME LXXXI
AUGUST TO DECEMBER, 1941
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 July 1 and October 1 and to the Department of
Zoology, Columbia University, New York City, during the re-
mainder 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, 1941
PAGE
ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY 1
VON BRAND, THEODOR AND NORRIS W. RAKESTRAVV
Decomposition and Regeneration of Nitrogeneous Organic
Matter in Sea Water. IV 63
MENDOZA, GUILLERMO
The Reproductive Cycle of the Viviparous Teleost, Neotoca
bilineata, a Member of the Family Goodeidae 70
BROWN, FRANK A., JR., AND ONA CUNNINGHAM
Upon the Presence and Distribution of a Chromatophorotropic
Principle in the Central Nervous System of Limulus 80
SCHARRER, BERTA
Neurosecretion. IV. Localization of neurosecretory cells in
the central nervous system of Limulus 96
OBRESHKOVE, VASIL
The Action of Acetylcholine, Atropine and Physostigmine on
the Intestine of Daphnia magna 105
HARVEY, ETHEL BROWNE
Vital Staining of the Centrifuged Arbacia punctulata Egg. ... 114
ANDERSON, B. G., AND H. L. BUSCH
Allometry in Normal and Regenerating Antennal Segments in
Daphnia 119
MALUF, N. S. RUSTUM
Experimental Cytological Evidence for an Outward Secretion
of Water by the Xephric Tubule of the Crayfish 127
MALUF, N. S. RUSTUM
Micturition in the Crayfish and Further Observations on the
Anatomy of the Nephron of this Animal 134
SCHRADER, FRANZ
Chromatin Bridges and Irregularity of Mitotic Coordination in
the Pentatomid Peromatus notatus Am. and Serv 149
54140
iv CONTENTS
PAGE
No. 2. OCTOBER, 1941
PARKER, G. H.
The Responses of Catfish Melanophores to Ergotamine 163
COE, WESLEY R.
Sexual Phases in Wood-boring Mollusks 168
GOLDIN, A., AND L. G. EARTH
Regeneration of Coenosarc Fragments Removed from the
Stem of Tubularia crocea 177
TYLER, ALBERT
The Role of Fertilizin in the Fertilization of Eggs of the Sea
Urchin and Other Animals 190
STUNKARD, HORACE W.
Specificity and Host-relations in the Trematode Genus
Zoogonus 205
HESS, WALTER N.
Factors Influencing Moulting in the Crustacean, Crangon
armillatus 215
CLAFF, C. L., V. C. DEWEY AND G. W. KIDDER
Feeding Mechanisms and Nutrition in Three Species of
Bresslaua 22 1
MALUF, N. S. R.
Secretion of Inulin, Xylose and Dyes and its Bearing on the
Manner of Urine-formation by the Kidney of the Crayfish . . . 235
HOLLINGSWORTH, JOSEPHINE
Activation of Cumingia and Arbacia Eggs by Bivalent Cations 261
PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS PRESENTED AT
THE MARINE BIOLOGICAL LABORATORY, SUMMER OF 1941 . . . 276
No. 3. DECEMBER, 1941
ROOT, R. W., AND L. IRVING
The Equilibrium between Hemoglobin and Oxygen in Whole
and Hemolyzed Blood of the Tautog, with a Theory of the
Haldane Effect 307
WENRICH, D. H.
Observations on the Food Habits of Entamoeba muris and
Entamoeba ranarum 324
OSBORN, C. M.
Studies on the Growth of Integumentary Pigment in the Lower
Vertebrates. I. The origin of artificially developed melano-
phores on the normally unpigmented ventral surface of the
summer flounder ( Paralichthys dentatus) 341
CONTENTS v
PAGE
OSBORN, C. M.
Studies on the Growth of Integumentary Pigment in the Lower
Vertebrates. II. The role of the hypophysis in melanogenesis
in the common catfish (Ameiurus melas) 352
TYLER, A., AND K. O'MELVENY
The Role of Antifertilizin in the Fertilization of Sea-urchin
Eggs 364
CARLSON, L. D.
Enzymes in Ontogenesis (Orthoptera). XVIII. Esterases in
the grasshopper egg 375
BODINE, J. H., AND T. H. ALLEN
Enzymes in Ontogenesis (Orthoptera). XIX. Protyrosinase
and morphological integrity of grasshopper eggs 388
LAFLEUR, L. J.
The Founding of Ant Colonies 392
KAYLOR, C. T.
Studies on Experimental Haploidy in Salamander Larvae. II.
Cytological studies on androgenetic eggs of Triturus viri-
descens 402
SCOTT, A.
Reversal of Sex Production in Micromalthus 420
RYAN, F. J.
The Time-Temperature Relation of Different Stages of De-
velopment 43 1
Vol. LXXXI, No. 1 August, 1941
THE
BIOLOGICAL BULLETIN
PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY
THE MARINE BIOLOGICAL LABORATORY
FORTY-THIRD REPORT, FOR THE YEAR 1940—
FIFTY-THIRD YEAR
ERRATA
The editor wishes to call attention to the following
errata in the June, 1941 issue of this journal:
Page 445. line 1: " 591 m, " should read « 491 m^
Page 453, line 40: il testes " should read
(== skeleton + skin).
_ ~^.~*. ....!<_ io.pei5, iv-tu .TTTTT7TT7T7TTTT" 42
8. General Scientific Meetings, 1940 43
9. Members of the Corporation 48
I. TRUSTEES
EX OFFICIO
FRANK R. LILLIE, President of the Corporation, The University of Chicago.
CHARLES PACKARD, Director, Columbia University.
LAWRASON RIGGS, JR., Treasurer, 120 Broadway, New York City.
PHILIP H. ARMSTRONG, Clerk of the Corporation, Syracuse University Medi-
cal College.
EMERITUS
H. C. BUMPUS, Brown University.
G 1ST r^ATTfTKt; Pnliimhia TTnivf>r«;itv
G. N. CALKINS. Columbia University.
E. G. CONKLIN, Princeton Universit
CASWELL GRAVE, Washington Unive
R. A. HARPER, Columbia University.
E. G. CONKLIN, Princeton University.
CASWELL GRAVE, Washington University.
1
*
Vol. LXXXI, No. 1 August, 1941
THE
BIOLOGICAL BULLETIN
PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY
THE MARINE BIOLOGICAL LABORATORY
FORTY-THIRD REPORT, FOR THE YEAR 1940—
FIFTY-THIRD YEAR
I. TRUSTEES AND EXECUTIVE COMMITTEE (AS OF AUGUST 13,
1940) 1
STANDING COMMITTEES 2
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 OK THE MANAGING EDITOR OK THK BIOLOGICAL
BULLETIN 12
VII. REPORT OF THE DIRECTOR 17
Statement 17
Addenda :
1. Memorials of Deceased Trustees 21
2. The Staff, 1940 2(>
3. Investigators and Students, 1940 29
4. Tabular View of Attendance 40
5. Subscribing and Cooperating Institutions, 1940 . . 40
6. Evening Lectures, 1940 41
7. Shorter Scientific Papers, 1940 42
8. General Scientific Meetings, 1940 43
9. Members of the Corporation 48
I. TRUSTEES
EX OFFICIO
FRANK R. LILLIE, President of the Corporation, The University of Chicago.
CHARLES PACKARD, Director, Columbia University.
LAWRASON RIGGS, JR., Treasurer, 120 Broadway, New York City.
PHILIP H. ARMSTRONG, Clerk of the Corporation, Syracuse University Medi-
cal College.
EMERITUS
H. C. BUMPUS, Brown University.
G. N. CALKINS, Columbia University.
E. G. CONKLIN, Princeton University.
CASWELL GRAVE, Washington University.
R. A. HARPER, Columbia University.
1
MARINE BIOLOGICAL LABORATORY
Ross G. HARRISON, Yale University.
H. S. JENNINGS, University of California.
C. E. McCLUNG, University of Pennsylvania.
T. H. MORGAN, California Institute of Technology.
G. H. PARKER, Harvard University.
W. B. SCOTT, Princeton University.
TO SERVE UNTIL 1944
H. B. BIGELOW, Harvard University.
R. CHAMBERS, Washington Square College, New York University,
W. E. GARREY, Vanderbilt University Medical School.
S. O. MAST, Johns Hopkins University.
A. P. MATHEWS, University of Cincinnati.
C. W. METZ, University of Pennsylvania.
H. H. PLOUGH, Amherst College.
W. R. TAYLOR, University of Michigan.
TO SERVE UNTIL 1943
W. C. ALLEE, The University of Chicago.
B. M. DUGGAR, University of Wisconsin.
L. V. HEILBRUNN, University of Pennsylvania.
LAURENCE IRVING, Swarthmore College.
J. H. NORTHROP, Rockefeller Institute.
W. J. V. OSTERHOUT, Rockefeller Institute.
A. H. STURTEVANT, California Institute of Technology.
LORANDE L. WOODRUFF, Yale University.
TO SERVE UNTIL 1942
DUGALD E. S. BROWN, New York University.
E. R. CLARK, University of Pennsylvania.
OTTO C. GLASER, Amherst College.
E. N. HARVEY, Princeton University.
M. H. JACOBS, University of Pennsylvania.
F. P. KNOWLTON, Syracuse University.
FRANZ SCHRADER, Columbia University.
B. H. WILLIER, Johns Hopkins University.
TO SERVE UNTIL 1941
W. R. AMBERSON, University of Maryland School of Medicine.
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.
EXECUTIVE COMMITTEE OF THE BOARD OF TRUSTEES
FRANK R. LILLIE, Ex. Off. Chairman.
CHARLES PACKARD, Ex. Off.
LAWRASON RIGGS, JR., Ex. Off.
ACT OF INCORPORATION
L. V. HEILBRUNN, to serve until 1941.
A. C. REDFIELD, to serve until 1941.
P. B. ARMSTRONG, to serve until 1942.
W. C. ALLEE, to serve until 1942.
THE LIBRARY COMMITTEE
E. G. CONKLIN, Chairman.
WILLIAM R. AMBERSON.
C. O. ISELIN, II.
C. C. SPEIDEL.
A. H. STURTEVANT.
WILLIAM R. TAYLOR.
THE APPARATUS COMMITTEE
E. N. HARVEY, Chairman.
H. C. BRADLEY.
M. H. JACOBS.
C. L. PARMENTER.
A. K. PARPART.
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.
G. Farlow, A. D. Phillips, and B. H. Van Vleck, their associates and sue-
MARINE BIOLOGICAL LABORATORY
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; (b) 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
define the duties of all the officers and agents; and may remove them, or
REPORT OF THE TREASURER 5
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 Labora-
tory for the year 1940.
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 Corporation.
At the end of the year 1940, the book value of the Endowment Fund
in the hands of the Central Hanover Bank and Trust Company, as
Trustee, was
General Fund, Securities (market $807,499.95) $856.629.50
Interest in Real Estate 24,921.89
Cash 30,648.97
$912,200.36
Library Fund, Securities (market $150,077.06) $164,294.79
Real Estate 20,000.00
Cash 10,862.62
$195.157.41
ft MARINE BIOLOGICAL LABORATORY
The income collected from these funds during the year was :
General Endowment $35.674.49
Library 8.463.30
$44,137.79
The income in arrears on these funds at the end of the year was:
Arrears General Fund $12,253.69
Arrears Library Funds 4.325.00
$16.578.69
Arrears at the end of 1939 $15.322.81
a falling behind of $ 1.255.88
General Biological Supply House, Inc.: The dividends from the Gen-
eral Biological Supply House, Inc. totalled $18,542.00, an increase of
$3.556 over 1939.
Bar Neck Property: The rental from the Bar Neck property which
is based on the net profit of the garage was $5,097.64, an improvement
of about $1.500 over the prior year, during which the absence of the
drawbridge adversely affected the business of the garage.
In addition, the notes given for the acquisition of the Bar Neck prop-
erty are now paid off so that the entire income from this property can
now be used for current expenses of the Laboratory.
Retirement Fund : A total of $3.710 was paid in pensions and $923.20
advanced from current funds in prior years was repaid. This fund at
the end of the year consisted of :
Participations in mortgages $ 8,154.39
Interest in Real estate 2.301.88
Cash . . . 5,048.06
$15.504.33
Plant Assets: The land (exclusive of Gansett and Devil's Lane
Tracts), the buildings, equipment and library represent an investment
of $1,867,005.60
less reserve for depreciation 564,225.03
or a net of $1,302.780.57
Income and Expenses: Income exceeded expenses (including de-
preciation of $25,648.22) by $8,035.14.
RKPORT OF THE TREASURER /
There was expended from current funds for plant account a net
of $16702.27 and in payment of note indebtedness $3,500, and $2,500
was transferred to the Reserve Fund.
The total damage caused by the hurricane on September 21, 1938,
finally liquidated during 1940 was $30,152.47
of which 20,000.00
was met by the grant of the Carnegie Corporation (1939) and the bal-
ance of $10,152.47 w*as paid from current funds or charged off.
At the end of the year the Laboratory had no indebtedness on notes
or mortgages. It owed on accounts payable $3,689.51, against which it
had accounts receivable of $11,667.91 and cash in its general bank ac-
counts of $13,359.26.
The Rockefeller Foundation made a grant of $110,400 for an addi-
tion to the library. During 1940, there was received $64,776.62 on
this grant, of which $39,851.12 was expended in the year.
Following is the balance sheet, the condensed statement of income
and outgo and the surplus account all as set out by the auditors.
EXHIBIT A
MARINE BIOLOGICAL LABORATORY BALANCE SHEET,
DECEMBER 31, 1940
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 $1,107,357.77
Securities and Cash— Minor Funds— Schedule II.. 9,194.41 $1,116,552.18
Plant Assets:
Land— Schedule IV $ 111,425.38
Buildings— Schedule IV 1,277,685.06
Equipment— Schedule IV 179,181.15
Library— Schedule IV 298,714.01
$1,867,005.60
Less Reserve for Depreciation 564,225.03 1,302,780.57
Cash in Building Fund 24,925.50
Cash in Reserve Fund : 2.524.65 1,330,230.72
Current Assets :
Cash $ 13.359.26
Accounts-Receivable 11,667.91
Inventories :
Supply Department $ 38,976.75
Biological Bulletin 11.069.82 50,046.57
MARINE BIOLOGICAL LABORATORY
Investments :
Devil's Lane Property $ 45,099.78
Gansett Property 6,030.81
Stock in General Biological Sup-
ply House, Inc 12,700.00
Other Investment Stocks 17,770.00
Securities, Real Estate, and Cash
—Retirement Fund — Schedule
V 15,504.33 97,104.92
Prepaid Insurance 3,445.81
Items in Suspense 172.40 $ 175,796.87
Liabilities
Endowment Funds :
Endowment Funds— Schedule III $1,105,900.37
Reserve for Amortization of Bond
Premiums 1,457.40 $1,107,357.77
Minor Funds— Schedule III 9,194.41 $1,116.552.18
Plant Liabilities and Surplus :
Donations and Gifts— Schedule III $1,104,666.73
Other Investments in Plant from Gifts and Cur-
rent Funds 225,563.99 $1,330,230.72
Current Liabilities and Surplus :
Accounts— Payable $ 3,689.51
Current Surplus— Exhibit C 172,107.36 $ 175,796.87
EXHIBIT B
MARINE BIOLOGICAL LABORATORY INCOME AND EXPENSE,
YEAR ENDED DECEMBER 31, 1940
Total Net
Expense Income Expense Income
Income :
General Endowment Fund $ 35,674.49 $ 35,674.49
Library Fund 8,463.30 8,463.30
Instruction $ 10,283.63 9,670.00 $ 613.63
Research 6.086.18 15,068.00 8,981.82
Evening Lectures 32.90 32.90
Biological Bulletin and Membership
Dues 8,834.37 9,725.93 891.56
Supply Department— Schedule VI. 27,345.79 32,376.45 5,030.66
Mess— Schedule VII 25.081.97 24,227.77 854.20
Dormitories— Schedule VIII 23,658.88 13,060.53 10,598.35
(Interest and Depreciation
charged to above 3 Depart-
ments— See Schedules VI, VII,
and VIII) 24,040.85 24,040.85
REPORT OF THE TREASURER
Dividends, General Biological Sup-
ply House, Inc 18,542.00 18,542.00
Dividends, Crane Company 400.00 400.00
Rents :
Bar Neck Property 5,097.64 5,097.64
Howes Property 117.80 160.00 42.20
Tanitor House 24.07 360.00 335.93
Danchakoff Cottages 307.77 715.00 407.23
Sale of Library Duplicates 80.26 80.26
Apparatus Rental 1,226.53 1,226.53
Sundry Income 57.50 57.50
Maintenance of Plant :
Buildings and Grounds 25,121.47 25,121.47
Chemical and Special Apparatus
Expense 15,833.22 15,833.22
Library Expense 7,675.89 7,675.89
Workmen's Compensation Insur-
ance 538.14 538.14
Truck Expense 466.96 466.96
Bay Shore Property 77.40 77.40
Great Cedar Swamp 19.20 19.20
General Expenses :
Administration Expense 12,426.(>4 12,426.64
Endowment Fund Trustee and
Safe-keeping 1,014.45 1,014.45
Interest on Notes— Payable 87.50 87.5(1
Bad Debts 228.60 228.<xi
Reserve for ni-pm-iation 25,648.22 25,648.22
$166,870.26 $174,905.40 $101,236.83 $109,271.97
Excess of Income over Expense
carried to Current Surplus — Ex-
hibit C 8,035.14 8,035.14
$174,905.40 $109,271.97
EXHIBIT C
MARINE BIOLOGICAL LABORATORY, CURRENT SURPLUS ACCOUNT,
VKAR ENDED DECEMBER 31. 1940
Balance, January 1, 1940 $163,206.29
Add:
Excess of Income Over Expense for Year as shown in
Exhibit B $ 8,035.14
Reserve for Depreciation Charged to Plant Funds 25,648.22
Transfer from Reserve for Repairs and Repacements
on account of Hurricane Water-Damage 1.072.68 $ 34,756.04
$197,962.33
10 MARINE BIOLOGICAL LABORATORY
Deduct :
Payments from Current Funds during Year
for Plant Assets as shown in Schedule
IV,
Buildings $ 1,426.71
Equipment 5,465.65
Library 10,104.08 $16,996.44
Less Received for Plant Assets Disposed
of $ 162.43
Loss on Equipment Charged Off Due to
Hurricane Water-Damage 131.74 294.17
$16,702.27
Payment of Plant Note— Payable $ 3,500.00
Transfer to Plant Reserve Fund 2,500.00
Pensions Paid $ 3,710.00
Less Retirement Fund Income 557.30 3,152.70 $ 25,854.97
Balance, December 31, 1940— Exhibit A $172,107.36
Respect fully submitted,
LAWRASON R1GGS, JR..
Treasurer.
V. REPORT OF THE LIBRARIAN
The $18,850.00 appropriated for the Library by the Marine Biological
Laboratory for 1940 was expended before the end of the year for books,
$766.28; serials, $2863.57; binding, $957.55; express, $73.97; supplies,
$268.85; salaries, $7200.00; back sets, $2653.35; sundries, $11.48; and
a reserve fund of $3977.18 was retained to pay for current serials and
back sets, ordered, but not yet received on account of the European war
conditions. A balance of $77.77 reverted to the Laboratory plus $80.26
from the sale of duplicates by the Library. An examination of our rec-
ord over the past decade shows a total of $5970.67 that has so reverted,
$4419.49 from the ten yearly budgets assigned by the Executive Com-
mittee to Library expenditures and $1551.18 from the Library's sale of
duplicates. This is an average reversion of $597.00 for each of the ten
years. The explanation to account for a variation from year to year
from overspending to underspending is too various to give here, but in
general, it is due to caution taken to buy only under advantageous condi-
tions. By calling attention to the sum that has reverted to the Labora-
tory during this ten-year period, it is the hope of the Librarian that on
the occurrence of a very favorable opportunity for buying, the Labora-
tory will meet this opportunity by placing the necessary sum at the dis-
REPORT OF THE DIRECTOR 11
posal of the Library. It would, in fact, be a fine idea to begin a reserve
fund for this purpose.
The Woods Hole Oceanographic Institution appropriation, which is
outright and is carried over from one year to the next, was, for 1940,
$600.00, plus $82.42 remaining from 1939. Of this $555.11 was ex-
pended and reported to the Director. A delayed set ordered from Eng-
land in August at 50 pounds has arrived in March 1941, before this
report goes to press.
In connection with plans made for extending the Library volumes
into the addition (the moving will be completed before the end of April,
1941), a count of periodical sets gave the interesting figure of a total
of 2366 titles. (These will require the shelving space of the entire top
floors, now the fourth and third, and the new section of the reading-room
floor, now the second floor. Books will remain in the old part of the
second floor and the reprints will extend throughout the first floor.)
Of these 2366 titles, only 1257 are received currently ; 422 are subscrip-
tions, 403 (15 new) of the Marine Biological Laboratory and 39 (0
new) of the Woods Hole Oceanographic Institution; 609 are exchanges,
544 (6 new) with the "Biological Bulletin" and 65 (1 new) with the
Woods Hole Oceanographic Institution publications ; 195 are gifts to
the former institution and 11 to the latter. An unusual number of
books, 142, was bought by the Marine Biological Laboratory Library
as selected by the investigators from a list accumulated by the Librarian
throughout the previous five years, while the Woods Hole Oceanographic
Institution purchased 12 ; authors presented 8 books to the Marine Bio-
logical Laboratory and 2 were received on exchange ; publishers gave 43,
and the Woods Hole Oceanographic Institution received 2 books from
publishers. The record for the filling in of back sets shows 22 com-
pleted; purchased by the Marine Biological Laboratory, 15, and by the
Woods Hole Oceanographic Institution, 2, by exchange with the " Bio-
logical Bulletin," 3, by exchange of duplicates 1, and by gift, 1 ; partially
completed back sets total 49 ; by purchase, for the Marine Biological
Laboratory, 18, for the Woods Hole Oceanographic Institution, 2, by
exchange with the " Biological Bulletin," 3, by gift to the former institu-
tion, 2, to the latter, 1, and by exchange of duplicates, 23. Reprint addi-
tions number 3528; 1614 current of 1939, 637 of 1940, and 1277 of pre-
vious dates. The present holdings of the Library number 47,697 bound
volumes and 1 16,305 reprints.
12 MARINE BIOLOGICAL LABORATORY
VI. THE REPORT OF THE MANAGING EDITOR
OF THE BIOLOGICAL BULLETIN
The Biological Bulletin is the property of the members of the Cor-
poration of the Marine Biological Laboratory. It is the one tangible
return which they receive in exchange for their membership fees, whether
they return each year to the laboratory or not. It seems proper that you
should receive, at least from time to time, some report on its progress,
its policies and problems.
The annual reports of the Laboratory contain only occasional refer-
ences to the Bulletin, recording appointments to the editorial board and
such matters of fact. Fortunately there is preserved in the Tenth Re-
port, for the years 1903-1906, a prospectus issued in 1902 announcing
the resumption of publication of the Bulletin, which had been interrupted
for a year. With the completion of the eightieth volume, after thirty-
nine years of continuous publication, it is not unfitting to reexamine this
prospectus and see to what extent its promise has been realized.
The Biological Bulletin was preceded by the Zoological Bulletin, of
which two volumes were published in 1897 and 1898 under the editor-
ship of Professor C. O. Whitman, then Director of the Laboratory, and
Professor W. M. Wheeler. It was intended to be a companion journal
to the Journal of Morphology and to take in shorter papers with simple
illustrations, where relatively rapid publication of original contributions
was desirable. Following the completion of two volumes, it was suc-
ceeded in 1899 by Volume I of the Biological Bulletin. The title page
of the new Biological Bulletin shows it to have been edited by the Di-
rector and members of the staff of the Marine Biological Laboratory.
Volume II was completed in June, 1901.
After the lapse of a year, Volume III appeared, bearing the title:
Biological Bulletin of the Marine Biological Laboratory, Woods Hole,
Mass., thus clearly establishing the relation of the publication to the
Laboratory. The treasurer's report for 1902 shows also that for the first
time the relationship was a material one. The editorial staff consisted
of E. G. Conklin, The University of Pennsylvania; Jacques Loeb, The
University of Chicago; T. H. Morgan, Bryn Mawr; W. M. Wheeler,
The University of Texas; C. O. Whitman, The University of Chicago;
E. B. Wilson, Columbia University ; and Frank R. Lillie appeared for
the first time as Managing Editor. Dr. Lillie continued to edit the Bul-
letin for 25 years, when in 1927 he was succeeded by Dr. Carl R. Moore.
With two other members of the original staff he still serves as a member
of the editorial board.
REPORT OF THE MANAGING EDITOR 13
The prospectus announcing the resumption of publication stated that:
' The Bulletin will be published as heretofore, under the auspices
of the Marine Biological Laboratory, and its scope will include Zoology,
General Biology, and Physiology. It will contain original articles in
these fields and also occasional reviews, and reports of work and lectures
at the Marine Biological Laboratory. Preliminary statements of impor-
tant results will be made a special feature. The Bulletin will be open,
as heretofore, to contributions from any source."
I think it is fair to say that the scope of the Bulletin still includes
Zoology, General Biology, and Physiology and that the character of its
contents remains essentially unchanged except as it reflects changing
emphasis and new developments within these fields. The question of
scope is. nevertheless, one of the most perplexing problems of policy
with which the managing editor must deal. The objects of the Labora-
tory and the curiosity of its clientele are broad, and it seems proper that
the contents of the Bulletin should reflect this catholic interest. It is
not undesirable in this day of specialization that one journal at least
should present a rather broad cross-section of biology as a whole. On
the other hand, practical considerations require some limitation, even
within the fields specified in the prospectus, and there is no easier way
for an editor to dispose of an unwelcome manuscript than to rule it in-
appropriate. There is also an evil temptation which besets every editor
to allow the selection of papers to reflect too closely his own particular
interests and prejudices. I think that in the selection of papers, the
guiding principle should be to produce a journal which will interest as
widely as possible those who support it with their subscriptions. These
are primarily the members of this Corporation, and secondarily, the bio-
logical libraries throughout the world. The character of both of these
groups argues for as great a diversity as possible. Worthy papers differ
greatly in the breadth of their interest. Many papers contain material
of value to specialists which are of little interest to other biologists.
"Where specialists maintain their own journals, as is true, for example, in
mammalian physiology, or genetics, it seems preferable that such papers
should be published in these places. For many years it was the policy
of the Bulletin not to publish taxonomic papers since taxonomy was
not a part of the program of the Marine Biological Laboratory. How-
ever. I feel that today those who are still following the older disciplines
deserve some encouragement and that the policy of the Bulletin should
be more lenient, at least in connection with papers dealing with the local
fauna and with groups of organisms which are not covered by specialized
journals.
14 MARINE BIOLOGICAL LABORATORY
It is significant that the founders of the Biological Bulletin did not
call it the Marine Biological Bulletin, though it was published under the
auspices of a marine laboratory. It is evident that they came to Woods
Hole not so much to study the sea, but because they found there good
material for the study of more general problems, and that they intended
that the Bulletin should represent all aspects of biology. In this charac-
teristic there has been no change. It is, however, natural that the journal
should deal to a large extent with the biology of marine organisms since
these are the creatures on which many of your studies are made, and it
seems reasonable to assume that you will be interested in papers of all
sorts dealing with these organisms and with the waters in which they
live. Thus the Bulletin should retain a distinctly salty flavor and should
welcome especially work done at other marine stations.
The prospectus states that the Bulletin will be open to contributions
' from any source." An examination of 778 papers published between
1930-1940 shows that 29 per cent originated from this Laboratory;
22 per cent were contributions from other marine laboratories, including
9 per cent derived from the Woods Hole Oceanographic Institution
These included contributions from : the Mt. Desert Island Biological
Laboratory, the Scripps Institution of Oceanography, the Duke Univers-
sity Marine Laboratory, the William Kerckhoff Marine Biological Labo-
ratories of the California Institute of Technology, the Hopkins Marine
Station, the U. S. Bureau of Fisheries, the Atlantic Biological Station
at St. Andrews, the Chesapeake Biological Laboratory, the University
of Washington Oceanographic Laboratories, and the New York Aquar-
ium ; the Naples Zoological Station, the Marine Biological Laboratory
at Plymouth, the Bermuda Biological Station, the Bergens Museum
Biologiske Stasjon, the Pacific Biological Station at Nanimo, and the
Misaki Marine Biological Station at Kanagawa-ken. The remaining
49 per cent came from various university laboratories and may in part
have been based on work done here or at other marine stations. This
distribution conforms well to the principles discussed above.
While the bulk of the material in the Bulletin consists of original
articles, some attempt was made a few years ago to print the lectures
given at the Laboratory. Because of the difficulty in securing the manu-
scripts and also because of the general pressure for space, this practice
was discontinued. The publication of reviews has not been made a fea-
ture of the Bulletin and there is probably no reason why it should in
view of the special journals now devoted to this purpose. An occasional
paper bringing together material of particular local interest would be
of value to workers in the Laboratory. I have in mind as an example
Dr. Harvey's paper on " Physical and chemical constants of the egg of
REPORT OF THE MANAGING EDITOR 15
Arbacia punctulata " (Biological Bulletin, April, 1932). The promise
that preliminary statements of important results would be made a special
feature of the Bulletin is now fulfilled by the publication of abstracts of
the papers delivered at the scientific meetings during the summer.
The prospectus continues :
' There is in America no journal that takes the place of the Biolo-
giscJies Centralblatt or the Anatomischer Anzeigcr in Germany, although
there is abundance of material to support such a publication. It is hoped
that the Bulletin may occupy this field, and meet the need for rapid
publication of results ; the editors, therefore, undertake to issue one
number each month, making two volumes a year, if the material offered
is sufficient."
At the time the Bulletin was founded, the American journals par-
ticipating in its field were: the Journal of Morphology, the American
Journal of Physiology, the Annals of Botany, the American Naturalist,
the Botanical Gazette and the Journal of Comparative Neurology. The
establishment shortly thereafter of the Anatomical Record, the Ameri-
can Journal of Anatomy and the Journal of Experimental Zoology,
and the subsequent appearance of many others has largely eliminated
this special raison d'etre. The hope that the Bulletin might meet the
need for rapid publication of results has not always been fulfilled, for it
was recorded in the twenty-seventh report, for the year 1924, that ". . .
lately, in common with many other biological research journals, it had
been falling behind in promptness of publication so that about a year
elapsed between the receipt of manuscripts and their appearance in
print." In the hope of correcting this situation the Bulletin was en-
larged to 900 pages and the subscription price increased from $6.00 to
$9.00 per year, and the fees of members of the Corporation were in-
creased proportionately. This change reduced the time required for
publication to at most six months. At the present time the Bulletin is
run on the theory that the only way to ensure prompt publication is to
accept for publication no more papers than one can afford to publish at
once. A journal operating on a fixed income has no more chance of
catching up with an accumulation of papers it cannot afford to publish
at once than does a man in like circumstances who has allowed himself
to get some months behind in paying his bills. It is better to require a
few papers less suited to one's purpose to find a publisher elsewhere than
to delay the entire stream of publication chronically. During the past
11 years no paper has remained in the editor's hands more than two
months before going to the printer except under special circumstances,
as when occasionally delays occur in securing the necessary reports from
16 MARINE BIOLOGICAL LABORATORY
referees. In order to facilitate this practice, it is customary not to ar-
rive at a decision concerning the disposition of any paper until the accu-
mulated material can be considered together at the time of going to press.
The Bulletin was originally offered at a price of $3.00 per volume of
300 pages. It is interesting to note that this is exactly the cost to the
subscriber per page of the present issues. Because of the low price, the
length of articles was originally limited to 25 pages and lithographic
prints were excluded. The cost of illustrations above $10 was charged
to the author. These limitations are no longer exactly exercised though,
naturally, longer papers must be discouraged if opportunities for pub-
lication are to be widely distributed. The criterion is that no paper
should be longer than is necessary to adequately present its contribution
and short and long papers alike should be scrutinized from this point of
view. This is sometimes an unwelcome task, but on the whole, I have
found our contributors uncommonly cooperative and goodnatured.
They can usually be made to appreciate that concise presentation is read
with understanding. It is no longer the practice to charge authors for a
part of the cost of necessary illustration provided they are content with
line cuts and halftones and comply with the general principles discussed
above.
The prospectus closes :
'The Bulletin will undoubtedly meet a real need; but the responsi-
bility for its success rests with American biologists, and the editors,
therefore, confidently appeal to them for their support. This can most
practically be given in the two forms of subscriptions and contributions
to its pages." The need for the Bulletin and its success, as well as the
support which it has received from American biologists, is amply attested
by its contents. The Index which was published at the completion of
Volume LX listed approximately 1200 titles. An index which is now
being prepared of the last 20 volumes records an additional 660 titles of
original articles and 652 titles of abstracts, making a total of some 2500
contributions. An examination of the original articles appearing be-
tween 1930-1940 showed that 40 per cent of these were written by mem-
bers of the Corporation. The list of Corporation members contains very
few productive workers in fields appropriate to the Bulletin who are not
contributing to its pages.
On the financial side, the Bulletin is supported in three ways. Of
1100 subscriptions, 300 go to members of the Corporation in return for
their membership fees. The remaining subscriptions are divided about
equally between paid subscriptions from libraries and exchanges. The
treasurer's reports show that with this support the Bulletin just about
REPORT OF THE DIRECTOR 17
breaks even or sometimes shows a small profit. This is due in a sense
to the accountant's art and requires a word of explanation. The income
from exchanges represents a transfer of Library funds to the Bulletin
in payment for issues used to secure exchanges. Thus, in a sense, the
Library helps to subsidize the Bulletin and the sum involved does not
represent cash income. In return, however, the Library receives 656
items in exchange, or approximately one-half of its current list of
periodicals. It is this fortunate association of the Bulletin with the Li-
brary which enables it to make ends meet. The large number of ex-
changes greatly widens the distribution of the papers published in the
Bulletin.
Respectfully submitted,
ALFRED C. REDFIELD,
Managing Editor.
VII. THE REPORT OF THE DIRECTOR
To THE TRUSTEES OF THE MARINE BIOLOGICAL LABORATORY :
Gentlemen: I present herewith a report of the fifty-third session of
the Marine Biological Laboratory, for the year 1940.
1. Present Conditions. The grievous conditions which are now dis-
turbing the world have thus far affected but little the activities of this
Laboratory. In the season just past investigators occupied almost all
of the available space, and students filled the courses. The attendance
was practically equal to that of 1937, the largest in the history of the
institution. Except for an occasional lack of current foreign journals,
and of scientific apparatus usually purchased abroad, we continued our
work in 1940 without serious difficulty. This fortunate state of affairs
cannot be expected to continue. Evidences of the change are already
apparent. During the season of 1941 several of our investigators will
give up their usual lines of research and will work at home on problems
connected with national defense, while others will carry on similar re-
search at Woods Hole. Apparatus, formerly obtained without delay, is
now difficult if not impossible to secure. Thus far, the Government has
made no request for space and facilities to be devoted to defense meas-
ures. We shall continue our usual activities of instruction and research
so long as no emergency arises which might temporarily interrupt them.
2. The Library Addition. We may congratulate ourselves that in
spite of the uncertainties which have surrounded us during the past year,
we have completed one part of the extensive building program which was
18 MARINE BIOLOGICAL LABORATORY
discussed at the last Trustees' meeting. The generosity of the Rocke-
feller Foundation made possible the immediate erection of the much-
needed extension of our Library. It will be recalled that, under the
terms of the gift of $110,400, the Laboratory accepted the responsibility
of obtaining from other sources at least $25,000 to be used primarily
for filling gaps in the present files of journals. A most liberal inter-
pretation of these terms permitted us to proceed with actual construction
before this sum was in hand.
Work was begun in August, 1CHO under the direction of the archi-
tect, Mr. F. V. Bulfinch. On his advice, the Executive Committee
voted to give the contract for the building to the Sawyer Construction
Company of Boston, without waiting for bids to be submitted by other
companies. The wisdom of this action was at once apparent, for exca-
vation began immediately, even before the architects' plans were fully
completed. The structural steel and other essential materials were ob-
tained quickly and without difficulty. Had we waited six weeks or
more for competitive bidding to be completed, it is very probable that
their delivery would have been greatly delayed, if not indefinitely post-
poned. The construction company awarded contracts, after competitive
bidding, for much of the inside work, such as the plumbing, heating,
electrical installations, and the book stacks. The building was enclosed
before cold weather set in, and was heated satisfactorily by means of a
new oil furnace, a part of the Library project. It may be added that
those parts of the Brick Building where heat was required were main-
tained at a comfortable temperature. The final cost of the building and
its equipment was substantially less than the first estimates of the archi-
tect and the contractor. With the balance we have been able to rebuild
the Eel Pond wall, considerably damaged by the building operations,
install an electric book lift in the shaft provided in the old Library but
never used, pave the road and parking spaces close to the new wing, and
re-grade the lawn.
Those who use the Library will find many improvements over the
old conditions. The journals and books are now spread out over three
floors only, an arrangement which greatly reduces the amount of stair
climbing. In the new wing there are many well-lighted desks, some of
which will be reserved for those who spend all of their time among the
books, and others which will be for general use. In the basement will
be housed, temporarily it is hoped, apparatus for sterilizing glassware,
for distilling water and for other services which heretofore have been
widely scattered throughout the Brick Building. There are also two
dark-rooms.
REPORT OF THE DIRECTOR 19
The space now available will serve the rapidly growing Library for
many years. For this happy situation we are deeply indebted to the
Rockefeller Foundation which now, as in the past, has so greatly fur-
thered the work of the Laboratory. To the Library Committee and
Mrs. Montgomery must be given credit for the planning of the book
stacks, the amply spaced study tables, and other features which all read-
ers will appreciate. Finally, thanks are due to Mr. Bulfinch and Mr.
Sawyer, who have so successfully designed and constructed the building
of which we may well be proud.
We take even greater satisfaction in its contents. The Library is
an indispensable part of our equipment. Although the number of vol-
umes is not large, the selection of books and serials has been made with
such skill that we now have on our shelves practically all of the impor-
tant journals, and we are adding to the number every year. In the An-
nual Report of 1889, a committee consisting of E. S. Minot, W. T. Sedg-
wick, and C. O. Whitman, made a double appeal ". . . first, to the pub-
lic, for $1,500 to complete the more important sets of journals, and
secondly, to all biologists, for reprints of their articles. We do this with
confidence, because we believe that nowhere in this country would such
a library be more useful or valuable than in the Marine Biological Labo-
ratory." At that time the library consisted of 343 volumes, 23 reprints,
and 23 serials, of which many were not current subscriptions. From
this modest beginning it has grown in importance until it is now one of
the most important biological libraries in the world. The increase in
the number of volumes, serials, and reprints during the past twenty
years is shown in the following figures, taken from the Librarian's
reports.
Volumes Serials Separates
1920 10,243 153 8,532
1925 15,000 500 25,000
1930 31,510 1,060 66,231
1935 40,180 1,271 91,641
1940 47,897 1,257 116,305
3. Committee on Instruction. The primary purpose of this Labora-
tory is to encourage research by qualified investigators and to increase
their number by preparing students to undertake original work. To
determine whether the courses of instruction are fulfilling this purpose,
the Committee on Instruction, under the able leadership of Dr. Alice,
has made a careful study of the situation. They were guided in their
deliberations by the following principles adopted by the Executive
Committee.
20 MARINE BIOLOGICAL LABORATORY
1. That instructorships are to be regarded as aids to research.
2. That the duty of the instructors consists of research and teaching,
and that they consequently are to remain in residence for a period
longer than the duration of the course.
3. That instructorships should be distributed widely among American
institutions of learning.
4. That long tenure of instructorships should be discouraged.
5. That it is better to have instructors who are specialists in the courses
in which they teach.
6. That the Director be authorized to appoint a Standing Committee on
Instruction to report to the Executive Committee each year.
The Committee met weekly during the summer, discussing these
problems with the directors of the various courses and with many other
interested investigators. Their general conclusion was that the work
of instruction is on a fairly satisfactory basis. The instructors are ac-
tively engaged in productive research, many of them in the general sub-
jects which they are called upon to teach. Not all, however, are in resi-
dence at Woods Hole for a substantial part of the summer. They are
drawn from 23 colleges and universities, of which 12 are eastern, 5 are
in the midwest, 4 in the south, and one each from the far west and
Canada. The tenure of instructorships averages about 5 years; that of
the directors, excluding their previous service as instructors, about 9
years. The Committee felt that a more frequent change of directors
would be desirable.
The Committee also recommended that in place of Dr. Irving, who
resigned after five successful years as head of the Physiology Course,
Dr. A. K. Parpart be appointed. And furthermore, that Dr. Calkins'
desire to be relieved of the course in Protozoology be granted, and that
this course be discontinued. It was with regret that this action was
taken. Under Dr. Calkins the Protozoology Course has for many years
occupied an outstanding place in our summer work of instruction. A
large proportion of his students, drawn from all parts of the world, have
made significant contributions to biology, a lasting tribute to the training
and inspiration which they received from him. The Committee were
convinced that no one could replace him, and that since there are now
many excellent courses in Protozoology offered in various parts of the
country, the need for continuing such a course here is not imperative.
I wish to express the thanks of the Trustees and Corporation to the
members of this Committee, and to the other Standing Committees who
have during the year coped successfully with many difficult problems.
REPORT OF THE DIRECTOR 21
4. Election of Officers and Trustees. At the meeting of the Cor-
poration held August 13, 1940 the following Trustees were elected Trus-
tees Emeritus :
Caswell Grave, Washington University
Ross G. Harrison, Yale University
C. E. McClung, University of Pennsylvania
The new Trustees elected at that meeting were :
Dugald E. S. Brown, Class of 1942
C. W. Metz, Class of 1944
H. H. Plough, Class of 1944
5. There are appended as parts of this report :
1. Memorials of deceased Trustees.
2. The Staff, 1940.
3. Investigators and Students, 1940.
4. A Tabular View of Attendance, 1936-1940.
5. Subscribing and Cooperating Institutions, 1940.
6. Evening Lectures, 1940.
7. Shorter Scientific Papers, 1940.
8. The General Scientific Meeting, 1940.
9. Members of the Corporation, 1940.
Respectfully submitted,
CHARLES PACKARD,
Director.
1. MEMORIALS OF DECEASED TRUSTEES
MEMORIAL TO DR. M. M. METCALF
BY DR. R. A. BUDINGTON
It is altogether fitting that the Corporation of the Marine Biological
Laboratory, at its annual meetings, should pause to pay such salutation
and honor as it may to those recently removed by death, and who over
many years supported the Laboratory by scientific work, wise counsel,
and energetic endorsement.
Such a Corporation member was Maynard Mayo Metcalf, who died
last April 19th after a very prolonged illness, which began suddenly while
he was at work in this building. His age was seventy-two years.
Dr. Metcalf's chief biological mentors were Prof. Albert A. Wright
at Oberlin (Wright was one of the very early workers at Woods Hole),
and Prof. W. K. Brooks of the Hopkins, under whom he took the
MARINE BIOLOGICAL LABORATORY
doctorate in 1893. His academic appointments as teacher were as or-
ganizer and head of the Department of Zoology at Goucher College,
1893-1906; at Oberlin he reorganized the corresponding department and
directed it from 1906 till 1914; from 1926 till 1933 he was research
associate with rank of Professor at the Johns Hopkins University. Dur-
ing the year 1924-25 he was chairman of the Division of Biology and
Agriculture of the National Research Council, Washington.
Among Metcalf's earliest published studies were some on morpho-
logical and embryological features of Amphineura and gastropods; but
thereafter for several years his attention was given to the morphology,
physiology, phylogeny, and taxonomy of the Tunicata with major em-
phasis on pelagic forms. He presented very comprehensive collections
of these to the National Museum. His third and most arduous series of
studies dealt with the morphology, taxonomy, and cytology of the Opa-
linidae; these led him to far-reaching analysis of specific host-parasite
relations, with deductions therefrom as to the ancient distribution of
Amphibia, as well as to evidences of former land connections between
now-separated continents.
All his life an outstanding charaacteristic of Metcalf, which should
be mentioned in any summary of his scientific work, was that of giving
credit to collaborators. Especially in his later years was assistance neces-
sary; and all such received appropriate acknowledgment in the publica-
tions involved.
Metcalf's publications include: papers exceeding 120 in number; a
book, "Organic Evolution" (Macmillan) ; and three large monographic
volumes on the opalinids. The most recent of these was issued by the
Smithsonian Institution as a Bulletin of the National Museum last spring.
He was elected to membership in 28 American, 3 British, and 3
French learned societies, and was a member of the Authors Club,
London. For 45 years he was a summer frequenter of the Woods Hole
Laboratories, and a member of the Board of Trustees of the Marine
Biological Laboratory from 1896 till his death — 44 years. Few men
indeed have been as deeply sincere in their solicitude for and belief in
the functions of this Laboratory as was Maynard Metcalf. Directly or
indirectly he assisted many a student, in financial or other ways, to come
here for study and research ; and mention should here be made of his
gift of his large collection of reprints to our library.
As a man he was chronically of discriminating judgment, positive
opinions, and uncompromising integrity. He was thoroughly human of
the finest grade ; an optimist ; an idealist ; a dispenser of cheer, with rare
generosity of spirit, and capacity for friendship. He will not be for-
gotten.
REPORT OF THE DIRECTOR 23
MEMORIAL TO DR. H. McE. KNOWER
BY DR. Ross G. HARRISON
Henry McElderry Knower died in Baltimore on January 10, 1940,
at the age of 71, after a long and distressing illness, which was borne
with the courage, patience, and good humor that characterized his whole
life.
He was born in Baltimore on August 5, 1868 and was educated in
schools in that city and at the Johns Hopkins University, where he received
the A.B. degree in 1890 and the Ph.D. in 1896. After graduation Knower
held an instructorship for one year at Williams College and subsequently
was for ten years on Doctor Mall's staff in the Department of Anatomy
at the Johns Hopkins Medical School. From there he went to the
University of Toronto as lecturer in 1909 and the following year to the
University of Cincinnati as Professor and Head of the Department of
Anatomy. After his resignation in 1924 he served as a visiting pro-
fessor at the University of Georgia, later as Professor of Anatomy at the
University of Alabama (1927-29) and as Associate Professor of Anat-
omy in the Albany Medical College. His last appointment was that of
Research Associate in Biology in Yale University.
Knower spent the summer of 1896 at the Marine Biological Labora-
tory as an investigator and was elected to membership in the Corpora-
tion. The following year he was on the staff of the Invertebrate course.
In 1908 he became one of the permanent members of the Woods Hole
summer colony and a regular attendant at the Laboratory, until the
failure of his health made that impossible. From 1909 till 1919 he
served as librarian of the Marine Biological Laboratory, and it was
during his administration that the library began its period of rapid
growth. It was well arranged and catalogued, particularly after its
removal to the Crane Building made that possible.
Knower first became interested in the embryology of termites when
in Jamaica as a student. This was the subject of his doctoral disserta-
tion, but on entering the Department of Anatomy at Johns Hopkins in
1899 his interest shifted to the development of the vascular system, the
study of which became his life work. Much of his research in this field
was done at Woods Hole. He developed very delicate methods of in-
jection and accumulated a great collection of exquisitely injected em-
bryos. Fortunately he was able to complete one of the major install-
ments of this work shortly before his death.
His services to scientific publication in this country were unusual.
Through his energy and enterprise the foundation of the American
Journal of Anatomv was greatly hastened, and its establishment came
24 MARINE BIOLOGICAL LABORATORY
just at the time when most needed. Over twenty years of his life were
devoted to this undertaking, which he served with devotion and skill as
Secretary of the Editorial Board. In 1906 he initiated the publication
of the Anatomical Record, first as a supplement to the American Journal
of Anatomy but soon to become an independent journal with its own edi-
torial board.
In all of his relations Knower was steadfast and sincere. He was
good humored, sympathetic with youth and wise in his counsel. Through
his death many of us here have lost a warm, devoted and genial friend.
The Corporation of the Marine Biological Laboratory desire to record
their sorrow at his death, their sense of personal loss, and their apprecia-
tion of his many contributions to biological science and his services to
the institution.
MEMORIAL TO DR. CHARLES ZELENY
BY DR. FERNANDEZ PAYNE
Charles Zelcny. Professor of Zoology at the University of Illinois,
died at his home in Urbana December 21, 1939. He was born at
Hutchinson, Minnesota, September 17, 1878, and spent his early boy-
hood days there. Later his parents moved to Minneapolis where he
entered the University of Minnesota and graduated in 1898. He re-
mained as a graduate and received his M.S. in 1901. The next year he
was a graduate student at Columbia University, working with T. H.
Morgan and E. B. Wilson, and the following year he worked at the
Naples Zoological Station. Returning to America in 1903, he entered
Chicago University where he obtained the Ph.D. in 1904. He came to
Indiana University as an instructor in the summer of 1904. Here he
advanced rapidly and held the rank of Associate Professor at the time
of call to the University of Illinois in 1909. Beginning at Illinois as
aji Assistant Professor, he was promoted the next year to the rank of
Associate Professor and in 1915 to a Professorship. Upon the retire-
ment of Professor H. B. Ward in 1933, he was made head of the De-
partment of Zodlogy and chairman of the Division of Biological Sci-
ences. Because of ill health, he had retired from his executive duties in
1938.
On May 29, 1911, he married Ida Benedicta Ellingson, of St. Morris,
Wisconsin. Mrs. Zeleny and a son, Charles, Jr., survive.
Dr. Zeleny 's family is unique in that three of his brothers are
scientists of note. Anthony Zeleny, now retired, was Professor of
Physics at the University of Minnesota ; John Zeleny is Professor of
Physics at Yale; and Frank Zeleny is an engineer with the Burlington
Railway.
REPORT OF THE DIRECTOR 25
As is true with every great man, chronological facts such as those
enumerated tell but little of the life of Charles Zeleny. They are cold,
external. It was the writer's good fortune to have been a student in
Dr. Zeleny's first class in embryology taught at the Biological Station
in the summer of 1904. For the next three years, our associations were
intimate. We worked together, ate at the same table, played together and
tramped through the woods and fields together. The fact that one was
teacher, the other student entered but little into our thinking. The
friendship formed in those early years remained to the end. As a friend
he was true, somewhat reserved, seldom talked of his own personal af-
fairs, possessed a subtle, sometimes mischievous wit, appreciated by those
who know him best. Seldom did he complain about anything. Bitter-
ness, if present, was kept hidden.
As a teacher he was kind, helpful, encouraging, stimulating. As a
zoologist his papers in the fields of regeneration, experimental embryol-
ogy and genetics speak for themselves. They rank among the best
contributions of his time. Originality in thinking stands out prominently
in all his work.
In recognition of his attainments, he was elected vice-president of
section F of the A. A. A. S. in 1932, and president of the American
Society of Zoologists in 1933.
Dr. Zeleny's death at the early age of 61 years is not only a loss to
his relatives and friends, but to science.
MEMORIAL TO CAPTAIN JOHN J. VEEDKK
BY DR. F. R. LILLIE
John J. Veeder, Captain of the fleet of the Marine Biological Lab-
oratory from 1890 to 1933, was born on the island of Cuttyhunk January
27, 1859. Like all Cuttyhunkers, he was accustomed to the manage-
ment of boats from early years, and acquired a most intimate knowledge
of the shoals, tides, currents and weather conditions of Vineyard Sound
and Buzzards Bay. He married and moved to Woods Hole in 1881.
The Marine Biological Laboratory was founded in 1888, and as Dr.
Bumpus has written me, " The summer of 1890 found the steam launch
" Sagitta " proudly added to the fleet of two old green dories that had
been inherited from the Annisquam Laboratory." It became necessary
to appoint a captain and John J. Veeder was called in for examination
by Dr. Gardiner. He was asked to "box the compass." Dr. Bumpus
relates, " The speed with which he went through the ritual settled the
matter then and there. Captain Veeder was promptly commissioned."
26 MARINE BIOLOGICAL LABORATORY
For a year, until George M. Gray was appointed, Captain Veeder acted
also as collector; and afterwards collaborated closely with the Supply
Department, became thoroughly familiar with the collecting grounds, and
located and set the fish traps of the Laboratory.
Captain Veeder was in charge of the class trips and picnics, and
though many thousands were carried in the years of his service, no one
was ever lost. He was a past master of the technique of the clambakes
which added so greatly to the enjoyment of the picnics. He kept his
eye on the weather and he always veteod a trip if his extraordinary
weather sense and wisdom warned him that the trip would be dangerous.
I cannot say how many times he came to the rescue of our amateur
sailors in distress, when marooned by bad weather or ignorance of tidal
currents ; and very frequently he and the crew went to the aid of small
craft grounded on shoals in the Hole or near the harbor.
He had the good old Cape Cod dignity and self-respect; he was a
shrewd judge of men in all walks of life, and met all on an equal basis.
He never regarded his position merely as a job; whatever was " for the
good of the Laboratory," as he used to say, was always cheerfully and
skilfully performed. He acted as interpreter of the Laboratory to the
town folk or in town meetings, and was helpful in maintaining the good
relations which we have always valued.
2. THE STAFF, 1940
CHARLES PACKARD, Associate Director, Assistant Professor of Zoology,
Institute of Cancer Research, Columbia University.
ZOOLOGY
I. INVESTIGATION
GARY N. CALKINS, Professor of Protozoology, Emeritus, Columbia Uni-
versity.
E. G. CONKLIN, Professor of Zoology, Emeritus, Princeton University.
CASWELL GRAVE, Professor of Zoology, Emeritus, Washington University.
H. S. JENNINGS, Professor of Zoology, University of California.
FRANK R. LILLIE, Professor of Embryology, Emeritus, The University of
Chicago.
C. E. McCLUNG, Professor of Zoology, Emeritus, University of Pennsyl-
vania.
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.
LORANDE L. WOODRUFF, Professor of Protozoology, Yale University.
REPORT OF THE DIRECTOR 27
II. INSTRUCTION
T. H. BISSONNETTE, Professor of Biology, Trinity College.
P. S. CROWELL, JR., Instructor in Zoology, Miami University.
A. M. LUCAS, Associate Professor of Zoology, Iowa State College.
W. E. MARTIN, Assistant Professor of Zoology, DePauw University.
S. A. MATTHEWS, Assistant Professor of Biology, Williams College.
J. S. RANKIN, JR., Instructor in Biology, Amherst College.
A. J. WATERMAN, Assistant Professor of Biology, Williams College.
JUNIOR INSTRUCTORS
E. R. JONES, JR., Professor of Biology, College of William and Mary.
N. T. MATTOX, Instructor in Zoology, Miami University.
PROTOZOOLOGY
I. INVESTIGATION
/
(See Zoology)
II. INSTRUCTION
GARY N. CALKINS, Professor of Protozoology, Columbia University.
VIRGINIA DEWEY, Assistant in Zoology, Vassar College.
G. W. KIDDER, Assistant Professor of Biology, Brown University.
EMBRYOLOGY
I. INVESTIGATION
(See Zoology)
II. INSTRUCTION
HUBERT B. GOODRICH, Professor of Biology, Wesleyan University.
W. W. BALLARD, Assistant Professor of Biology and Anatomy, Dartmouth
College.
DONALD P. COSTELLO, Assistant Professor of Zoology, University of North
Carolina.
VIKTOR HAMBURGER, Assistant Professor of Zoology, Washington Uni-
versity.
OSCAR SCHOTTE, Associate Professor of Biology, Amherst College.
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. GARREY, Professor of Physiology, Vanderbilt University Medical
School.
M. H. JACOBS, Professor of Physiology, University of Pennsylvania.
RALPH S. LILLIE, Professor of General Physiology, The University of
Chicago.
ALBERT P. MATHEWS, Professor of Biochemistry, University of Cincinnati.
MARINE BIOLOGICAL LABORATORY
II. INSTRUCTION
Teaching Staff
LAURENCE IRVING, Professor of Biology, Swarthmore College.
ROBERT CHAMBERS, Professor of Biology, New York University.
KENNETH C. FISHER, Assistant Professor of Experimental Biology, Uni-
versity of Toronto.
RUDOLF HOBER, Visiting Professor of Physiology, University of Pennsyl-
vania.
C. LADD PROSSER, Assistant Professor of Zoology, University of Illinois.
JAMES A. SHANNON, Assistant Professor of Physiology, New York Uni-
versity Medical College.
F J. M. SICHEL, Instructor in Physiology, University of Vermont, College
of Medicine.
BOTANY
I. INVESTIGATION
S. C. BROOKS, Professor of Zoology, University of California.
B. M. DUGGAR, Professor of Physiological and Economic Botany, Univer-
sity of Wisconsin.
D. R. GODDARD, Assistant Professor of Botany, University of Rochester.
E. W. SINNOTT, Professor of Botany, Barnard College.
II. INSTRUCTION
WM. RANDOLPH TAYLOR, Professor of Botany, University of Michigan.
B. F. D. RUNK, Instructor in Botany, University of Virginia.
RUFUS H. THOMPSON, Teaching Assistant, Stanford University.
GENERAL OFFICE
F. M. MACNAUGHT, Business Manager.
POLLY L. CROWELL, Assistant.
EDITH BILLINGS, Secretary.
RESEARCH SERVICE AND GENERAL MAINTENANCE
SAMUEL E. POND, Technical Mgr. T. E. LARKIN, Superintendent.
G. FAILLA, X-ray Physicist. LESTER F. Boss, Technician.
ELBERT P. LITTLE, X-ray Technician. W. C. HEMENWAY, Carpenter.
J. D. GRAHAM, Glassblower. R. S. LILJESTRAND
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.
REPORT OF THE DIRECTOR 29
3. INVESTIGATORS AND STUDENTS
Independent Investigators, 1940
ABELL, RICHARD G., Instructor, University of Pennsylvania. School of Medicine.
ABRAMOWITZ, A. A., Research Assistant, Harvard University.
ADAMS, MARK H., Assistant in Chemistry, Rockefeller Institute for Medical Re-
search.
ADDISON, W. H. F., Professor of Normal Histology and Embryology, University
of Pennsylvania, School of Medicine.
ALBAUM, HARRY G., Instructor in Biology, Brooklyn College.
ALEXANDER, LLOYD E., Assistant Professor of Biology, Fisk University.
ALLEE, W. C.. Professor of Zoology, The University of Chicago.
AMBERSON, WILLIAM R., Professor of Physiology, University of Maryland, School
of Medicine.
ANDERSCH, MARIE, Associate Professor, Woman's Medical College of Pennsyl-
vania.
ANDERSON, RUBERT S., Biophysicist, Memorial Hospital, New York City.
ANGERER, CLIFFORD A., Instructor in Physiology, Ohio State University.
ARMSTRONG, PHILIP B., Professor of Anatomy, Syracuse University, College of
Medicine.
AVERY, GEORGE S., Professor of Botany and Director of the Connecticut Arbore-
tum, Connecticut College.
BAKER, HORACE B., Professor of Zoology, University of Pennsylvania.
BALLENTINE, ROBERT, Research Fellow, Princeton University.
BALL, ERIC G., Associate in Physiological Chemistry, Johns Hopkins University,
School of Medicine.
BALLARD, W. W., Assistant Professor of Biology and Anatomy, Dartmouth College.
BARRINGTON, E. J. W., Lecturer in Zoology, University College, Nottingham,
England.
BARTH, L. G., Assistant Professor of Zoology, Columbia University.
BARTLETT, JAMES H., JR., Associate Professor of Theoretical Physics, University
of Illinois.
BISSONNETTE, T. HUME, Professor of Biology, Trinity College.
BLINKS, L. R., Professor of Biology, Stanford University.
BLISS, A. F., Assistant in Biophysics, Columbia University.
BLOCK, ROBERT, Research Assistant, Osborn Botanical Laboratory, Yale University.
BODIAN, DAVID,. Fellow in Anatomy, Johns Hopkins University, School of Medi-
cine.
BOELL, EDGAR J., Instructor in Zoology, Yale University.
BOTSFORD, E. FRANCES, Assistant Professor of Zoology, Connecticut College.
BOWEN, WILLIAM J., Instructor, Johns Hopkins University.
BRADLEY, HAROLD C., Professor of Physiological Chemistry, University of Wis-
consin.
BRILL, EDMUND R., Graduate Student of Biology, Harvard University.
BRONFENBRENNER, J. J., Professor of Bacteriology and Immunology, Washington
University, School of Medicine.
BRONK, D. W., Director, Johnson Research Foundation.
BROOKS, MATILDA M., Research Associate in Biology, University of California.
BROOKS, S. C., Professor of Zoology, University of California.
BROWN, DUGALD E. S., Assistant Professor of Physiology, New York University.
BROWN, FRANK A., JR., Associate Professor of Zoology, Northwestern University.
BRUCKE, ERNEST VON, Research Associate, Harvard University Medical School.
BUCHSBAUM, RALPH, Instructor in Zoology, The University of Chicago.
BUCK, JOHN B., Instructor, University of Rochester.
BUDINGTON, R. A., Professor of Zoology, Oberlin College.
30 MARINE BIOLOGICAL LABORATORY
BULLOCK, THEODORE H., Sterling Fellow, Yale University.
CABLE, RAYMOND M., Associate Professor of Parasitology, Purdue University.
CALKINS, GARY N., Professor Emeritus of Protozoology, Columbia University.
CAROTHERS, E. ELEANOR, Research Associate in Zoology, University of Iowa.
CHAMBERS, EDWARD, Medical Student, Washington Square College, New York
University.
CHAMBERS, ROBERT, Research Professor of Biology, Washington Square College,
New York University.
CHENEY, RALPH H., Chairman, Biology Department, Long Island University.
CHURNEY, LEON, Harrison Research Fellow, University of Pennsylvania.
CLAFF, C. LLOYD, Research Associate in Biology, Brown University.
CLARK, ELEANOR LINTON, University of Pennsylvania.
CLARK, ELIOT R., Professor of Anatomy, University of Pennsylvania, School of
Medicine.
CLARK, LEONARD B., Assistant Professor of Biology, Union College.
CLEMENT, ANTHONY C., Assistant Professor of Biology, College of Charleston.
CLOWES, G. H. A., Director of Research, Eli Lilly and Company.
COLE, KENNETH S., Associate Professor of Physiology, Columbia University.
COLWIN, ARTHUR L., Instructor, Queens College.
COMMONER, BARRY, Tutor in Biology, Queens College.
COOPER, KENNETH W., Instructor, Princeton University.
COPELAND, D. EUGENE, Assistant in Biology, Harvard University.
COPELAND, MANTON, Professor of Biology, Bowdoin College.
CORI, CARL F., Professor of Pharmacology, Washington University, School of
Medicine.
CORI, GERTY T., Research Associate, Washington University, School of Medicine.
CORNMAN, IVOR, Teaching Fellow, Washington Square College, New York Uni-
versity.
COSTELLO, DONALD P., Assistant Professor of Zoology, University of North Caro-
lina.
Cox, EDWARD H., Professor of Chemistry, Swarthmore College.
CRAMPTON, HENRY E., Professor of Zoology, Barnard College, Columbia Uni-
versity.
CROASDALE, HANNAH T., Technical Assistant, Dartmouth College.
CROUSE, HELEN V., Fellow in Zoology, University of Missouri.
CROWELL, SEARS, Assistant Professor of Zoology, Miami University.
CURTIS, W. C., Professor of Zoology, University of Missouri.
DILLER, IRENE C., Research Associate, University of Pennsylvania.
DILLER, WILLIAM F., Assistant Professor, University of Pennsylvania.
DONNELLON, JAMES A., Assistant Professor of Biology, Villanova College.
DOYLE, WILLIAM L., Assistant Professor of Biology, Bryn Mawr College.
DuBois, EUGENE F., Professor of Medicine, Cornell University Medical College.
DURYEE, WILLIAM R., Visiting Assistant Professor of Biology, Washington Square-
College, New York University.
EVANS, GERTRUDE, Instructor, Beloit College.
EVANS, LLEWELLYN THOMAS, Assistant Professor of Zoology, University of
Missouri.
EVANS, TITUS C., Research Assistant Professor, State University of Iowa.
FAILLA, G., Physicist, Memorial Hospital, New York City.
FISHER, KENNETH C., Assistant Professor of Experimental Biology, University
of Toronto.
FRIES, E. F. B., Assistant Professor, College of the City of New York.
FRISCH, JOHN A., Professor of Biology, Canisius College.
GARREY, W. E., Professor of Physiology, Vanderbilt University, School of Medi-
cine.
GIESE, ARTHUR C., Rockefeller Foundation Fellow, Stanford University.
REPORT OF THE DIRECTOR 31
GOODRICH, H. B., Professor of Biology, Wesleyan University.
GRAEF, IRVING, Associate Professor of Pathology, New York University, College
of Medicine.
GRANICK, SAM, Assistant, Rockefeller Institute for Medical Research.
GRANT, RONALD, Lecturer in Zoology, McGill University.
GRAVE, CASWELL, Professor of Zoology, Washington University.
GRIFFITHS, RAYMOND B., Instructor, Princeton University.
GUTTMAN, RITA, Tutor in Physiology, Brooklyn College.
HAMBURGER, VIKTOR, Associate Professor, Washington University.
HARNLY, MORRIS H., Associate Professor of Biology, Washington Square College,
New York University.
HARRISON, JOHN E., Research Associate, State University of Iowa.
HARTMAN, FRANK A., Professor of Physiology and Chairman of Department,
Ohio State University.
HARVEY, ETHEL B.. Research Investigator, Princeton University.
HARVEY, E. NEWTON, Henry Fairfield Osborn Professor of Physiology, Princeton
University.
HAUGAARD, G., Assistant, Carlsberg Laboratory, Denmark.
HAYWOOD, CHARLOTTE, Associate Professor of Physiology, Mount Holyoke College.
HEILBRUNN, L. V., Associate Professor of Zoology, University of Pennsylvania.
HIBBARD, HOPE, Professor, Oberlin College.
HICKSON, ANNA KELTCH, Research Chemist, Eli Lilly and Company.
HIESTAND, WILLIAM A., Associate Professor of Physiology, Purdue University.
HOBER, RUDOLF, Visiting Professor of Physiology, University of Pennsylvania,
School of Medicine.
HODGE, CHARLES, 4TH, Assistant Professor, Temple University.
HOWE, H. E., Editor, Industrial and Engineering Chemistry, Washington, D. C.
HUNNINEN, ARNE V., Professor of Biology, Oklahoma City University.
HUNTER, LAURA N., Assistant Professor, Pennsylvania College for Women.
IRVING, LAURENCE, Professor of Biology, Swarthmore College.
JACOBS, MERKEL H., Professor of General Physiology, University of Pennsylvania.
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., Professor of Biology, College of William and Mary.
KABAT, ELVTN A., Instructor in Pathology, Cornell University Medical College.
KAISER, SAMUEL, Instructor, Brooklyn College.
KALCKAR, H. M., Rockefeller Research Fellow, Institute of Medical Physiology,
University of Copenhagen.
KATZIN, LEONARD L, Research Worker, University of California.
KAYLOR, CORNELIUS T., Instructor in Anatomy, Syracuse University, College of
Medicine.
KIDDER, GEORGE W., Assistant Professor of Biology, Brown University.
KINDRED, JAMES E., Professor of Anatomy, University of Virginia.
KNOWLTON, FRANK P., Professor of Physiology, Syracuse University, College of
Medicine.
KOPAC, M. J., Visiting Assistant Professor of Biology, Washington Square Col-
lege, New York University.
KORR, IRVTN M., Instructor in Physiology, New York University, College of
Medicine.
KRAHL, M. E., Research Chemist, Eli Lilly and Company.
KRAATZ, C. P., Instructor in Physiology and Pharmacy, The University of Chicago
Medical School.
KRIETE, BERTRAND C., Graduate Assistant, University of Cincinnati.
KUNITZ, MOSES, Associate Member, Rockefeller Institute for Medical Research.
LANCEFIELD. DONALD E. Associate Professor of Biology, Queens College.
MARINE BIOLOGICAL LABORATORY
LEUCHTENBERGER, CECILIE, Assistant in Pathology, Mount Sinai Hospital.
LEUCHTENBERGER, RUDOLF, Assistant in Pathology, Mount Sinai Hospital.
LILLIE, FRANK R., Professor of Embryology, Emeritus, The University of Chicago.
LILLIE, RALPH S., Professor of General Physiology, The University of Chicago.
LLOYD, DAVID P. C., Assistant in Physiology, Rockefeller Institute.
LOEWI, OTTO, Research Professor in Pharmacology, New York University, College
of Medicine.
LOVELESS, MARY H., Instructor, Cornell University Medical College.
LUCAS, ALFRED M., Associate Professor, Iowa State College.
LUCRE, BALDUIN, Professor of Pathology, University of Pennsylvania, School of
Medicine.
LUDWIG, FRANCIS W., Villanova College.
LYNN, WILLIAM G., Fellow of the Rockefeller Foundation, Osborn Zoological
Laboratory, Yale University.
MACKNIGHT, ROBERT H., Instructor, Northwestern University.
McCLUNG, C. E., Director, Zoological Laboratory, University of Pennsylvania.
MCDONALD, MARGARET R., Fellow, Rockefeller Institute for Medical Research.
MARRAZZI, AMEDEO S., Assistant Professor of Pharmacology, New York Univer-
sity, College of Medicine.
M AKSLAND, DOUGLAS A., Assistant Professor of Biology, Washington Square
College, New York University.
MARTIN, WALTER E., Assistant Professor of Zoology, DePamv University.
MAST, S. O., Professor of Zoology, Johns Hopkins University.
MATHEWS, ALBERT P., Professor Emeritus, Biochemistry, University of Cincinnati.
MATTHEWS, SAMUEL A., Assistant Professor, Williams College.
MATTOX, N. T., Instructor in Zoology, Miami University.
MAYOR, JAMES W., Professor of Biology, Union College.
MAZIA, DANIEL, Assistant Professor of Zoology, University of Missouri.
MEGLITSCH, PAUL A., Instructor, Wright Junior College.
MELNICK, JOSEPH L., Finney-Howell Research Foundation Fellow, Yale University.
MENKIN, VALY, Instructor in Pathology, Harvard University Medical School.
MICHAELIS, LEONOR, Member, Rockefeller Institute.
MILLER, JAMES A., Instructor in Anatomy, University of Michigan.
MILLER, RUTH N., Associate Professor of Anatomy, Woman's Medical College of
Pennsylvania.
MILLIKAN, GLENN A., Assistant Professor, Cornell University Medical College.
MORGAN, ISABEL M., Rockefeller Institute.
MORGAN, LILIAN V., Pasadena, California.
MORGAN, T. H., Professor of Biology, California Institute of Technology.
MORRILL, C. V., Associate Professor of Anatomy, Cornell University Medical
College.
MOSER, FLOYD, Research Associate, University of Pennsylvania.
NACHMANSOHN, DAVID, Research Fellow, Laboratory of Physiology, Yale Uni-
versity Medical School.
NASH, CARROLL B., Instructor in Zoology, University of Arizona.
NAVEZ, ALBERT E., Science Department, Milton Academy.
NONIDEZ, Josi1 F., Professor of Anatomy, Cornell University Medical College.
NORTHROP, JOHN H., Member, Rockefeller Institute for Medical Research.
O'BRIEN, JOHN P., Graduate Student, Johns Hopkins University.
OLSON, MAGNUS, Instructor in Zoology, University of Minnesota.
ORR, PAUL R., Assistant Professor, Brooklyn College.
OSTERHOUT, Wr. J. V., Member Emeritus, Rockefeller Institute for Medical Re-
search.
OXFORD, ALBERT E., Rockefeller Foundation Fellow in Biochemistry, Rockefeller
Foundation.
PACKARD, CHARLES, Assistant Professor, Cancer Research, Columbia University.
REPORT OF THE DIRECTOR 33
PARK, THOMAS, Assistant Professor of Zoology, The University of Chicago.
PARKER, ALICE E., Instructor in Anatomy, Child Research Council, and University
of Colorado Medical School.
PARKER, G. H., Professor of Zoology, Emeritus, Harvard University.
PARMENTER, CHARLES L., Professor, University of Pennsylvania.
PATRICK, RUTH, Associate Curator, Academy of Natural Sciences.
PERLMANN, GERTRUDE E., Research Assistant, Harvard University Medical School.
PERROT, MAX, Visiting Fellow, Princeton University.
PIRENNE, MAURICE H., Belgian American Educational Foundation.
PLOUGH, HAROLD H., Professor of Biology, Amherst College.
POND, SAMUEL E., Technical Manager, Marine Biological Laboratory.
PRICE, DOROTHY, Research Associate in Zoology, The University of Chicago.
PROSSER, C. LADD, Assistant Professor of Zoology, University of Illinois.
RABINOWITCH, E., Research Associate, Massachusetts Institute of Technology.
RANKIN, JOHN S., JR., Instructor in Biology, Amherst College.
RAY, O. M., Instructor in Physiology, North Dakota Agricultural College.
Ris, HANS, Assistant in Zoology, Columbia University.
ROGERS, CHARLES G., Professor of Comparative Physiology, Oberlin College.
ROOT, CLINTON W., Assistant Professor of Zoology, University of Syracuse.
ROSENE, HILDA F., Assistant Professor of Physiology, University of Texas.
ROSE, S. MERYL, Assistant, Columbia University.
Rous, PEYTON, Member, Rockefeller Institute for Medical Research.
RUEBUSH, TRENTON K., Instructor, Yale University.
RUGH, ROBERTS, Associate Professor, Washington Square College, New York
University.
RUNK, B. F. D., Instructor in Biology, University of Virginia.
RUSSELL, ALICE M., Instructor in Zoology, University of Pennsylvania.
SALOMON, KURT, Research Fellow, Yale University Medical School.
SAYLES, LEONARD P., Assistant Professor, College of the City of New York.
SCHAEFKER, A. A., Professor, Temple University.
SCHAEFFER, MoKKis, Research Associate, Bureau of Laboratories, New York I ><
partment of Health.
SCHARREK, BKKTA, Voluntary Research Worker, Rockefeller Institute.
SCHARREK, ERNST, Fellow, Rockefeller Institute.
SCHECHTER, VICTOR, Instructor, College of the City of New York.
SCHOLANDER, P. F., Rockefeller Fellow, Swarthmore College.
SCHOTTE, OSCAR E., Associate Professor of Biology, Amherst College.
SCHRAM, MILDRED W. S., Secretary, International Cancer Research Foundation.
SI'OTT. ALLAN C., Assistant Professor of Biology, Union College.
SKI.SAM, MILLICKNT E., Columbia University.
SHANNON, JAMES A., Assistant Professor of Physiology, New York University
College of Medicine.
SHAPIRO, HERBERT, Instructor in Physiology, Vassar College.
SHAW. MMOI.K, Senior Bacteriologist, New York State Department of Health.
SICHEL, ELSA KEIL, Head of the Science Department, Vermont State Normal
School.
SICHEL, F. J. M., Assistant Professor of Physiology. L'liiversity of Vermont,
College of Medicine.
SLIFER, ELEANOR H., Assistant Professor, State University of Iowa.
SMITH, CARL C., Research Associate in Medicine, University of Cincinnati.
SMITH, JAY A., Head of Biology Department, Springfield College.
SMITH, MARSHALL E., Student, Johns Hopkins University, School of Medicine.
SPKIDEL, CARL C., Professor of Anatomy, University of Virginia.
STEINBACH, HENRY B., Assistant Professor of Zoology, Columbia University.
STERN, KURT G., Research Assistant Professor, Yale University, School of Medi-
cine.
34 MARINE BIOLOGICAL LABORATORY
STILWELL, E. FRANCES, Instructor, Woman's Medical College of Pennsylvania.
STOREY, ALMA G., Professor of Botany, Mount Holyoke College.
STUNKARD, HORACE W., Professor of Biology and Head of Department, New York
University.
SUMMERS, FRANCIS M., Instructor, College of the City of New York.
TASHIRO, SHIRO, Professor of Biochemistry, University of Cincinnati.
TAYLOR, WM. RANDOLPH, Professor of Botany, University of Michigan.
THOMPSON, RUFUS H., Teaching Assistant, Stanford University.
TORDA, CLARA, Research Worker, Johnson Foundation.
TOWNSEND, GRACE, Professor of Biology, Great Falls Normal College.
TUCKER, GORDON H., Instructor, Duke University.
TURNER, C. L., Professor of Zoology, Northwestern University.
VENNESLAND, BIRGIT, Research Fellow, Harvard University Medical School.
WATERMAN, A. J., Assistant Professor, Williams College.
WEISS, PAUL A., Associate Professor of Zoology, The University of Chicago.
WENRICH, D. H., Professor of Zoology, University of Pennsylvania.
WHALEY, W. GORDON, Instructor, Columbia University.
WHITAKER, D. M., Professor of Biology, Stanford University.
WHITING, ANNA R., Guest Investigator, University of Pennsylvania.
WHITING, P. W., Associate Professor of Zoology, University of Pennsylvania.
WICHTERMAN, RALPH, Assistant Professor of Biology, Temple University.
WILBUR, KARL M., Instructor, University of Pennsylvania.
WILLIER, B. H., Chairman. Division of Biological Sciences, University of Rochester.
WOLF, E. ALFRED, Associate Professor of Biology, University of Pittsburgh.
WOLF, OPAL M., Assistant Professor, Goucher College.
WOLFSON, CHARLES, Instructor in Anatomy, University of Kansas.
WOODRUFF, LORANDE L., Professor of Protozoology and Director of the Osborn
Zoological Laboratory, Yale University.
WRINCH, DOROTHY, Member of Chemical Faculty, Johns Hopkins University.
YOUNG, ROGER A., Graduate Student, University of Pennsylvania.
ZWILLING, EDGAR, Teaching Assistant, Columbia University.
Beginning Investigators, 1940
ALSUP, FRED W., Graduate Student, University of Pennsylvania.
ARENA, JULIO F. DE LA, Latin American Fellowship, John Simon Guggenheim
Foundation.
BARNES, MARTHA R., Assistant in Zoology, University of Illinois.
BLOCK, EDWARD H., The University of Chicago.
BOUGIOVANNI, ALFRED, Graduate Student, Villanova College.
BROOMALL, ANNABELLE, Graduate Student, University of Pittsburgh.
CARSON, HAMPTON L., JR., Instructor in Zoology, University of Pennsylvania.
CASS, RUTH E., Instructor in Biology, Russell Sage College.
Ciu, RUTH E., Student of Graduate School, University of Michigan.
COBB, SIDNEY, Student, Harvard University Medical School.
COMPTON, ALFRED D., JR., Master in Biology, The Choate School.
COOPER, RUTH EDNA SNYDER, Princeton University.
CUNNINGHAM, ONA, Northwestern University.
DEAN, PETER M., Princeton University.
DsLiEE, ELVIRA, Fellow in Medicine, New York University, College of Medicine.
DENT, JAMES N., Graduate Student, Johns Hopkins University.
DIENNES, PRISCILLA, Student, Yale University, School of Medicine.
DONOVAN, MARY K., Villanova College.
DOWLING, DELPHINE L., Instructor in Botany, Vassar College.
DRESSLER, ELSIE L., Graduate Student in Genetics, University of Pittsburgh.
EDGERLEY, ROBERT H., Graduate Assistant, Ohio State University.
REPORT OF THE DIRECTOR 35
EVANS, DAVID, Assistant Professor, University of Mississippi.
EVERETT, GUY M., Graduate Student, University of Maryland, School of Medicine.
FRANCIS, M. CATHERINE, Instructor, Hallahan High School.
FRANK, SYLVIA R., Graduate Resident Scholar, Columbia University.
GABRIEL, MORDECAI L., Assistant in Zoology, Columbia University.
GAYER, H. KENNETH, Graduate Assistant, Washington University.
GIDDINGS, C. BLAND, Graduate Student Assistant, University of Cincinnati, College
of Medicine.
GILBERT, WILLIAM J., Graduate Assistant in Department of Botany, University of
Michigan.
CLANCY, ETHEL A., Tutor, Queens College.
GOLDIN, ABRAHAM, Graduate Student, Columbia University.
GOULDING, HELEN J., Graduate Research Student, University of Toronto.
HARRIS, DANIEL L., Instructor, University of Pennsylvania.
HEATH, JAMES P., Student and Teaching Assistant, Stanford University.
HENDLEY, CHARLES D., Assistant in Zoology, Columbia University.
HENSON, MARGARET, Teaching Fellow in Biology, Washington Square College,
New York University.
HERGET, CARL M., Research Fellow, Russell Sage Institute of Pathology.
HINCHEY, M. CATHERINE, Graduate Student, University of Pennsylvania.
HOBSON, LAWRENCE B., 5423 Woodlawn Avenue, Chicago, Illinois.
HOLZ, A. MARIE, Graduate Student, Columbia University.
JENKINS, DALE W., Ridgway Fellow, The University of Chicago.
JOSEPH, M., Nativity High School, Scranton, Pennsylvania.
LAWRENCE, MARIA, Graduate Student, Villanova College.
LUCKMAN, CYRIL E., Graduate Student, University of Pennsylvania.
MACHAFFIE, R., Graduate Student, Columbia University.
MOLTER, JOHN A., University of Pennsylvania.
MOOG, FLORENCE, Instructor, University of Delaware.
NACE, PAUL, Student, Columbia University.
NETSKY, MARTIN, Medical Student, University of Pennsylvania.
PIERSON, BERNICE F., Graduate Student, Johns Hopkins University.
RYAN, ELIZABETH J., Assistant in Zoology. Columbia University.
RYAN, FRANCIS J., Assistant in Zoology, Columbia University.
SAMORODIN, ALBERT J., Graduate, University of Minnesota.
SHERMAN, FRED G., Laboratory Assistant, Northwestern University.
SNEDECOR, JAMES, Graduate Assistant, Indiana University.
TERRY, ROBERT L., Graduate Student in Zoology, University of Pennsylvania.
THIVY, MRS. FRANCESCA, Post-graduate Student, University of Michigan.
WHITELEY, ARTHUR H., Graduate Student, University of California.
WIERCINSKI, FLOYD J., Graduate Student, University of Pennsylvania.
WILDE, CHARLES E., JR., Dartmouth College.
WILLIAMS, J. LECOQ, Graduate Assistant, New York University.
ZORZOLI, ANITA, Graduate Student, Columbia University.
Research Assistants, 1940
ALLEY, ARMINE, Demonstrator, McGill University.
ARMSTRONG, CHARLES W. J., Demonstrator in Biology, University of Toronto.
ARMSTRONG, MARY, Milton Academy.
BADGER, ELIZABETH, Research Assistant, University of Cincinnati.
BAKER, LINVILLE A., Research Assistant, Eli Lilly and Company.
BAKER, RICHARD F., Research Associate, Columbia University.
BELFER, SAMUEL, Research Assistant, University of Wisconsin.
BENEDICT, DORA, Milton Academy.
BOWSER, E. R., JR., Student, University of Pittsburgh.
36 MARINE BIOLOGICAL LABORATORY
BRINK, FRANK, JR., Research Assistant, Johnson Research Foundation.
BROUNELL, KATHARINE A., Research Assistant, Ohio State University.
BURT, RICHARD L., Graduate Assistant, Brown University.
BUTLER, PHILIP A., Assistant, Northwestern University.
CALABRISI, PAUL, Instructor in Anatomy, George Washington Medical School.
CARDIFF, MARGARET, Assistant, Swarthmore College.
COHEN, IRVING, Research Assistant, Washington Square College, New York Uni-
versity.
CRAWFORD, JOHN D., Milton Academy.
CURTIS, HOWARD J., Rockefeller Fellow, Columbia University.
DERINGER, MARGARET K., Student, Johns Hopkins University.
DEWEY, VIRGINIA C., Graduate Student. Brown University.
DuBois, ARTHUR, Milton Academy.
DYTCHE, MARYON M., Graduate Assistant, University of Pittsburgh.
EDER, HOWARD, Student, Harvard University Medical School.
EGAN, RICHARD W., Undergraduate Assistant, Canisius College.
FERGUSON, FREDERICK P., Graduate Assistant, Wesleyan University.
FINKEL, ASHER J., Research Assistant in Zoology, The University of Chicago.
FRASER, DORIS A., Research Assistant, University of Pennsylvania.
GETTEMANS, JOHN F., Laboratory Assistant, Rockefeller Institute.
GRAHAM, JUDITH E., Graduate Student, The University of Chicago.
GRAND, C. G., Research Associate, Washington Square College, New York Uni-
versity.
GRINNELL, STUART W., Research Associate, Swarthmore College.
GWARTNEY, RICHARD H., DePauw University.
HAYASHI, TERU, Graduate Assistant, University of Missouri.
HEMSTEAD, GEORGE W., Student, Union College.
HERSKOWITZ, IRWIN, Graduate, Brooklyn College.
HOBER, JOSEPHINE, Philadelphia, Pennsylvania.
ITO, TETSUJI, Research Fellow, New York University, College of Medicine.
JACOBS, JOYE, Assistant, University of Maryland, School of Medicine.
JAKUS, MARIE A., Graduate Assistant, Washington University.
JONES, WILLIAM D., Graduate Student, University of Pennsylvania.
KALMANSON, GEORGE M., Research Fellow, Washington University.
KEEFE, EUGENE L., Research Assistant, Washington University.
KLEIN, ETHEL, Research Assistant, University of Pennsylvania.
LEWIS, LENA A., Research Assistant, Ohio State University.
McVAY, JEAN, Assistant, Northwestern University.
MARRAZZI, ROSE, Herter Fellow in Department of Pharmacology, New York Uni-
versity, College of Medicine.
MARTIN, PHYLLIS COOK, Assistant Professor of Biology, Pennsylvania College for
Women.
MARTIN, ROSEMARY D. C., Assistant in Biology, University of Toronto.
MERWIN, RUTH M., Research Assistant in Zoology, University of Chicago.
MEYERHOF, BETTINA, Research Assistant, Johns Hopkins University Medical
School.
MILFORD, JOHN J., Graduate Assistant, New York University.
NEUBECK, CLIFFORD E., University of Pittsburgh.
O'BRIEN, F. DONAL, Canisius College.
O'NEAL, JOHN D., Graduate Student, University of Pittsburgh.
PAPANDREA, D. A., Student, Albany Medical College.
RAMSDELL, PAULINE A., Research Assistant, Johns Hopkins University Medical
School.
RIMMLER, LUDWIG, JR., Research Assistant, Syracuse University, College of Medi-
cine.
ROI.LASOX, H. DUNCAN, JR., Williams College.
REPORT OF THE DIRECTOR 37
RONKIN, RAPHAEL R., Student, University of California.
SCHAEFFER, OLIVE K., Research Assistant.
SHELDEN, FREDERICK F., Instructor in Physiology, Ohio State University.
SKOW, ROYCE K., Research Assistant, Stanford University.
SPRATT, NELSON T., JR., Research Assistant, University of Rochester.
TRINKAUS, J. PHILIP, Assistant, Wesleyan University.
WALTHER, ROWLAND F., Research Assistant, Ohio State University.
\YELLINGTON, DOROTHY, Research/ Assistant, New York University.
WILLIAMSON, ROBERT R., Student, The University of Chicago.
WOODWARD, ARTHUR, JR., Teaching Fellow, New York University.
WORKMAN, GRACE, Research Assistant, University of Toronto.
WULFF, VERNER J., Northwestern University.
ZIMMERMAN, ALICE C, Graduate Student, Brown University.
Students, 1940
BOTANY
ANDERSON, JOE N., Student, University of Michigan.
BROWN, DONALD H., Student, Dartmouth College.
BROWN, DOROTHY M., Science Instructor, St. Luke's Hospital, Xew York City.
BUCHANAN, NATALIE V., Student, Wellesley College.
CAMPBELL, VIRGINIA, Wheaton College.
Ciu, RUTH E., University of Michigan.
MAcCosBE, HENRIETTA E., Instructor in Botany and /<>c">l<>gy, Pennsylvania State
College.
MORGAN, DELBERT T., JR., Kent State University.
SANDERS, JOAN, Smith College.
SILVER, SAMUEL, Graduate Student, College of the City of New York.
EMBRYOLOGY
ALPER, CARL, Student Assistant, Brothers College, Drew University.
ATKINSON, WILLIAM B., Graduate Student, University of Virginia.
BELANGER, LEONARD F., Assistant in Histo-embryology, University of Montreal.
CASS, RUTH E., Instructor, Russell Sage College.
DuBois, REBECKAH, Student, Vassar College.
FETTER, DOROTHY, Instructor, Brooklyn College.
FINCKE, ROBERT T., Graduate Teaching Assistant, Indiana University.
FOULKS, JAMES G., Graduate Teaching Assistant, University of Rochester.
FRIEDMAN, ROBERT S., Graduate Student, Harvard University.
GOLDMAN, PHILIP W., Graduate Student, Harvard University.
HALSTED, GEORGE O., Princeton University.
HARTMANN, J. FRANCIS, Assistant in Histology and Embryology, Cornell Uni-
versity.
HARTUNG, ERNEST W., JR., Harvard University.
HEATH, JAMES P., Stanford University.
HENDERSON, JOHN M., McGill University.
HOPPER, ARTHUR F., JR., Laboratory Assistant, Yale University.
JOHNSON, VIRGIL O., Technician, University of Oklahoma.
JOLLY, MARGIE, DePauw University.
JONES, SARAH R., Graduate Assistant, Connecticut College.
KARELSEN, JUNE VAN RAALTE, Undergraduate, Oberlin College.
KRANTZ, MARION, Student, Bennington College.
LEE, RICHARD E., Harvard University.
LUDWIG, FRANCIS W., Villanova College.
MARINE BIOLOGICAL LABORATORY
MCFARLAND, WILLIAM, Student, Washington and Jefferson College.
MILLER, GERALD, Student, Oberlin College.
NICHOLS, MYRON McCALL, Laboratory Assistant, DePauw University.
POND, SIDNEY M., Wesleyan University.
ROBINSON, EDWIN J., JR., Teaching Fellow, Washington Square College, New
York University.
SAMORODIN, ALBERT J., Graduate, University of Minnesota.
SAWYER, CHARLES H., Assistant in Biology, Yale University.
SHERMAN, FREDERICK G., Laboratory Assistant, Northwestern University.
STEELE, KENNETH C, Dartmouth College.
SWEENY, FRANK P., Amherst College.
YANCEY, MAUDE J., Student, North Carolina College for Negroes.
PHYSIOLOGY
BAYLOR, EDWARD R., Student, University of Illinois.
BLANCHARD, BARBARA D., Teacher, Placer Junior College, California.
CARLEEN, MILDRED H., Graduate Assistant, Mount Holyoke College.
CHIDSEY, JANE L., Assistant Professor, Wheaton College.
DAVIES, PHILIP W., Johnson Foundation Scholar, University of Pennsylvania.
EDGERLEY, ROBERT H., Graduate Assistant, Ohio State University.
EDWARDS, GEORGE A., Graduate Assistant, Tufts College.
EVERETT, GUY M., Graduate Teaching Assistant, University of Maryland Medical
School.
Fox, RUTH P., Assistant, Vassar College.
HENRY, RICHARD J., University of Pennsylvania, School of Medicine.
HOHWIELER, HAROLD J., Graduate Assistant, Washington University.
HOLTON, GEORGE W., Wesleyan University.
JACKSON, BLANCHE E., Fellowship Student, Radcliffe College.
JAKUS, MARIE A., Graduate Assistant, Washington University.
KASSERMAN, WALTER H., Washington and Jefferson College.
NORMAN, GEORGE R., Student, Wabash College.
ORMSBEE, RICHARD A., Graduate Assistant, Brown University.
RATHBUN, EDITH N., 88 Fosdyke Street, Providence, Rhode Island.
SCHOLANDER, PER FREDRiK, Research Associate, University of Oslo.
STOKES, ALLEN W., Harvard University.
WOLF, MARY H., Student, Duke University.
WOODWARD, ARTHUR, JR., Graduate Assistant, Wesleyan University.
PROTOZOOLOGY
BEAM, CARL A., Student, Brown University.
CARROLL, KENNETH M., Student, Franklin and Marshall College.
COSGROVE, WILLIAM B., Student, Cornell University.
DODGE, FRANCES, Student, Gettysburg College.
HARRIGAN, MARY K., Special Instructor in Biology, Simmons College.
MACDONALD, KATHERINE C., Graduate Student, McGill University.
MARCHAND, DORIS, Teacher, St. Catherine's School, Richmond, Virginia.
INVERTEBRATE ZOOLOGY
ADAMS, ESTHER F., Instructor in Biology, Moberly Junior College.
ALLEN, JEAN, Miami University.
BEEMAN, ELIZABETH A., Graduate Assistant in Zoology, Mount Holyoke College.
BERGSTROM, WILLIAM H., Student, Amherst College.
REPORT OF THE DIRECTOR
39
BOVING, BENT G., Assistant, Swarthmore College.
BRUSH, HELEN V., Assistant in Zoology, Vassar College.
BURNS, JOHN E., Graduate Laboratory Assistant, Wesleyan University.
CAIRNS, MALCOLM G., New Jersey State Teachers College, Montclair, New Jersey.
CLARK, ARNOLD M., Student, University of Pennsylvania.
COE, GRACE L., Student, New Jersey State Teachers College, Montclair, New
Jersey.
DENT, JAMES N., Graduate Assistant in Zoology, Johns Hopkins University.
EDWARDS, GENE C, Student, Wabash College.
FITZGERALD, LAURENCE R., State University of Iowa.
GIBBS, ELIZABETH, Undergraduate, Wheaton College.
GOODRICH, MARY W., Student, Wheaton College.
GRAVETT, HOWARD T., Associate Professor of Biology, Elon College.
HALE, BARBARA, Student, Radcliffe College.
HILDEBRANDT, WALLACE H., Undergraduate Instructor, Canisius College.
HOLDSWORTH, ROBERT P., JR., Austin Teaching Fellow, Harvard University.
HORWITZ, DIANA C., Teacher, Hyde Park High School, Hyde Park, Massachusetts.
HOYT, JANE M., Barnard College.
JAMES, MARIAN F., Graduate Fellow, University of Illinois.
KILLOUGH, JOHN H., Graduate Student, Johns Hopkins University.
KLINE, IRENE T., Duke University.
KREEGER, FLORENCE BROOKS, Graduate Assistant in Biology, Newcomb Collar.
LAMOREUX, WELFORD F., Assistant Professor, Cornell University.
LERNER, ELEANOR, Brooklyn College.
LEVITZKY, EDWARD, Student, Rutgers University.
MACRAE, ROBERTA M., Graduate Assistant, Wellesley College.
MCKENZIE, HELEN E., Seton Hill College.
MARBARGER, JOHN P., Graduate Student, Johns Hopkins University.
MEANS, OLIVER W., JR., Yale University.
MICKLEWRIGHT, HELEN L., Student, Wilson College.
MUSSER, RUTH E., Student, Goucher College.
NOCE, MILDRED W., Student, Southwestern College.
POWERS, SAMUEL R., JR., Swarthmore College.
PUTNAM, WILLIAM S., Graduate Assistant, Amherst College.
REEVES, WALTER P., JR., Graduate Student, University of Alabama.
ROYLE, JANE G., Graduate Assistant in Anatomy and Invertebrate Zoology, Bryn
Mawr College.
SAMUELS, ROBERT, University of Pennsylvania.
SAUNDERS, GRACE S., Hunter College.
SCHNABEL, MARGARET J., Student, Oberlin College.
SCOTT, GEORGE T., Assistant, Harvard University.
SHANK, MARGARET L., Student, New Jersey State Teachers College, Montclair.
New Jersey.
SMITH, FERN W., Student, Smith College.
SMITH, FREDERICK E., Massachusetts State College.
SMITH, JULIA P., Student, University of Rochester.
STIFLER, MARGARET C., Assistant, Goucher College.
STONE, FRED L., University of Rochester.
SYNER, JAMES C., Student, Springfield College.
WALKER, WARREN F., JR., Student, Harvard University.
WHEELER, BERNICE M., Instructor, Westbrook Junior College.
WHITE, FRANCIS M., Graduate Assistant in Biology, Purdue University.
WOLAVER, JOHN H., JR., Student, DePauw University.
WRIGHT, MARGARET R., Student, Yale University.
40
MARINE BIOLOGICAL LABORATORY
4. TABULAR VIEW OF ATTENDANCE, 1936-1940
1936 1937 1938 1939 1940
INVESTIGATORS — Total 359 380
Independent . . 226 256 246
Under Instruction 76 53
Research Assistants 57 61 81
STUDENTS — Total 138
Zoology 55 57 54
Protozoology 17 16 10
Embryology 34 35
Physiology 22 16 22
Botany 10 9 12
TOTAL ATTENDANCE 497 524 512
Less Persons registered as both students
and investigators 24 13 1(>
473 511 496
INSTITUTIONS REPRESENTED — Total 158 165 151
By Investigators 120 134 125
By Students 79 67
SCHOOLS AND ACADEMIES REPRESENTED
By Investigators 4
By Students 3 1
FOREIGN INSTITUTIONS REPRESENTED
By Investigators 9 16 14
By Students 5 3
352
213
60
79
133
55
12
36
21
9
485
14
162
132
1
386
253
62
71
128
55
7
34
22
10
514
471 507
148
112
79
1
1
5. SUBSCRIBING AND COOPERATING INSTITUTIONS
1940
Amherst College
Barnard College
Biological Institute, Philadelphia, Penn-
sylvania
Bowdoin College
Brooklyn College
Brown University
Bryn Mawr College
Canisius College
College of Physicians and Surgeons
Columbia University
Cornell University
Cornell University Medical College
DePauw University
Duke University
Fisk University
Goucher College
Harvard University
Harvard University Medical School
Hunter College
Industrial and Engineering Chemistry,
of the American Chemical Society
Johns Hopkins University
Johns Hopkins University Medical
School
Eli Lilly and Company
Long Island University
Massachusetts State College
Memorial Hospital, New York City
Mount Holyoke College
Mount Sinai Hospital, New York City
Newcomb College
New York State Department of Health
New York University
New York University College of Medi-
cine
New York University Washington
Square College
North Carolina College for Negroes
Northwestern University
Oberlin College
Ohio State University
Princeton University
Purdue University
Radcliffe College
Rockefeller Foundation
REPORT OF THE DIRECTOR
41
Rockefeller Institute for Medical Re-
search
Russell Sage College
Rutgers University
Smith College
Springfield College
Stanford University
State University of Iowa
Syracuse University
Syracuse University College of Medi-
cine
Tufts College
Union College
University of Chicago
University of Cincinnati
University of Illinois
University of Kansas
University of Missouri
University of Pennsylvania
University of Pennsylvania School of
Medicine
University of Pittsburgh
University of Rochester
University of Virginia
Vanderbilt University Medical School
Vassar College
Villanova College
Wabash College
Washington University
Washington University Medical School
Wellesley College
Wesleyan University
Wheaton College
Wilson College
Yale University
Yale University Medical School
6. EVENING LECTURES, 1940
Friday, July 5
DR. LEONOR MICHAELIS " Oxidation and Reduction in Organic
and Biological Chemistry."
Friday, July 12
DR. KENNETH V. THIMANN " Hormones and the Physiology of
Growth in Plants."
Friday, July 19
DR. K. S. COLE " Electrical Properties of the Cell
Membrane."
Friday, July 26
DR. D". H. WENKICH " Chromosomes in Proto/oa."
Wednesday, July 31
MR. GEORGE C. LOWER " Local Marine Life in Color."
Friday, August 2
DR. ERIC G. BAM " Catalysts of Biological Oxidations,
their Composition and Mode of
Action."
Thursday, August 8
DR. L. J. MILNE " Animated Diagrams of Biological
Processes."
Friday, August ()
PROF. F. O. SCUM ITT " Modern Concepts of Protoplasmic
Organization."
Friday, August 16
DR. ALFRED S. KOMKK " The Phylogeny and Structure of the
Lower Vertebrates."
Friday, August 23
DR. DUGALD BROWN " The Regulation of Metabolism in
Contracting Muscle."
42 MARINE BIOLOGICAL LABORATORY
Friday, August 30
DR. CURT STERN " On Dependent Growth and Form of
the Testes in Various Species of
Drosophila."
Saturday, August 31
DR. PER HOST " Arctic Seal Hunting in the White
Sea and in Greenland Waters."
7. SHORTER SCIENTIFIC PAPERS, 1940
Tuesday, July 9
MR. EDWARD L. CHAMBERS " Inter-relations between Egg-Nucleus,
Sperm-Nucleus and Cytoplasm of
the Asterias Egg."
DR. DANIEL MAZIA '' Digestion Studies on Salivary Chro-
mosomes."
DR. M. J. KOPAC " Some Properties of the Residue
from Rapidly Disintegrated Ar-
bacia Egg Cytoplasm."
Tuesday, July 16
DR. S. C. BROOKS "Ion Intake by Living Cells."
DR. L. I. KATZIN " The Use of Radioactive Tracers in
the Determination of Irreciprocal
Permeability of Biological Mem-
branes."
DR. K. C. FISHER " Urethane and the Respiration of
Yeast Cells."
DR. M. M. BROOKS " Spectrophotometric Determinations
on Hemoglobin and its Deriva-
tives."
Tuesday, July 23
DR. NELSON J. SPRATT, JR '' An in vitro Analysis of the Organi-
zation of the Eye-forming Area in
the Early Chick Blastoderm."
DR. ERNEST SCHARRER " On the Determination of the Vascu-
lar Pattern of the Brain of the
Opposum."
DR. PAUL WEISS " Functional Properties of Trans-
planted and Deranged Parts of the
Central Nervous System of Am-
phibians."
Tuesday, July 30
DR. B. H. WILLIER "A Study of Feather Color Patterns
Produced by Grafting Melano-
phores During Embryonic Devel-
opment."
DR. H. B. GOODRICH " The Cellular Basis of the Color Pat-
tern in some Bermuda Coral Reef
Fish."
REPORT OF THE DIRECTOR 43
Tuesday, August 6
DR. ALBERT E. OXFORD " Production of a Complex Nitrog-
enous Compound Related to Ty-
rosine by a Species of Penicil-
lium."
DR. KURT SALOMON " Studies on Erythrocruorin (Inver-
tebrate Hemoglobin)."
DR. KURT G. STERN,
DR. JOSEPH L. MELNICK AND
DR. DELAFIELD DuBois " Photochemical Spectrum of the Pas-
teur Enzyme."
Tuesday, August 13
DR. A. C. GIESE " Effects of Ultra-violet Light on
Respiration of the Luminous Bac-
teria."
DR. IVOR CORNMAN " Effects of Ether upon the Develop-
ment of Drosophila melanogaster."
DR. BERTA SCHARRER " Neurosecretory Cells in Cock-
roaches."
DR. G. HAUGAARD " The Mechanism of the Glass Elec-
trode."
Tuesday, August 20
DR. W. GORDON WHALEY " Developmental Changes in Apical
Meristems."
DR. HARRY G. ALBAUM AND
DR. BARRY COMMONER " The Relation between the Four-
Carbon Acid Respiratory System
and the Growth of Oat Seedlings."
DR. R. K. SKOW AND
L. R. BLINKS " Respiratory Changes following
Stimulation in Nitella."
DR. L. R. BLINKS " The Relation of Potassium to the
Bio-electric Effects of Tempera-
ture and Light in Valonia."
8. GENERAL SCIENTIFIC MEETINGS, 1940
Tuesday, August 27
DR. S. O. MAST AND
DR. W. T. BOWRN " The Hydrogen Ion and the Osmotic
Concentrations of the Cytoplasm
in Vorticella Similis Stokes, as In-
dicated by Observations on the
Food Vacuoles."
DR. M. H. JACOBS AND
MR. W. D. JONES " The Reversibility of Certain Arti-
ficially Induced Changes in the
Permeability of the Erythrocyte."
44 MARINE BIOLOGICAL LABORATORY
DR. E. J. BOELL,
DR. R. CHAMBERS,
Miss E. A. CLANCY,
DR. K. G. STERN AND
Miss B. MEYERHOF " Oxidase Activity and Respiration of
Cells and Cell Fragments."
DR. E. J. BOELL AND
DR. L. L. WOODRUFF " Respiratory Metabolism of Mating
Types in Paramecium Calkinsi."
DR. ERIC G. BALL AND
Miss PAULINE A. RAMSDELL " Squid Ink, a Study of its Composi-
tion and Enzymatic Production."
DR. A. E. OXFORD " Observations on the Occurrence of
Simple Ethereal Sulphates in Ma-
rine Algae."
DR. E. J. W. BARRINGTON " Blood-sugar and the Problem of the
Pancreas in Lampreys."
DR. A. E. NAVEZ AND
MR. A. DuBois " Fatty Acid Compounds in the Un-
fertilized Egg of Arbacia punc-
tulata."
MR. C. B. GIDDINGS " Distribution of Plasmalogen in Cer-
tain Invertebrate Forms."
DR. G. H. PARKER " Lipoids and their probable Relation
to Melanophore Activity."
MR. SAMUEL BELFKK,
MR. B. BAILEY,
DR. H. C. BRADLEY, AND
MR. HOWARD EDER " Studies of the Distribution of the
Autolytic Mechanism."
DR. CARL C. SMITH " The Effect of Various Cholinergic
Drugs on the Radula Protractor
Muscle of Busycon canalicu-
latum."
DR. E. J. BOELL AND
DR. D. NACHMANSOHN '' Choline Esterase in Nerve Fibers."
DR. R. G. ABELL AND
DR. IRVINE H. PACK. " Vascular Reactions to Renin and
Angiotonin."
MR. J. CRAWFORD,
Miss D. BENEDICT, AND
DR. A. E. NAVEZ " Factors Affecting the Frequency of
Contraction of the Heart of Venus
mercenaria."
MR. CHARLES E. WILDK, JK " Determining Factors in the Regen-
eration of Hyclractinia."
DR. EDGAR ZWILLING '' Time of Determination and Domi-
nance in Tubularian Reconstitti-
tion."
REPORT OF THE DIRECTOR 45
DR. S. MERYL ROSE ............. "A Regeneration-Inhibiting- Substance
Released by Tubularia Tissue."
DR. L. G. EARTH ................ " The Role of Oxygen in Regenera-
tion of Tubularia."
DR. HARRY G. ALBAUM .......... " The Growth of Oat Coleoptiles after
Seed Exposure to Different Oxy-
gen Concentrations."
DR. W. GARDNER LYNN .......... " Results of Transplantation of the
Pituitary Anlage to the Thyroid
Region in Amblystoma."
Wednesday, August 28
DR. T. C. EVANS ................ " Oxygen Consumption of Arbacia
Eggs Following Exposure to
Roentgen Radiation."
DR. T. C. EVANS ................ " Effects of Roentgen Radiation on
the Jelly of Arbacia Egg. I. Dis-
integration of the Jelly."
DR. M. E. SMITH AND " Effects of Roentgen Radiation on
DR. T. C. EVANS ................ the Jelly of the Arbacia Egg. II.
Changes in pH of Egg Media."
MR. E. P. LITTLE AND
DR. T. C. EVANS ................ "I May in First Cleavage of Arbacia
Eggs Following Roentgen Irra-
diation of Zygotes."
DR. GRACE TOWNSEND ........... "Concerning Sensitivity of Cells to
X-Ray."
DR. GRACE TOWNSKND ........... " Laboratory Ripening of Arbacia in
Winter."
DR. ETHEL BROWNE HARVEY ..1. "A Note on Determining the Sex of
Arbacia punctulata."
DR. ETHEL BROWNE HARVEY .II. "Centrifugal Speed and the Arbacia
^
DR. ETHEL BROWNE HARVEY ITT. " Colored Photographs of Stratified
Arbacia punctulata Eggs Stained
with Vital Dyes."
DR. HERBERT SHAPIRO ........... " Elongation and Return in Spherical
Cells."
MR. IVOR CORN MAN ............. " Echinochrome as the Sperm-activat-
ing Agent in Egg-water."
MR. TERU H AVASH i ............. "A Relation between the Dilution
Medium and the Survival of
Spermatozoa of Arbacia punctu-
lata."
DR. WM. H. F. ADDISON ......... " The Occurrence of Cartilage at the
Bifurcation of the Common Caro-
tid Artery in an Adult Dog."
DR. HOPE HIBBARD .............. " Cytoplasmic Morphology in the Giz-
zard of Gallus domesticus."
46 MARINE BIOLOGICAL LABORATORY
PAPERS READ BY TITLE
MR. FRED W. ALSUP " Further Studies of Photodynamic
Action in the Eggs of Nereis lim-
bata."
MR. C. W. J. ARMSTRONG AND
DR. KENNETH C. FISHER "A Quantitative Study of the Effect
of Cyanide and Azide on Carbonic
Anhydrase."
DR. FRANK A. BROWN, JR., AND
DR. ALISON MEGLITSCH " Upon the Sources in the Insect Head
of Substances which Influence
Crustacean Chromatophores."
DR. RALPH H. CHENEY " Myofibrillar Modifications in the
Caffeinized Frog Heart."
DR. LEONARD B. CLARK " Effects of Visible Radiation on Ar-
bacia Eggs Sensitized with Rho-
damine B."
DR. A. C. CLEMENT " Effects of Cyanide on Cleavage in
Eggs of Ilyanassa and Crepidula."
DR. D. P. COSTELLO " The Cell Origin of the Prototroch
of Nereis limbata."
DR. TAMES DONNELLON " Blood Clotting in Callinectes sapi-
dus."
DR. LLEWELLYN T. EVANS " Effects of Light and Hormones upon
the Activity of Young Turtles,
Chrysemys picta."
DR. LLEWELLYN T. EVANS " Effects of Testosterone Propionate
upon Social Dominance in Young
Turtles, Chrysemys picta."
DR. KENNETH C. FISHER AND
MR. RICHARD J. HENRY " The Use of Urethane as an Indi-
cator of " Activity " Metabolism
in the Sea Urchin Egg."
MR. MORDECAI L. GABRIEL " The Inflation Mechanism of Sphe-
roides maculatus."
Miss E. A. GLANCY " Micromanipulative Studies on the
Nuclear Matrix of Chironomus
Salivary Glands."
DR. JOHN E. HARRIS " The Reversible Nature of the Potas-
sium Loss from Erythrocytes dur-
ing Storage of Blood at 2-5° C."
DR. ARNE V. HUNNINEN AND
DR. RAYMOND M. CABLE " Studies on the Life History of Ani-
soporus Manteri sp. nov. (Tre-
matoda : Allocreadiidae)."
DR. CORNELIUS T. KAYLOR " Histological Studies on the Problem
of Edema in Haploid Triturus
pyrrhogaster Larvae."
HI-TORT OF THE DIRECTOR 47
DR. BALDUIN LUCRE,
DR. ARTHUR K. PARPART, AND
MR. R. A. RICCA "Do Carcinogenic Compounds affect
Cell Permeability? "
DR. W. G. LYNX " The Development of the Skull in the
Non-aquatic Larva of the Tree-
toad, Eleutherodactylus nubicola."
DR. \Y. G. Lv\\ " The Embryonic Origin and Develop-
ment of the Pharyngeal Deriva-
tives in Eleutherodactylus nubi-
cola."
SISTER MARIA LAURENCE MAKER . " Preliminary Report on Effect of In-
dole Acetic Acid on Growth of
Chlamydomonas."
DR. H. SHAPIRO " Further Studies on the Metabolism
of Cell Fragments."
DR. CARL C. SMITH,
Miss BLANCHE JACKSON AND
DR. C. LADD PROSSKK " Responses to Acetylcholine and Cho-
linesterase Content of Cerebratu-
lus."
DR. A. J. WATERMAN " Response of the Heart of the Com-
pound Ascidian, Perophora Viri-
dis, to Pilocarpine, Atropine and
Nicotine."
DEMONSTRATIONS
Wednesday, August 28
DR. W. H. F. ADDISON " Corrosion Preparations of the Bran-
chial Circulation in the Dogfish."
DR. E. SCHARRER " Vascularization of the Extramedul-
lary Nerve Cells of the Puffer,
Spheroides Maculatus."
DR. E. R. CLARK AND
MRS. ELEANOR LINTON CLARK . . . . " The Microscopic Study of Living
Tissues in Transparent Chambers
Installed in Rabbits' Ears."
MR. E. P. LITTLE " Color and Luminescence Produced
by Roentgen Rays in Glass and
Chemicals."
DR. E. J. BOELL " The Cartesian Diver Ultramicro-
Respirometer."
PER F. SCHOLANDER,
DR. S. W. GRINNELL AND
DR. L. IRVING " Apparatus for Measurement of Re-
spiratory Metabolism and Circula-
tion Changes."
48 MARTNF. P.IOI.OGICAL LABORATORY
0. MEMBERS OF THE CORPORATION, 1<>40
1. LIFE MEMBERS
ALLIS, MR. E. P., JR., Palais Carnoles, Mcnton, France.
ANDREWS, MRS. GWENDOLEN FOULKE, Baltimore, Marylaiul.
BILLINGS, MR. R. C., 66 Franklin Street, Boston, Massachusetts.
CALVERT, DR. PHILIP P., University of Pennsylvania, Philadelphia,
Pennsylvania.
CONKLIN, PROF. EDWIN G., Princeton University, Princeton, New
Jersey.
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 Street, Boston, Massachusetts.
JACKSON, MR. CHAS. C., 24 Congress Street, Boston, Massachusetts.
KING, MR. CHAS. A.
LEWIS, PROF. W. H., Johns Hopkins University, Baltimore, Maryland.
LOWELL, MR. A. L., 17 Quincy Street, Cambridge, Massachusetts.
MEANS, DR. J. II., 15 Chestnut Street, Boston, Massachusetts.
MOORE, DR. GEORGE T., Missouri Botanical Gardens, St. Louis, Mis-
souri.
MORGAN, MR. J. PIERPONT, JR., Wall and Broad Streets, New York
City, New York.
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, New York.
NOYES, Miss EVA J.
PORTER, DR. H. C., University of Pennsylvania, Philadelphia, Pennsyl-
vania.
SEARS, DR. HENRY F., 86 Beacon Street, Boston, Massachusetts.
SHEDD, MR. E. A.
THORNDIKE, DR. EDWARD L., Teachers College, Columbia University,
New York City, New York.
TREADWELL, PROF. A. L., Vassar College, Poughkeepsie, New York.
TRELEASE, PROF. WILLIAM, University of Illinois, Urbana, Illinois.
WALLACE, LOUISE B., 359 Lytton Avenue, Palo Alto, California.
2. REGULAR MEMBERS
ABRAMOWITZ, DR. ALEXANDER A., Biological Laboratories, Harvard
University, Cambridge, Massachusetts.
ADAMS, DR. A. ELIZABETH, Mount Holyoke College, South Hadley,
Massachusetts.
REPORT OF THE DIRECTOR 49
ADDISON, DR. W. H. F., University of Pennsylvania Medical School,
Philadelphia, Pennsylvania.
ADOLPH, DR. EDWARD F., University of Rochester Medical School,
Rochester, New York.
ALBAUM, DR. HARRY G., 3115 Avenue I, Brooklyn, New York.
ALLEE, DR. W. C., The University of Chicago, Chicago, Illinois.
AMBERSON, DR. WILLIAM R., Department of Physiology, University
of Maryland, School of Medicine, Lombard and Greene Streets,
Baltimore, Maryland.
ANDERSON, DR. RUBERT S., Memorial Hospital, 444 East 58th Street,
New York City, New York.
ANGERER, DR. CLIFFORD A., Department of Physiology, Ohio State Uni-
versity, Cleveland, Ohio.
ARMSTRONG, DR. PHILIP B., College of Medicine, Syracuse University,
Syracuse, New York.
AUSTIN, DR. MARY L., Wellesley College, Wellesley, Massachusetts.
BAITSELL, DR. GEORGE A., Yale University, New Haven, Connecticut.
BAKER, DR. H. B., Zoological Laboratory, University of Pennsylvania,
Philadelphia, Pennsylvania.
BALLARD, DR. WILLIAM W., Dartmouth College, Hanover, New Hamp-
shire.
BALL, DR. ERIC G., Department of Biological Chemistry, Harvard Uni-
versity Medical School, Boston, Massachusetts.
BARD, PROF. PHILIP, Johns Hopkins Medical School, Baltimore, Mary-
land.
BARRON, DR. E. S. GUZMAN, Department of Medicine, The University
of Chicago, Chicago, Illinois.
BARTH, DR. L. G., Department of Zoology, Columbia University, New
York City, New York.
BEADLE, DR. G. W., School of Biological Sciences, Stanford University,
California.
BEAMS, DR. HAROLD W., Department of Zoology, State University of
Iowa, Iowa City, Iowa.
BECKWITH, DR. CORA J., Vassar College, Poughkeepsie, New York.
BEHRE, DR. ELINOR H., Louisiana State University, Baton Rouge,
Louisiana.
BIGELOW, DR. H. B., Museum of Comparative Zoology, Cambridge,
Massachusetts.
BIGELOW, PROF. R. P., Massachusetts Institute of Technology, Cam-
bridge, Massachusetts.
BINFORD, PROF. RAYMOND, Guilford College, Guilford, North Carolina.
BISSONNETTE, DR. T. HUME, Trinity College, Hartford, Connecticut.
50 MARINE BIOLOGICAL LABORATORY
BLANCHARD, PROF. KENNETH C, Washington Square College, New
York University, New York City, New York.
BODINE, DR. J. H., Department of Zoology, State University of Iowa,
Iowa City, Iowa.
BORING, DR. ALICE M., Yenching University, Peking, China.
BRADLEY, PROF. HAROLD C., University of Wisconsin, Madison, Wis-
consin.
BRONFENBRENNER, DR. JACQUES J., Department of Bacteriology, Wash-
ington University Medical School, St. Louis, Missouri.
BROOKS, DR. S. C., University of California, Berkeley, California.
BROWN, DR. DUGALD E. S., New York University, College of Medicine,
New York City, New York.
BROWN, DR. FRANK A., JR., Department of Zoology, Northwestern Uni-
versity, Evanston, Illinois.
BUCKINGHAM, Miss EDITH N., Sudbury, Massachusetts.
BUCK, DR. JOHN B., Department of Zoology, University of Rochester,
Rochester, New York.
BUDINGTON, PROF. R. A., Winter Park, Florida.
BULLINGTON, DR. W. E., Randolph-Macon College, Ashland, Virginia.
BUMPUS, PROF. H. C., Duxbury, Massachusetts.
BYRNES, DR. ESTHER F., 1803 North Camac Street, Philadelphia, Penn-
sylvania.
CALKINS, PROF. GARY N., Columbia University, New York City, New
York.
CANNAN, PROF. R. K., New York University College of Medicine, 477
First Avenue, New York City, New York.
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, Iowa.
CARPENTER, DR. RUSSELL L., Tufts College, Tufts College, Massachu-
setts.
CARROLL, PROF. MITCHEL, Franklin and Marshall College, Lancaster,
Pennsylvania.
CARVER, PROF. GAIL L., Mercer University, Macon, Georgia.
CATTELL, DR. McKEEN, Cornell University Medical College, 1300 York
Avenue, New York City, New York.
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, New York.
REPORT OF THE DIRECTOR 51
CHENEY, DR. RALPH H., Biology Department, Long Island University,
Brooklyn, New York.
CHIDESTER, PROF. F. E., Auburndale, Massachusetts.
CHILD, PROF. C. M., Jordan Hall, Stanford University, California.
CHURNEY, DR. LEON, Zoological Laboratory, University of Pennsyl-
vania, Philadelphia, Pennsylvania.
CLAFF, MR. C. LLOYD, Department of Biology, Brown University, Prov-
idence, Rhode Island.
CLARK, PROF. E. R., University of Pennsylvania Medical School, Phila-
delphia, Pennsylvania.
CLARK, DR. LEONARD B., Department of Biology, Union College, Sche-
nectady, 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, Colum-
bia University, 630 West 168th Street, New York City, New York.
COLE, DR. LEON J., College of Agriculture, Madison, Wisconsin.
COLLETT, DR. MARY E., Western Reserve University, Cleveland, Ohio.
COLTON, PROF. N. S., Box, 601, Flagstaff, Arizona.
COONFIELD, DR. B. R., Brooklyn College, Bedford Avenue and Ave-
nue H, Brooklyn, 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, New York.
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, New York.
CURTIS, PROF. W. C., University of Missouri, Columbia, Missouri.
DAN, DR. KATSUMA, Misaki Biological Station, Misaki, Japan.
DAVIS, DR. DONALD W., College of William and Mary, Williamsburg,
Virginia.
MARINE BIOLOGICAL LABORATORY
DAWSON, DR. A. B., Harvard University, Cambridge, Massachusetts.
DAWSON, DR. J. A., The College of the City of New York, New York
City, New York.
DEDERER, DR. PAULINE H., Connecticut College, New London, Con-
necticut.
DILLER, DR. WILLIAM F., 4501 Larchwood Avenue, Philadelphia, Penn-
sylvania.
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, New York.
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, New York.
EDWARDS, DR. D. J., Cornell University Medical College, 1300 York
Avenue, New York City, New York.
ELLIS, DR. F. W., Monson, Massachusetts.
FAILLA, DR. G., Memorial Hospital, 444 E. 68th Street, New York City,
New York.
FAURE-FREMIET, PROF. EMMANUEL, College de France, Paris, France.
FERGUSON, DR. JAMES K. W., Department of Pharmacology, University
of Toronto, Ontario, Canada.
FIGGE, DR. F. H. J., Yale University, School of Medicine, New Haven,
Connecticut.
FISCHER, DR. ERNST, Department of Physiology, Medical College of
Virginia, Richmond, Virginia.
FISHER, DR. KENNETH C., Department of Biology, University of
Toronto, Toronto, Canada.
FLEISHER, DR. MOYER S., 20 North Kingshighway, St. Louis, Missouri.
FORBES, DR. ALEXANDER, Harvard University Medical School, Boston,
Massachusetts.
FRISCH, DR. JOHN A., Canisius College, Buffalo, New York.
FRY, DR. HENRY J., Old Danbury Road, Westport, Connecticut.
FURTH, DR. JACOB, Cornell University Medical College, 1300 York
Avenue, New York City, New York.
GAGE, PROF. S. H., Cornell University, Ithaca, New York.
UF.PORT OF THE DIRECTOR -^
GALTSOFF, DR. PAUL S., 420 Cumberland Avenue, Somerset, Chevy
Chase, Maryland.
CARREY, PROF. W. E., Vanderbilt University Medical Sehool, Nashville,
Tennessee.
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.
GODDARD, DR. D. R., Department of Botany, University of Rochester,
Rochester, New York.
GOLDFORB, PROF. A. J., College of the City of New York, Convent Ave-
nue and 139th Street, New York City, New York.
GOODRICH, PROF. H. B., Wesleyan University, Middletown, Connecticut.
GOTTSCHALL, DR. GERTRUDE Y., 10 West 86th Street, New York City,
New York.
GRAHAM, DR. J. Y., University of Alabama, University, Alabama.
GRAVE, PROF. B. H., DePumv 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, New York.
GUTHRIE. DR. MARY J., University of Missouri, Columbia, Missouri.
GUYER, PROF. M. F., University of Wisconsin, Madison, Wisconsin.
HADLEY, DR. CHARLES E., State 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 Uni-
versity, St. Louis, Missouri.
HANCE, D. ROBERT T., Department of Biology, Duquesne University,
Pittsburgh, Pennsylvania.
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, New York.
HARPER, PROF. R. A., Columbia University, New York City, New York.
HARRISON, PROF. Ross G., Yale University, New Haven, Connecticut.
HARTLINE, DR. H. KEFFER, Cornell University Medical College, 1300
York Avenue, New York City, New York.
HARTMAN, DR. FRANK A., Hamilton Hall, Ohio State University, Co-
lumbus, Ohio.
54 MARINE BIOLOGICAL LABORATORY
HARVEY, DR. ETHEL BROWNE, 48 Cleveland Lane, Princeton, New Jer-
sey.
HARVEY, DR. E. NEWTON, Gnyot Hall, Princeton University, Princeton,
New Jersey.
HAYDEN, DR. MARGARET A., Wellesley College, Wellesley, Massachu-
setts.
HAYES, DR. FREDERICK R., Zoological Laboratory, Dalhousie Univer-
sity, Halifax, Nova Scotia.
HAYWOOD, DR. CHARLOTTE, Mount Holyoke College, South Hadley.
Massachusetts.
HAZEN, DR. T. E., Barnard College, Columbia University, New York
City, New York.
HECHT, DR. SELIG, Columbia University, New York City, New York.
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., National Cancer Institute, Bethesda, Mary-
land.
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, Russell Sage College,
Troy, New York.
HINRICHS, DR. MARIE, Department of Physiology and Health Educa-
tion, South Illinois Normal University, Carbondale, Illinois.
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., University of Pennsylvania Medical School, Phil-
adelphia, Pennsylvania.
HOLLAENDER, DR. ALEXANDER, c/o National Institute of Health, Lab-
oratory of Ind. Hygiene, Bethesda, Maryland.
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, New York.
HOWE, DR. H. E., 2702 36th Street, N. W, Washington, D. C.
REPORT OF THE DIRECTOR
ROWLAND, DR. RUTH B., Washington Square College, New York Uni-
versity, Washington Square East, New York City, New York.
HOYT, DR. WILLIAM D., Washington and Lee University, Lexington,
Virginia.
HYMAN, DR. LIBBIE H., 85 West 166th Street, New York City, New
York.
IRVING, PROF. LAURENCE, Swarthmore College, Swarthmore, Pennsyl-
vania.
ISELIN, MR. COLUMBUS O'D.. Woods Hole, Massachusetts.
JACOBS, PROF. MERKEL H., School of Medicine, University of Pennsyl-
vania, Philadelphia, Pennsylvania.
JENKINS, DR. GEORGE B., 30 Gallatin Street, N. W., Washington, D. C.
JENNINGS, PROF. H. S., Department of Zoology, University of Cali-
fornia, Los Angeles, California.
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.
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.
KNOWLTON, PROF. F. P., Syracuse University, Syracuse, New York.
KOPAC, DR. M. J., Washington Square College, New York University,
New York City, New York.
KORR, DR. I. M., Department of Physiology, New York University, Col-
lege of Medicine, 477 First Avenue, New York City, New York.
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, New York.
LANCEFIELD, DR. D. E., Queens College, Flushing, New York.
LANCEFIELD, DR. REBECCA C., Rockefeller Institute, 66th Street and
York Avenue, New York City, New York.
LANGE, DR. MATHILDE M., Wheaton College, Norton, Massachusetts.
56 MARINE BIOLOGICAL LABORATORY
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.
LOEB, PROF. LEO, Washington University Medical School, St. Louis,
Missouri.
LOWTHER, MRS. FLORENCE DEL., Barnard College, Columbia University,
New York City, New York.
LUCAS, DR. ALFRED M., Zoological Laboratory, Iowa State College,
Ames, Iowa.
LUCAS, DR. MIRIAM SCOTT, Department of Zoology, Iowa State Col-
lege, Ames, Iowa.
LUCKE, PROF. BALDUIN, University of Pennsylvania, Philadelphia,
Pennsylvania.
LYNCH, DR. CLARA J., Rockefeller Institute, 66th Street and York Ave-
nue, New York City, New York.
LYNCH, DR. RUTH STOCKING, Maryland State Teachers College, Tow-
son, Maryland.
*/
LYNN, DR. WILLIAM G., Department of Zoology, Johns Hopkins Uni-
versity, Baltimore, Maryland.
MACDOUGALL, DR. MARY S., Agnes Scott College, Decatur, Georgia.
MACLENNAN, DR. RONALD F., 1588 South Cedar Avenue, Oberlin, Ohio.
McCLUNG, PROF. C. E., University of Illinois, Urbana, Illinois.
MCGREGOR, DR. J. H., Columbia University, New York City, New
York.
MACKLIN, DR. CHARLES C., School of Medicine, University of Western
Ontario, London, Canada.
MAGRUDER, DR. SAMUEL R., Department of Anatomy, Tufts Medical
School, 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, New York.
MARTIN, PROF. E. A., Department of Biology, Brooklyn College, Bed-
ford Avenue and Avenue H, 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.
REPORT OF THE DIRECTOR 57
MAZIA, DR. DANIEL, Department of Zoology, University of Missouri,
Columbia, Missouri.
MEDES, DR. GRACE, Lankenau Research Institute, Philadelphia, Penn-
sylvania.
MEIGS, DR. E. B., Dairy Division Experimental Station, Beltsville,
Maryland.
MEIGS, MRS. E. B., 1736 M Street, N. W., Washington, D. C.
METZ, PROF. CHARLES W., University of Pennsylvania, Philadelphia,
Pennsylvania.
MICHAELIS, DR. LEONOR, Rockefeller Institute, 66th Street and York
Avenue, New York City, New York.
MILLER, DR. J. A., Department of Anatomy, University of Michigan,
Ann Arbor, Michigan.
MITCHELL, DR. PHILIP H., Brown University, Providence, Rhode
Island.
MOORE, DR. CARL R.T 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, New York.
MOSER, DR. FLOYD, Department of Biology, University of Alabama,
University, Alabama.
NAVEZ, DR. ALBERT E., Department of Biology, Milton Academy, Mil-
ton, Massachusetts.
NEWMAN, PROF. H. H., 1951 Edgewater Drive, Clear water, Florida.
NICHOLS, DR. M. LOUISE, Rosemont, Pennsylvania.
NOBLE, DR. GLADWYN K., American Museum of Natural History, New
York City, New York.
NONIDEZ, DR. JOSE F., Cornell University Medical College, 1300 York
Avenue, New York City, New York.
NORTHROP, DR. JOHN H., The Rockefeller Institute, Princeton, New
Jersey.
OKKELBERG, DR. PETER, Department of Zoology, University of Michi-
gan, Ann Arbor, Michigan.
OPPENHEIMER, DR. JANE M., Department of Biology, Bryn Mawr Col-
lege, Bryn Mawr, Pennsylvania.
OSBURN, PROF. R. C., Ohio State University, Columbus, Ohio.
OSTERHOUT, PROF. W. J. V., Rockefeller Institute, 66th Street and
York Avenue, New York City, New York.
OSTERHOUT, MRS. MARIAN IRWIN, Rockefeller Institute, 66th Street
and York Avenue, New York City, New York.
58 MARINE BIOLOGICAL LABORATORY
PACKARD, DR. CHARLES, Columbia University, Institute of Cancer Re-
search, 630 West 168th Street, New York City, New York.
PAGE, DR. IRVINE H., Lilly Laboratory Clinical Research, Indianapolis
City Hospital, Indianapolis, Indiana.
PAPPENHEIMER, DR. A. M., Columbia University, New York City, New
York.
PARKER, PROF. G. H., Harvard University, Cambridge, Massachusetts.
PARMENTER, DR. C. L., Department of Zoology, University of Pennsyl-
vania, Philadelphia, Pennsylvania.
PARPART, DR. ARTHUR K., Princeton University, Princeton, New Jer-
sey.
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, New
York.
POND, DR. SAMUEL E., Marine Biological Laboratory, Woods Hole,
Massachusetts.
PRATT, DR. FREDERICK H., Boston University, School of Medicine, Bos-
ton, Massachusetts.
PROSSER, DR. C. LADD, University of Illinois, Urbana, Illinois.
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, New York.
RICE, PROF. EDWARD L., Ohio Wesleyan University, Delaware, Ohio.
REPORT OF THE DIRECTOR 5()
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, New York.
ROGERS, PROF. CHARLES G., Oberlin College, Oberlin, Ohio.
ROMER, DR. ALFRED S., Harvard University, Cambridge, Massachusetts.
ROOT, DR. R. W., Department of Biology, College of the City of New
York, Convent Avenue and 139th Street, New York City, New
York.
ROOT, DR. W. S., College of Physicians and Surgeons, Department of
Physiology, 630 West 168th Street, New York City, New York.
RUEBUSH, DR. T. K., Osborn Zoological Laboratory, Yale University,
New Haven, Connecticut.
RUGH, DR. ROBERTS, Department of Biology, Washington Square Col-
lege, New York University, New York City, New York.
SASLOW, DR. GEORGE, Boston City Hospital, 818 Harrison Avenue,
Boston, Massachusetts.
SAYLES, DR. LEONARD P., Department of Biology, College of the City
of New York, 139th Street and Convent Avenue, New York City
New York.
SCHAEFFER, DR. ASA A., Biology Department, Temple University, Phil-
adelphia, Pennsylvania.
SCHECHTER, DR. VICTOR, College of the City of New York, 139th Street
and Convent Avenue, New York City, New York.
SCHMIDT, DR. L. H., Christ Hospital, Cincinnati, Ohio.
SCHOTTE, DR. OSCAR E., Department of Biology, Amherst College, Am-
herst, Massachusetts.
SCHRADER, DR. FRANZ, Department of Zoology, Columbia University,
New York City, New York.
SCHRADER, DR. SALLY HUGHES, Department of Zoology, Columbia Uni-
versity, New York City, New York.
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, New
York.
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 West 168th Street, New York City,
New York.
60 MARINE BIOLOGICAL LABORATORY
SHAPIRO, DR. HERBERT, Department of Physiology, Vassar College,
Poughkeepsie, New York.
SHULL, PROF. A. FRANKLIN, University of Michigan, Ann Arbor,
Michigan.
SHUMWAY, DR. WALDO, University of Illinois, Urbana, Illinois.
SICHEL, DR. FERDINAND J. M., University of Vermont, Burlington,
Vermont.
SICHEL, MRS. F. J. M., State Normal School, Johnson, Vermont.
SINNOTT, DR. E. W., Osborn Botanical Laboratory, Yale University,
New Haven, Connecticut.
SIVICKIS, DR. P. B., Pasto Deze 130, Kaunas, Lithuania.
SLIFER, DR. ELEANOR H., Department of Zoology, State University of
Iowa, Iowa City, Iowa.
SMITH, DR. DIETRICH CONRAD, Department of Physiology, University
of Maryland School of Medicine, Lombard and Greene Streets,
Baltimore, Maryland.
SOLLMAN, DR. TORALD, Western Reserve University, Cleveland, Ohio.
SONNEBORN, DR. T. M., Department of Zoology, Indiana University,
Bloomington, Indiana.
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, New York.
STEINBACH, DR. HENRY BURR, Columbia University, New York City,
New York.
STERN, DR. CURT, Department of Zoology, University of Rochester,
Rochester, New York.
STEWART, DR. DOROTHY R., Skidmore College, Saratoga Springs, New
York.
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, New York.
STUNKARD, DR. HORACE \V., New York University, University Heights,
New York.
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, New York.
REPORT OF THE DIRECTOR f'1
SUMVVAI.T, DR. MARCAKKT, National Institute <>i Health, Washington,
D. C.
SVVETT, DR. FRANCIS IT., Duke University Medical School, Durham,
North Carolina.
TAFT, DR. CIIARI.KS If., JR., University of Texas Medical School, Gal-
vcston, Texas.
TASHIRO, DR. SIIIRO, Medical College, rniversity of Cincinnati, Cin-
cinnati, Ohio.
TAYLOR, DR. WILLIAM R., University of Michigan, Ann Arbor, Michi-
gan.
TENNENT, PROF. D. H., Bryn Mawr College, Bryn Mawr, Pennsyl-
vania.
TEWINKEL, DR. L. K.. Department of Zoology, Smith College, North-
ampton, Massachusetts.
TURNER, DR. ABBY H., Department of Physiology, Mount Holyoke Col-
lege, South Haclley, Massachusetts.
TURNER, PROF. C. L., Northwestern University, Evanston, Illinois.
TYLER, DR. ALBERT, California Institute of Technology, Pasadena,
California.
UHLENHUTH, DR. EDUARD, University of Maryland, School of Medi-
cine, Baltimore, Maryland.
UNGER, DR. W. BYERS, Dartmouth College, Hanover, New Hampshire.
VISSCHER, DR. J. PAUL, W'estern Reserve University, Cleveland, Ohio.
WAITE, PROF. F. C., Western Reserve University Medical School,
Cleveland, Ohio.
WALD, DR. GEORGE, Biological Laboratories, Harvard University, Cam-
bridge, Massachusetts.
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.
WTENRICH, DR. D. H., University of Pennsylvania, Philadelphia, Penn-
sylvania.
WHEDON, DR. A. D., North Dakota Agricultural College, Fargo, North
Dakota.
WHITAKER, DR. DOUGLAS A., P. O. Box 2514, Stanford University,
California.
WHITE, DR. E. GRACE, Wilson College, Chambersburg, Pennsylvania.
62 MARINE BIOLOGICAL LABORATORY
WHITING, DR. PHINEAS W., Zoological Laboratory, University of
Pennsylvania, Philadelphia, Pennsylvania.
WHITNEY, DR. DAVID D., University of Nebraska, Lincoln, Nebraska.
WICHTERMAN, DR. RALPH, Biology Department, Temple University,
Philadelphia, Pennsylvania.
WIEMAN, PROF. H. L., University of Cincinnati, Cincinnati, Ohio.
WILLIER, DR. B. H., Department of Biology, Johns Hopkins University,
Baltimore, Maryland.
WTILSON, DR. J. W., Brown University, Providence, Rhode Island.
WITSCHI, PROF. EMIL, Department of Zoology, State University of
Iowa, Iowa City, Iowa.
\VOLF, DR. ERNST, Biological Laboratory, Harvard University, Cam-
bridge, Massachusetts.
WOODRUFF, PROF. L. L., Yale University, New Haven, Connecticut.
WOODWARD, DR. ALVALYN E., Zoology Department, University of Mich-
igan, Ann Arbor, Michigan.
YNTEMA, DR. C. L., Department of Anatomy, Cornell University Medi-
cal College, 1300 York Avenue, New York City, New York.
YOUNG, DR. B. P., Cornell University, Ithaca, New York.
YOUNG, DR. D. B., 7128 Hampden Lane, Bethesda, Maryland.
DECOMPOSITION AND REGENERATION OF NITRO-
GENOUS ORGANIC MATTER IN SEA WATER
IV. INTERRELATIONSHIP OF VARIOUS STAGES; INFLUENCE OF
CONCENTRATION AND NATURE OF PARTICULATE MATTER 1
THEODOR VON BRAND AND NORRIS W. RAKKSTRAW
(1'roin the ITixids Hole Oceanographic Institution, ll'oods Hole, Mass.)
In previous investigations (1937, 1939, 1940) it has been demon-
strated that the decomposition of participate organic matter in sea water
proceeds in well-defined steps, the main stages being the formation of
ammonia, nitrite and nitrate. The appearance of these substances is due
to the successive development of different bacterial floras acting upon
the original substratum of organic matter. In nature, however, a mix-
ture of the various floras will probably occur, with frequent or continu-
ous addition of new decomposing material. It seemed desirable, there-
fore, to study the interrelationship of the different stages of the cycle
and the results obtained when two or more stages occur simultaneously.
For this purpose a 20-liter carboy of filtered sea water from Woods
Hole Harbor, to which washed diatoms (Nitzschia Clostcriuin} were
added, was kept at room temperature in the dark. In order to deter-
mine what effect the bacterial flora present at various stages would have
on the decomposition of fresh organic matter, portions of the culture
were withdrawn at various times during the decomposition cycle. New
participate organic matter was added to these subcultures, as indicated
below, after which they were put in fresh containers in the dark. The
parent culture (No. 42) and the various subcultures (42A to 42H) were
analyzed regularly for the different forms of nitrogen and the changes
found are shown in Fig. 1.
The first subculture. No. 42A, was separated from the parent culture
when the ammonia in the latter had reached its maximum and when the
first trace of nitrite had appeared; the second subculture (42B) a few
days later, when the nitrite formation was well under way; and the
third (42C), when the nitrite had about reached its maximum. Later,
when the nitrite began to decline in subculture No. 42B and when nitrate
formation had begun, a portion of it was used in preparing a new sub-
culture (42G). To all these subcultures new participate matter was
added in the form of living, washed diatoms.
1 Contribution No. 292 from the Woods Hole Oceanographic Institution.
63
64
THEODOR VOX BRAND AND N. W. RAKESTRAW
300
CDAYS 10 30 4,0 50 6,0 70 8O DAYS 50 60 70 80
DAYS
3O 40 50 60 70 80
DAYS 60 70 80 90
FIG. 1. Interrelationship of different stages of the decomposition cycle. Time-
in days. Different forms of nitrogen in micrograms (gamma) per liter. The
original culture, No. 42, consisted of filtered sea water with washed diatoms
(Nitzschia Closterium) added. Decomposition in the dark. Subcultures A, B,
etc., separated at times indicated by arrows and with new participate organic matter
added.
Figure 1 shows that nitrite formation in the first three subcultures
(42A, B and C) was in no way interfered with by the addition of new
diatoms, but proceeded at normal speed without interruption. In each
case ammonia rose only slightly higher than in the original culture, No.
ORGANIC DECOMPOSITION AND REGENERATION 65
42, indicating that the ammonia formed from the new decomposing
diatoms was at once oxidized to nitrite. Finally, the nitrite disappeared
from all cultures in the usual way, appearing quantitatively as nitrate.
A somewhat different result was obtained in subculture 42G, which
was prepared by adding new organic matter to a portion of 42B when
the latter was approaching the end of the nitrite stage. As before, am-
monia remained low throughout the whole time. During the first week
nitrite disappeared exactly as in the mother-culture from which it had
been prepared (42B), but after this it increased again, reaching a new
maximum ten days later. Apparently the nitrite- forming flora was on
the decline when this subculture was begun but was able to recover under
the influence of the newly-formed products of decomposition. Nitrate
formation seems to have occurred throughout this subseries. It is prob-
able that we, have here a case in which ammonia-, nitrite- and nitrate-
forming floras were active at the same time.
In the cultures so far described living diatoms were used as a source
of new organic matter ; consequently, vigorous ammonia formation was
not actually under way at the start of each subculture. In the next two
cultures organic matter was introduced which was already in the
ammonia- formation stage. Fresh diatoms were added to a fresh quan-
tity of harbor water (No. 42D). After 12 days in the dark, when
ammonia was being formed rapidly, portions of this culture were with-
drawn and mixed with equal amounts of older cultures in various stages
of the decomposition cycle. Thus, subculture 42E consisted of an equal
mixture of 42D and 42C, the latter taken when the nitrite had reached
its maximum. In this case the ammonia introduced with culture 42D
disappeared rapidly, with a corresponding rise in nitrite. Evidently the
nitrite-forming flora of culture 42C was still active when the new,
partially-decomposed organic matter was added.
Subculture 42F was prepared by separating a portion of the original
culture, No. 42, when nitrite had begun to diminish, and adding an equal
amount of 42D containing organic matter in the stage of ammonia for-
mation. Both ammonia and nitrite disappeared rapidly, in contrast to
the last preceding subculture, 42E, probably due to the fact that the
nitrate- forming flora in the parent culture was at that time the most
potent one.
The last culture, 42H, behaved in a somewhat similar manner. This
consisted of a portion of culture 42A, separated at a time when the nitrite
was about half converted to nitrate. To this was added a large number
of partially decomposed diatoms, centrifuged from a culture which had
stood for six days in the dark. A relatively small increase in ammonia
was observed during the first days, with a subsequent rapid decrease.
66 THEODOR VON BRAND AND N. W. RAKESTRAW
Nitrite was present somewhat lunger than in 42A. but never reached a
very high level. It is likely that during this whole time nitrate formation
proceeded rapidly.
The following conclusions may be drawn from the study of culture
42 and its subcultures : Ammonia formation does not interfere with the
formation of either nitrite or nitrate, in such concentrations as we ob-
served. The strict sequence of processes in our normal decomposition
experiments can therefore hardly be due to any inhibiting action of
ammonia or other initial products of decomposition upon nitrite or
nitrate formation. More likely is it connected with a very slow devel-
opment of the oxidizing floras. Doubtless, however, some other, hitherto
unrecognized factor must also be involved. A slow increase in the
nitrate-forming population, for example, is alone insufficient to explain
why it should require weeks, or even months, for the first traces of
nitrate to appear, whereas once the process has started the nitrate maxi-
mum may be reached in five days.
These observations are not necessarily inconsistent with experiments
we have described previously, involving deep sea water, in which there
was evidence of a retarding influence on the development of the oxidiz-
ing floras. This influence has not yet been explained, but seems to be
connected with some unknown special property of the deep sea water
used.
The course which the decomposition will take, when new organic
material is added, will depend upon the flora which predominates. In
general, a shortening of the cycle will occur, as far as the newly added
material is concerned. The original culture, No. 42, took 55 days to
complete its cycle. In the various subcultures the mean time from the
addition of new organic matter to the end of the cycle was 36 days, or
41 days if one includes the initial period of decomposition of the added
organic matter before its addition to Series 42E, F and H.
DURATION OF THE CYCLE
As pointed out in previous papers, the time required for the de-
composition cycle varies considerably in different series. It seemed pos-
sible that the initial concentration of organic matter might be a deter-
mining factor in this connection and Series 47 to 50 were set up to
investigate this point. The four cultures contained amounts of par-
ticulate nitrogen varying from 185 7 to 768 y per liter. As shown in
Fig. 2, this factor seems to be of some, though not of very great im-
portance. The rate of disappearance of participate nitrogen was nearly
the same in each case. In the higher concentrations the ammonia maxi-
ORGANIC DECOMPOSITION AND REGENERATION
67
mum was reached a little earlier and nitrite appeared and disappeared
more rapidly. The total time for the cycle varied from dl days in the
highest concentration to 88 days in the lowest.
PARTICULATE AC-— -o .' \
700
60O
500
400
300
200
(OO
DAYS
FIG. 2. Series 47 to 50. Influence of varying concentration of participate
matter. Filtered sea water with different amounts of Nitzschia Clostcrium added.
Decomposition in the dark. Time in days. Different forms of nitrogen in micro-
grams (gamma) per liter.
The nature of the suspended organic matter also determines the
duration of the cycle, as the next series show. Series 43 and 45 (Figs.
3 and 4) were set up with the same harbor water ; in 43 was suspended
a small amount of mixed plankton, in 45 a large amount of yeast. In
No. 43 the nitrogen cycle proceeded in the normal way, but in Xo. 45
nitrite appeared only very slowly, with no formation of nitrate when the
experiment was terminated after 5^> months.
It has been shown that the length of the decomposition cycle depends
upon the source of the water and it has been suggested that this might
involve the action of growth-promoting substances upon the bacterial
flora. Series 44 and 46 (Figs. 3 and 4) were planned as an approach
to this question. Two samples of harbor water, the same as in Nos. 43
68
THEODOR VON BRAND AND N. W. RAKESTRAW
and 45, were evaporated to dry ness and the salt residues ignited at
600-700° C. for 5 hours, to destroy organic matter. The salts were dis-
solved in the original volume of distilled water, with a little HC1. and
the pH brought back to between 7.5 and 8.2 with NaOH. To the two
(Nos. 44 and 46) were added amounts of mixed plankton and yeast,
respectively, corresponding to the quantities in Series 43 and 45. In
DAYS
100
FIG. 3. Series 43 and 44. Mixed plankton added to filtered sea water (No.
43) and to a "synthetic" water made by redissolving the ignited salt residue of
evaporated sea water (No. 44). Time in days. Different forms of nitrogen in
micrograms (gamma) per liter.
FIG. 4. Series 45 and 46. Yeast added to filtered sea water (No. 45) and to
a " synthetic " water made by redissolving the ignited salt residue of evaporated
sea water (No. 46). Time in days. Different forms of nitrogen in micrograms
(gamma) per liter.
both 44 and 46 the formation of ammonia was much slower than in
the untreated water of Series 43 and 45, an effect even more pronounced
on the formation of nitrite, which had not reached its maximum at the
termination of the experiments. This apparently indicates that some
" growth-promoting factor " had been eliminated from the water by the
process of ignition, but further investigation will be necessary before a
definite conclusion can be reached.
ORGANIC DECOMPOSITION AND REGENERATION M
SUM MA in
1. With a recurrent supply of participate organic matter, the forma-
tion of ammonia, nitrite and nitrate may take place simultaneously.
The process which predominates will depend upon the stage at which
the new organic matter is introduced.
2. The nature of the suspended particulate matter is of considerable
importance in determining the total duration of the decomposition cycle,
but the level of its original concentration is only a minor determining
factor.
3. There is some evidence of a " growth-promoting " factor, nor-
mally effective in the decomposition cycle, but which can be destroyed
by high temperature.
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, T., N. W. RAKESTRAW AND C. E. RENN, 1939. Further experiments
on the decomposition and regeneration of nitrogenous organic matter in
sea water. Biol. Bull, 77 : 285-296.
VON BRAND, T., AND N. W. RAKESTRAW, 1940. Decomposition and regeneration of
nitrogenous organic matter in sea water. III. Influence of temperature
and source and condition of water. Biol. Bull.. 79: 231-236.
THE REPRODUCTIVE CYCLE OF THE VIVIPAROUS
TELEOST, NEOTOCA BILINEATA, A MEMBER
OF THE FAMILY GOODEIDAE
III. THE GERM CELL CYCLE
GUILLERMO MENDOZA
(Trom tJie Department of Zoology, University College, Northwestern Unn'crsity)
INTRODUCTION
In previous articles on the reproductive cycle of Neotoca bilineata,
the writer has described the breeding cycle and the somatic cycle of the
ovary (Mendoza, 1939. 1940). It was shown that, in Neotoca, as is true
for many viviparous teleosts, the height of the breeding season occurs
during the spring and early summer ; broods are spaced approximately
44 days apart. During the breeding cycle, the soma of the ovary was
shown to undergo very marked cyclic changes. These changes include
(a) an increased tumescence of the ovarian stroma, (b) a secretory acti-
vation of the internal ovarian epithelium, and (c) marked changes in
the free cellular elements. These conditions are particularly prominent
during the middle stages of gestation and later recede to the more normal
resting condition. In order to complete the analysis of the reproductive
cycle, a study was made of the germ cells and their cyclic variation during
gestation. The present account, therefore, presents such an analysis of
the germ cells. The detailed description of the germ cells, follicles, their
changes during growth, and the fate of the atretic and evacuated follicles,
etc., will be considered in a later report.
Previous accounts of the reproductive cycle of viviparous teleosts
have made little or no reference to the germ cell cycle, an omission that
is difficult to justify since, obviously, the phenomenon is an important
phase of the reproductive cycle. It is true, of course, that in many inves-
tigations of viviparous teleosts the paucity of material prevented a de-
tailed analysis of the complete gestation cycle. Among recent accounts,
the ones on Jcnynsia (Fitsroyia) lineata1 (Scott, 1928) and XiphopJwrus
hclleri (Bailey, 1933) contain no reference to the germ cell cycle whereas
1 E. J. MacDonagh of the Museo de La Plata has kindly informed me that the
name Jcnynsia lincata is considered preferable over the name Fitsroyia lincata. In
two publications (1934, 1938), MacDonagh uses the name Jcnynsia; in the latter,
he quotes correspondence with C. L. Hubbs of the University of Michigan in which
the latter supports the use of the name Jenynsia lincata.
70
THE GERM CELL CYCLE IN NEOTOCA 71
Turner (1938a) mentions it but briefly in his description of the repro-
ductive cycle of Cymatogastcr aggregates. However, Turner does de-
vote considerable attention to the germ cells in his general study of poe-
ciliid fishes (Turner, 1937) and in a special article on the poeciliid,
Brachyrhaphis cpiscopi (Turner, 1938fr). In general, nevertheless, a
complete count and measurement of the germ cells during gestation has
not been made in a viviparous teleost. Hence it is the purpose of the
present account to supplement a preliminary description (Mendoza,
1938) by considering in greater detail the cyclic variation of the germ
cells during gestation.
MATERIALS
The present analysis of the germ cell cycle is based on a study of
the gonads used in the preceding study on the somatic cycle. Fourteen
of these ovaries were chosen at well-spaced intervals during the resting
and gestation periods. Detailed cell counts were made in one lateral
half of each of the above ovaries ; these cell counts, involving a total of
4686 oocytes, form the basis of the present analysis. The brief descrip-
tion of the germ cells and follicles is based on gonads fixed in Bouin's
fluid and stained with iron hematoxylin. In a considerable number of
sections, Mallory's triple connective tissue stain was used. A detailed
account of the ovaries used and the method of treating the data is given
later.
DESCRIPTION OF THE GERM CELLS
As is true for all the Goodeidae, the germinal tissue is confined to
the lobulated, ovigerous folds in the ovary. In Neotoca there are two
such folds, one on either side of the median sagittal septum. The germ
cells are confined solely to the ovigerous folds and are more or less evenly
distributed throughout the gonad except at the extreme anterior and
posterior ends.
In general, the oocytes of Neotoca are essentially similar to those
of other viviparous teleosts. The eggs are spherical, attain a maximal
size of 180-200 micra in diameter and are characterized by the absence
of large masses of yolk. The cytoplasm is coarsely granular and con-
tains the scattered, flocculent, albumen-like yolk. The nucleus of the
fully-grown oocyte is granular, oxyphylic in reaction, and contains typical
'' lampbrush " chromosomes. Numerous vacuolated nucleoli may appear
within the nucleus. Surounding the oocyte is a follicle composed of a
single row of tall columnar cells tightly pressed together. External to
the follicle is a thin sheath of connective tissue fibers in which appears
a plexus of capillaries.
72
( - 1 ' I LLERMO MENDOZA
GERM CELL CYCLE
A detailed account of the germ cell cycle during gestation was ob-
tained by using fourteen females (see Table I), chosen at well-spaced
intervals before and during gestation. In each female a count was made
of every oocyte, germ cell nest, and atretic follicle in one of the two
ovigerous folds. Each oocyte counted was measured and placed in one
of six groups depending upon its diameter (see Table II). The groups
TABLE I
Females used in the analysis of the germ cell cycle.
Number of
Female
Stage of Gestation
Number of
Female
Stage of Gestation
6
Non-gravid ovary
12
Embryos 4.5 mm. in
length
20
Non-gravid ovary-
8
4.5 " "
i i
22
Early segmentation
23
6.0 " "
i I
19
Late segmentation
27
6.0 " "
t t
47
Embryos 1.5 mm. in length
9
7.0 " "
1 i
16
it 9V * ' * ' ' *
11
7.2 " "
1 i
44
3.5 " "
1
9.0 " "
were chosen arbitrarily to facilitate counting and measuring the cells ;
the actual limits of each group were determined largely by the ocular
micrometer units at that particular magnification. Since the total num-
ber of oocytes in each group would vary with the size of the gonad, the
average number of cells per half-section of ovary was obtained. Thus,
an average figure \vas obtained that could be compared with those of
other gonads regardless of size differences. In order to further reduce
the possibilities of individual vaiations of different ovaries, counts were
made, wherever possible, of two ovaries for each stage of gestation.
Thus six of the eight representative stages chosen are based on the aver-
age figures between two different ovaries ; only in two stages, IV (3.5
mm.) and VIII (9.0 mm.) are the figures based on a single ovary. All
in all, fourteen ovaries were examined histologically, 5633 sections were
checked for oocytes, and 4686 germ cells were counted and measured (see
Table II). Graph I is based on these figures. From a careful study
of these graphs and figures certain definite and interesting conclusions
wcne obtained.
CONCLUSIONS ON THE GERM CELL CYCLE
Continuous Production of Oocytes
There is no evidence of a complete cessation of egg production during
gestation. Nests occur abundantly at all times although, with the excep-
THE GERM CELL CYCLE IN NEOTOCA
73
TABLE II
The germ cell count during gestation.
In order to facilitate the analysis of the germ cells an arbitrary segregation was
made of the oocytes into germ cell nests and six other groups on the basis of size.
The six groups and the diameter in micra of the oocytes involved are indicated
below. Similarly, gestation and the resting period were divided into eight arbitrary-
stages. The pre-fertilization or resting period forms Stage I; thereafter the different
stages are distinguished by the stage of development or size of the contained young.
Birth normally follows immediately after Stage VIII. In all Stages except IV and
VIII, two ovaries were used for the analysis of the germ cells. By using two ovaries
it was hoped to get a more typical picture. In the two exceptional cases only one
ovary was available for each, but since both ovaries were normal in every respect
it is assumed that the cell counts also are typical. For each cell group there are two
figures: the whole numbers are the total numbers of cells of each particular group
in the different stages of gestation; the numbers in decimals represent the average
number of cells of each group per half-section of the ovary.
XlltH-
ber
of
Ovary
Stage of
Gestation
Num-
ber of
Sec-
tions
Xum-
ber
of
Nests
Groups of Oocytes
I
lO-36/i
II
40-72/1
ill
76-108M
IV
11 2-1 4V
V
148-180M
VI
184-21 6M
0, 20
I
Resting
651
136
.2149
560
.8889
121
.1993
54
.0861
29
.0461
41
.0635
4
.0061
19, 22
II
Segmen-
tation
640
169
.2641
496
.7750
178
.2781
57
.0891
32
.0500
26
.0406
4
.0063
16,47
III
2.0 mm.
437
83
.1899
253
.5789
79
.1878
39
.0892
26
.0595
14
.0320
44
IV
3.5 mm.
154
29
.1883
86
.5584
29
.1883
14
.0909
8
.0521
6
.0389
8, 12
V
4.5 mm.
507
129
.2544
245
.4832
92
.1815
33
.0651
23
.0454
12
.0237
23, 27
VI
6.0 mm.
977
106
.1085
681
.6970
162
.1658
54
.0553
65
.0665
14
.0144
9,11
VII
7.2 mm.
1417
153
.1079
304
.2145
143
.1009
50
.0353
25
.0176
12
.0085
1
.0007
1
VIII
9.0 mm.
850
128
.1506
493
.5800
72
.0847
22
.0259
14
.0165
13
.0153
Totals
5633
933
3118
876
323
222
138
9
tion of the rise in number of nests in Stage V, it can be stated that nests
of germ cells decrease in number until the latter part of gestation ; at that
time they are approximately half as abundant as in pre-fertilization
stages.
74 GUILLERMO MENDOZA
Time of Onset of Egg-production
The onset of the wave of egg-production for the following brood
is not a prominent one as is true for some of the poeciliids described by
Turner (1937). Following the general decrease in number of nests and
small oocytes during gestation, there is a rather sharp rise again at the
end of gestation when the current brood is about ready for birth. It is
unlikely that these minute oocytes are the ones destined for fertilization
and the formation of the following brood. Rather it is more probable
that the rise in number of small oocytes is the first indication of a general
activation which results in an increase in the number of eggs of all groups
before the following fertilization period. Hence, it is more likely that
eggs of Groups III and IV will grow sufficiently during the resting pe-
riod to form the bulk of eggs to be fertilized for the succeeding brood.
Growth of these larger eggs, however, must of necessity occur in the
interval between the expulsion of the current brood and the fertilization
of the next group of eggs since there is no indication of an increase in
number among these larger groups (III and IV) before the end of ges-
tation. The only other noticeable increase in the number of eggs before
the end of gestation apparently occurs in Group V eggs, and that increase
is but a slight one.
Variation in Xitmber of Eggs u'ith Size
Regardless of the stage of gestation, oocytes of the 10-36 /x group are
the most numerous. Following that there is a regular and almost perfect
drop in the number of cells in each succeeding!}- larger group so that,
actually, cells of maximal size, 184-216 /x, are the smallest in number.
Only two exceptions occur to this generalization. The first, in Stage
VI of gestation, is caused by an abnormally large count of cells of Group
1 12-144 /A in ovary number 27 ; this may be purely an individual variation.
The second exception is one that is readily understood. In Stage I, pre-
ceding gestation, cells of Group 148-180 /x include the bulk of the cells
that are to be fertilized and hence appear in large numbers. The number
is so large, in fact, that it exceeds that of the next smaller group of
cells. Group TV. Furthermore, it is interesting to note that, during the
stage of segmentation, since a large percentage of the cells of Group \
were fertilized, the count once more drops below that of Group IV eggs
and remains so during the rest of gestation.
Decrease in Number of Eggs During Gestation
Xot only do eggs decrease in number with increase in size in any
one ovary but eggs of all sizes also decrease in number during gestation.
THE GERM CELL CYCLE IN NEOTOCA 75
The cell count for each size group is maintained fairly well until the
middle period of gestation ; from that time on there is a noticeable drop
in the number of cells to one-third or one-quarter of their number in
the middle stages of gestation. In keeping with this observation, there
is a noticeable increase in atretic follicles during the latter half of
gestation.
Maximal Sice of Eggs
Normally, oocytes attain maximal size during the resting period of
the ovary and remain until stages of segmentation. Following that there
is a noticeable absence of large eggs. This condition is verified further
by the large number of degenerating follicles of maximal size which are
found in pre-gestation stages. Absence of large degenerating follicles
and large eggs in the later stages of gestation indicates failure of eggs
normally to attain maximal size during that period.
Super-fetation
It has been determined before (Turner. 1933 ; Mendoza, 1939) that
superfetation normally does not occur among the growing embryos. It
is interesting that, in keeping writh this fact, there is no evidence among
growing oocytes of a phenomenon similar to superfetation ; rather there
is a continuous gradation in the size of oocytes in all ovaries.
1'ariatiou in Xinnhcr of Oocytes
The fluctuation in number of oocytes of different sizes during gesta-
tion varies with the size of the cells. Larger eggs vary but little in
number during gestation ; smaller oocytes, however, fluctuate widely in
number. The almost perfect inverse relation between the size of oocytes
and fluctuation in number is clearly evident in Table III.
Fcrccittugc of Eggs Fertilized
Failure to find fertilization occurring before the expulsion of the
previous brood is explainable, in part at least, by the presence of only
20-25 per cent as many large eggs at the end of gestation as there are
in the resting ovary immediately preceding the time for fertilization.
An indication of the large number of eggs prepared for fertilization
is obtained from the fact that despite the large number of atretic follicles
in the gonad prior to fertilization, there still is a greater number of large
eggs present at that time than at any other stage of gestation. It appears
likely that fully 40-50 per cent of the large eggs available at time of
76
GUILLERMO MENDOZA
fertilization actually are activated to start development. This is verified
by the knowledge that a normal brood averages from fifteen to twenty
young, a number which is approximately half of the total number of
O 0.6
i •
VARIATION IN THE NUMBER OF OOCYTES
OF DIFFERENT SIZES DURING GESTATION
STAGE OF GESTATION
EXPLANATION OF GRAPH I
The graph represents in diagrammatic form the variation of germ cells during
gestation. Each curve represents the number of cells of a limited size range during
each of the stages of gestation. The different curves are identified by the diameters
of the eggs involved. The group of the smallest oocytes is called Group I ; groups
of successively larger eggs are called respectively Groups II, III, etc. The stages
of gestation are plotted on the horizontal axis (see Table II for the different
stages) ; the numbers of oocytes of each size group are plotted on the vertical axis.
The vertical scale is plotted equally for all curves with exception of the one for
Group I ( 10-36 /"•). The values for Group I were so great that, in order to facili-
tate plotting all curves on the same graph, only the number of cells per half-section
are plotted. All other curves are plotted on the basis of number of cells per whole
section. The latter figure is hypothetical and was derived by multiplying by two
the actual count of cells per half-section obtained during the investigation.
eggs available at time of fertilization (see Table II). The marked drop
in the number of eggs of Group 148-180 /JL (see Graph I) following
fertilization (between Stages I and II) is a further indication of the
large number of eggs fertilized. The fact that eggs over 184 p. remain
THE GERM CELL CYCLE IN NEOTOCA 7
constant in number until after stages of segmentation probably indicates
that growth of the next smaller group of eggs continues until after ferti-
lization and thus maintains the same level, replacing those which had been
fertilized. Normally, all eggs fertilized develop completely for degen-
eration of developing embryos is very scarce.
Atretic Follicles
With reference to the atretic follicles, only two generalizations are
justified : (1) large degenerating follicles may occur throughout gestation
but are more numerous in early and middle stages of gestation ; (2) there
is a noticeable increase in the number of small atretic follicles during the
TABLE III
Size of Oocytes
Variation in Number of
Oocytes per Half Section
Extent of Fluctuation
per Half Section
10- 36 M
.2145-.8889
.6744
40- 72 n
.0847-.2781
.1934
76-108 n
.0259-.0909
.0650
11 2-144 ju
.0165-.0665
.0500
148-1 80 »
.0085-.0635
.0550
184-2 16 M
.0007-.0063
.0056
latter half of gestation, an observation that agrees with the general de-
crease of oocytes of all sizes. On the whole, atresia of the eggs and
their follicles does not undergo a cyclic behavior as evident as that found
in the development of the oocytes.
DISCUSSION
The only serious discussion of the variation of germ cells during
gestation in viviparous teleosts is that of Turner (1937) on the ovo-
viviparous poeciliid fishes. In that article he compares a large number
of poeciliids and makes several classifications on the basis of (1) number
of broods of young in the ovary and (2) the relationship of the growing
oocytes to the stage of gestation. It is evident that Neotoca, a true
viviparous fish, cannot be classified with any of the poeciliids reviewed
by Turner in his interesting article.
In the Gambusia affinis type the growing oocytes are held back until
the birth of the current brood. Following that, they grow very rapidly
from 0.5 mm. to as much as 1.5 mm. in order to attain maximal size
before the next fertilization period.
78 GUILLERMO MENDOZA
In the Lcbistes reticulatus type, the oocytes are more or less grouped
into different sizes, the largest eggs being nearly of maximal size at the
end of gestation.
In the Quintana atrisona type, fertilization follows immediately upon
birth of the previous brood, necessitating that eggs be fully grown and
matured at time of birth of the preceding brood.
Two further types are those of Poecilistcs plcurospilus and Heteran-
dria formosa in which superfetation occurs, requiring that different sets
of oocytes grow, mature, and be fertilized while one or more broods of
young are still growing within the ovary. Even in Heterandria where
there are six levels of embryos. Turner recognizes one or two definite
waves of eggs.
Neotoca, on the other hand, is unique in that there are no such waves
or groups of embryos or oocytes. The present analysis shows a condi-
tion different from any described by Turner for the poeciliids. Here
the oocytes show a continuous gradation in size throughout gestation.
Unlike the Gatnbusia type, growth of oocytes is not inhibited completely,
merely somewhat retarded ; throughout gestation there is a variable num-
ber of eggs of maximal size. Because of this, there is no necessity for
a period of marked growth in the interval between birth of young and the
following fertilization period. The different sizes of oocytes merely con-
tinue their growth over the non-gestation period. It is true, however,
that in Neotoca there is a general activation of oocytes of all si/.es in the
resting interval because each size group shows a doubling or tripling in
the number of cells before the following fertilization period.
Furthermore, since there arc eggs of maximal size at the end of
gestation in Neotoca, it cannot be compared to Lcbistes. A second point
of difference is that in Lebistcs definite groups of oocytes are recognized ;
in Neotoca the gradation seems to be complete.
Neotoca resembles Quintana atrizona more than the others since in
both forms there are fully grown eggs at the end of gestation ; however,
Neotoca differs in that fertilization does not occur until seven days later
whereas in the poeciliid, fertilization follows in a few hours. Further-
more, in Quintana, apparently the full complement of eggs is present at
the end of gestation ; in Neotoca, however, only between 20-25 per cent
of the large eggs are present.
Finally, the condition of superfetation as in Poecilistcs and Heter-
andria has been made possible in part by the maturing of eggs before the
expulsion of the growing young from the ovary and in part by the re-
moval of a physiological block that prevents such growth and fertiliza-
tion. In view of this, superfetation technically could occur in Neotoca
since there are eggs fully grown throughout gestation but, in addition,
THE GERM CELL CYCLE IN NEOTOCA 79
there still is some physiological obstacle that normally prevents copulation
and fertilization. Despite these normal conditions, Turner has reported
(1940) occasional examples of superfetation in Neotoca. Furthermore,
there was one ovary known to the writer in which several eggs had been
fertilized within a few hours after the release of a previous brood but
the eggs were retained most abnormally within the follicles and not
evacuated as is normally true. This observation, in addition to Turner's
finding of occasional cases of superfetation, shows that the phenomenon
normally does not occur but may, in exceptional cases, get started.
These exceptional cases in Neotoca apparently are always abortive.
If the germ cell cycle as it occurs in Neotoca is true of most or all
Goodeidae, certainly an interesting difference occurs between two large
and important families of viviparous teleosts.
LITERATURE CITED
BAILEY, R. J., 1933. The ovarian cycle in the viviparous teleost Xiphophorus hel-
leri. Biol. Bull., 64 : 206-225.
MACDONAGH, E. J., 1934. Distribution geografica de los peces argentinos. Rev.
del Museo de La Plata, 34 : 21-170.
— , 1938. Contribution a la sistematica y etologia de los peces fluviales argen-
tinos. Rev. del Museo de La Plata (Nueva serie), 1: 119-208.
MENDOZA, G., 1938. El ciclo ovarico de la Neotoca bilineata. Rev. de Biologia y
Med., No. 3 : 20-25.
— , 1939. The reproductive cycle of the viviparous teleost, Neotoca bilineata, a
member of the family Goodeidae. I. The breeding cycle. Biol. Bull., 76:
359-370.
— , 1940. The reproductive cycle of the viviparous teleost, Neotoca bilineata, a
member of the family Goodeidae. II. The cyclic changes in the ovarian
soma during gestation. Biol. Bull., 78 : 349-365.
SCOTT, M. I. H., 1928. Sobre el desarrollo intraovarial de Fitzroyia lineata (Jen.)
Berg. Anal. Museo Hist. Nat. de Buenos Aires. 34: 361-424. (Ictio-
logia, Publ. No. 13.)
TURNER, C. L., 1933. Viviparity superimposed upon ovo-viviparity in the Good-
eidae, a family of cyprinodont teleost fishes of the Mexican Plateau. Jour.
Morph., 55: 207-251.
— , 1937. Reproductive cycles and superfetation in poeciliid fishes. Biol. Bull.
72: 145-164.
— , 1938a. Histological and cytological changes in the ovary of Cymatogastcr
aggregatus during gestation. Jour. Morph., 62 : 351-373.
— , 19386. The reproductive cycle of Brachyrhaphis episcopi, an ovoviviparous
poeciliid fish, in the natural tropical habitat. Biol. Bull., 75 : 56-65.
— , 1940. Superfetation in cyprinodont fishes. Copeia, No. 2: 88-91.
UPON THE PRESENCE AND DISTRIBUTION OF A
CHROMATOPHOROTROPIC PRINCIPLE IN THE
CENTRAL NERVOUS SYSTEM OF LIMULUS *
FRANK A. BROWN, JR., AND ONA CUNNINGHAM
(From llic Marine Biological Laboratory, Woods Hole, and flic Department
of Zoology, Northwestern University}
Certain definite glandular bodies in arthropods have been shown to
produce hormone substances. The more important of these are the
crustacean sinus gland, located in the eyestalks of a majority of decapod
crustaceans, and the corpora allata and the corpora cardiaca located in
the vicinity of the esophagus in insects. These glands appear to be
concerned with chromatic adaptations, growth, molt, reproduction, meta-
morphosis, and certain other functions.
Recently it has been pointed out that certain portions of the nervous
system act in an endocrine capacity. This has been demonstrated by
the work of Kopec (1922), Brown (1933), Fraenkel (1935), Hosoi
(1934), Brown and Ederstrom (1940), Wigglesworth (1940). Fur-
thermore, histological studies of the nervous system of invertebrates as
well as vertebrates have shown certain cells and cell clusters whose
cytoplasm is definitely filled with granules or colloid, very strongly sug-
gesting glandular activity (See Scharrer and Scharrer. 1940). This
latter paper also describes the presence of such neurosecretory cells in
Limulus.
With these facts in mind, we attempted to discover and measure an
endocrine activity of certain tissues in the arachnid Limulus. The only
work which had been done previously was that of Snyder-Cooper ( 1938 ) .
She was unable to discover any endocrine activity of the eyes, optic
nerves, or central nervous system of Limulus, using the chromatophore
system of Palaemonetes vulgaris as a test object. Since there are many
chromatophore types in the crustaceans and recent work has demon-
strated that the chromatophores show fundamental differences in their
responses to known endocrine materials, it appeared worthwhile to re-
investigate the problem using Limulus with a number of chromatophore
types other than those of Palaemonetes as an index of the presence of
an active chromatophoric substance.
1 This investigation was supported by a research grant from the graduate
school of Northwestern University.
80
CHROMATOPHOROTROPIC PRINCIPLE IN LIMULUS 81
The experiments reported here are restricted to a consideration of
the activity of the central nervous system, because it appeared to be the
most likely place of origin of an endocrine substance should any occur
within the group, especially so since a portion of the nervous system has
been shown to be active in both the other two classes of arthropods in-
vestigated. In this report a chromatophorotropic activity of the nervous
system of Lhnnlus will be described and it will be demonstrated con-
clusively that the active principle found within the nervous system is not
uniformly distributed throughout the nervous tissue but shows a definite
differential distribution. It may be seen in the paper following upon this
one (Scharrer, 1941) that this differential distribution can be correlated
with the distribution of neurosecretory cells within the central nervous
system of the same species.
MATERIALS AND METHODS
The experiments were commenced at the Marine Biological Labora-
tory at Woods Hole, Massachusetts, where freshly-caught Limulns and
Uca were available, and completed at Evanston, Illinois, using Limulus
and Uca which had been shipped from Woods Hole.
For the preparation of extracts of the nervous system, the live
Limulus was quickly opened up, the nervous system removed and placed
in sea water in a shallow container. The lateral nerves were trimmed
away, leaving only their short stubs attached to the large nerve ring and
the longitudinal chain of abdominal ganglia. In order to determine the
effectiveness of various regions of the central nervous system, the system
was cut with a scalpel into seven portions : section 1 included the anterior
portion of the nerve ring; section 2, the lateral portions; section 3, the
posterior portion of the nerve ring; sections 4, 5, 6 and 7 included re-
spectively the first, second, third, and terminal ganglionic masses of the
longitudinal cord. The relative positions of these cuts through the
nervous system can be seen in Fig. 1. Each of the seven portions of
the central nervous system was placed in a separate mortar and permitted
to dry briefly ; the nerve masses were then triturated thoroughly with
pestles, in 2 cc. of sea water. It is appreciated that the total volume of
extract obtained for the various nerve sections was somewhat different,
due to the different sizes of the nerve masses. However, since the larg-
est portion of the nervous system used in our experiments weighed less
than .04 gram (except in one animal), this error was not considered an
appreciable one. The extracts were then brought to a boil in order to
precipitate out protein materials from the solution. The clear super-
natant fluid was then used for assay purposes.
82
F. A. BROWN AND ONA CUNNINGHAM
With each experimental series a control solution was prepared, con-
sisting of a piece of muscle or digestive tract wall or gonad of approxi-
mately the same size as the largest nerve portion, extracted and treated
in the same manner as the experimental solutions.
A sample of each extract, including the control, was injected into
three blinded Uca pugnax, each Uca receiving an injection of approxi-
mately .05 cc. The injection was made into the basal segment of the
third or fourth thoracic appendage.
L
2
FIG. 1. Diagram of Limulus central nervous organs showing the sections of
the system which were separately assayed for the chromatophorotropic principle.
Five experimental series were run. The chromatophore index for
both black and white chromatophores was recorded at the beginning of
each experiment and readings were taken at 15, 30, 45, 60 and 90
minutes. In the first experiment a large Limulus, approximately 30 cm.
in length from the anterior end of the cephalothorax to the base of the
telson, served as the source of nervous tissue. In the remaining four
experiments smaller specimens of Limulus (about 12 cm. from anterior
tip to base of telson) provided the nervous tissue.
CHROMATOPHOROTROPIC PRINCIPLE IN LIMULUS 83
RESULTS
The results of these five experiments are shown in tabular form in
Tables la and Ib. These tables give only the average chromatophore
index for the three animals injected with each of the extracts, with the
indices for the black and white chromatophores of course averaged sepa-
rately. In these tables is shown also what has been called the coefficient
of effectiveness of the various extracts. This coefficient of effectiveness
we realize has only relative significance. It was calculated in the follow-
ing manner: the sum of the averaged chromatophore indices for each of
the two pigments at 15, 30, 45, 60 and 90-minute intervals was obtained.
Since an extract having no effect upon the black chromatophores would
leave these chromatophores with a chromatophore index of 1 (complete
contraction) at each interval — hence a sum of 5 — it was considered rea-
sonable to subtract the constant 5 from the sum obtained following injec-
tions of active extracts. Similarly, since an extract which would leave
the white chromatophores in an initially full dispersed condition (5)
would yield a sum of 25, a true index of the effect of an active extract
upon the white chromatophores would be the difference between the sum
of the average indices and 25. - In brief, the coefficient of effectiveness
for the black pigment is taken to be x - - 5 and the coefficient of effective-
ness of an extract in concentrating the white pigment is taken to be
25 — .r. In both of these instances x is equal to the sum of the averaged
indices. This we believe to be a fair indication of the effectiveness of the
extract since it takes into consideration both the magnitude and rate of
the response, and, in many cases, duration of the response as well.
Another step was taken to make all the data of the five experiments
comparable by obviating the differences which might exist as a result of
the different sizes of Limulus used for the experiments. This was done
by stating the effectiveness of the various portions of the nervous system
in terms of the percentage of the effectiveness of Part III (the posterior
portion of the ring), which was found in the earliest experiments to be
obviously far more effective than any other portion of the nervous sys-
tem. Thus, in Table I in the column " relative effectiveness " Part III
has been arbitrarily assigned an activity of 100 and percentages have
been calculated, on the basis of their coefficients of effectiveness, repre-
senting the effectiveness of the remaining portions of the nervous system
in proportion to Part III.
2 One difficulty arose which appeared to have no simple solution, namely, that
the white chromatophores sometimes initially had their white pigment partially
concentrated. It may readily be understood that to the extent to which this is
true, the demonstrated differences in concentration of the substance in the nervous
system assayed will be minimized.
84
F. A. BROWN AND ONA CUNNINGHAM
The results of these experiments are summarized in Table II, in
which the relative effectiveness of all the parts of the nervous system
and of the control solution have been assembled and averaged. Inspec-
tion of these data indicates clearly that Part III is the most active, then.
TABLE la
Effect of Extracts upon Uca White
Exp.
0
15
30
45
60
90
Sum
Coeffi-
cient of
Effec-
tiveness
(25-.v)
Relative
Effect
I
5.0
2.3
1.5
1.2
1.3
1.5
7.7
17.2
100.0
II
5.0
2.5
2.5
1.75
1.25
1.0
9.0
16.0
93.0
1 I"
5.0
5.0
2.8
2.0
1.5
3.0
1.5
1.8
1.0
2.0
1.0
2.0
7.7
10.7
17.2
14.2
100.0*
82.5
V
5.0
1.8
2.5
2.5
2.5
2.5
11.7
13.2
76.6
c
5.0
5.0
5.0
5.0
5.0
5.0
25.0
0.0
I
5.0
1.5
1.4
1.4
1.3
2.2
7.7
17.3
102.2
II
4.8
2.2
1.9
1.5
1.2
1.1
7.9
17.1
101.2
III
5.0
2.0
1.7
1.4
1.2
1.8
8.1
16.9
100.0*
IV
5.0
3.2
3.1
2.8
2.7
3.5
15.3
9.7
57.4
V
4.5
2.5
2.2
2.0
1.8
2.2
10.7
14.3
84.5
c
4.0
4.2
4.2
4.1
4.0
4.0
20.5
4.5
26.6
I
4.7
2.7
2.7
2.7
2.7
3.0
13.8
11.2
66.2
II
4.7
2.0
1.8
1.5
1.3
1.7
8.3
16.7
98.7
III
5.0
2.3
1.8
1.6
1.3
1.1
8.1
16.9
100.0*
in (y
5.0
5.0
2.8
3.0
2.3
3.0
2.5
3.4
2.7
3.8
2.8
4.5
13.1
17.7
11.9
7.3
70.4
43.2
VI
4.7
3.3
3.3
3.1
3.0
3.4
16.1
8.9
52.6
VII
3.4
2.0
1.8
1.8
1.8
2.3
9.7
15.3
90.5
c
4.5
4.5
5.0
4.7
4.5
4.5
23.2
1.8
10.7
I
3.7
1.8
1.8
1.7
1.5
1.5
8.3
16.7
91.3
II
2.8
1.5
1.5
1.3
1.1
1.1
6.5
18.5
101.0
III
2.3
1.7
1.5
1.3
1.1
1.1
6.7
18.3
100.0*
IV yV
3.5
3.8
2.8
2.8
2.8
2.7
2.7
2.7
2.5
2.7
2.8
3.1
13.6
14.0
11.4
11.0
62.0
60.1
VI
3.0
2.8
2.7
2.7
2.8
2.8
13.8
11.2
61.1
VII
3.2
2.7
2.7
2.8
3.0
3.0
14.2
10.8
59.0
c
2.8
3.7
3.7
3.7
3.7
3.7
18.5
6.5
35.5
I
2.8
1.5
1.3
1.4
1.5
1.3
7.0
18.0
102.2
II
3.2
2.0
1.7
1.4
1.3
1.5
7.9
17.1
97.2
III
3.2
1.7
1.5
1.4
1.3
1.5
7.4
17.6
100.0*
v IV
V
2.7
3.3
2.7
3.2
2.5
3.2
2.4
3.1
2.3
3.0
2.6
3.0
12.5
15.5
12.5
9.5
71.0
54.0
VI
4.3
3.5
3.0
3.0
3.0
3.2
15.7
9.3
52.9
VII
3.0
2.3
2.2
2.4
2.7
2.7
12.3
12.7
72.3
c
3.3
3.2
3.3
3.7
4.0
3.5
17.7
7.3
41.5
CHROMATOPHOROTROPIC PRINCIPLE IN L1MULUS
85
TABLE II >
Effect of Extracts upon Uca Black
Exp.
0
15
30
45
60
90
Sum
Coeffi-
cient of
Effec-
tiveness
Relative
Effect
I
1.0
2.5
2.5
3.3
2.7
3.0
14.0
9.0
81.8
II
1.0
2.0
1.8
2.2
2.7
3.3
12.0
7.0
63.6
I HI
1.0
2.3
2.5
2.7
4.3
4.2
16.0
11.0
100.0*
IV
1.0
2.0
2.5
2.7
2.5
2.8
12.7
7.7
70.0
V
1.0
3.0
2.5
3.0
3.2
2.3
14.0
9.0
81.8
c
1.0
1.0
1.0
1.0
1.0
1.0
5.0
0.0
0.0
1
1.0
1.7
1.8
1.8
1.8
1.7
8.8
3.8
38.8
II
1.0
1.5
2.6
3.5
4.5
3.0
15.1
10.1
103.0
III
1.0
1.8
2.5
3.7
4.4
2.4
14.8
9.8
100.0*
11 IV
1.0
1.8
1.6
1.3
1.1
1.0
6.8
1.8
18.3
V
1.0
1.3
1.3
1.3
1.3
1.0
6.2
1.2
12.2
c
1.0
1.0
1.0
1.0
1.0
1.0
5.0
0.0
0.0
I
1.0
2.2
2.3
2.3
2.2
2.7
11.7
6.7
58.8
II
1.0
1.5
2.5
3.0
3.3
2.7
13.0
8.0
70.2
III
1.0
1.5
3.2
3.7
4.2
3.8
16.4
11.4
100.0*
IV
1.0
1.5
1.7
1.4
1.2
1.0
6.8
1.8
15.8
III v
1.0
1.5
1.3
1.1
1.0
1.0
5.9
0.9
7.9
VI
1.0
1.3
1.2
1.0
1.0
1.0
5.5
0.5
4.4
VII
1.0
1.5
1.5
1.3
1.0
1.0
6.3
1.3
11.4
c
1.0
1.0
1.0
1.0
1.0
1.0
5.0
0.0
0.0
I
1.3
2.3
3.5
3.2
3.0
3.2
15.2
10.2
104.1
II
1.0
1.3
1.8
2.7
3.7
4.3
13.8
8.8
88.6
III
1.0
1.8
2.3
3.2
4.2
3.3
14.8
9.8
100.0*
IV
1.0
1.7
1.7
1.7
1.7
2.0
8.8
3.8
38.8
iv v
1.0
1.8
1.8
1.7
1.7
1.1
8.1
3.1
31.6
VI
1.0
1.1
1.7
1.4
1.1
1.0
6.3
1.3
13.3
VII
1.0
1.5
1.3
1.2
1.0
1.0
6.0
1.0
10.2
c
1.0
1.2
1.0
1.0
1.0
1.0
5.2
0.2
2.0
I
1.0
2.0
2.8
2.8
2.8
3.2
13.6
8.6
90.5
II
1.0
2.2
2.8
3.2
3.8
2.6
14.6
9.6
101.0
III
1.0
1.8
3.0
3.2
3.5
3.0
14.5
9.5
100.0*
V IV
1.0
1.3
1.5
1.3
1.1
1.0
6.2
1.2
12.6
V
1.0
1.0
1.0
1.0
1.2
1.0
5.2
0.2
2.1
VI
1.0
1.2
1.2
1.2
1.0
1.0
5.6
0.6
6.3
VII
1.0
1.5
1.5
1.5
1.5
1.3
7.3
2.3
24.2
c
1.0
1.0
1.0
1.0
1.0
1.0
5.0
0.0
0.0
in order, Part II and Part I, and finally the nerve tissue of the ganglia
of the longitudinal cord. With these data only, it is obviously impossible
to determine the concentration of active principle within the various parts
of the nervous system, since the portions varied considerably in size.
86
F. A. BROWN AND ON A CUNNINGHAM
TABLE II
Relative Effects of Parts of Central Nervous System
On White
Exp.
I
II
in
IV
V
VI
VII
Control
I
100.0
93.0
100.0
82.5
76.6
—
—
0.0
II
102.2
101.2
100.0
57.4
84.5
—
—
26.6
III
66.2
98.7
100.0
70.4
43.2
52.6
90.5
10.7
IV
91.3
101.0
100.0
62.0
60.1
61.1
59.0
35.5
V
102.2
97.2
100.0
71.0
54.0
52.9
72.3
41.5
Av.
92.4
98.2
100.0
68.7
63.7
55.5
73.9
22.8
On Black
Exp.
I
11
III
IV
V
VI
VII
Control
I
81.8
63.6
100.0
70.0
81.8
—
— .
0.0
II
38.8
103.0
100.0
18.3
12.2
—
—
0.0
lit
58.8
70.2
100.0
15.8
7.9
4.4
11.4
0.0
IV
104.1
83.6
100.0
38.8
31.6
13.3
10.2
1.0
V
90.5
101.0
100.0
12.6
2.1
6.3
24.2
0.0
Av.
74.8
84.3
100.0
31.1
27.1
8.0
15.3
0.2
To make this calculation, it was necessary to know two more facts : first.
the volume of the various parts of the nervous system extracted, and
second, the relation between the concentration of active principle within
an extract and the calculated coefficients of effectiveness.
In order to answer the first problem, the various parts of the nervous
system used were individually weighed prior to their extraction. The
results of these weighings are found in Table III.
TABLE III
Weights of Parts
Xo.
Part
Exp. IV
Wgt. Gins.
Exp. V
Wgt. Cms.
Average
I
Anterior nerve ring
.0291
.0350
.032
II
Lateral nerve ring
.0310
.0347
.033
III
Posterior nerve ring
.0219
.0306
.026
IV
First ganglion
.0047
.0044
.0046
V
Second ganglion
.0041
.0030
.0036
VI
Third ganglion
.0032
.0061
.0047
VII
Fourth ganglion
.0021*
.0073
.0047
Control
Muscle
.0186
.0246
.022
* Part of ganglion was lost in the preparation of the ganglion for weighing,
therefore average should be higher.
CHROMATOPHOROTROPIC PRINCIPLE IN LIMULUS 87
An experiment was then designed to determine the relationship be-
tween the coefficients of effectiveness and the concentration of the active
principle in the extracts. In two of the preceding- five experiments, a
portion of the extract prepared from Part III was set aside in order to
determine the effects of known dilutions upon the two chromatophoric
types of Uca. In this experiment the extract of Part III was diluted
to half its original concentration, then one-fourth, one-eighth, one-
sixteenth, one-thirty-second, and one-sixty-fourth. Each dilution stage
was injected into three Uca just as in the original assay experiments and
the coefficient of effectiveness of each dilution was calculated in the same
way. The results were expressed as percentages, keeping the original
concentration of Part III of the nervous system as 100 per cent with the
various dilution stages decreasing according to their coefficients. The
results of this experiment with respect to the white chromatophores are
seen in Table IVa. and for the black chromatophores in Table IVb.
These data were used for calculating the relative concentration of
active principle throughout the nervous system of Limulus, as follows:
a graph was constructed, the abscissa of which represented the logarithm
of the relative concentration and the ordinate the effectiveness in terms
of percentage of the original concentration. The results are plotted in
Fig. 2. The best smooth curves possible have been drawn through the
two series of eight points. With the aid of these plots, it was possible
to determine the relative concentration of active chromatophorotropic
principle by locating the percentage response on the graph and reading
the log of the relative concentration on the abscissa. Using this tech-
nique, the relative concentration of active principle for the various por-
tions of the nervous system used in this experiment were calculated.
In Fig. 3 we have plotted together, upon the same abscissa (the seg-
ments of the nervous system) but on different ordinates, the weights of
the various experimental sections of the nervous system and the apparent
relative total quantity of active principle in each part of the nervous
system. We have assumed that the specific gravity of all portions of the
nervous system is roughly constant, which seems reasonable.
Now the apparent relative quantity of active principle in each part
of the nervous system was divided by the weight in grams of that par-
ticular portion, and a figure was obtained which indicates the relative
concentration of the active principle in these portions. These calcula-
tions are summarized in Table V. Inspection of this table indicates that
Part III of the nervous system has double the concentration of Part II
and nearly four times the concentration of Parts I, IV, V and VII, and
nearlv ten times the concentration of Part VI.
88
F. A. BROWN AND ONA CUNNINGHAM
TABLK IVa
Effect of Dilution on White
15
30
45
60
90
Sum
Coeff.
Percentage
Relative
Effect
Exp. IV
1
1.7
1.5
1.3
1.1
1.1
6.7
18.3
100.0*
1/2
2.0
1.7
1.5
1.3
1.5
8.0
17.0
93.0
1/4
2.0
1.5
1.6
1.7
2.2
9.0
16.0
87.5
1/8
1.7
1.8
1.8
1.8
1.7
8.8
16.2
88.5
1/16
2.2
2.5
2.6
2.7
2.3
12.3
12.7
69.4
1/32
2.3
2.7
2.5
2.3
2.5
12.3
12.7
69.4
1/64
3.7
3.3
3.7
4.0
4.0
18.7
6.3
34.4
0
3.7
3.7
3.7
3.7
3.7
18.5
6.5
35.5
Exp. V
Average
1
1.7
1.5
1.4
1.3
1.5
7.4
17.6
100.0* 100.0
1/2
1.8
1.5
1.4
1.3
1.3
7.3
17.7
100.8 96.9
1/4
2.2
2.3
2.0
1.7
1.5
9.7
15.3
87.0 87.3
1/8
2.2
2.0
1.9
1.8
2.2
10.1
14.9
84.7 86.6
1/16
2.2
2.2
2.0
1.8
1.8
10.0
15.0
85.3 77.4
1/32
3.5
3.2
3.2
3.2
3.2
16.3
8.7
49.5 59.5
1/64
3.0
2.5
2.6
2.6
2.6
13.3
11.7
66.5 50.5
0
3.2
3.3
3.7
4.0
3.5
17.7
7.3
41.5 38.5
TABLE IVb
Effect of Dilution on Black
15
30
45
60
90
Sum
Coeff.
Percentage
Relative
Effect
Exp. IV
1
1.8
2.3
3.2
4.2
3.3
14.8
9.8
100.0*
1/2
2.2
. 2.3
2.7
3.2
3.2
13.6
8.6
87.7
1/4
2.8
3.8
3.5
3.2
3.0
16.3
11.3
113.0
1/8
2.3
2.3
2.4
2.5
2.3
11.8
6.8
69.4
1/16
1.8
1.7
1.6
1.5
1.3
7.9
2.9
29.6
1/32
1.5
1.3
1.3
1.3
1.2
6.7
1.7
17.3
1/64
1.2
1.3
1.3
1.3
1.2
6.3
1.3
13.3
0
1.0
1.2
1.0
1.0
1.0
5.2
0.2
2.0
Exp. V
Average
1
1.8
3.0
3.3
3.5
3.0
14.3
9.3
100.0* 100.6
1/2
2.0
2.3
2.3
2.3
2.6
11.5
6.5
69.9 78.8
1/4
1.5
1.5
1.6
1.7
1.7
8.0
3.0
32.2 72.6
1/8
1.7
1.7
1.9
2.2
1.7
9.2
4.2
45.1 57.2
1/16
1.5
1.5
1.6
1.8
1.7
8.1
3.1
33.3 31.5
1/32
1.3
1.3
1.5
1.7
1.5
7.3
2.3
24.7 21.0
1/64
1.0
1.0
1.0
1.3
1.0
5.3
0.3
3.2' 8.2
0
1.0
1.0
1.0
1.0
1.0
5.0
0.0
0.0 1.0
CHROMATOPHOROTROPIC PRINCIPLE IX L1MULUS
89
- 0 5 —1.0 - 1.5 - 2.0
LOG CONCENTRATION OF ACTIVE PRINCIPLE
- 00
FIG. 2. The relationship between the log concentration of the active principle
and the relative effectiveness upon the Uca white (dashed line) and black (solid
line) pigments.
-O32
E HI T3L ~3. 3ZL
PARTS OF NERVOUS SYSTEM
211
FIG. 3. Plotted together for comparison are the apparent relative quantity of
white pigment concentrating principle (dashed line), of black pigment dispersing
principle (dot-dash line), and weights of each of the seven assayed parts of the
Liinuliis nervous svstem.
90
F. A. BROWN AND ONA CUNNINGHAM
TABLE Y
Part
Apparent Rel.
Quant. Black
Disp. Principle
Apparent Rel.
Quant. White
Cone. Principle
Weight
of Part
(gms.)
Rel. Cone.
Black Disp.
Principle
Rel. Cone.
White Cone.
Principle
Average
I 0.32
0.32 0.032
10.0
10.0
10.0
II
0.47
0.75 0.033
14.3
22.8
18.6
III
1.00
1.00 0.026
38.5
38.5
38.5
IV
0.053
0.043
0.0045
11.8
9.6
10.7
V
0.045
0.034
0.0035 12.()
9.7
11.3
VI
0.016
0.022
0.0047 3.4
4.7
4.1
VII
0.024 0.054
0.0047 5.1
11.5
8.3
Effects of the Chromatophorotropic Principle of Limulus Nervous
System upon Certain Other Chromatophore Types
In the light of the work of Snyder-Cooper (1938) in which she
found no apparent Chromatophorotropic effects of injection of Limulus
nervous system extracts upon Palaemonetes chromatophores, we believed
it worthwhile to repeat her experiments. We were also unable to show
any response of either the red or the white chromatophores of Palaemo-
netes to these extracts.
Extracts of the nervous system of Limulus were tested upon isolated
chromatophores of Cambarus, according to the technique of Brown and
TABLE VI
Uca
Cambarus
Palaemonetes
Black
White
Red White
Red
White
Sinusgland
Cambarus
D
D
D
D
C
C
C
C
C
O
o
D
C
O
c
C
C
O
D
O
Sinusgland
Uca
Nervous system
Cambarus
Corpus cardiacum
insect* .
Brain
insect
Nervous system
Limulus ...
CHROMATOPHOROTROPIC PRINCIPLE IN LIMULUS (M
Meglitsch (1940) and it was found that the chromatophorotropic prin-
ciple of Limulus was very effective in concentrating white pigment, but
was entirely without effect upon the red. Furthermore, as Fig. 4 indi-
cates, the relative effectiveness of Parts I through VII was approxi-
mately the same upon Cambarus white chromatophores as upon Uca
black and white.
5
3
I
X 5
LJ
§3
cr
O
CL
O
t 3
o
cr
u
o o — -o — -o
o o — -o o o
o o o — -o — -o
o o — -o — -o
5 -0 o — o — -o — -o
.321
o o o o — -o
o — o — o o — -o
3ZL
o — -o — o — o o
CONTROL
15 30 45 600 15 50 45
TIME IN MINUTES
60
FIG. 4. Relative effects of the parts of Limulus nervous system upon crayfish
(Cambarus immums) white chromatophores (solid line) and red ones (dashed
line). Five upon the ordinate indicates a fully dispersed pigment mass, and one, a
fully concentrated pigment mass.
DISCUSSION
Comparison of the Chromatophorotropic Material of Limulus with
Chromatophorotropic Materials of Other Arthropods
Table VI has been prepared to show the effects of various arthropod
organ extracts upon six types of crustacean chromatophores. This table
is admittedly incomplete and although it would be both interesting and
instructive to have the gaps filled, it is still possible to draw certain con-
clusions from it as it is.
92 F. A. BROWN AND ONA CUNNINGHAM
In comparing the action of the Limulus nerve cord extract with that
of the sinus gland extracts of Uca and of Cambarus, we see that upon
Palacmonetes red and white chromatophores the Limulus extract has no
effect, whereas definite and characteristic effects are produced by sinus
gland. Upon Cambarus red, sinus gland exercises a strong concentrat-
ing influence; this is apparently entirely lacking in the L'uniilns extract.
Upon Cambarus white chromatophores, on the other hand, both the ex-
tracts are effective but result in opposite responses of the chromatophore.
Similarly, the two extracts have opposite actions upon Uca white chro-
matophores,3 but upon Uca black the activity of the two substances is
qualitatively the same.
It seems to us reasonable to assume that this is a similar response
of the chromatophore to two chemically different materials, in other
words, a non-specific chromatophore reaction. We assume this inas-
much as our experiments suggest that both the white-concentrating ac-
tion and the black-dispersing action were produced by the same material.
At first it may seem rather extraordinary that Limulus should be
suspected of having a chromatophorotropic material because of the ab-
sence of functional chromatophores in this group of animals, but many
other organisms without physiological color change (cockroaches, etc.)
possess active corpora cardiaca and sinus glands, and there is abundant
evidence accumulated that the chromatophorotropic action of these or-
gans is only one of a number of functions, many of which are far more
basic in the life processes of the animals than that of chromatic adapta-
tion. We have utilized the chromatophore response as a test method
with a full appreciation of this fact.
There are some who will contend that the materials with which we
dealt are nothing more than materials resulting from the mechanical
destruction of nerve tissue and possess no normal endocrine function
within the organism. This seems highly unlikely considering a number
of observations such as the restriction of a specific material to the com-
missural ganglion of Crago and the restriction of a specific action to the
corpora cardiaca of insects, and finally, in this research, to a definite
demonstration that the material is not uniformly distributed throughout
the nervous system, some portions of the nervous system showing
roughly ten times the concentration of active principle shown by others.
There is some evidence, however, that the material in question in Limulus
is not produced by a single locus within the nervous system and then
3 Abramowitz (1937) states that Uca eyestalk extract concentrates white pig-
ment in Uca pugna.v. We have been unable to confirm this observation of Abramo-
witz and, on the contrary, find that Uca eyestalk extract has a definite and striking
dispersing action upon white pigment, just as seen in Palacmonetes and Cambariis.
CHROMATOPHOROTROPIC PRINCIPLE IN LIMULUS 93
distributed out from this center by diffusion because it was demonstrated
in a brief experiment that the longitudinal commissure connecting the
posterior end of the nerve ring with the first ganglion of the longitudinal
chain showed significantly lower concentration of active principle (prac-
tically no effect) than either the posterior portion of the nerve ring or
the ganglion at its opposite end. In the case of diffusion, a smoothly
gradual decline in activity would be expected. The increased activity
at the posterior tip of the central nervous system also argues against the
diffusion of the material out from a single center. Therefore we are
inclined to believe that this differential distribution of activity in the
nervous system is the result of a differential distribution of cells actively
engaged in the production of the substance.
Tlic Probable Number of Hormonal Substances in Liinnlns
Nervous System
If one examines Fig. 3 one is impressed with the parallel nature of
quantitative distribution of the black and white pigment-concentrating
principles. The differences which occur are not only readily within the
experimental error but also are even astonishingly close to one another.
On the basis of these data there is no justification for any assumption
other than that these two pigments are being affected by one and the
same substance. An examination of Fig. 4, showing the effects of the
various parts of the nervous system of Liinnlns upon the white chro-
matophores of Cambarus, shows a quantitative gradation of activity of
the various parts quite parallel to those shown in Fig. 3 for the Uca
chromatophores. Again there is apparently no reason for assuming any-
thing other than that the substance active upon Cambarus white chro-
matophores is the same substance responsible for influence on Uca chro-
matophores. We realize the danger of drawing any conclusions upon
negative evidence and hence conclude only that there is no suggestion for
more than one chromatophorotropic principle in Llmulus nervous sys-
tem. However, just as Snyder-Cooper failed to show the presence of
any chromatophorotropic principle in Limulus using Palacmonetes red
and white chromatophores, so is it quite possible that utilizing other
chromatophores than we have tried will demonstrate the presence of
other hormones than we have been able to demonstate.
The action of Limulus extract is qualitatively unlike that of extracts
of the commissural ganglia and other nervous organs in Cambarus, as
shown by their effect upon Cambarus red chromatophores. The Cam-
barus extracts show an extremely potent activity in concentrating the red
pigment while Limulus extracts show no effect. On the other hand, the
94 F. A. BROWN AND ONA CUNNINGHAM
effects of these two extracts are identical upon Cambarns white chromato-
phores. Two alternative explanations are possible : ( 1 ) that the white-
concentrating principle from these two sources is similar and that Cam-
barus nervous system possesses in addition a red pigment-concentrating
principle, or (2) one could assume that each extract possesses a single
principle which is structurally different in the two cases. Again, Limulus
nerve organ extract differs from extracts of the corpora cardiaca of
insects in having a different action upon both red and white chromato-
phores of Cambarus. Finally, in comparing the activity of insect brain
and the activity of Limulus nervous system, one finds a qualitatively
similar action of these two extracts upon both red and white chromato-
phores of Cambarus. Neither possesses an effect on the Cambarus red
pigment and both exercise a white pigment-concentrating action. Of
course it is too soon even to suspect that these latter two substances are
identical and further conclusions cannot be drawn until more properties
of these two substances have been shown to be identical.
A consideration of these results brings us to a complete realization
that a unitary theory of hormonal control of chromatophores in crus-
taceans— and even more in arthropods in general — is completely un-
tenable. There are undoubtedly several different chromatophorotropic
materials found within the various groups, but it is not beyond the realm
of possibility that certain threads of similarity or continuity can be woven
through various active tissues and their secreted principles in this phylum
of animals.
It can be calculated readily that the posterior portion of circum-
esophageal nerve ring of Limulus is still effective when diluted in nearly
5000 times its volume of salt solution. The active secreting cells un-
doubtedly occupy a very small percentage of the volume of the nervous
tissue and consequently, in terms of the ratio of neuro-glandular tissue
to volume of extract, the maximal dilution value would be in the hun-
dreds of thousands or even millions.
SUMMARY
1. A principle influencing pigment concentration in Uca chromato-
phores is found in extracts of the central nervous system of Limulus
polyphemus. This principle is not uniformly distributed through the
central nervous system of Limulus but is concentrated in the ganglionic
masses, with the greatest quantity in the posterior portion of the circum-
esophageal nerve ring. The lateral portions of the nerve ring show
approximately one-half the concentration of the posterior portion and
CHROMATOPHOROTROPIC PRINCIPLE IN LIMULUS
all the remaining portions of the nervous system show from one-quarter
to one-tenth the concentrations of the posterior portion of the nerve ring.
2. The distribution of the principle influencing Uca white pigment
appears to be identical with that producing dispersion of the Uca black
pigment and concentration of Cainbants white pigment. Hence it is
concluded that all three of these effects are brought about by one and
the same principle.
3. Certain physiological properties of the chromatophorotropic mate-
rial from the nervous system of L'unulns were compared with corre-
sponding properties of certain other invertebrate hormones and it was
found that the Linnilus chromatophorotropic principle is physiologically
unlike any other known arthropod hormone substance with the possible
exception of insect brain extract.
4. It is calculated that an extract of the posterior portion of the
circumesophageal nerve ring is still effective when diluted in nearly 5000
times its volume of salt solution.
LITERATURE CITED
ABRAMOWITZ, A. A., 1937. The comparative physiology of pigmentary responses
in the Crustacea. Jour. E.rfcr. Zoo!.. 76 : 407-422.
BROWN, F. A., JR.. 1933. The controlling mechanism of chromatophores in Palae-
monetes. Proc. Xiit. .Ictid. Sci. Washington, 19: 327—329.
BROWN, F. A., JR. AND H. E. EDERSTROM, 1940. Dual control of certain black
chromatophores of Crago. J our. E.vpcr. Zoo!., 85 : 53-69.
BROWN, F. A., JR. AND A. MEGLITSCH, 1940. Comparison of the chromatophoro-
tropic activity of insect corpora carcliaca with that of crustacean sinus
glands. Biol. Bull.. 79: 409-418.
FRAENKEL, G., 1935. A hormone causing pupation in the blowfly Calliphora
erythrocephala. Proc. Roy. Soc. London, Scr. B, 118: 1-12.
Hosoi, T., 1934. Chromatophore-activating substance in the shrimps. Jour. Fac.
Sci. Jinf>. Univ. Tokyo, Section IV. Zoology, 3: 265-270.
KOPEC. S., 1922. Studies on the necessity of the brain for the inception of insect
metamorphosis. Biol. Bull.. 42: 323-342.
SCHARRER, B., 1941. Neurosecretion. IV. Localization of neurosecretory cells in
the central nervous system of Limulus. Biol. Bull.. 81 : 96.
SCHARRER, E., AND B. SCHARRER, 1940. Secretory cells within the hypothalamus.
Res. Pnhl. Ass. Nerv. Mcnt. Dis., 20: 170-194.
SxvDER-CoopER, RUTH, 1938. Probable absence of a chromatophore activator in
Limulus polyphemus. Biol. Bull., 75: 369 (Abstract).
WIGGLES WORTH, V. B., 1940. The determination of characters at metamorphosis
in Rhodnius prolixus (Hemiptera). Jour. E.rf^cr. Biol., 17: 201-222.
NEUROSECRETION
•
IV. LOCALIZATION OF NEUROSECRETORY CELLS IN THE CENTRAL
XKRVOUS SYSTEM OF LIMULUS
BERTA SCHARRER
(From the Department of Anatomy, Western Reserve University and the Marine
Biological Laboratory, Woods Hole, Mass.)
From an investigation, still under way, of the neuroglandular cells
in the central nervous system of the horseshoe crab (Limulus}, mention
of which has been made in a previous review article (Scharrer and
Scharrer, 1940), some data concerning the occurrence, localization, and
numerical distribution of these cells are reported here. The publication
of these data appears to be timely in view of the findings of Brown and
Cunningham, reported in the preceding paper of this journal (1941).
These authors demonstrate the presence of a chromatophorotropic prin-
ciple within the nervous centers of Limulns and calculate the concen-
tration of this substance in different, separately tested portions of the
nervous system. They conclude that the chromatophorotropic principle
is probably produced in definite groups of cells within the central nervous
system. The agreement between the results of the physiological work
of Brown and Cunningham and the morphological findings to be re-
ported here suggests the possibility that in Limulus the neurosecretory
cells are the source of the chromatophorotropic principle.
MATERIAL AND METHODS
Altogether forty adult male and female specimens of Limulus poly-
phemus were studied. Most of the material was preserved during the
summer months of 1937, 1939, and 1940 at the Marine Biological Lab-
oratory, Woods Hole.1 Additional specimens were obtained alive from
the New York Aquarium,2 and were preserved for histological study at
various intervals during the years 1938-1940. Furthermore, a few
young specimens of Limulus polyphemus and two female L'umthis inoluc-
canus, one of them from Penang (Malay Peninsula) fixed in the summer
1 For the use of research facilities during these periods the author is indebted
to the Rockefeller Foundation and to the Rockefeller Institute for Medical Re-
search, New York.
- I am obliged to Dr. C. W. Coates for his friendly assistance in obtaining this
material.
96
NEUROSECRETORY CELLS IX I.1MULUS 9/
of 1938, the other from the mangrove swamps at Chandipur (Orissa,
India). :i were included in this study.
In order to obtain comparable results, the same histological tech-
nique was used in all cases. The central nervous system, which in adult
specimens is of considerable size, was carefully dissected out and was
fixed in Zenker-formol. It was subsequently embedded in celloidin
(nitrocellulose) and horizontal serial sections of 20 p. were stained with
Foot's modification of Masson's trichrome method.
The various degrees of neurosecretory activity to be found in dif-
ferent parts of the central nervous system as well as in the different
specimens of L'unnlus studied were estimated by counting in every sec-
tion all cells containing secretory colloid. Thus undoubtedly a certain
percentage of the cells was counted more than once because the vacuoles
containing colloid are often large enough to appear in more than one
section of 20 p. thickness. This is not to be considered an error of
consequence, because this method of recording colloid whenever it ap-
pears in the sections, even if it belongs to the same cell, takes account
of the volume of colloid as a whole rather than of the actual number of
cells containing colloid inclusions. For purposes of comparing the secre-
tory activity of different regions of the central nervous system this
method appears to be satisfactory.
OBSERVATIONS
The Histoloc/ical Appearance of the N euro glandular Cells
It is not intended here to describe the cytological characteristics of
cells considered as having a glandular function or to investigate the steps
in which the transformation of a nerve cell into a gland cell takes place.
In the present study the concern is only with such cells as are believed
to represent the fully developed type of neurosecretory cell characteristic
of Linniliis. This cell type is fairly uniform and easily recognizable.
The cells contain large masses of a colloidlike substance (Fig. 1 ) which
appears homogeneous in sections treated in the manner described before.
The substance stains green with the light green component of the Masson
trichrome stain and seems to have physical properties not unlike those of
the colloid of the thyroid gland (for corresponding parallels see also
Hanstrom, 1941). This similarity is also suggested by vacuoles in the
periphery of the colloid masses in the cells of Limulus which remind the
observer of similar vacuoles seen in sections of thyroid colloid. Appar-
ently this colloid mass pushes the nucleus and the cytoplasm aside and
3 The efficient cooperation of Dr. Baini Prashad, Director of the Indian
Museum, Calcutta. India, is gratefully acknowledged.
98
B. SCHARRER
takes up a large space in the cell. These conspicuous colloid-carrying
cells were counted.
Cells of this kind have also been found in the ganglia of cockroaches
(Scharrer, 1941). However, in the cockroach they represent one of
several kinds of neurosecretory cells, whereas in Linntlns only this one
type is encountered. There are in Limulns cells the appearance of which
suggests that they represent phases preceding the fully developed " ma-
ture " neurosecretory cell but their description is not undertaken here,
since their relation, if any, to the colloid-containing cells is still undeter-
mined. From a histological point of view they certainly seem insignifi-
cant as a possible source of secretion material when compared with the
FIG. 1. Three neurosecretory cells, each in its capsule, from the circum-
csophageal ring of Liuntliis polyphcmus with partly vacuolated colloid indicated in
solid black. Zenker-formol. nitrocellulose, 20 A1, Masson.
cells containing the large masses of colloid. The contrast between the
ordinary nerve cells, including those in which differences in stainability
of the cytoplasm etc. suggest that they may be in a state of transforma-
tion into glandular elements, and the cells counted here as neurosecretory
cells is always so definite that no doubt arises as to which cells should IK-
included in the counts. Liunthis is particularly favorable in this respect
for the kind of investigation carried out here.
It should be repeated that the cells containing a homogeneous mass
of colloid with varying numbers of marginal vacuoles are always observed
when the histological technique described before is used. The fact that
the appearance of the colloid is somewhat different after treatment by
different methods is of no concern here where only the amount and
distribution of the secretory elements are of interest.
NEUROSECRETORY CELLS IN LIMULUS
The Localisation of tlic N euro glandular Cells within the Central
Nervous System
The central nervous system of Linniliis consists of the circumesopha-
geal ring, situated in the cephalothorax, and the abdominal ganglia (Fig.
2). The ring contains the "brain" and a number of thoracic ganglia
of the ventral cord, designated in Fig. 2 as Xos. 1-8, beginning with the
cheliceral ganglia. There are, in addition, eight pairs of abdominal
ganglia, only the first four or five of which are well defined, the re-
maining pairs being fused together (Patten and Redenbaugh, 1900).
With the exception of the corpora pedunculata which make up about
three-fourths of the brain, all parts of the central nervous system of
Linntlns contain neurosecretory elements among the ordinary nerve cells.
The distribution of the neurosecretory cells varies, however, with respect
to the different ganglia and their total number shows the greatest varia-
tions from specimen to specimen.
A most " active " neurosecretory region is the posterior part of the
circumesophageal ring, i.e. the area of the thoracic ganglia No. 6 and
No. 7. Here the neuroglandular elements are found in clusters which
may constitute a considerable proportion of the total mass of cells in the
ganglion. These clusters of glandlike cells are arranged symmetrically
with respect to the mid-sagittal plane (Fig. 3).
More anteriorly in the ring the neuroglandular cells become less and
less frequent. They may even be absent in these portions, particularly
in those specimens which, on the whole, show less neurosecretion than
others. If present, neuroglandular cells in ganglia No. 1 to No. 5 appeal-
single or in small groups of two or three. Their approximate number
and position are the same on both the left and right side of the ring.
Of all neurosecretory cells found in the circumesophageal ring only
about one-tenth or less lie in the two anterior thirds, the majority being
concentrated in the posterior third of the whole ring.
The abdominal ganglia, counted together, contain roughly on the1
average twice or three times as man}- neurosecretory elements as the
corresponding ring. The numbers of neuroglandular cells counted in
the circumesophageal ring and in the abdominal ganglia of four speci-
mens are given here as examples:
l/ircumcsopliageal ring 117 206 264 516
Abdominal ganglia 340 442 861 1978
Thus as a rule and within limits, from the degree of neurosecretory ac-
tivity in a given ring the activity in the abdominal ganglia of the same
specimen can be predicted. The individual abdominal ganglia do not
100
B. SCHARRER
BRAIN
CIRCUM-
ESOPHAGEAL
DINS
VENTDAL
CORD
ABDOMINAL
GANGLIA
FIG. 2. Diagram of the central nervous system of Limnliis. Numbers 1-8 are
the ganglia of the circumesophageal ring. The areas where neurosecretory cells
may be found are dotted. Coarser dots indicate a higher degree of neuroglandular
activity than finer dots.
NEUROSECRETORY CELLS IN LIMULUS
101
seem to differ significantly from each other in number of neurosecretory
cells.
Individual I'ariations of Neurosecretory Activity
Whereas the central nervous system of a few young specimens of
Liniulus Polyphemus (width of carapace from 5 to 8 cm.) does not con-
3^" '" ;;W ("
I
MSC
NC
FIG. 3. Horizontal section through posterior part of the circumesophageal
ring of Limulus polyphemus. NSC, neurosecretory cells; NC, nerve cells. The
colloid of the neurosecretory cells which appears green with the histological tech-
nique used is indicated by solid black. Note symmetry of neurosecretory cell
groups. Zenker-formol, nitrocellulose, 20 ,"., Masson.
tain neuroglandular cells, these cells are present in all adults studied thus
far. This concerns animals from different geographical sources as well
as from two different species. Furthermore, neurosecretion is found
in hoth male and female Limulus.
The degree of neurosecretory activity, however, varies considerably
from specimen to specimen. In some, the entire central nervous system
is found to contain only one or two neuroglandular cells, in others ovet
102 B. SCHARRER
a thousand such cells may be counted by the method described. The
highest count thus far made, 2494, was in a large female. Between these
extremes are such counts as 457, 648, 1125. These figures are given
only to show the wide variation in view of which the errors necessarily
involved in the calculation method employed here appear of minor
importance.
The question may be asked next whether there exist any relations
between the degree of neurosecretory activity and certain known factors.
The following observations were made:
(1) A 24-hour cycle of secretion does not seem to exist. The his-
tological appearance of neuroglandular cells is the same in different
specimens fixed at various hours of the day.
(2) Neurosecretion in Limulus is not restricted to one time of the
year, such as, for instance, the breeding season. None of the summer
specimens contains more neuroglandular cells than the animals with the
highest counts fixed in January, March, or November.
(3) The degree of neurosecretory activity in males and females is
not essentially different. On the average, however, smaller numbers of
neuroglandular cells are found in males, but this may be explained by
the smaller average size of the male central nervous system.
(4) So far the only factor of some importance appears to be tin-
age of the animals, as expressed by the size, i.e. the width of the carapace.
As a rule, the larger specimens show a more active state of neurosecre-
tion. The Limulus with the highest count of neurosecretory elements
(almost 2500) was the largest specimen studied, a female of 32 cm.
width which contained many and large eggs. On the other hand, none
of five females under 23 cm. width showed more than about five secreting
cells. Also among the males the largest specimen examined yielded the
highest count but that does not exclude the fact that one or another small
male may be encountered in a comparatively active state of neurosecre-
tion. Thus, for instance, in the smallest male among a dozen studied,
having a carapace-width of 16.5 cm., the relatively high number of 64S
secreting cells was found. For comparison it may be noted that in
two other male specimens these counts were made : carapace 19 cm., 5
cells ; carapace 20 cm., 205 cells.
Although the existing evidence is not entirely conclusive, the extent
to which nerve cells are engaged in secretory activity seems to run grossly
parallel with age as expressed in the size of the animal. This fact has
to be taken into account in attempts to influence experimentally the ratio
of neuroglandular cells. Such experiments as have been carried out
up to the present time were unsuccessful. In one extensive series ex-
tracts were made from the circumesophageal ring of the central nervous
NEUROSECRETORY CELLS IN LIMULUS 103
system of Limit/its by grinding it with sand and sea water. The ex-
tracts from several, for instance five, different specimens of varying
sizes and different sex, were pooled and injected into the body cavity of
male and female specimens over varying periods of time. Approximate
estimates were made of the total number of rings injected into each
experimental animal. Thus, for instance, each of several animals got
the equivalent of 17 rings over a period of one month. The counts made
in such animals kept well within the limits of the normal variation :
<$ 21 cm. 1073 $ 23 cm. 1
21 " 617 23.5 " 22
22 " 446 28 " 492
From the number of experiments done by the method described it
can be safely concluded that sea water extracts from the neurosecretory
cells of Limulus do not influence the number of cells engaged in secre-
tion. It may be added also that no change in the histological appearance
of these cells has been observed.
Similarly ineffectual were injections of pilocarpin. Of a 1 per cent
solution in two specimens as much as 19 cc. were administered by means
of two injections, both given on one day. Smaller amounts were in-
jected in others. Again, the counts and the histological appearance of
the cells gave no indication that pilocarpin acts on the neuroglandular
cells in the concentrations used here which, in view of their general effect
on the animals, may be considered as near the toxic ones.
DISCUSSION
The data reported here on the occurrence and distribution of neuro-
secretory cells in the central nervous ganglia of Limulus correlate well
with the findings of Brown and Cunningham (1941). The two authors
describe a chromatophorotropic principle in extracts from all parts of
the central nervous system of Limulus polyphemus. From the effect
on crustacean chromatophores they conclude that the concentration of the
active material varies with respect to different, separately tested parts
of the central nervous system. The present histological study demon-
strates the presence of nerve cells offering the picture of gland cells in
all portions of the nervous system tested by Brown and Cunningham.
The distribution of these elements in the nervous tissue varies in dif-
ferent regions, and this variation corresponds well with the distribution
of the chromatophorotropic material found by these two authors. This
correlation becomes particularly evident in the different portions of the
circumesophageal ring. Its posterior third for which Brown and Cun-
ningham report the greatest concentration of the active principle con-
104 B. SCHARRER
tains the majority of neurosecretory elements present in the ring. In the
lateral sectors where the relative chromatophorotropic activity shows a
considerable decrease, many fewer cells are found to contain secretory
colloid. The anterior portion of the ring with relatively the lowest ac-
tion on crustacean chromatophores contains glandular cells only occa-
sionally. The connectives between the circumesophageal ring and the
abdominal ganglia did not yield the chromatophorotropic principle;
neither colloid nor neuroglandular cells are found in these connectives.
Considering that the estimate of the concentration of the chromato-
phorotropic principle in the nervous system of Limulus must necessarily
be approximate, and that the cell counts may mean little in terms of
function, the correlation between physiological and anatomical findings
demonstrated here seems all that can be expected.
Within its obvious limitations this correlation consequently suggests
that the neurosecretory cells in the central nervous system of Limulits
may be considered as the source of the chromatophorotropic principle.
If this proves to be correct the functional significance of neurosecretion
assumes a new aspect. Thus far only one function has, with good evi-
dence, been attributed to neuroglandular cells, namely the production of
a hormone controlling molting in insects (Wigglesworth, 1940).
SUMMARY
The occurrence, localization, and quantitative distribution of neuro-
secretory cells in the central nervous system of Linmlns have been de-
scribed. These anatomical findings are in good agreement with the
physiological data of Brown and Cunningham (1941), who demonstrate
the presence and distribution of a chromatophorotropic principle in the
nervous system of this animal. Therefore, the neurosecretory cells of
Limulus may be considered as the source of a substance influencing color
change in crustaceans.
LITERATURE CITED
BROWN, F. A., JR., AND O. CUNNINGHAM, 1941. Upon the presence and distribu-
tion of a chromatophorotropic principle in the central nervous system of
Limulus. Biol. Bull., 81 : 80.
HANSTROM, B., 1941. Einige Parallelen im Bau und in der Herkunft der inkre-
torischen Organe der Arthropoden und der Vertebraten. Lunds Univ.
Arsskrift N.F. Avd. 2., 37, No. 4: 1-19.
PATTEN, W., AND W. A. REDENBAUGH, 1900. Studies on Limulus. II. The nervous
system of Limulus polyphemus, with observations upon the general anat-
omy. Jour. Morph., 16: 91-200.
SCHARRER, B., 1941. Neurosecretion. II. Neurosecretory cells in the central nervous
system of cockroaches. Jour. Comp. Neur., 74 : 93-108.
SCHARRER, E., AND B. SCHARRER, 1940. Secretory cells within the hypothalamus.
Res. Publ. Ass. nerv. ment. Dis., 20: 170-194.
WIGGLESWORTH, V. B., 1940. The determination of characters at metamorphosis
in Rhodnius prolixus (Hemiptera). Jour, exper. Biol., 17: 201-222.
THE ACTION OF ACETYLCHOLINE, ATROPINE AND
PHYSOSTIGMINE ON THE INTESTINE OF
DAPHNIA MAGNA
VASIL OBRESHKOVE
(From Bard College, Columbia University']
INTRODUCTION
The effects of acctylcholine when administered to animals under ex-
perimental conditions have recently afforded valuable information which
suggests the possibility that acetylcholine is acting as a chemical trans-
mitter of nervous impulses from nerve endings to certain organs of the
body. In view of the excitatory action of acetylcholine which has been
demonstrated on the heart of crustaceans (Welsh, 1939 a and b} and
the influence of this substance on the autotomy of certain members of
this group of animals (Welsh and Haskin, 1939), it appears worth while
to investigate the action of this drug on the intestine of a cladoceran, a
problem heretofore unexplored. Although there is nothing in Cladocera
which corresponds morphologically to the autonomic nervous system in
vertebrates, the intestine of these animals is subject to accelatory and
inhibitory nervous influences. If the intestine of Daphnia magna, for
example, is touched with a very fine glass needle at the bend of the
digestive tube where the intestine enters the stomach, the heart imme-
diately stops beating and the posterior region of the intestine commences
to exhibit powerful intestinal contractions. After a certain period, de-
pending on the strength of the mechanical stimulus applied, the heart
renews its activity and the intestine reestablishes its normal muscular
contractions. If acetylcholine is involved in the transmission of nervous
impulses to this organ, it should be possible to obtain some evidence of
the action of this substance and other substances with which it has been
said to be associated, when they are administered to this animal.
METHODS
Daphnia magna young, when in their second instar, were used ex-
clusively for the experimental work. The animals at this stage measure
about 1 mm. in length, they are more transparent than the adult indi-
viduals and hence the changes produced in the course of the experimen-
tation can be easily observed under the microscope. The mothers from
105
106 VASIL OBRESHKOVE
which the young were obtained for the experiments were reared at 25°
C. in bottles containing the standard amount of the culture medium
(Banta, 1921). The daily examination of the animals and the other
methods employed in rearing the organisms in the laboratory (Obresh-
kove, 1930) enabled us at all times in the course of the experimental
work to secure animals which were of the same age. A careful selection
of the animals was necessary, because of the endeavor which was made
to measure the period which elapsed between the addition of the par-
ticular chemical substances under investigation and the characteristic
changes which they produced. The chemical substances employed were
acetylcholine chloride, physostigmine (eserine) and atropine. Care was
taken to use freshly diluted chemicals. The acetylcholine was adjusted
to pH 5.7.
The animals were subjected to experimentation separately. A single
individual was transferred to a micro culture slide with polished spherical
concavity 18 mm. in diameter and approximately 3 mm. deep. The cul-
ture medium surrounding the animal was removed and immediately after
this there was added the chemical substance whose action on the animal
was to be studied. The amount of solution employed in each depression
slide was kept the same and in each case it was just sufficient to cover
the animal without permitting it to carry on extensive locomotive move-
ments. This procedure enabled us to make continuous observations on
a single individual under the microscope. The animal is seen at all
times to ingest solid particles and fluid with which it comes in contact in
the depression slide. With each opening of the mouth, a quick and
powerful peristaltic wave of the esophagus forces the ingested material
into the stomach, a process which can be easily observed under the micro-
scope. Normally about 40 such peristaltic waves occur each minute.
It is suggested, therefore, that the drugs employed in this work were
administered orally.
EXPERIMENTAL RESULTS
The intestine of untreated animals usually exhibits movements which
are more or less rhythmic in nature. There is a gentle surging back and
forth of the nutritive material and only when the animal is in the act
of evacuating the contents of the intestine is one able to observe peristaltic
and antiperistaltic waves in the musculature of the organ itself. At such
times the forward peristalsis becomes more noticeable than the reverse
peristalsis, the anus opens and the animal excretes only a small portion
of the intestinal contents. This act is repeated at irregular intervals
which varv from 30 seconds to more than 1 minute in some cases. At no
ACTION OF ACETYLCHOLINE 107
time, however, is the intestine entirely empty, for in the depression slide
the animal is continuously reengulfing the materials which it has evac-
uated.
THE ACTION OF ACETYLCHOLINE
When a Daphnia iiun/na young is treated with acetylcholine, a very
distinct change occurs in the intestine. The muscular peristaltic and
antiperistaltic contractions of the organ become extremely violent and
when stronger solutions are employed the entire contents of the intestine
are emptied in a little more than a minute. The time which elapses be-
tween the application of the drug and the appearance of the first vigorous
muscular contraction varies very definitely with the concentration of the
drug employed. From an inspection of Table I it is seen that with
acetylcholine 1 X 10~2 this occurs on the average in less than 20 seconds
and with acetylcholine 1 X 10~3 this period is increased to 27.4 seconds.
There is not a gradual development in the establishment of the violent
intestinal activity. When, the drug becomes effective, it exhibits its
effectiveness to the fullest extent with an abrupt initial powerful con-
tractile wave of considerable amplitude. After treatment with acetyl-
choline 1 X 10~3 and subsequent transference to water, vigorous forward
and reverse peristalsis will continue in some cases for as long as 20 or
30 minutes. Acetylcholine 1 X 10~2 with lapse of time produces high
intestinal tone and contracture.
When Daf>lniia inagna young are treated with acetylcholine 1 ] ' 10~4,
the time which elapses between the addition of the drug and the first
appearance of the characteristic effect produced is on the average 10.7
minutes for the group of experiments presented here (Table I, column
3). With further dilution of the drug this period becomes longer.
With acetylcholine 1 ; ; W ' the time varies from 50 to 137 minutes
(Table I, column 4). showing a definite and considerable increase over
the time of reaction obtained with the higher concentrations of the drug.
THE ACTION OF ATROPINE
Atropine was found to antagonize the action of acetylcholine. When
Daphnia inagna are treated with acetylcholine until the characteristic
powerful action of the intestine is established and then the solution is
replaced by atropine, the effects of acetylcholine are quickly abolished.
The powerful contractions, which would otherwise persist for many min-
utes, not only disappear but in many individuals after the atropine has
become fully effective there is no longer any evidence of intestinal mus-
cular contractions. Atropine 10 - abolishes the effect of acetvlcholine of
108
VASIL OBRESHKOVE
the same concentration in less than 20 seconds, but atropine was found
to be effective even in dilutions of 1 ' [ 10~9. The range of effectiveness
beyond this concentration of the drug was not investigated. The results
obtained with acetylcholine 1 X 10~3 and atropine 1 X 10~s are shown in
Table II. The rapidity with which acetylcholine 1 X 10~3 produced its
characteristic action on the intestine is shown here to be no different from
that previously recorded in this paper (Table I). Atropine 1 X 10~5.
on the other hand, repeatedly abolished the effects of acetylcholine within
20 to 52 seconds. Table II also shows that following the abolishing of
the powerful intestinal contractions by atropine, a stronger solution of
TABLE I
Onset of vigorous intestinal contractions in Daphnia magna after treatment
with acetylcholine of various concentrations. The time of action is expressed in
seconds or minutes and represents the period elapsing from the addition of the drug
to the appearance of the characteristic effect.
Acetylcholine
1X10-2
Acetylcholine
lX10-»
Acetylcholine
1X10-"
Acetylcholine
1X10-'
seconds
seconds
minutes
minutes
20
25
10.8
119
20
35
8.2
137
22
40
8.2
123
19
30
14.4
125
22
30
12.3
74
15
20
9.3
113
16
22
11.7
110
17
25
12.2
124
19
20
9.3
50
24
27
10.8
69
Average 19.4
27.4
10.7
104.4
acetylcholine (1 X 10~2) reestablished the previous effect of acetylcho-
line, the average time for this being 21.8 seconds — a reaction time char-
acteristic for this concentration of the drug (compare with Table I).
Acetylcholine and atropine of the dilutions employed in this work
produced no lethal effect on the animals. Likewise atropine, when it
was repeatedly administered to the same individual after treatments
with acetylcholine, had no paralytic effect on the musculature of the
intestine. To test this, a single individual was subjected to experi-
mentation in the following way. The animal was treated with acetyl-
choline 1 " [ 102. Immediately after the appearance of strong intestinal
contractions, the drug was removed and replaced with atropine 1 X 10~5.
After the abolishing of the muscular contractions, the animal was again
treated with acetylcholine and then atropine of the same dilutions as pre-
ACTION OF ACETVI.i HoLlNl-. 10«)
TABLE II
Onset of vigorous intestinal contractions in Daphnia magna after treatment
with acetylcholine; the time of abolishing the acetylcholine effect by atropine; and
the time of reestablishment of strong contractions by acetylcholine following atropine.
Acetylcholine 1 XIO"3 Atropine 1 Xlfl-s Acetylcholine 1 X10~2
Time of action
Time of abolishing of acetylcholine
Time of action acetylcholine effect 10~2 after atropine
seconds
seconds
seconds
29
30
20
26
30
25
25
40
22
26
52
18
34
22
35
38
36
32
22
38
20
38
20
18
24
38
20
32
34
22
28
35
18
34
42
18
34
38
20
43
42
20
22
40
19
Average 30.3
35.8
21.8
TABLE III
The effects of repeated treatment of a single Daphnia magna with acetylcholine
and atropine at regular intervals of a few seconds.
Acetylcholine 1 X10~2 Atropine 1 XIO"5
Time of abolishing
Time of action acetylcholine effect
seconds seconds
20 90
20 110
18 80
14 80
16 95
13 70
14 110
14 80
28 52
18 85
Average 17.7 85.2
110 VASIL OBRESHKOVE
viously employed. This procedure was repeated on the same individual
for ten times and the results which were obtained are shown on Table
III. It is evident from an inspection of the table that the drugs con-
tinued after each application to produce their characteristic effects. At-
ropine 1 ] [ 10' :' under the conditions employed in this set of experi-
ments required on the average 85.2 seconds to produce its characteristic
effect, in comparison with 35.8 seconds (Table II), which was required
for the drug of this dilution to block the effect of acetylcholine 1 ] [ IQ~3.
This difference in the reactivity is apparently due to the fact that the
treatment with atropine in this particular set of experiments was pre-
ceded by a stronger solution of acetylcholine (1 ] [ 10~-) than heretofore
employed in studying the antagonistic effect of atropine.
TABLE IV
Onset of vigorous intestinal contractions in Daphnia magna after treatment with
acetylcholine (1 X 10~7) following the administration of physostigmine (1 X 10~4 for
2 minutes) and the action of physostigmine 1 X 10~4 when administered alone.
Time of action of
acetylcholine 1 X10~7 Time of action of
after eserinization physostigmine 1 X10"4
seconds minutes
45 9.9
47 10.0
52 10.2
58 10.0
41 9.7
55 10.3
70 9.4
37 10.1
42 9.7
30 9.6
51 9.9
41 10.4
97 10.7
52 9.5
34 9.5
Average 50.1 9.9
THE ACTION OF PHYSOSTIGMINE
Physostigmine (eserine) causes in DapJinia viayna intensification
and prolongation of the effects of acetylcholine. Likewise, after eserini-
zation of animals, the acetylcholine becomes effective on the intestine in a
shorter period of time. Fifteen animals which were treated with physo-
stigmine 1 X 10"4 for 2 minutes and then with acetylcholine 1 ] [ 10~T
yielded results which are shown in Table IV. The reaction time for
eserinized individuals in the production of vigorous muscular contrac-
ACTION OF ACETYLCHOLINE HI
tions when treated with acetylcholine 1 X 10~7 is shown to be on the
average 50.1 seconds as compared with 104.4 minutes when acetylcholine
of the same concentration is employed alone (see Table I).
The intestine of Daphnia magna responds to a treatment of physo-
stigmine when employed alone in the same way as it does to acetylcholine.
When 15 animals were treated with physostigmine 1 ] '. 10 ', vigorous
intestinal contractions appeared in about 10 minutes (Table IV, column
2). This relatively strong concentration-of physostigmine was employed
because the utilization of this strength revealed certain manifestations
in the course of the action of the chemical substance which were not
observed when higher dilutions were employed. The animal under the
influence of the drug becomes immediately immobile. The wall of the
intestine becomes opaque due to an extreme contraction of the muscular
fibers and the intestine enters into a state of contracture. After 2 or 3
minutes the organism gradually begins to recover its normal swimming
movements and the intestinal wall commences to reestablish its normal
state. In time there appear extremely powerful intestinal contractions.
These contractions with lapsed time become more intensified and persist
for a considerably longer period than when acetylcholine alone is admin-
istered to the animals. This period was often observed to extend over
one hour after the drug is replaced by water.
DISCUSSION
The action of acetylcholine, atropine and physostigmine on the intes-
tine of Daphnia magna is such that it strongly suggests the possibility
that this organ is controlled by cholinergic nerves. Acetylcholine, when
applied in the concentrations employed in this work, was shown to in-
tensify the intestinal activity. This action of acetylcholine was shown
to be antagonized by atropine and augmented and prolonged by physo-
stigmine. These and other observations recorded in this paper are in
accord with the role which has been ascribed to these substances in
physiological processes where nervous impulses are involved and where
acetylcholine is believed to act as a transmitter of nervous impulses.
The sudden appearance of vigorous muscular contractions of the
intestine under the influence of acetylcholine have enabled us to obtain
certain data pertaining to the time which elapses between the application
of the chemical substance and the onset of the specific effect produced.
It is of considerable interest and importance to note that whereas acetyl-
choline in concentrations of 1 " ; 10~2 and 1 X 10~3 produces vigorous
intestinal contractions in less than 30 seconds, with further dilution of
the drug this period is considerably prolonged before the accelerating
112 VASIL OBRESHKOVE
response of the intestine to acetylcholine is noted. With acetylcholine
1 ; ; 10~4 the period becomes, on the average, 10.7 minutes and with
acetylcholine 1 X 10~7 the time which elapsed between the addition of
the chemical substance and the appearance of the characteristic response
was shown to be on the average 104.4 minutes. Latent periods of such
extreme magnitudes are not in accordance with our present knowledge
pertaining to the action of chemical substances which are thought to act
as chemical transmitters of nervous impulses.
In view of the observation recorded in this paper it may be assumed
that the effectiveness of acetylcholine is dependent on the rate of pene-
tration and diffusion of the drug to the site of action, and on the rate
of destruction. Acetylcholine 1 ] [ 10~T, however, when preceded by
physostigmine 1 ] [ 10~* produces vigorous intestinal contractions in
Daphnia magna in less than one minute. This indicates that acetylcho-
line of this relatively weak concentration reaches the site of action quickly
and that the rate of penetration and the rate of diffusion in this particular
instance are not primarily factors. However, it is possible that the rapid
destruction of the acetylcholine when unprotected by physostigmine is
responsible for the long delays preceding the onset of its characteristic
action.
Artemov and Mitropolitanskaja (1938) have demonstrated the pres-
ence in whole Daphnia of an acetylcholine-like substance. As yet, how-
ever, no one has undertaken to demonstrate the presence or absence of
choline esterase in this group of animals. The questions of how acetyl-
choline, if present in Daphnia magna, is bound in the tissues and how
it is protected must wait further investigations before they are answered.
SUMMARY
1. Acetylcholine produces in Daphnia magna vigorous intestinal con-
tractions which persist for some time after they are established.
2. The period which elapses between the addition of the acetylcholine
and the onset of the characteristic effect is definitely dependent on the
concentration of the drug employed.
3. Atropine blocks the action of acetylcholine.
4. Physostigmine causes intensification and prolongation of the ef-
fects of acetylcholine.
5. Acetylcholine, when it is preceded by physostigmine, causes in
Daphnia magna a considerable reduction in the time which elapses be-
tween the administration of the drug and the appearance of the vigorous
intestinal contractions.
ACTION OF ACETYLCHOLINE 113
LITERATURE CITED
ARTEMOV, N. M., AND R. L. MITROPOLITANSKAJA, 1938. Content of acetylcholine-
like substances in the nerve tissue and of choline esterase in the hemolymph
of crustaceans. Bull, de Biol. ed de Med. Exper. U. R. S. S., 5 : 378-381.
BANTA, A. M., 1921. A convenient culture medium for daphnids. Science, NS.,
53 : 557-558.
OBRESHKOVE, V., 1930. Oxygen consumption in the developmental stages of a
cladoceran. Physiol. Zool., 3 : 271-282.
WELSH, J. H., 1939a. Chemical mediation in crustaceans. I. The occurrence of
acetylcholine in nervous tissues and its action on the decapod heart. Jour.
Expcr. Biol., 16 : 198-219.
\\ i i sir, J. H., 1939&. Chemical mediation in crustaceans. II. The action of
acetylcholine and adrenalin on the isolated heart of Panulirus argus.
Physiol. Zool., 12: 231-237.
WELSH, J. H., AND H. H. HASKIN, 1939. Chemical mediation in crustaceans. III.
Acetylcholine and autotomy in Petrolisthes armatus (Gihbes). Biol. Bull.,
76 : 405-415.
VITAL STAINING OF THE CENTRIFUGED ARBACIA
PUNCTULATA EGG
ETHEL BROWNE HARVEY
(From tJie Marine Biological Laboratory, Woods Hole, and the Biological
Laboratory, Princeton University)
The stratification and parts of the Arbacia punctnlata egg obtained
by centrifugal force are shown in Plate I (from E. B. Harvey, 1936).
The size of the parts and degree of stratification varies with the cen-
trifugal force; the greater the force, the larger the red half and the less
marked the stratification (E. B. Harvey, 1941). In any experimental
work with the halves and quarters, it is of importance to know exactly
what materials are present. This is best done by the use of vital dyes
which stain the different materials differentially.
Table I contains a list of vital dyes used, arranged alphabetically, and
the effect of each dye on the various materials in the egg. In all cases,
the egg was viable after staining, since it could be fertilized and at least
begin development. Different brands of the same dye have been found
in some cases to differ considerably both in staining capacity and in
toxicity. In general, the dyes put out by the National Medicinal Prod-
ucts or the National Aniline and Chemical Co. gave the best results, but
for Nile blue sulphate and thionin, Griibler's were better.
It was found better to stain the eggs first by allowing them to stand
about one-half hour in a dilute solution of the dye in sea water, and then
centrifuge them, because the mitochondrial layer disappears in 5-10
minutes after centrifuging and it often takes longer than that for the
dye to be taken up by a centrifuged egg. No accurate measure of the
amount of dye used was made, but it was soon learned how deeply the
sea water should be tinged for the dye to be efficacious but not toxic.
Some of the dyes are readily soluble in sea water, others (Bismarck
brown, neutral red, Nile blue, safranin O, thionin) must be dissolved
in distilled water and a drop of this added to the sea water ; some vital
dyes (e.g. cresyl violet, Victoria blue) were found not to be sufficiently
soluble even in distilled water. No acid dye was found to enter the cell.
The jelly forms a layer around the egg which in Arbacia punctulata
is 20-30 /j. thick. It is invisible under the microscope unless outlined
by particles of India ink or stained, since it is of the same refractive
index as the sea water. When it is present, the eggs are well separated
from each other; when the eggs are contiguous, it means that the jelly
114
VITAL DYES ON CENTRIFUGED ARBACIA EGGS
115
has disappeared, and the eggs are then usually not in optimum condition.
The jelly is destroyed by X-rays or by a small amount of acid in the
sea water (1 drop of N/10 HC1 + 50 cc. of sea water). It is some-
times centrifuged off while the eggs are rotating, though it may remain,
TABLE I
Arbacia punctulata. Vital dyes.
Dye
Jelly
Oil
Clear Layer '
Mitochon-
dria
Yolk
Pigment
Remarks
Bismarck brown
0
0
Yellow (upper
part more
intense)
Yellow
Yellow
Brown
Slightly
soluble in
sea water
Brilliant cresyl
blue
0
0
0
0
Blue
Blue
Very
innocuous
Chrysoidin
0
0
Light yellow
(upper part
more intense)
Light
yellow
Yellow
Reddish
brown
Gentian violet
0
0
0
Purple
0
0
Janus dark
blue B
Purple
0
0
0
0
0
Janus green
( =diazin green)
Purple
0
0
Blue
0
0
Rather toxic
Methyl green
0
0
0
Purple
0
0
Methyl violet
0
0
Upper part
violet
Purple
Purple
(later)
Purple
(later)
Methylene blue
0
0
0
0
Blue
Blue
Very
innocuous
Neutral red
0
0
Pinkish yellow
(lower part
more intense)
Pinkish
yellow
Brick red
Blood red,
almost
black
Slightly
soluble in
sea water
Nile blue
sulphate
0
0
Light blue
(upper part
more intense)
Light blue
Blue
Bluish
brown to
blue black
Slightly
soluble in
sea water
Rhodamine
0
0
Pink (upper
part more
intense)
Pink
Pink
Deep red
Very
innocuous
Safranin O
Yellow
(few cases)
0
0
Pink
(after 1-2
hours)
0
Blood red
Not soluble
in sea water
Thionin
Pinkish
(few cases)
0
0
Lavender
(few cases)
0
0
Not soluble
in sea water
Toluidin blue
Pinkish
lavender
0
Pinkish
lavender
Lavender
Lavender
Purple to
blue black
More intense
if stained
after cent.
somewhat elongate, on well centrifuged elongate eggs, or even around
the two separated half -eggs when close together, or it may remain around
one half-egg. It is best to determine its reaction to dyes on uncentri-
fuged eggs. The jelly stains purple with Janus green and Janus dark
blue B, and pinkish lavender with toluidin blue ; in a few cases it stained
yellow with safranin 0, and pinkish with thionin.
116 ETHEL BROWNE HARVEY
The oil cap is not stained by any of the vital dyes. A slight tinge
of color was observed in some cases, e.g. with Bismarck brown, chrysoidin
and Nile blue, but it is probable that the slight color was in the matrix
and not in the oil drops themselves.
The nucleus is not stained by any of the vital dyes.
It has been stated that the clear layer does not stain in the living egg
(Lucke, 1925), and this is certainly true of many dyes. There is no
doubt, however, that some dyes do stain the clear layer, not very in-
tensely, while the egg is still living, as could be told by its subsequent
development after fertilization. A comparison of the stained egg along-
side a control egg in fresh sea water showed whether the clear layer was
really stained. The clear layer stains yellow with Bismarck brozvn and
chrysoidin, blue with Nile blue, pink with rhodaminc, pinkish yellow with
neutral red and pinkish lavender with toluidin blue. With some dyes
there is a decided difference in the intensity of the stain in the upper
and lower portions of the clear layer, indicating a stratification of mate-
rials within the layer. With Bismarck brown and chrysoidin, which in
general act similarly, Nile blue and rhodamine, the upper portion of the
clear layer stains more intensely. With neutral red, the lower portion
stains more intensely. With methyl violet, only the upper portion stains
(violet}. This difference in different regions of the clear layer is more
marked when the eggs are stained first and then centrifuged. Although
the clear layer is optically empty in the living unstained egg, and no
granules can be distinguished in the unstained or vitally stained egg,
nevertheless in fixed material, this layer is filled with very fine granules,
deeply staining (blue) with Heidenhain's iron hematoxylin (E. B.
Harvey, 1940).
The best mitochondrial stain is methyl green, which stains the mito-
chondria purple and stains no other granules, so that the mitochondria
appear as a purple band across the egg. Gentian violet also stains the
mitochondria differentially (purple}. Methyl violet stains the mito-
chondria purple, like methyl green, but it stains other granules as well.
It may be that the purple stain of the methyl green is due to a con-
tamination of this dye with methyl violet or crystal violet, but every
brand of methyl green tried has given the same result. Janus green,
which has been advocated especially by Cowdry as a mitochondrial stain,
stains the mitochondria blue, but all brands have been found rather toxic,
some brands more so than others. Safranin O was found to stain the
mitochondria pink after some time, and thionin in a few cases stained
them lavender. Other dyes stained the mitochondria, but also stained,
somewhat more intensely, the underlying yolk (see Table I). As men-
tioned above, the mitochondrial layer disappears 5-10 minutes after
VITAL DYES ON CENTRIFUGED ARBACIA EGGS
117
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118 ETHEL BROWNE HARVEY
removal from the centrifuge, so that observations on it must be made
quickly. It is also the last layer to be formed in centrifuging, and in
some batches of eggs is not at all sharply defined.
Yolk and pigment can be easily distinguished from each other in the
unstained egg by the color. They are usually both stained with the
same dye, the pigment more intensely and at first more reddish. They
are stained blue with brilliant crcsyl blue and methylene blue, which in
general act alike, and Nile blue; velloi^-in-oicn with Bismarck brozvn and
chrysoidin; red with neutral red and rhodamine ; lavender-purple with
toluidin blue. With safranin 0, the pigment is stained blood-red and
the yolk is unstained.
Considerable information as to the chemical structure of the mate-
rials in the egg might be obtained from a study of the dye reactions,
since the chemical composition of the various dyes is known. For the
chemistry and preparation of the dyes and other data, the reader is re-
ferred to Rowe's Color Index (1924). Schultz' Farbstofftabellen (1934)
and Conn's Biological Stains (1940). For the rate of penetration of the
dyes (into gelatin), see Mollendorff's excellent article in Abderhaldcn
Handbuch dcr biologischen Arbcitsmethoden, Abt. V, Teil 2, Heft 2
(1921).
SUMMARY
A table is given of the action of various vital dyes on the different
materials in the centrifuged egg of Arbacia punctulata. The jelly sur-
rounding the egg is stained with Janus green, Janus dark blue B, (pur-
ple) and toluidin blue (pinkish lavender). The clear layer is slightly
stained with Bismarck brown and chrysoidin (yellow), Nile blue (blue),
toluidin blue (pinkish lavender), rhodamine (pink), and neutral red
(pinkish yellow). The mitochondrial layer is differentially stained with
methyl green and gentian violet (purple) and Janus green (blue).
Yolk and pigment are stained with brilliant cresyl blue, methylene blue
and Nile blue (blue), toluidin blue (purple), rhodamine and neutral
red (red), Bismarck brown and chrysoidin (yellow-brown). With saf-
ranin, the pigment is stained blood red, the yolk is unstained.
LITERATURE CITED
HARVEY, E. B., 1936. Parthenogenetic merogony or cleavage without nuclei in
Arbacia punctulata. Biol. Bull., 71 : 101-121.
HARVEY, E. B., 1940. A comparison of the development of nucleate and non-
nucleate eggs of Arbacia punctulata. Biol. Bull., 79 : 166-187.
HARVEY, E. B., 1941. Relation of the size of "halves" of the Arbacia punctulata
egg to centrifugal force. Biol. Bull., 80: 354.
LUCRE, B., 1925. Observations on intravitam staining of centrifuged marine eggs.
Proc. Soc. Expcr. Biol. Mcd., 22: 305-306.
ALLOMETRY IN NORMAL AND REGENERATING
ANTENNAL SEGMENTS IN DAPHNIA
BERTIL GOTTFRID ANDERSON AND HARVEY LOUIS BUSCH
(From the Biological Laboratory, Western Reseri'c University')
The growth of a part in relation to that of the whole body in many
animals follows the law of allometry and may he expressed by the equa-
tion
y== bx<*
where y is the size of the part; x, the size of the whole; and b and a
are constants (Huxley, 1932 and Huxley and Teissier, 1936). Regen-
eration has been " regarded as an acceleration of normal growth proc-
esses " (Przibrara, 1919 and 1926). Regenerative growth might there-
fore also be expected to follow the law of allometry. Using the data of
Zeleny (1908) on the gulf -weed crab, Portunus sayi, Huxley (1931)
demonstrated that the above equation holds for the size of the chela in
relation to the body as a whole and also for the amount of regeneration
of the chela during any one instar in relation to the size of the animal
during that instar. More recently others, especially Paulian (1938),
have shown that in many arthropods the amount of regeneration of a
part during any one instar in relation to the size of the body follows the
law of allometry. Paulian has also pointed out that normal and re-
generating antennae in Gammarus pule.v and in Caraitsiits inorosus in-
crease exponentially with time. Inasmuch as the relative growth equa-
tion is derivable on the assumption that the parts increase exponentially
with time (Huxley, 1932 and Lumer, 1937), we may conclude that
regeneration of the antennae in Gammarus pnlc.v and in Carausius nwro-
sus also follows the law of allometry.
In the above cases we are dealing with regeneration that tends to be
complete. Do these relations hold in a form where regeneration is not
complete ? In seeking a solution to the problem Daphnia magna is well
suited as an experimental animal. After amputation of an antenna
regeneration is limited to the restoration of the most proximal segment
injured and the formation of setae. The amount of regeneration varies
with the level of injury within the segment (Anderson, 1935).
The present study is a determination of the relations of the growth
of normal and regenerating antennal segments to that of the animal as
a whole in Daf>lmia magna.
119
120
B. G. ANDERSON AND H. L. BUSCH
EXPERIMENTAL PROCEDURE
Females from a single clone of Daphnia magna Straus were used.
The culture medium was pond water rich in organic matter. Individ-
uals were isolated within six hours after their release from the mothers
and placed in watch glasses with a few drops of culture medium. Just
enough of a saturated solution of chloretone was added to make the
animals immobile. Each animal was placed on its left side with the
left antenna stretched out in front of the body. Those animals that
were used to study the growth of normal segments were then drawn
with the aid of camera lucida. After the drawings were made the
animals were placed in individual vials containing about sixty cubic
centimeters of fresh culture medium. In the case of those animals that
FIG. 1. Diagram showing the method of making measurements. T — total
length, longest dimension of the body exclusive of the spine. 5" — segment length
taken on the central axis of tin- segment.
were used to study regeneration, the first segment of the ventral ramus
of the left antenna was severed by applying pressure with a needle which
had been ground to a chisel edge. The level of amputation was varied
in each instance. After the operation these animals were also placed in
individual vials containing fresh culture medium. Several hours later
the operated animals were again placed in watch glasses and immobilized
with chloretone. They were then drawn in the same manner as were the
unoperated animals. Care was taken to denote exactly the extent of
the brown area just proximal to the level of amputation, for this is
injured tissue that is cast off at the next molt (Anderson, 1935). After
being drawn, they were replaced in their respective vials. From this
point on both normal and operated animals were treated alike. Each
animal was drawn during each successive instar up to and including the
tenth. Inasmuch as the animals change in size only at ecdysis and im-
AI.I.OMETRY IX DAPHNIA
121
mediately thereafter ( Agar, 1930), drawings were made at any time
during the instar. Every time after an animal was drawn it was placed
in fresh culture medium. The experiment was run at room tempera-
ture (18°-26° C).
Measurements of the total length of the animals and the length of
the antennal segments were made from the drawings. The total length
of the animal was taken as the distance from the base of the spine to
the most anterior point on the head. The length of the antennal seg-
ment was taken on the central axis of the segment. These measurements
are illustrated in Fig. 1, and conform to those made by Anderson (1932,
1935) in other studies on Daphnia inagjia.
mm.
.40
.10
0.8 1.0 1.5 2.0 3.0 4.0 mm.
TOTAL LENGTH
FIG. 2. Double logarithmic plot of the relations between the lengths of the
normal segment and the total lengths during each of the first nine instars.
RELATIVE GROWTH OF THE NORMAL ANTENNAL SEGMENT
The relation of the logarithms of the mean lengths of the first seg-
ment of the ventral ramus of the left antenna to the logarithms of the
mean total lengths of eighteen animals for the first nine instars is
shown in Fig. 2. The points fall approximately in a straight line. We
may therefore conclude that the law of allometry holds and the relation
between the length of the segment and the total length may be expressed
by the equation
y = b.\-a
(1)
122
B. G. ANDERSON AND H. L. BUSCH
where 3; is the length of the segment and .r is the total length of the
animal. The values of the constants b and a are 0.145 and 0.74, respec-
tively. These were determined by the method of least squares. The
value of a being less than unity indicates that the antennal segment
grows at a lower rate than the body as represented by the total length.
The antenna as a whole also grows more slowly than does the body as
mm.
40
30
.20
.15
08
\06
.04
.03
.02
.015
0.8 1.0 1.5 2.0 3.0
TOTAL LENGTH
4.0
mm.
FIG. 3. Double logarithmic plots of the relations between the lengths of re-
generating segments and the total lengths during each of the first nine instars for
animals with antennae amputated at different levels. The level of injury is desig-
nated by the Roman numerals whose values are given in Table I. The dasli line
designated by Ar is that for the normal segments shown in Fig. 2.
is evidenced by the fact that the young at birth have antennae whose
length is greater in proportion to the body than do the adults. The
antennal segments, however, maintain the same proportions to each
other throughout life.
Huxley (1931) has shown that the limbs of sheep grow less rapidly
than the body after birth. This is also true for the macaques (Lumer
ALLOMETRY IN DAPHNIA
123
and Schultz, 1941) and probably for all mammals where the young at
birth are able to run along with their mothers and perhaps for many
other animals that depend on their means of locomotion for protection
and 'or food-getting.
REGENERATION
The nature of regenerated antennae has been adequately described by
others and need not be repeated here. Regeneration is limited to the
restoration of the most proximal segment injured and the formation of
new setae (see Anderson, 1935). The amount of regeneration is quan-
titatively related to the level of injury within the segment.
The operated animals were divided into six classes on the basis of
the length of the intact portion of the segment during the latter part of
the instar of amputation. The relations of the logarithms of the mean
TABLE I
The values of b and a for the relations of the length of the regenerating first
segment of the ventral ramus of the left antenna to the total length of the animal.
Class
Level of Injury *
Number of Cases
b
a
I
0.076-0.110
8
0.103
1.01
II
0.060-0.070
7
0.068
1.14
III
0.046-0.053
7
0.058
1.42
IV
0.040-0.042
8
0.047
1.40
V
0.027-0.030
9
0.039
1.49
VI
0.010-0.023
7
0.018
1.02
* Length of the intact portion of the segment in millimeters during the instar
of amputation. The values of b and a were determined by the method of least
squares.
lengths of the amputated segments to the logarithms of the mean total
length of the animals for the instar of amputation and the next eight
instars for each class are shown in Fig. 3. While the points for any
one class do not fall along a straight line as closely as do those for the
relations in unoperated animals (Fig. 2), they do approximate a straight
line. The law of allometry may be considered applicable and the rela-
tions can be expressed by the equation (1). The values of the constants
are given in Table I.
The value of the constant a in the equation (1) is the ratio of the
percentage increase of v to the percentage increase in .r. Since x always
represents the total length of the animals, both normal and operated, in
the 'relations described above, the values of a are directly comparable.
Examination of Fig. 3 and Table I shows that the value of a increases
124
B. G. ANDERSON AND H. L. BUSCH
as the level of injury reaches a lower point in the segment until a certain
level is reached after which the value of a decreases. This relation is
brought out graphically in Fig. 4. Another point worthy of note is that
in Fig. 3 the curves with one exception tend to converge at a point where
the total length would he about five millimeters, the maximum size that
the animals reach. From this it is apparent that as long as the level of
injury is above the critical level, the growth rate of the regenerating
antennal segments is such that they approach the length of the normal
segment simultaneously as full growth of the animals is attained.
Somewhat analogous results have been found by others. Zeleny
(1905, 1909) found that the rate of regeneration of an organ in many
1.5
1.3
a LI
0.9
0.7
I I I I I
J L
J I I
.02 .04 .06 .08 .10 .12 .14 mm.
LEVEL OF INJURY
FIG. 4. The relation between the value of a in the equation
y = bxa
and the level of injury. The symbols correspond to those used in Fig:. 3.
animals increases with the degree of injury up to an optimum, after
which the rate decreases. Zeleny's work is not directly comparable
inasmuch as he was concerned with the rate of regeneration of an organ
when that one only was removed in comparison with the rate when
several others were removed in addition. The results of Paulian (1938)
are more directly comparable. He amputated the antennae of Gaiu-
marus pule.v and Carausius morosus, and inspection of his figures (Figs.
14 and 15, pages 320 and 322) indicates that the rate of growth of the
antennae increased as the level of amputation approached the proximal
end. Whether or not a critical level might be reached beyond which
the rate decreases, was not determined in his experiments. Further, the
amputated antennae reach the size of the normal at different times, the
time taken varies directly with the amount removed.
ALLOMETRY IN DAPHN1A 125
THE SIGNIFICANCE OF THE CONSTANT b
The question of the biological significance of the constants b and u
in the law of allometry have been subject to considerable discussion.
Huxley (1932) and Needham (1934) have stated that the constant b
is of little biological importance. The value of the constant b is that of
V when .v = = \. As a consequence, its value changes with the unit of
measure employed while the actual relations of 3* and .r remain the same.
Again the unit chosen is usually such that b is an extrapolated value of v.
Because of these arbitrary factors the significance of b has remained
elusive. Recently Lumer, Anderson, and Hersh (1941) have pointed
out that if b is to have biological significance, the unit of measure chosen
should be one given by the organism. They suggest that the most satis-
factory unit would be the size of a standard part at the beginning of a
developmental period, but where this cannot readily be ascertained an
approach to it could be made by taking the smallest value of the standard
part given by the data as unity. In this way b would be an actual value
of v. This is in line with the proposal of Huxley and Teissier (1936)
that b should be called the " initial-growth index," for indeed that is
what it becomes as far as the data are concerned when the above sug-
gestions are followed. The constant a has presented no such difficulties.
Since it is the ratio of the percentage growth rates of the parts y and .r,
it is constant regardless of the unit of measure used, a has therefore
been considered of relatively greater importance than b.
Lumer, Anderson, and Hersh (1941) have shown how the constant b
may be made a more tangible entity in that it can be given in terms of
the organism. As such it has significance. The question still remains
as to the degree of its importance. If the value of b, i.e., the initial
ratio of y to x, could be altered experimentally, and if as a consequence
the value of a would change, we could conclude that the value of b de-
termines the value of a; b would then have a greater biological impor-
tance than heretofore supposed.
This is precisely what we have done in the experiments described in
this paper. We have amputated the antenna and so reduced the length
of the segment. The ratio of the intact portion of the segment during
the instar of amputation to the total length of the animal is given by b,
since the total length during that instar is approximately one millimeter
and the millimeter is the unit of measure. Following amputation, the
growth rate of the antennal segment is changed so that new values of
a result. Further, as b decreases a increases until b reaches a particular
value, after which a also decreases as is shown in Table I and Fig. 4.
The constant b, in the sense in which we have employed it, serves as a
measure of the conditions at the beginning of the developmental period.
126 B. G. ANDERSON AND H. L. BUSCH
and as these conditions differ, so also do the consequent rates of develop-
ment as represented by the constant a.
SUMMARY
The law of allometry
y = /?.ra
was found to be applicable to both normal and regenerating antennal
segments in Daphnia magna.
The growth rate of the regenerating segments increases as the level
of injury approaches the proximal end of the segment until a critical
point is reached, after which the rate decreases. As long as the level
of injury is distal to the critical level, the growth rate is such that the
regenerating segments tend to approach the length of the normal seg-
ment simultaneously as full growth of the animals is attained.
The significance of the constant b in the law of allometry is discussed.
CITATIONS
AGAR, W. E., 1930. A statistical study of regeneration in two species of Crustacea.
Brit. Jour. E.vpcr. Biol, 7 : 349-369.
ANDERSON, B. G., 1932. The number of pre-adult instars, growth, relative growth,
and variation in Daphnia magna. Biol. Bull., 63 : 81-98.
ANDERSON, B. G., 1935. Antennal regeneration in Daphnia magna. Ohio Jour.
Set., 35: 105-111.
HUXLEY, J. S., 1931. Notes on differential growth. Am. Nat., 65 : 289-315.
HUXLEY, J. S., 1932. Problems in Relative Growth. Methuen and Company,
London.
HUXLEY, J. S., AND G. TEISSIER, 1936. Terminology of relative growth. Nature,
137 : 780-781.
LUMER, H., 1937. The consequences of sigmoid growth for relative growth func-
tions. Groivth, 1 : 140-154.
LUMER, H., B. G. ANDERSON, AND A. H. HERSH, 1941. On the significance of the
constant b in the law of allometry y = b.ra. Am. Nat. (in press).
LUMER, H., AND A. H. SCHULTZ, 1941. Relative growth of the limb segments and
tail in macaques. Human Biol. (in press).
NEEDHAM, J., 1934. Chemical heterogony and the ground-plan of animal growth.
Biol. Rev., 9: 79-109.
PAULIAN, R., 1938. Contribution a 1'etude quantitative de la regeneration chez les
Arthropodes. Proc. Zool. Soc., London, Ser. A, 108 : 297-383.
PRZIBRAM, H., 1919. Tierische Regeneration als Wachstumsbeschleunigung. Arch.
f. Entwmcch., 45 : 1-38.
PRZIBRAM, H., 1926. Transplantation and regeneration: their bearing on develop-
mental mechanics. Brit. Jour. E.vpcr. Biol., 3 : 313-330.
ZELENY, C., 1905. The relation of the degree of injury to the rate of regeneration.
Jour. E.vpcr. Zoo!., 2 : 347-369.
ZELENY, C., 1908. Some internal factors concerned with the regeneration of the
chelae of the gulf-weed crab (Portunus sayi). Papers from the Tortugas
Laboratory. Carnegie Institution of Washington, 2: 103-138.
ZELENY, C., 1909. The relation between degree of injury and rate of regeneration
— additional observations and general discussion. Jour. E.vpcr. Zool., 7 :
513-561.
EXPERIMENTAL CYTOLOGICAL EVIDENCE FOR AN
OUTWARD SECRETION OF WATER BY THE
NEPHRIC TUBULE OF THE CRAYFISH *
N. S. RUSTUM MALUF
(From the Department of Zoology, The Johns Hopkins University and the
Department of Tropical Medicine, The Tnlane University)
INTRODUCTION
When a crayfish is in freshwater, its normal habitat, it does not
drink, but water diffuses into its body through the gills (Maluf, 1937,
1940). An internal aqueous and saline steady state is maintained, in
spite of a constant inward diffusion of water, because of the unvarying
capacity of its kidneys to manufacture urine that is markedly hypotonic
to the blood (Schlieper and Herrmann, 1930; Herrmann, 1931).
The concentration of chloride in the luminal fluid of the coelomosac
and labyrinth of the nephron is equal to that in the blood, but that in the
tubular fluid is markedly lower than that in the blood (Peters, 1935).
The hypotonicity of the urine must therefore be clue either to an active
resorption of salts by the tubule or to an outward secretion of a hypo-
tonic liquid by the tubule.
The tubule of a 35-gram animal is approximately 3 cm. long and
about 2 mm. in greatest breadth.2 The ventral, i.e., proximal, half of
the tubule consists of flat cells without apical secretory globules. The
dorsal, i.e. distal, coil is composed of relatively large columnar cells with
a distinct mitochondrium and, generally, with large clear apical vacuoles
which bulge into the lumen of the tubule (Maluf, 1939).
Because the nephron of the crayfish does not possess a tenuous
syncytium, such as the glomerular capsule of the vertebrate nephron,
filtration seems unlikely as a major process of urine-formation. Ac-
cordingly, this is an attempt to find whether the apical vacuoles of the
distal coil of the tubule represent an outward secretion of water.
The experimental attack is partly based on the observation of Herr-
mann (1931) that, as the salinity of the external medium is raised, the
rate of urinary flow falls and the osmotic pressure of the urine simul-
1 This work was performed when the author was Johnston Research Scholar
in the Department of Zoology, The Johns Hopkins University. To Professor S.
O. Mast much obligation is due for numerous kindnesses.
2 In the 1939 paper this was misprinted as "2 cm."
127
128 N. S. RUSTUM MALUF
taneously increases. As shown by constancy in weight, the total quantity
of water in the crayfish is the same in freshwater as in salinities up to
272 mM. NaCl per liter, which is initially hypertonic to the blood. This
indicates that the decrease in the rate of urinary flow, with rising ex-
ternal salinity, is not due to a decrease in haemocoelic pressure which, as-
suming that filtration does occur, might cause a decrease in the rate of
filtration. There is, furthermore, no apparent basis for the supposition
that the haemocoelic pressure undergoes a localized fall in the vicinity of
the kidneys as the salinity of the external medium is raised.
From the above it might be expected that, when the crayfish is in a
medium in which inward diffusion of water can be only very small and
in which the rate of urinary flow is accordingly depressed, the apical
vacuoles of the nephric tubule will tend to disappear.
METHODS
The test animals were immersed in 210 mM. NaCl per liter of fresh-
water, a solution in which they can remain vigorous indefinitely. Al-
though this concentration is somewhat hypertonic to the blood at the
outset (see Lienemann, 1938, for the normal osmotic pressure of the
blood of Cambarus clarkii), some water, probably only a negligible quan-
tity, diffuses inwardly because, as Herrmann (1931) showed, the osmotic
pressure of the blood eventually exceeds that of the external medium.
Parenthetically, the invariable hypertonicity of the blood, as compared
with the external medium, is probably mainly because the urine is always
hypotonic to the blood regardless of the osmotic pressure of the external
medium (Herrmann). Integumental uptake of salt from the exterior
is a relatively minor factor, as can be readily calculated (data of Liene-
mann, 1938, and Maluf, 1940).
At the end of one to several days the animals were sacrificed and
their kidneys removed with minimum handling and fixed in unneutralized
formol-sublimate for several hours, washed in running tap-water over-
night, dehydrated with dioxane (50 per cent, 75 per cent, and two
changes of 100 per cent), imbedded in paraffin with a melting point of
about 49° C., sectioned 8 ju, thick, and stained with eosin and methylene
blue-borax.
RESULTS
A seven-day stay of three animals in 210 mM. NaCl per liter abol-
ished almost all the apical vacuoles from the cells of the distal coil of
the nephric tubule (Fig. 1) whereas the majority of the corresponding
cells of the three controls, which had been in freshwater, possessed large
WATER SECRETION IN CRAYFISH 129
apical vacuoles which bulged into the lumen of the tubule (Fig. 2).
One of the test animals exhibited an exceptional number of vacuoles for
an animal in 210 mM. NaCl but the vacuoles were small and scanty as
compared with those of the controls. The photographs were taken from
areas at random. The data were analyzed objectively as follows : In
this experiment two to three slides were prepared containing serial
sections of the pair of kidneys from each individual (16 slides in all) ;
the labels were covered so as to remove every vestige of external identi-
fication ; the slides were shuffled. The examination of each slide never
exceeded one or two minutes and was made under low power (100 X)-
In 15 slides out of 16, the identification of the series to which the prep-
aration belonged (freshwater or 210 mM. NaCl) was correct. Meas-
urements did not show a correlation between the height of the cells
and the existence of apical vacuoles.
The experiment was repeated with six larger animals and a duration
of three days. Here, too, the difference between the three test animals
in 210 mM. NaCl per liter (Fig. 3) and the controls (Fig. 4) was pro-
nounced. The objective analysis, identical with that above described,
showed a correct identification of 20 slides out of 22. Here, too, exten-
sive measurements indicated no correlation between the height of the
cells and the presence of vacuoles. The three-day experiment was re-
peated with confirmatory results : the two test animals showing prac-
tically no apical vacuoles whereas the two controls displayed apical
vacuoles in the majority of cells of the distal half of the tubule.
Even a 24-hour stay in 210 mM. NaCl produced a practically com-
plete abolition of the apical vacuoles (Fig. 5) although the interior of
the cells was considerably vacuolated. Nearly all of the corresponding
cells of the controls in freshwater exhibited large clear apical vacuoles
(Fig. 6). Figures 5 and 6 are at a lower magnification than the other
photographs and thus exhibit a larger field. The objective analysis
showed a correct identification of 8 slides out of 8. Subjection to 210
mM. NaCl for less than 24 hours was not attempted.
After vacuole-formation has presumably been practically abolished
by an 168-hour stay in 210 mM. NaCl, the vacuoles reappear upon
returning the crayfish to freshwater. In this experiment there were two
tests and two controls.
The fact that a large fraction of the cells of the distal half of the
tubule of the controls invariably exhibited large apical vacuoles in itself
shows that the almost complete absence of such vacuoles in slightly
hypertonic NaCl is not an artefact of histological technique. The distal
half of the tubule of a live animal was dissected out of the kidney in
130 N. S. RUSTUM MALUF
crayfish-saline.3 Fragments teased out of this part of the tubule and
suspended in a hanging drop of crayfish-saline on a coverslip, gave an
ample picture of the vacuoles.
HISTORICAL STATEMENT AND DISCUSSION
The nephric tubule of the decapod kidney was first discovered by
Neuwyler (1841), who did not understand the function of the "green
glands." Only within the present decade have we come to realize
the importance of the crustacean kidney in the aqueous and ionic
regulation of the bodily fluids. Grobben (1881) was the first to
observe that the nephric tubules of freshwater Crustacea and Annelida
are markedly longer than those of corresponding marine forms and that
length of tubule is not correlated with bodily size. He did not theorize
as to the significance of these facts but remarked that, "It therefore ap-
pears that the length of the urinary canal goes parallel with life in fresh-
water." Richard (1891) came to an identical conclusion with regard to
copepod Crustacea. Rogenhofer (1905, 1909) confirmed Grobben and
found that differences in the nephric dimensions of marine and fresh-
water Crustacea are not due to differences in cellular size. Rogenhofer
failed to alter the length of the tubule of the freshwater isopod, Asclliis
aqnaticus, in one generation by gradually bringing the isopod to a salinity
of 2 per cent in one year. Delia Valle (1893) believed that the differences
PLATE I *
EXPLANATION OF FIGURES
FIG. 1. Epithelium of a portion of the distal half of the nephric tubule of a
crayfish which had been in 210 mM. NaCl for seven days, ha, haemocoele and
blood-vessels; LU, lumen of tubule. Triple Mallory's ; daylight bulb; Zeiss lens.
Animal about 10 grams.
FIG. 2. Control to Fig. 1 ; animal in freshwater. i'a, large apical vacuoles.
Animal about 10 grams.
FIG. 3. Epithelium of a portion of the distal half of the nephric tubule of a
crayfish which had been in 210 mM. NaCl for three days. Methylene blue-eosin ;
red filter ; Zeiss lens. Animal about 30 grams.
FIG. 4. Control to Fig. 2; animal in freshwater. Animal about 30 grams.
FIG. 5. Epithelium of a portion of the distal half of the nephric tubule of a
crayfish which had been in 210 mM. NaCl for 24 hours. Methylene blue-eosin;
red filter ; Zeiss lens. Animal about 13 grams.
FIG. 6. Control to Fig. 5 ; animal in freshwater. Animal about 13 grams.
3 The saline was based on the most acceptable data on the concentration of
inorganic electrolytes in the blood of the crayfish (see Maluf, 1940, for references)
and was as follows (g./l.) : NaCl, 7.81; CaCL, 1.31; MgCU, 0.82; KC1, 0.70;
buffered at pH 7.5 with 0.5 cc. M/5 NaoHPO4/NaH2PO4. A A of about 0.66° C.
is assumed (see Lienemann, 1938, and Schlatter, 1941).
4 The writer is much indebted to Dr. Charles E. Brambel, The Johns Hopkins
University, for kind personal instruction in photomicrography.
WATER SECRETION IN CRAYEISH
131
PLATE
132 N. S. RUSTUM MALUF
in tubular length are of phylogenetical origin rather than a direct
environmental effect. Marchal (1892) observed that the nephrons of
the lobster and other marine decapods have no tubule. He suggested
that the external medium, — freshwater and sea-water, — may be a deter-
mining factor but remarked that the estuarine crab, Tclphusa, has no
nephric tubule even though it frequents freshwater.
In 1930, Schlieper and Herrmann found that the urine of the crayfish
is markedly hypotonic to the blood and that the urine of the shore-crab,
Carcimts inacnas, and of the estuarine crab, Tclplmsa fluviatilis — neither
of which possess nephric tubules — is isotonic with the blood. They
suggested that the nephric tubule is responsible for the hypotonic urine
of the crayfish and that it acts by resorbing salts from a filtrate formed
at the coelomosac. Herrmann (1931) and Peters (1935), in Schlieper's
laboratory, demonstrated that the tubule is of paramount importance in
osmoregulation. Peters suggested that the apical vacuoles of the distal
coil may indicate a resorption of salts from lumen to blood. Peters'
theory presupposes that a filtrate is formed somewhere in the nephron
proximal to the tubule. Peters made the important discovery that only
in the tubule is the concentration of chloride of the presumptive urine
lower than that of the blood. His results do not show, howyever, in
which part of the tubule this is true.
The facts in this paper suggest that the vacuoles represent an out-
ward secretion of water in compensation for that which diffuses in-
wardly. Physiological data indicate that the crayfish nephron is para-
mountly if not entirely a secretory organ (Maluf, 1941). The hypo-
tonic urine of this animal may thus be the result of an outward secretion
of a liquid markedly hypotonic to the blood and the rate of water-
secretion by the tubule may be determined by a hormone.
SUMMARY
The majority of the cells of the distal half of the nephric tubule of
the crayfish exhibit large, clear apical vacuoles at their luminal borders
when the animal is in freshwater, its normal medium.
If the crayfish remains in a saline medium which is initially slightly
hypertonic to the blood, for twenty-four hours or more, these vacuoles
completely disappear. The condition is reversible upon return of the
animal to freshwater. (The crayfish can maintain its vigor indefinitely
in 210 mM. Nacl per liter, which is initially slightly hypertonic to the
blood. )
WATER SECRETION IN CRAYFISH 133
REFERENCES
GROBBEN, C., 1881. Die Antennendriise der Crustaceen. Arb. Zool. Inst. Univ.
Wicn 11. Stat. in Tricst, 3: 93-110.
HERRMANN, FRANZISKA, 1931. liber den Wasserhaushalt des Flusskrebses (Pota-
mobius astacus Leach). Zcitschr. vcrgl. Physiol., 14: 479-524.
LIENEMANN, LOUISE J., 1938. The green glands as a mechanism for osmotic and
ionic regulation in the crayfish (Cambarus clarkii Girard). Jour. Cell.
Comfy. Physiol., 11 : 149-159.
MALUF, N. S. R., 1937. The permeability of the integument of the crayfish
(Cambarus bartoni) to water and electrolytes. Biol. Centralbl., 57: 282-
287.
— , 1939. On the anatomy of the kidney of the crayfish and on the absorption
of chlorid from freshwater by this animal. Zool. Jalirb., Abt. f. allgetn.
Zool. u. Physiol. d. Ticrc, 59 : 515-534.
— , 1940. The uptake of inorganic electrolytes by the crayfish. Jour. Gen.
Physiol., 24: 151-167.
— , 1941. The secretion of inulin, xylose, and dyes and its bearing on the manner
of urine-formation by the kidneys of the crayfish. (In press.)
MARCHAL, P., 1892. Recherches anatomiques et physiologiques sur 1'appareil
excreteur des crustaces decapodes. Arch. Zool. e.vpcr. ct gen., 10 (ii) :
57-275.
NEUWYLER, HERRN, 1841. Anatomische Untersuchungen ueber den Flusskrebs.
Verhandl. dcr schwciz. naturforsch. Gcsellsch. bei Hirer Versammlung zu
Zurich, 26th meeting, pp. 176-185.
PETERS, H., 1935. Uber den Einfluss des Salzgehaltes im Aussenmedium auf den
Bau und die Funktion der Exkretionsorgane dekapoder Crustaceen. (Nach
Untersuchungen an Potamobius fluviatilis und Homarus vulgaris.)
Zeitschr. Morph. u. Okol. d. Ticrc, 30: 355-381.
RICHARD, J., 1891. Recherches sur le systeme glandulaire et sur le systeme nerveux
des copepodes libres d'eau douce, suivies d'une revision des especes de ce
groupe qui vivent en France. Ann. Sci. nahtr., Zool. et Paleon., 12 (vii) :
113-270.
ROGENHOFER, A., 1905. Uber das relative Grossenverhaltnis der Nierenorgane bei
Meeres- und Siisswassertieren. Verhandl. dcr kaiserlich-kdnig. zool.-bot.
Gescllsch. in Wicn, 55: 11 (abstract).
— , 1909. Zur Kenntnis des Baues der Kieferdriise bei Isopoden und der Groszen-
verhaltnisses der Antennen- und Kieferdriise bei Meeres- und Stisswasser-
krustazeen. Arb. zool. Inst. Univ. Wicn u. zool. Stat. in Triest, 17:
139-156.
SCHLATTER, M. J., 1941. Analyses of the blood serum of Cambarus clarkii, Pachy-
grapsus crassipes and Panulirus interruptus. Jour. Cell. Comp. Physiol.,
17: 259-261.
SCHLIEPER, C., AND FRANZISKA HERRMANN, 1930. Bezieliungen zwischen Bau und
Funktion bei den Exkretionsorganen dekapoder Crustaceen. Zool. Jahrb.,
Abt. f. Anat. u. Out. dcr Ticrc, 52: 624-630.
DELLA VALLE, A., 1893. Gammarini del Golfo di Napoli. (In Fauna u. Flora des
Golfes von Naepel, vol. 20, xi + 948 pp., fo., plus atlas with 16 pis. See
pp. 70-72 for the excretory organs.)
MICTURITION IN THE CRAYFISH AND FURTHER
OBSERVATIONS ON THE ANATOMY OF THE
NEPHRON OF THIS ANIMAL
N. S. RUSTUM MALUF
{From the Department of Zoology, The Johns Hopkins University, and the
Department of Tropical Medicine, The Tnlane University}
Preliminary to studies on renal function in the crayfish (Maluf,
1940, 19416), it is necessary to know how urine is retained in the blad-
ders and how discharged. Nothing has been indicated, until the present,
as to how urine is retained. There is, furthermore, no adequate study
of the anatomical features surrounding the urinary outlet of decapod
Crustacea. As a result of this deficit, investigators of renal function
in the crayfish have punctured the membranous operculum at the nephro-
pore prior to collecting urine by suction (Marchal, 1892; Boivin, 1929;
Herrmann, 1931; Scholles, 1933; Lienemann, 1938). It is not clear
why the opercula were destroyed. From Marchal's diagrams it appears
that removal of the opercula would tear the ureters and lead into the
haemocoele and that, consequently, the urine would be contaminated with
blood. Marchal and Boivin, however, stated that the liquid they col-
lected was limpid, clear, and almost colorless and practically uncon-
taminated with blood. The chemical analyses of Herrmann, Scholles,
and Lienemann show that the concentration of inorganic electrolytes in
the liquid collected from the excretory orifices was markedly lower than
in the blood. The fact that the distal portion of the bladder contacts the
base of the excretory eminences at most of its circumference (Fig. 2, B)
apparently explains how the urine collected by the afore-mentioned
investigators did not contain an appreciable quantity of blood. The
urine aspirated by Picken (1936), by piercing the operculum with a fine
hypodermic needle, was doubtless, at times at least, notably contaminated
with blood as shown by the strongly positive xanthoproteic reaction and
by the large discrepancies, in this respect, with regard to the urine from
both kidneys. Thus, in one instance, the urine from the right kidney
gave a negative xanthoproteic test while that from the left gave a strong
reaction. The writer found that urine collected from Cambarus clarkii
by suction from intact nephropores invariably gave a weak xanthoproteic
but a negative biuret reaction.
134
MICTURITION IN THE CRAYFISH 135
The review of Burian and Muth (1924) may leave one with the
impression that the communication between the coelomosac and labyrinth
" is closed by a sphincter muscle, and any passage of fluid from the
labyrinth into the coelomic sac appears to be prevented by a valve-like
arrangement of cells" (Picken, 1936). Examination of the literature
left the writer dubious about the existence of a sphincter between the
coelomosac and labyrinth. The present paper shows that, at least in
Canibarus clarkii, there is no sphincter or valve between coelomosac and
labyrinth or between nephric tubule and bladder.
Weismann (1874), Grobben (1881), Schlieper (1935), and Peters
(1935) believed that a blood-ultrafiltrate is formed in the coelomosac.
The writer has made a detailed histological examination of this part of
the nephron to find out whether the histological facts support the hy-
pothesis of filtration.
The results in this paper refer to Canibarus clarkii, the swamp cray-
fish.
MICTURITION
The Retention of Urine
Because the bladders are normally distended with urine and because
urine only occasionally leaves the nephropores of undisturbed unheated
animals seen under a microscope, urine must be adequately retained in
the bladders. The volume of retained urine was sometimes as much as
4 per cent of the fresh weight of the animal.
On the ventral surface of the basal segment of each second antenna
is the whitish excretory eminence (Fig. 5, e) in the central depression
of which is a convex, finely corrugated, flexible, thin membrane, o, known
as the operculum. Because the operculum does not cover anything ex-
ternal, the name is inaccurate. The convexity of the operculum is main-
tained by blood-pressure, as the opercula invariably collapse after thor-
oughly bleeding the animal. In contrast to the rest of the excretory
eminence, the operculum is very sensitive to contact as shown by the
resulting generalised motor response. The operculum is invaginated at
its anterior border, thus forming a narrow crescentic slit (Figs. 2, 3, 5,
and 6, a) which is the actual excretory orifice, or nephropore. The in-
vagination proceeds at a sharp angle posteriorly, forming the short flat
ureter (Figs. 2 and 3, ur).
The rounded flexible contour of the operculum is inessential. An
animal with both opercula damaged by puncture was under observation
for about a month, at the end of which time its opercula were still col-
lapsed. The ureters, however, were not damaged, as shown by subse-
quent dissection.
136 N. S. RUSTUM MALUF
The ureter (Figs. 2 and 3, ur) is short, dorso-ventrally flattened,
and parallel to the operculum. Fine spindle-shaped fibers (Figs. 2 and
3, /') containing elongate nuclei (13 /A long in crayfish-saline) extend
from the dorsal wall of the ureter to the basal margins of the excretory
eminence. With care, the whole dorsal wall of the ureter, including the
fibers, may be dissected and mounted.
The fibers are unstriated (observed at 970 X while in fresh saline or
after being fixed in formalin and stained with haematoxylin or
Wright's) and are apparently identical with those which stretch between
the distal extremity of the bladder and the integument (Fig. 2, /, /").
These ureteral fibers apparently act as a sphincter and their discovery
answers the question as to how urine is retained in the bladders. Be-
cause a gentle outflow of urine has been seen in devisceratecl inverted
animals, the bladder must be elastic and the ureteral sphincter evidently
normally retains urine in the bladder by tonic contraction.
Similar fibers occur, circularly arranged in considerable numbers,
on the haemocoelic surface of the most proximal portion of the bladder,
to a very much slighter degree on the main body of the bladder, and
also on the main stem of the renal artery. Spindle-shaped unstriated
fibers have been observed on the bladder of the American lobster by
Waite (1899).
PLATE I
FIG. 1. Dorsal aspect of the opening into the left second antenna and sur-
rounding exoskeleton, showing the distal portion of the bladder wedged between
the proximal antennal muscles, am, articular membrane between antenna and
cephalothorax ; B, distal portion of bladder ; bas, basipodite ; c , lateral wall of the
cephalothorax ; comp, compressor muscles of the antenna ; cox, coxopodite ; dep\-\,
depressor branches of the antenna ; lev, levator muscle of the antenna ; prom, pro-
motor muscle of the antenna ; rem, remoter muscle of the antenna ; s, sternum.
FIG. 2. Sagittal section through the distal portion of the bladder, ureter, and
nephropore. B, distal portion of the bladder ; c, connective tissue ; /, /', /", un-
striated fibers ; /', ureteral sphincter ; n, nephropore ; o, operculum ; s, coxopodite ;
ur, ureter.
FIG. 3. Dorsal aspect of the ureter and the depression of the coxopodite which
corresponds to the eminence of the ventral aspect, a, nephropore, shown in broken
lines because it is ventral to the ureter; f, ureteral syncytium ; ur, ureter.
FIG. 4. Dorsal aspect of the brain, a, nerve-stems passing into the lumen of
the second antenna ; c\, c?, individual nerve-fibers issuing from the roots of the
former ; Ic, longitudinal connectives ; m, median nerve ; oc, oculomotor nerve ; op,
optic nerve ; P, protocerebrum ; T, tritocerebrum ; te, " tegumentary " nerves. The
root of the nerve to the first antenna issues from the ventral surface of the brain
and is thus not shown here.
FIG. 5. Ventral aspect of the region surrounding the right nephropore. a,
crescentic nephropore ; c, excretory eminence of the basal segment of the second
antenna ; o, operculum ; s, coxopodite ; u, droplet of urine.
MICTURITION IN THE CRAYFISH
137
PLATE I
(All figures refer to Cambarus clarkii; the animals of Figs. 1, 4, and 5
measured about 7.5 cm. from rostrum to end of telson.)
138 N. S. RUSTUM MALUF
In spite of numerous careful dissections, the writer has not been
able to find any fibers inserting on the operculum. This conforms with
Marchal's (1892) observations on the crab, Mala. Schmidt (1915),
who gave a comprehensive and well-illustrated account of the somatic
musculature of the European crayfish, did not mention any special
muscles of micturition. The region between the operculum (Fig. 2, o)
and the ureter, ur, is occupied by connective tissue and does not contain
spindle-fibers.
At the posterior margin of the excretory eminence the ureter bends
sharply anteriorly, enlarges in girth, and continues as the bladder (Fig.
2, B). Upon emerging- from the excretory eminence (Fig. 2), the
bladder passes through a mass of antennal muscles (Fig. 1).
The Expulsion of Urine
The animal was drained of moisture, water was sucked from the
branchial chambers, and the anterior border of the chambers plugged
with absorbent cotton-wool to prevent remaining water from flowing
over the opercula. The opercula were observed under magnifications of
22.5 and 112.5 X-
The outflow of urine in air occurs anteriad, i.e. in the plane of
the ureter. At times the urine issues from the orifice for a short
distance and is then sucked back. Slight pressure on the operculum
with a blunt instrument frequently induces urinary outflow. A pipette
of suitable size and carrying suction (about 10 mm. Hg) may produce
urination even for some time after the use of suction. The urine issues
as a series of generally spherical droplets. The suction does not injure
the operculum. The latter does not undergo movement except for a
scarcely perceptible motion only as the urinary droplet attains maximal
size. This is doubtless a passive effect. Marchal (1892) stated that, in
the crab Mala, movements of the opercula accompany the discharge of
urine; it is probable that in Maia, too, the motion is passive. Marchal
stated that muscles do not insert on the operculum of Maia. The writer
confirms this for the crayfish.
Not infrequently, while the animal was held dorsum down and both
nephropores were apparent, fine jets of urine abruptly spurted from both
orifices sometimes to a distance of a foot or more. Every jet consisted
of droplets in quick succession. On one occasion the occurrence was
especially striking in that a series of jets to at least a foot followed one
another rapidly. Although the spurts from both nephropores generally
were not entirely simultaneous, the writer cannot recollect any instance
in which urine spurted from one nephropore and not from the other
MICTURITION IN THE CRAYFISH 139
within a brief interval of time. Such powerful and sudden jets cannot
be accounted for by the very sparsely distributed unstriated fibers of the
bladder. Other decapods act similarly. In a single instance the estu-
arine crab, Callincctcs hastatus, immediately on being grasped spurted
urine to a distance of about 9 cm. from both nephropores simultaneously.
Marchal (1892) noted a distance of 2 cm. from a shrimp and Herrick
(1909) "an inch or more" from the American lobster on being held.
Herrick ascribed the phenomenon to contractility of the bladder but
evidently made no observations to support this supposition.
Whether the sparsely scattered vesicular fibers contribute to the dis-
charge of urine is still unknown. The bladder was subjected to electrical
induction shocks of high and low frequency, led through fine Ag-AgCl
electrodes, both while distended with urine in situ and when isolated and
under slight stretch in the longitudinal or in the transverse direction
between two points in crayfish-saline. Contraction was never observed
even under a magnification of 22.5 X- The induction shocks were capa-
ble of causing cardiac tetanus, contraction of the dorso-anterior and
-posterior dilators of the crop-gizzard, of the dorso-posterior longitudinal
muscles of the crop-gizzard, and of the intact and isolated intestine, and
abduction and adduction of the claw of the cheliped. Electrical stimula-
tion of the bladder frequently produced strong generalised somatic mus-
cular contraction ; abrupt flexion of the abdomen and contraction of the
homolateral remoter of the second antenna (Fig. 1, rein.) were among
the main effects. Because the latter muscle is contiguous with the latero-
ventral surface of the bladder, its contraction generally falsely suggested
contraction of the bladder. Marchal (1892) briefly stated that he was
unable to elicit contraction of the bladder of Mala by electrical stimula-
tion.
Doubtless the major factor in the expulsion of urine is pressure
exerted on the bladder by the blood and crop-gizzard. The nephropores,
of animals drained from moisture, were often observed to remain dry
for forty minutes or more. The injection of 1 to 1.5 cc. of saline into
the haemocoele, between the chelipeds, i.e., in the vicinity of the bladders
and crop-gizzard, invariably resulted in an immediate outflow of urine
from both nephropores simultaneously. Merely puncturing the integu-
ment did not produce effects. If urination was occurring slowly, the
injection of 1 to 1.5 cc. of saline resulted in a marked increase in the
rate of outflow. It is conceivable that in some instances both bladders
may be entirely collapsed ; urination then would not be expected even
upon the injection of any amount of liquid. Bilateral compression of
the integument lateral to the bladders often produced an outflow of urine
or an increase in the rate of flow. The large crop-gizzard is partly
140 N. S. RUSTUM MALUF
wedged between the upper surfaces of the bladders. As direct mechani-
cal pressure on the bladders results in their collapse and in the expulsion
of urine, the movements of the crop-gizzard must be a factor in urination.
Innervation
Probably because the kidney and bladder are organs of the second
antennal somite, all nerve-fibers to the bladder issue from the trito-
cerebral lobe of the brain (Fig. 4, T). The anterior component of the
tegumentary nerves, tc, sends a branch to the integument beneath the
labyrinth ; the posterior component sends branches to some of the proxi-
mal muscles of the second antenna. About nine fibers issue in the
anterior cluster, clf which arises from the base of the root of the main
antennal nerve-trunks, a. Fibers from cl innervate the anterior and
posterior surface of the bladder. The cluster, c2, which consists of about
five fibers, innervates the posterior surface of the bladder and some of
the proximal muscles of the second antenna. Judging from the course
of c2, the sensitive operculum is probably furnished with afferent fibers
from Co rather than from r,. The nerve-fibers to the bladder are prob-
ably mainly afferent.
Repeated observation could not duplicate, in Cambarus, Keim's affir-
mation (1915; and quoted by Stoll, 1925) that in the European crayfish
there extends a nerve-fiber (" nervus glandulae viridis"), bilaterally,
from the suboesophageal ganglion to the labyrinth. Keim considered
Marchal's description of a renal innervation from the second antennal
nerve-bundles as incorrect. Marchal, however, stated that he " could
not find a nerve which passed directly to the antennal gland," i.e. without
first going to the bladder. Neuwyler (1841) disagreed with the laby-
rinthic auditory hypothesis of his eminent predecessors, as regards the
function of the "green glands," because he could never find a nerve-
supply to the glands.1 Wassiliew (1878), in one of the first papers on
the histology of the decapod kidney, stated that no nerves could be seen
to enter the kidney. The present writer's observations are in accord with
Wassiliew in this respect. The absence of a nerve-supply at the kidney
proper indicates that secretion by this organ is not influenced by the
1 To Ernst Haeckel (1857) credit is due for first suggesting that the green
glands are renal organs. Haeckel demonstrated the communication of the bladders
with the exterior and with the glands by introducing metallic mercury into the
bladders. He pointed out that the existence of an external orifice indicates that
the liquid in the bladder is a secretion which is eliminated. This observation, to-
gether with Neuwyler's discovery of the tubule and Gorup-Besanez and Will's
remark that guanine occurs in the green glands, led Haeckel to term these glands
urinary organs. Gorup-Besanez and Will, however, did not state the concentrations
"I guanine in urine and blood.
MICTURITION IN THE CRAYFISH 141
nervous system. This is supported by Maluf, Clarke, and Thompson
(1939), who were the first to show that, per unit volume of glomerular
filtrate, the rate of secretion of various substances is identical in the
clenervated and normal mammalian kidney.
THE ABSENCE OF A VALVE BETWEEN THE NEPHRIC TUBULE
AND THE BLADDER
The epithelial cells of the bladder, except those of the most proximal
portion, are never columnar. They may be highly vacuolated (Fig. 9,
A and B} or plain (Fig. 9, C) in the same bladder. The physiological
evidence indicates that the epithelium of the main body of the bladder
is non-secretory (Maluf, 19415).
Even though the columnar secretory epithelium of the distal half of
the nephric tubule (Fig. 7, dt and Maluf, 1939) merges imperceptibly
into the epithelium of the main body of the bladder (Maluf, 1939), the
tubule as an organ ends abruptly (Fig. 7), since the epithelium of the
bladder is not anastomosed and acutely involuted as is that of the tubule
(Fig. 8). The distal orifice of the tubule can be readily observed in situ
(Fig. 8) through the dorsal surface of the translucent distended bladder.
There is no valve between the tubule and bladder (Fig. 8) and no
evident constriction of the proximal end of the bladder. Sections show
no valve or sphincter at the distal orifice of the tubule.
The bladder of a 14-gram animal normally can distend to a diameter
of at least 8 mm. The hydrostatic pressure within the bladder must
then be somewhat greater than that of a column of water 8 mm. high
because the bladder is elastic (see above). This pressure is doubtless too
low to interfere with the outward secretion of water for which evidence
is presented in an accompanying paper (1941a).
THE ABSENCE OF A VALVE AND SPHINCTER BETWEEN THE
COELOMOSAC AND LABYRINTH
The entire series of sagittal and horizontal serial sections of two
kidneys, fixed in formol-sublimate and stained with haematoxylin and
methylene blue, was studied. No valve or fibers could be found. A
sagittal section at the communication of the lumina of labyrinth and
coelomosac is shown in Fig. 10.
THE EPITHELIUM OF THE COELOMOSAC
The epithelium of the coelomosac, like the rest of the nephron, is
single-layered. The appearance of more than one layer throughout a
142
N. S. RUSTUM MALUF
PLATE II
FIG. 6. Dorsal aspect of left nephropore, a, and membranous operculum.
Note diagonal position of the nephropore. Animal, 22 grams.
FIG. 7. Sagittal section through the kidney showing communication of the
distal extremity of the tubule, dt, with the bladder, />/. /, labyrinth ; p, peritoneum.
Solid black areas indicate blood-sinuses and blood-vessels. Animal, 35 grams.
FIG. 8. Dorsal aspect of the distal orifice of the tubule at its communication
with the bladder.
FIG. 9. Sections of the main body of a single bladder, be, blood-cells ; e,
epithelium of bladder ; p, peritoneum. Animal, 35 grams.
FIG. 10. Sagittal section through the kidney showing communication of the
coelomosac, coc, with the labyrinth, /. dt, distal portion of the tubule ; leu, leuco-
cyte. Solid black areas indicate blood-sinuses and blood-vessels. Animal. 35
grams.
MICTURITION IN THE CRAYFISH
143
O 2Of-
O
O
PLATE III
FIG. 11. "Living" portion of the coelomosac teased from the kidney in cray-
fish-saline and suspended in a hanging drop of crayfish-saline. The apical bulbous
protuberances of the cells are shown in relief.
FIGS. 12 to 17. Epithelium of the coelomosac from medium-sized individuals.
ha, haemocoele ; LU ' , lumen of coelomosac. See text.
144 N. S. RUSTUM MALUF
large part of the coelomosac, labyrinth, and tubule (Fig. 10) is doubtless
due to tangential sectioning.
The nephron of the crayfish has no tenuous syncytium, such as the
glomerular capsule of the vertebrate nephron. The epithelium of the
coelomosac, the most promixal organelle of the nephron, approaches
nearer to being a narrow syncytium than any other part of the nephron
(Figs. 12-15). In several of the individuals examined, however, the
cells were large and rounded throughout the coelomosac (Figs. 16 and
17; and Maluf, 1939).
The epithelium of the coelomosac varies considerably not only from
one individual to another but often, too, in a single animal (Figs. 11-17).
The cells may be either compact, rounded, and sometimes vacuolated, as
in Figs. 16 and 17, or more or less squamous with large protuberances
directed into the lumen (Figs. 12-15). The cells at the periphery may
be rounded while those toward the center are protuberant ; the reverse
has never been found. Frequently, either the protuberant or the
rounded cells occur exclusively. Both coelomosacs of an individual are
always identical.
The histological methods have been described in the previous paper
(Maluf, 1939). The protuberant type of cell is evidently not an arte-
fact because it has been observed in teased-out " living " fragments in a
hanging drop of crayfish-saline (Fig. 11). The composition of the
saline is stated elsewhere (1941 a).
Grabowska (1930) claimed that the secretion of the coelomosac con-
sists of a discharge of cells in their entirety, i.e. " holocrine " secretion.
If the cells are discharged as a whole, one would expect them to be
substituted by mitosis. In not one instance, out of numerous coelomo-
sacs examined, has the writer been able to find a mitotic figure. The
evidence for a discharge of globules from the apical region of these
cells is dubious because where rounded bodies have been seen " free,"
in the lumen of the coelomosac in sectioned preparations, these may have
been merely sections of the bulbous tipped protuberances. In contrast
to the rest of the nephron, the main lumen of the coelomosac generally
contains numerous leucocytes (Fig. 10, leu).
Upon teasing the kidneys of a crayfish on one occasion, the coelomo-
sacs were found packed with hard yellowish-brown irregular concretions
the size of which showed that they could not have been intracellular.
The largest stone was about 0.2 mm. in length. The material was
insoluble in cold and hot water and in absolute ethyl alcohol. The
alcohol decomposed the surrounding yellowish-brown organic material
and the white stones readily fell apart, upon contact, into minute needle-
like crystals which did not dissolve. The stones readily dissolved in
MICTURITION IN THE CRAYFISH 145
dilute HC1 with energetic release of a colorless gas and were slowly
soluble in 10 per cent NH4C1. There is very little doubt, therefore, thai
these concretions were CaCO:;. The individual had a highly melanized
abdominal venter and, hence, must have possessed well-developed cal-
careous gastroliths. Twenty-one crayfish with gastroliths were examined
and only one exhibited a similar condition. This was a single fairly
large concretion in the coelomosac of only one kidney ; other parts of
the kidney did not contain any stones. About thirty animals with a light
abdominal venter and without gastroliths were examined and in no
instance was any concretion found in the kidneys.
The concentration of calcium in the blood of the crayfish and crabs
remains fairly constant even immediately after molting (Paul and
Sharpe, 1916; Damboviceanu, 1930), i.e. even when there is occurring,
by way of the blood, a rapid transfer of calcium from the hepatopancreas
and/or gut to the integument. Oesterlen (1840) has suggested that the
formation of gastroliths may be a way of preventing a rise in the con-
centration of calcium in the blood. The above instances of renal calculi
may be exceptions that prove the possible rule that one of the functions
of the coelomosac is the secretion of calcium from the blood.
Weismann (1874) suggested that a blood-ultrafiltrate is formed
through the coelomosac of the crustacean nephron just as Ludwig (1844)
had presumed to occur through the glomerular capsule of the vertebrate
nephron. Grobben pointed out that the relatively simple coelomosac of
various amphipod crustaceans is attached to the integument by strands ;
this fact supports Weismann's belief inasmuch as effective resistance to
blood-pressure would thereby be offered by the coelomosac, which would
otherwise float in the haemocoele. Grobben also suggested that the
location of the coelomosac between the antennal muscles in phyllopod
Crustacea favors filtration. He nevertheless pointed out that, in copepod
Crustacea, the coelomosac lies freely at the entrance to the homolateral
second antenna and that these animals have no heart ; he also drew
attention to the fact that the phyllopod Crustacea have no heart and
that it is therefore questionable whether, in such instances, filtration can
occur and he ascribed the formation of urine in copepods and early-instar
phyllopods to secretion by the tubule — a conception which had just begun
to gain favor due to Heidenhain's (1874) experiments with the mamma-
lian kidney.
Certain teleological evidence centra-indicates filtration through the
coelomosac. Marshall and Smith (1930) and Marshall (1934) pointed
out that when fishes migrated from freshwater, where they evidently
arose, into the sea they had to conserve water. Some succeeded in
losing their glomeruli while others are still doing so. The crayfish, how-
146 N. S. RUSTUM MALUF
ever, has probably descended from a marine ancestor and is capable of
compensating for water which diffuses inwardly through the gills (Maluf,
1937) by manufacturing a hypotonic urine through the agency of its
nephric tubule. The crayfish nephron has a coelomosac but so does that
of the lobster — a strictly marine relative. Because the osmotic pressure
of the blood of the lobster is slightly hypertonic to that of the surrounding
sea water (Cole, 1940), the lobster, unlike the crayfish, does not absorb
water by diffusion from the exterior and hence does not have to maintain
a steady state by an outward secretion of water. The lobster has either
lost its nephric tubule or has never owned one. If the coelomosac were
a filtration-organdie one would expect it to show some signs of regres-
sion in the lobster ; but the coelomosac of this crustacean exhibits no
evidence of being on the decline. Physiological evidence (Maluf,
1941 b) indicates that the nephron of the crayfish is paramountly if not
entirely a secretory organ.
SUMMARY
1. The internal anatomical features surrounding the urinary outlet
of the crayfish are described in detail for the first time.
2. Urine is retained in the bladders evidently by the ureteral syncytium,
which is here described for the first time. There is no other way, con-
ceivable to the writer, by which urine can be retained. Fibers do not
insert on the operculum of the nephropore.
3. Urine is discharged by a localized rise in the haemocoelic pressure
and can be expelled by direct action of the crop-gizzard on the bladders.
Adequate electrical stimulation cannot cause contraction of the bladder
but often evokes generalized motor activity.
4. Occasional abrupt spurts of urine, which were almost simultaneous
from both nephropores, extended to the distance of a foot or more.
5. Destruction of the opercula before urinary collection has no
rationale.
6. The bladder is innervated by fibers from the tritocerebral lobe of
the brain. These fibers are doubtless mainly if not entirely afferent.
The kidney is not innervated.
7. There is no valve between the nephric tubule and the bladder.
8. There is no valve or sphincter between the coelomosac and the
labyrinth.
9. The epithelium of the coelomosac, the most proximal portion of
the nephron, has been studied in detail. ' Holocrine " secretion evi-
dently does not occur because no mitotic figures could be found. The
histological, chemical, and phylogenetical data contra-indicate filtration
through the coelomosac.
MICTURITION IN THE CRAYFISH 147
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CHROMATIN BRIDGES AND IRREGULARITY OF
MITOTIC COORDINATION IN THE PEN-
TATOMID PEROMATUS NOTATUS
AM. AND SERV.
FRANZ SCHRADER
(Front the Department of Zoology, Columbia University, .Y<\v York City)
INTRODUCTION
A .specimen of the pentatomid species Pcroiuatiis iwtatus obtained
in 1937 presents such significant modifications of the orthodox course of
meiosis, that a description and consideration of the most striking fea-
tures seem warranted. As will be seen, the individual in question clearly
is an exceptional case, but its departure from the normal is based on
fundamental changes that have altered its mitotic mechanism in a very
definite way. Apparently it is chiefly the relative timing of the various
mitotic processes that has been affected, and the chromosomes and the
spindle apparatus are, so to speak, out of step with each other. Their
behavior under these conditions is of some interest in the analysis of
mitosis in general.
MATERIAL
The specimen was caught on Barro Colorado Island in the Panama
Canal Zone, in March. 1937. Its testes were fixed in B 15 within two
hours of capture. It was close to the maximum size recorded for the
species, in good condition, and very active.
Peromatus notatus, like the other six species of the genus, is strictly
neotropical in its distribution. Examination of the sixteen specimens in
the collections of the U. S. National Museum and the American Museum
of Natural History shows that the species is subject to considerable
variation in form and color. Variability in form is, however, more or
less superficial and chiefly due to differences in the size and shape of the
pronotal spines. The usual chestnut-brown color is replaced by green
in some individuals from Panama (identified and labeled in the Ameri-
can Museum collection by H. G. Barber). It is a specimen of the latter
type caught on Barro Colorado Island in 1941 that has served for com-
parison in the present study. It offers a typically pentatomid spermato-
genesis which is almost indistinguishable from that of a specimen of
Pcrouiatiis tnincatus obtained in the same locality.
149
150 FRANZ SCHRADER
SPERMATOGONIA
The spermatogonial divisions of the exceptional individual show no
unusual features. The spindles conform to the common type, the chro-
mosomes divide normally, and successive spermatogonial cell generations
show no variation in chromosome number. The latter comprises the
usual set of 14 chromosomes, in which one pair is a little larger and one
pair somewhat smaller than the rest. The X is intermediate in size,
whereas the Y is about as large as a member of the smallest pair (Fig.
1).
MEIOTIC PRorHASEs TO DIAKINESIS
Up to late diakinesis, the meiotic prophase stages conform to the
usual pentatomid behavior. The sex chromosomes are heteropycnotic
and frequently, though not always, appear joined from leptotene to
diakinesis. There is a plasmosome which dwindles rapidly after the
pachytene stage.
It is not until toward the end of diakinesis that the first unusual fea-
ture is encountered. Just as in other pentatomids, the two centers at this
time move toward opposite sides of the nucleus. Both are in contact
with the nuclear membrane and when they have reached their final posi-
tion, the membrane underneath them is pulled or bulged outward.
This and the oval form of the nucleus, assumed in the direction of
the centriolar axis, have frequently been noted (as early as 1891 by Henk-
ing). The point to be noted in this instance, however, is that the centers
in the majority of cases are not on truly opposite points of the nucleus
but are closer to each other on one side than on the other (Fig. 2). It
is, of course, true that in other pentatomids also the position of the
centers is not always geometrically exact, but the position here clearly is
not accidental. This is borne out by the metaphase conditions that im-
mediately follow the breakdown of the nuclear membrane.
THE FIRST METAPHASE
The equator of the first spindle is in almost all cases displaced to
one side, so that a line through the two centers does not represent the
symmetrical axis of the mitotic figure as in other cases. In many cells
all the chromosomes form a plate that lies to one side of the centriolar
axis and hence the half spindle components are similarly displaced (Fig.
4). A few continuous fibres can sometimes be seen to stretch between
the centers without such displacement, indicating their relative inde-
pendence of the chromosomes. The latter rarely form a circle or round
CHKOMATIX BRIDGES IX PKKOMATUS 151
plate, but constitute a semicircle with the two sex chromosomes usually
but not always lying on the concave side (Fig. 3).
Despite this distortion of the mitotic apparatus, the tetrads divide in
orderly fashion (Fig. 6) and the sex chromosomes undergo an equation
division, just as in the normal Peromatus and other pentatomids. The
peculiar configuration of the chromosome plate, however, is mirrored in
the two daughter groups and may persist until middle anaphase (Fig. 5).
The initial movement of the dyads seems to occur without reference
to the center and hence show-s no effect of their askew7 position (Fig. 4).
This is, of course, what might be expected since in nearly all cases known
these first division stages of the chromosome appear to be autonomous.
The configuration of this first spermatocyte spindle challenges several
interpretations concerning the mitotic mechanism. If the poles of the
spindle are established by a mutual repulsion of two centrioles, it is very
difficult to conceive of anything but a symmetrical spindle structure re-
sulting therefrom. If the chromosomes assume their metaphase position
because they react to forces from the poles, it is again not easy to under-
stand why they should take such an " off center" position as they do.
The conclusion is unavoidable that the mitotic conditions are affected
by factors which normally are not present at this time.
FIRST Ax A PHASE TO SECOND ANAPHASE
In the normal Pcroiiiutus as well as in most other pentatomids so far
investigated, each of the centers carries two centrioles already at diakine-
sis. These two centrioles usually remain closely associated until telo-
phase, though occasionally they have separated by some 15° before the
end of anaphase (see. for instance, Paulmier's Fig. 29. 1899). The
movement is quickened at telophase and before the second division is
begun, the two centrioles are separated by 180°. There appears to be
no exception to the rule that in Heteroptera the polar axis of this second
division is at right angles to that of the first. This relation is especially
striking in those cases where the interzonal connections of the first divi-
sion continue to stain intensely, as in PacJiylis (Fig. 8. and also those of
other Heteroptera by Henking, 1891 ; Montgomery, 1898; and Paulmier,
1899).
The course followed in the present case is characterized by either one
of two departures from the normal procedure just described. In about
75 per cent of the cells there is a marked precocity in the movements
of the centrioles. Starting with little more separation than in normal
cases, they diverge quickly after the early anaphase and in most cases
have separated by 40°-45° before the anaphase movement of the chro-
152 FRANZ SCHRADER
mosomes has been completed (Fig. 7). Among the remaining cells
about half show no such precocious separation of the centrioles, but the
center as a whole may shift as much as 90° from the axial position of
the first division (Fig. 9). In short, in such cases both centrioles assume
the position of one of the poles of the second division, though the chro-
mosomes are still in late anaphase of the first.
It was a matter of some surprise to find that in every such instance
both centers moved to the same side of the anaphase cell. But this may
simply be the consequence of the asymmetry of the first spindle which
puts both centers closer to one side than the other to begin with. The
two extremes of centriolar behavior are bridged by intermediate condi-
tions which are not always easy to interpret. Thus the centrioles may
succeed in separating after the center as a whole has begun to shift, or
else one of the centrioles is for some reason held at the first pole and
only the other moves toward its position for the second division (Fig. 7).
Whatever the type of variation may be, one point is held in common
by all these cells. The processes that establish the achromatic figure of
the second division are decidedly in advance of the corresponding steps
in normal cells.
The precocity of the centers has marked effects on the behavior of
the chromosomes. This is, perhaps, no more than might be expected,
since they are still in the anaphase of the first division when the centers
are already in process of establishing the mechanism for the second.
The chromosomes show a definite response to the two poles which is
manifested most strikingly in a tendency to divide again at this early
PLATE I
Drawings made with Zeiss, 90 X objective and 20 X ocular. They were re-
duced % in reproduction.
FIG. 1. Three plates showing the 14 spermatogonial chromosomes.
FIG. 2. Diakinesis. The two centers are closer to each other on one side
than the other.
FIG. 3. First metaphase. Autosomal tetrads arranged in semicircle, with X
and Y on the inside.
FIG. 4. Side view of early anaphase, showing asymmetrical spindle. The
autonomy of the initial separation of chromosomes is attested by lack of orienta-
tion toward the centers.
FIG. 5. Polar view of two sister groups in first anaphase, still showing typical
arrangement.
FIG. 6. Middle anaphase of first division. The centrioles at each pole are
separated less than usual.
FIG. 7. Upper pole of a late anaphase of first division. The two centrioles
already have separated by about 45°, and there is no collocation of the chromosomes.
FIG. 8. Interphase in the coreid Pachylis, to show the characteristic relation
of the second to the first spindle in the Heteroptera.
CHROMATIN BRIDGES IN PEROMATUS 153
PLATE I
154 FRANZ SCHRADER
stage (Fig. 10). If this occurs before they have become dissociated
from the interzonal connectives, such peculiar configurations as shown in
Fig. 12 may result. In these as well as in less extreme cases the signifi-
cant feature lies in the marked elongation of the chromosomes.1
This occurs despite the fact that the two chromatids of each dyad
move in opposite directions toward the centrioles which are establishing
a new axis. In other words, though the demarcation between the two
chromatids is clearly indicated — as indeed it already is in diakinesis—
and though the attenuation of the chromatids evidently betokens forces
that tend to move them apart, they do not succeed in separating from
each other (Figs. 10-13). The attenuating process continues until the
chromosome body is torn into two pieces. The break apparently occurs
at random and usually not in the natural line of separation between the
chromatids (Figs. 13 and 14). Hence the amount of chromosome mate-
rial distributed to each pole is variable and certainly not normal.
During this time the centriolar movement is completed. As a result
the flexion that characterizes the spindles during the early part of this
division disappears and the spindles of the late second anaphase are
perfectly straight (Fig. 13).
Pl.ATK II
FIG. 9. Late anaphase of first division. Each of the centers (both show two
centrioles) has moved through 90° toward one pole of the second division.
FIG. 10. Centrioles of second division acting on chromosomes which are still
in the condition of the first anaphase. (In Figs. 10, 11, and 12 only one of two
sister cells is shown.) The demarcation between the chromatids is evident in
several dyads.
FIG. 11. Second division showing attenuation, with chromatic! demarcation
showing in several dyads.
FIG. 12. Second division. The centrioles have separated relatively little, and
the whole figure is strongly flexed as a result. Trace of interzonal connections of
first division still showing at lower left.
FIG. 13. Late anaphase of second division. The spindle has straightened out.
Chromatid demarcation still present in two of the dyads.
FIG. 14. Telophase of second division. There is no trace of collocation. The
abnormality of the chromosome division is evident.
FIG. 15. Late telophase. Chromosomes still scattered and already becoming
diffuse.
FIG. 16. Spermatid with four micronuclei, one Nebenkern, and one tail fila-
ment.
1 It will be seen that the side of the chromosome presented toward the pole in
the first division does not correspond to that of the second. This puzzling feature
is, however, encountered in all Heteroptera and does not constitute a peculiarity of
the present case. The explanation may lie in the fact that in the Hemiptera we are
dealing with a " diffuse " instead of a localized kinetochore, as Hughes-Schrader
and Ris fin press) have recently established.
CHROMATIN BRIDGES IN PEROMATUS 155
II
12
14
15
PLATE II
156 FRANZ SCHRADER
SECOND TELOPHASE TO SPERMATOZOA
Since the chromosomes of the first division are subject to the forces
of the second division while they are still in anaphase, nothing can be
said of their behavior under telophase conditions. Since there is only
one centriole at each pole of the second division, no comparable centriolar
disturbance takes place there and the chromosomes reach the telophase in
every case.
Instead of the collocation of chromosomes that is typical of normal
telophases, the chromosomes here actually tend to move further apart
or to repel each other (Fig. 14). This tendency is not overcome even
by the time that the chromatin becomes diffuse and as a result the prod-
ucts of the division lie more or less scattered in the cell (Fig. 15). Sep-
arate, small nuclei are formed from such masses of chromatin, and the
spermatid is always a multinucleate cell (Fig. 16). In most cases only
one Nebenkern is formed though in some instances two have been en-
countered. In no cell, however, does one find more than one axial fila-
ment and middle piece. These are associated with one of the nuclei
which is not necessarily the largest one.
Apparently even the smallest of the nuclei takes steps toward the
elongation that characterizes the formation of the sperm head. Later,
however, there is much degeneration, though some of the sperms appear
more or less normal.
The relative independence of mitotic phenomena in the cytoplasm and
in the nucleus is attested by the fact that all the manoeuvers of the
centers and the chromosomes do not hinder the division of the cytoplasm.
Separate and complete cells, more or less equal in size, are found both
after the first as well as the second division.
DISCUSSION
The relationship of the significant features of this case is not always
entirely clear, though it seems safe to assume that they are interconnected.
They may be listed as follows: 1. The asymmetry of the first division
figure. 2. The precocity in the behavior of the centers. 3. The attenua-
tion and irregular division of the chromosomes in the second division.
4. The formation of multinuclear spermatids.
( 1 ) . The asymmetry of the first division figure is difficult to explain.
If bipolarity is brought about merely by a mutual repulsion of two
centers, the latter should be separated by 180° on the diakinetic nucleus
and in the first metaphase. Again, the location of the chromosome plate,
if it rests on a system of repulsive or attractive forces correlated with
CHROMATIN BRIDGES IN PEROM ATI'S 157
those of the centers, should be on the axis formed by the latter. To
explain the askew position of the chromosomes, it might be suggested
that a primary spindle, comprised of fibres extending from pole to pole,
arises before the chromosomes have formed a metaphase plate. This
spindle then constitutes a core into which the chromosomes do not pene-
trate and hence they are disposed in the form of a semicircle around it.
But such a hypothesis does not touch the root of the matter, which lies
in the asymmetrical position of the centers themselves. And for this
nothing more can be said than that a factor or force, probably extraneous
to centers and chromosomes, is responsible.
(2). The extreme degree of separation of sister centrioles during
the first division is clearly an indication of precocity in their cycle. Not
so pertinent to this conception are those instances where the entire center,
including both centrioles, moves to one of the poles of the second division
(Fig. 9).
This might be attributed to the elongation of the spindle which pushes
both centers around the periphery to one side. Precocity would there be
expressed only in the development of astral rays and half-spindle fibres
which actually appear to be growing at a time when in normal cases
they are waning.
If, however, the movement of the undivided center is not thus acci-
dental, its shift to the axis of the second division must mean that this
pole is predetermined. This would imply that the centers are only
secondarily concerned. The evidence hardly permits of extensive hypo-
thetical considerations, but the early establishment of such a pole might
involve forces that also are responsible for the asymmetry of the first
division.
(3). But whether or not the centers are the primary agents in the
determination of polarity, their direct influence on the chromosomes is
not to be denied. This is strikingly shown in the premature second
division, where it appears that the precocity of the centriolar processes is
correlated with an exertion of forces that are normally not in evidence
until a later stage. Their influence is indicated by the fact that the
mitotic movement of chromosomes is toward the two centers from the
very start. The autonomous separation of chromatids which takes place
without reference to centers and which always comprises the first step
under normal conditions, does not take place at all.
The attenuation of the chromosomes suggests that they are subjected
to tensile forces. The failure of the chromatids to dissociate from eacli
other under such conditions must then indicate that they are not yet
completely reach- when the centriolar forces are exerted thus precociously.
158 FRANZ SCHRADER
The lag does not lie in the chromosome proper, for in this as well as in
normal cases all the chromatids are sharply demarcated from each other
already in the preceding diakinesis (Fig. 2). That this demarcation
persists into the second division is clearly shown in Figs. 10, 11 and 13,
and the conclusion hence is unavoidable that a separation is prevented by
other factors. The latter can be sought only in either the sheath or the
matrix of the chromosomes, and it is therefore this constituent which is
not yet ready for the division and holds the chromatids together.
It may be pointed out that the attenuation of chromosomes during
division is not at all rare and that its cause is by no means always the
same. It has been reported in cells that were subjected to X-ray or
radium treatment. It is then usually correlated with a tendency of
chromosomes to clump, and secondarily to translocations and inversions.
Such cases are difficult to analyze since so many of the mitotic processes
seem to be affected.
It has been described in tapetum cells (Steil, 1935) which show signs
of degeneration. The attendant irregularities may well arise from an
upset in the timing of the various mitotic processes as in the present
case, but the necessary details of behavior that would justify such a
conclusion are not available.
Bauer (1931) has reported it in Tipula and ascribes it to the presence
of supernumeraries. The disturbance is there correlated with an ad-
hesion of the chromosomes to each other.
It results from changes in the physical condition of the chromosomes,
which in at least one case arise from the mutation of a single gene
(Beadle, 1932). The "stickiness" which there characterizes the chro-
mosomes seems to be caused chiefly by changes in the matrix and it is
not impossible that the frequent attenuation during division is closely
akin to that observed in Peroniatus.
Lastly, it is a well-recognized characteristic of chromosome inver-
sions which have resulted in dikinetic or dicentric chromosomes. Such
" chromosome bridges " have played so striking a role in recent cyto-
genetic investigations that there has been a tendency to forget that not
all chromatin bridges need be of the same nature. Thus Gentscheff and
Gustafsson (1940) in their excellent study of meiosis in Hieraciuin
utilize Beadle's conception that fragmentation of his maize chromosomes
results from changes in the matrix, but quite ignore his explanation that
his chromosome bridges were due to stickiness and increase in viscosity.
Instead, they ascribe the very similar bridges in Hieraciuin to inversions
and thus seem to agree with Darlington (1937, p. 320), who does not
accept Beadle's convincing interpretation and states that " at anaphase
CHROMATIN BRIDGES IN PEROMATUS 159
several bridges are found, showing that the changes include inversions."
It need hardly be pointed out that in the present case of Peromatus
notatus, an explanation that rests on inversions is not tenable at all.
This is already strongly indicated by the fact that the first division shows
no bridges whatever, whereas they characterize all second divisions. To
explain this on the basis of inversions would necessitate that two cross-
overs of a very specific type take place, and that these occur in the
meiotic prophase of all cells. This would, moreover, result in a chromo-
some fragment which most assuredly is not present. Further, such a
hypothesis would assume an orthodox, localized kinetochore, whereas
here we are dealing with one of the diffuse type (Hughes-Schrader and
Ris, in press). Finally, it must be remembered that an inversion bridge
arises because bipolar tension is exerted upon a portion of a chromosome
which does not include the natural line of demarcation between chro-
matids and which therefore can be divided only by tearing. In contrast,
the bridges in Peromatus include the region where two chromatids,
sharply demarcated from each other, are placed end to end. Dissocia-
tion therefore should and would follow quite normally without attenua-
tion, if it were not hindered by the matrix or the sheath.
(4). Multinucleate spermatids arise because of the upset in the
timing of mitotic processes. The chromosomes of the second division
arrive at telophase when, in a sense, they are still in the anaphase condi-
tion. The mutual repulsion that characterizes them at the normal
metaphase and anaphase, is therefore still encountered here when they
have arrived at the poles. Hence there is the reverse of the usual collo-
cation, the chromosome bodies are scattered singly or in small groups
through the cell, and several micronuclei are found. The case for an
irregularity in the timing of the centers is further supported by the fact
that the actual division of the centrioles, albeit their movements are
precocious, is in itself quite normal and only one middle piece and one
axial filament are encountered in every multinucleate spermatid.
CONCLUSION
The nature of the case makes it rather futile to speculate on the
origin of the meiotic abnormalities just described. Practically nothing-
is known about the ecology of the genus, and the possibility of inter-
racial and interspecific crosses is purely hypothetical.
Clearly, however, the case is an exceptional one for the species. The
conditions basically affect the production of normal sperms and can have
no survival value. Indeed, the rather orthodox course of- spermato-
genesis in other specimens of Peromatus renders this certain.
160 FRANZ SCHRADER
But so far as this individual is concerned, the abnormality is a deep-
seated one since the absence of normal spermatids indicates that it has
persisted for some time. The conditions strongly suggest that at least
one of the mitotic processes has fallen out of step and that coordination
with the other processes becomes progressively more difficult in the suc-
cessive cell generations, from spermatogonia to spermatids. The dis-
turbance has no visible effect on the spermatogonia; has a well-defined
influence on the spindle mechanism of the first division without, however,
upsetting the essential aspects of orderly chromosome division ; renders
impossible a normal distribution of chromosomes in the second division ;
and culminates in spermatids that are definitely abnormal.
SUMMARY
1. The abnormal course of meiosis in a specimen of Peroiitatns no-
la fits is characterized by a series of well-defined irregularities.
2. The spindle of the first division shows both centers to one side
of the geometrical axis and the metaphase plate displaced to the opposite
side.
3. Before the chromosomes of the first division have reached the
poles, they are subjected to the forces involved in the second division.
4. The effect on the chromosomes is to attenuate them without bring-
ing about a normal division. The resulting configurations simulate
inversion bridges, though that is quite clearly not their nature.
5. The spermatids receive varying amounts of chromosome material
and are multinucleate.
6. It is suggested that this abnormal meiosis is due to an irregularity
in the timing of one of the mitotic processes. The indications are that
this process involves the movement of the centers.
REFERENCES
BAUER, H., 1931. Die Chromosomen von Tipula paludosa Meig. in Eibildung und
Spermatogenese. Zcitschr. f. Zcllforschung, 14: 138-193.
BEADLE, G. W., 1932. A gene for sticky chromosomes in Zea mays. Zcitschr. f.
hidukt. Abstimmungs- und J'crcrh.. 63: 195-217.
DARLINGTON, C. D., 1937. Recent Advances in Cytology. Blakiston Son and Co.,
Philadelphia.
GENTCHEFF, G., AND A. GUSTAFSSOX, 1940. The balance system of meiosis in
Hieracium. Hcrcditas, 26: 209-249.
HENKING, H., 1891. Ueber Spermatogenese und deren Beziehung zur EienUvick-
lung bei Pyrrhocoris apterus L. Zcitschr. f. wiss. Zool, 51 : 685-736.
HUGHES-SCHRADER, S., AND HANS Ris, 1941. The diffuse spindle attachment of
coccids, verified by mitotic behavior of induced chromosome fragments.
Jour. R.rpcr. Zool. Tn press.
i IIKitMATIN BRIDGES IN PEROMATUS
MONTGOMERY, T. H., 1899. The spermatogenesis in Pentatoma up to the forma-
tion of the spermatid. Zool. Jahrb. (Anat.), 12: 1-89.
PAULMIER, F. C, 1899. The spermatogenesis of Anasa tristis. Jour. Morph.,
(Suppl.), 15: 224-272.
STEIL, W. N., 1935. Incomplete nuclear and cell division in the tapetum of
Botrychium virginianum and Ophioglossum vulgatum. Am. Jour. Bot.,
22: 409-425.
Vol. LXXXI, No. 2 October, 1941
THE
BIOLOGICAL BULLETIN
PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY
THE RESPONSES OF CATFISH MELANOPHORES
TO ERGOTAMINE
G. H. PARKER
(From the Biological Laboratories, Harvard University}
Some eight years ago Bacq (1933), on the basis of experimental evi-
dence, reached the conclusion that ergotamine contracts denervated cat-
fish melanophores and expands normally innervated ones. It is now
known that the so-called contraction of catfish melanophores is the result
of a neurohumor from the concentrating nerve-fibers, very likely ad-
renaline, and that their expansion is due to two agents, intermedine from
the pituitary gland and a neurohumor, probably acetylcholine, from the
dispersing nerve-fibers (Chang, Hsieh, and Lu, 1939; Parker, 1940).
In consequence of these new discoveries it seemed desirable to repeat
Bacq's experiments with the view of bringing his rather remarkable re-
sults into relation with this newly acquired information.
The ergotamine tartrate used by Bacq is fortunately still to be had
in the American market. It is dispensed in 1 cc. glass ampules under
the name of gynergen (Sandoz Chemical Works) and in this form it is
extremely convenient for experiments on fishes. Three sets of catfishes
(Ameiurus nebulosus) were prepared for these tests, — pale, intermedi-
ate, and dark. The pale fishes, three in number in the initial set, were
kept in white-walled, illuminated vessels. Two caudal bands were cut
in each fish. By the end of six days these fishes were very pale and their
caudal bands were almost fully blanched. The axis and tip of each
band, however, were noticeably dark as observed and figured by Bacq
(1933). The three fishes of intermediate tint were kept in a gray, illu-
minated vessel. Their caudal bands after six days were very slightly
darker than the rest of their darkish tails. The three fishes of the dark
set wrere rendered very dark, coal-black, by complete blinding. It is
well known that catfishes assume this intense shade on double enucleation.
Notwithstanding the great depth of tint thus produced, the caudal bands
in these fishes were a shade darker than the rest of their very dark tails.
163
164 G. H. PARKER
Six days after the cutting of the caudal bands in the pale fishes these
bands were recut a little distal to the original incision. Since the part
of the band distal to the new cut did not change in tint as a result of the
recutting, it was concluded that so far as color changes were concerned
the nerves of such bands had degenerated. As the three fishes in any
given set, pale, intermediate, or dark, were very similar in color, one in
each set was reserved as a control and the other two were subjected to
tests. Two injections each of 0.25 cc. of gynergen separated by an in-
terval of about a quarter of an hour yielded the best results. These in-
jections were at times supplemented by a third. Two injections of 0.5
cc. of gynergen with an interval of fifteen minutes between them gave
more vigorous responses than the weaker injections, but they were usu-
ally followed some hours later by the death of the fish. I was unable
to obtain unquestionable responses with only a single injection of 0.25
cc. of gynergen as reported by Bacq. The catfishes used by me weighed
each about 50 grams. Bacq makes no statement as to the weight of his
specimens. Possibly he had smaller individuals than I had and therefore
obtained satisfactory responses with less ergotamine. In my procedure
any given catfish must have received into its body from the two injections
ordinarily given a total amount of 0.25 mg. of ergotamine tartrate judged
from the formula published by the Sandoz Chemical Works for their
preparation of gynergen.
Bacq's tests, which were carried out only on pale catfishes, consisted
in injecting into such a fish with a blanched caudal band 0.25 cc. of
gynergen whereupon the fish as a whole became dark, but the band re-
mained pale or even took on a somewhat lighter tint. My repetition of
such a test gave almost identical results. When a pale fish with two
blanched caudal bands was injected with the usual two doses of gynergen,
0.25 cc. each, with an intervening quarter of an hour, the fish began to
darken noticeably in about half an hour after the first injection and in
an hour to an hour and a half it had reached a full intermediate tint, its
maximum color change under the circumstances. As the tail darkened
the bands appeared to become paler as noted by Bacq, but whether this
was an actual blanching or a contrast phenomenon could not be settled
except by close scrutiny. When the bands on the tail of an injected fish
were closely compared with those on the uninjected pale control, the two
sets of bands were found to be in very close agreement. This was par-
ticularly well seen under a low power of the microscope. In both sets
of bands the pigment masses in the macromelanophores were rounded
bodies with short, blunt protuberances on their sides marking the roots
of the pigmented processes of the dispersed stage. The pigment masses
in the injected fishes appeared to be in no sense less dispersed than those
ERGOTAMINE AND MELANOPHORES 165
in the control fish, and yet when the bands were inspected by the unaided
eye those in the dark fishes appeared to be paler than those in the pale
control. In my opinion this apparent difference is purely an illusion due
to contrast. The dark surroundings of the pale bands in the injected
fishes made these bands appear paler than the pale bands in the pale con-
trol. I therefore conclude that, contrary to Bacq's view, ergotamine has
no effect on denervated melanophores with concentrated pigment. This
agent, however, does induce pigment dispersion in innervated color cells,
as stated by Bacq.
In one other respect my observations do not agree with those of Bacq.
In the majority of caudal bands that have been blanched in pale fishes
for some six days the axes and tips of these bands, as already stated, are
slightly dark. This feature was described and figured by Bacq, who
noted further that when catfishes showing these peculiarities were in-
jected with ergotamine the pale bands not only became paler but their
dark axes and tips also blanched. In my experience such was not the
case. After full doses of ergotamine had been allowed to act on the two
pale catfishes tested by me, the dark axes and tips in their caudal bands
were as visible after the injection as they had been before it or as they
were in the control.
In making these several comparisons the individual catfishes in the
course of inspection were necessarily much handled. As is well known,
this treatment induces such fishes to darken temporarily and it might be
supposed that this darkening could in some way have influenced the re-
sults just described. But both the control fish and the two injected indi-
viduals were handled to about the same degree and therefore should have
shown the same amount of change as a result of this treatment. More-
over, it has been demonstrated in a recent paper (Parker, 1940) that the
darkening already alluded to is a response mediated by the dispersing
nerves. Consequently it ought to play no part in the activities of a
denervated area such as a caudal band. There is therefore no reason to
suppose that the ordinary darkening of catfishes from handling could
have had any influence on the results herein recorded.
The tests on the three pale catfishes just described were repeated on
two other sets of pale individuals, one of two fishes and the other of
three. In both these sets the pale bands of the injected fishes showed no
more change in tint than did those of the first set and their bodies in
general darkened to intermediate. This agreement in three sets of re-
sults justifies the conclusion that, as Bacq maintained, ergotamine excites
innervated melanophores in catfishes to disperse their pigment to a point
where the fish attains an intermediate tint. It shows further that, con-
trary to Bacq's opinion, this agent does not induce a concentration of
166 G. H. PARKER
pigment in denervated melanophores whereby caudal bands in pale fishes
would become still paler. Ergotamine apparently has no influence what-
ever on denervated melanophores with concentrated pigment. It does
induce pigment dispersion in innervated melanophores.
When catfishes with caudal bands cut in their tails are kept for some
six days in a gray, illuminated vessel they take on, as already stated, an
intermediate tint in which the bands are as a rule slightly darker than
the rest of the fish. If these fishes now receive the usual injections of
ergotamine, two doses of gynergen, 0.25 cc. each, separated by a quarter
of an hour, they will either show no noticeable change in tint at all or
darken very slightly. This rule held for all three sets of catfishes tested,
including a total of nine individuals. In no instance was there any
evidence of the blanching of the denervated bands, but, contrary to what
might have been expected from Bacq's statements, these bands remained
usually a little darker than the rest of the tail. Bacq apparently never
tested fishes of intermediate tint with ergotamine. Had he done so, he
surely would have observed that when the ergotamine excited any change
at all it was a very mild darkening in the region of the innervated color
cells and not only no blanching but no change of color whatever in that
of the denervated cells.
What has been stated for catfishes of an intermediate tint may be
affirmed in general for those that are fully dark. As noted previously,
the caudal bands in such fishes are as a rule very slightly darker than
the rest of the tails in these individuals. After the usual injections of
ergotamine these color conditions either showed no change or the fish
as a whole darkened very slightly. In two instances this general darken-
ing was sufficient to make the tail slightly darker than the bands. As
comparisons with the control fish showed, this was a real darkening of
the tail and not a slight blanching of the band. Hence we are justified in
concluding, as in the instance of the intermediate fishes, that ergotamine
induces pigment dispersion in innervated melanophores but has no effect
on denervated ones.
From these several tests on pale, intermediate, and dark catfishes it
seems fair to conclude that ergotamine acts only on innervated melano-
phores by inducing them to disperse their pigment and has no effect what-
ever on denervated melanophores. The assumed blanching of these color
cells by this agent, as described by Bacq, is purely illusory. So far as
the end result is concerned, ergotamine is like intermedine or acetylcho-
line in that it causes catfishes to darken. But it does not act in the same
way as these two neurohumors do. They act directly on the melano-
phores (Parker, 1941), for they will induce denervated caudal bands to
darken. Ergotamine acts on melanophores indirectly, that is through
ERGOTAMINE AND MELANOPHORES 167
nerves, in that it excites at some central nervous station the dispersing
nervous elements which in turn excite the appropriate nervous terminals
to discharge acetylcholine. This neurohumor causes the melanophores
to disperse their pigment whereby the fish darkens. Ergotamine is a
good example of an indirect melanophoric agent as contrast with direct
ones such as intermedine, acetylcholine, and adrenaline. These will
activate denervated melanophores in caudal band. Ergotamine is inca-
pable of this activity.
•
SUMMARY
1. Ergotamine acts on only innervated melanophores by inducing
them to disperse their pigment. It is without effect on denervated me-
lanophores either with dispersed or with concentrated pigment.
2. It acts on innervated melanophores only indirectly, that is, through
their nerves. These are excited by ergotamine centrally to produce at
their melanophore terminals acetylcholine which causes the color cells
to disperse their pigment.
3. Ergotamine is a good example of an indirect excitant of melano-
phores as contrasted with direct excitants such as intermedine, acetyl-
choline, and adrenaline, all of which act directly on these color cells.
REFERENCES
BACQ, Z. M., 1933. The action of ergotamine on the chromatophores of the cat-
fish (Ameiurus nebulosus). Biol. Bull., 65: 387-388.
CHANG, H. C., W. HSIEH, AND Y. M. Lu, 1939. Light-pituitary reflex and the
adrenergic-cholinergic sympathetic nerve in a teleost. Proc. Soc. Exper.
Biol. Mcd., 40: 455-456.
PARKER, G. H., 1940. On the neurohumors of the color changes in catfishes and
on fats and oils as protective agents for such substances. Proc. Am.
Philos. Soc., 83 : 379^09.
PARKER, G. H., 1941. The method of activation of melanophores and the limita-
tions of melanophore responses in the catfish Ameiurus. Proc. Am. Pliilos.
Soc. (In press.)
SEXUAL PHASES IN WOOD-BORING MOLLUSKS
WESLEY R. COE
(From the Osborn Zoological Laboratory, Yale University, and the Scripps
Institution of Oceanography, University of California} x
*
It has been demonstrated previously that the well-known shipworm
Teredo navalis is typically protandric, nearly all individuals functioning
as males when young and later changing to the female phase (Coe, 1936;
Grave and Smith, 1936). Under favorable conditions the female phase
may be followed by an additional sequence of male and female phases.
In no other species of the wood-boring mollusks (Teredinidae) is the
sexual sequence fully known, although Yonge (1926) recognized pro-
tandry in Teredo norvegica and Siegerf oos ( 1908) concluded that it was
present in " Xylotrya gouldi " = Bankia fitnbriata (Jeffries).
During the past few years the writer has had the opportunity of in-
vestigating the biology of three species, Teredo navalis, T. diegensis and
Bankia setacea, with particular reference to the development of the go-
nads and the sequence of the sexual phases. The results of this study
may be briefly summarized and compared with previous reports on the
sexuality of the shipworms.
SEXUAL PHASES OF TEREDO NAVALIS
This widely distributed species occurs on both the Atlantic and Pacific
coasts of the United States and has been particularly destructive in past
years in San Francisco Bay. Individuals in the female phase are larvi-
parous, the fertilized eggs developing through about half the larval period
in the maternal gill chambers. A free-swimming period follows the dis-
charge of the larvae into the water. After settling upon a piece of wood,
transformation to the adult condition takes place and boring into the
wood begins (Kofoid and Miller, 1927; Grave, 1928).
The primary male phase becomes functional within four to six weeks
after the completion of the free-swimming larval condition in the warmer
season of the year or in warm localities, but may be delayed for six
months or more under colder conditions. The body is then only 20 to
30 mm. in length and about 2 mm. in diameter. The female phase may
begin at the age of eight to ten weeks.
1 Contributions from the Scripps Institution of Oceanography, No. 145.
168
SEXUAL PHASES IN WOOD-BORING MOLLUSKS 169
Growth is rapid under favorable conditions. At the end of one year
the body may have attained a length of 10 to 40 cm. and a diameter of
4 to 9 mm. In the meantime the individual has normally transformed
to the female phase and has produced perhaps 1,000,000 young. A
second sequence of male and female phases may have occurred.
Because of the long breeding season and the sequence of sexual
phases, the proportion of the two sexes in each piece of wood will obvi-
ously depend upon the ages represented in the colony. In a recently
attacked timber nearly every individual will be in the male phase. A
few weeks later, after the sexual transformation has occurred, 60 to 90
per cent of the original colony will be functioning as females.
But in the meantime there may have been daily additions of recently
arrived young from other pieces of wood, resulting in a continuing supply
of male-phase individuals. These, together with the few so-called true
males, and a small proportion of second male-phase individuals, are then
available for the fertilization of the eggs produced by such individuals as
are at that time functioning as females. Because of this overlapping of
sexual phases all the older colonies are at all times represented by both
sexual phases in varying proportions. Most of the smaller and conse-
quently younger individuals will be functioning as males, while most of
the larger, older individuals are in the female phase.
A rhythmical sequence of four sexual phases may be considered to
represent the normal life cycle but this is seldom realized because of an
earlier death due to parasites or to the exhaustion of the wood supply or
to other unfavorable environmental conditions. Most individuals die
after only two of these phases have been completed and many others
survive only the primary male phase.
There are some variations in this sequence, however, because a second
female phase may sometimes follow the first without an intervening male
phase. Other individuals, known as true males, retain the male phase
long after their contemporaries have changed to the female condition,
and this may sometimes mean throughout their entire lifetimes.
SEXUAL PHASES IN TEREDO DIEGENSIS
This species, like T. navalis, is protandric and larviparous, but the
two species differ considerably both in the conditions of sexuality and
in larval development.
T. diegcnsis Bartsch occurs abundantly and causes considerable dam-
age along the coast of Southern California, and has been reported as far
north as San Francisco (Kofoid and Miller, 1927). It is also found
at the Hawaiian Islands. On the coast of Southern California this spe-
170 WESLEY R. COE
cies breeds through all except the two or three cooler months of the year
and through the entire year when the winter is warmer than usual. At
the lower temperatures the larvae may remain within the maternal gill
chambers for several months before they are discharged.
Ovulation occurs at intervals of a few weeks, the later broods of
larvae often becoming established in the gills before an earlier brood has
left. This condition occurs throughout the year. With the exception of
young individuals and a few " true males " a sexually mature individual
without a brood of larvae is seldom found.
Large individuals, 120 mm. or more in length, may have more than
1000 larvae in the gill chambers, while dwarfs may have less than 100.
The bivalve larvae reach a shell length of 0.35 to 0.38 mm. before leaving
the gills. In this species, as in T. pcdicellata (Roch, 1940), the larvae
remain within the gill chambers until nearly ready for metamorphosis.
The free-swimming stage consequently lasts but a few hours if wood is
available for attachment. The total period of larval development is
about four weeks and is therefore of about the same length as in T.
navalis (Coe, 1933a). In this latter species, however, only about two
weeks of this time are spent in the maternal gill chambers, followed by a
free-swimming period of about the same duration.
Shortly after being set free in the water the larvae of T. dicgensis
attach themselves to any available piece of wood but do not immediately
penetrate the surface. Some of them may remain two weeks or more on
the surface of the wood before beginning to bore. Their stomach con-
tents show that minute particles of organic food materials are ingested
in the meantime. Because there is no necessity for feeding during the
brief free-swimming stage, this species may be reared from generation
to generation in the aquarium. By supplying a fresh piece of wood occa-
sionally the stock may be continued for at least several years. The
aquarium water evidently contains sufficient materials to supplement the
wood as sources of nourishment. After penetrating the wood the young
teredos grow rapidly and reach the primary male phase within four to
five weeks. The body is then about 8 to 12 mm. in length.
The primary gonad is composed of branching follicles filled with
large, vacuolated cells and having a few proliferating germinal cells scat-
tered along the walls of the follicles as shown for Bankia setacea (Fig.
I, A). This condition is closely similar to that described by Coe and
Turner (1938) for the developing gonads of Mya. As the germinal
cells increase in number they become differentiated into the two sexual
types of gonia and then further differentiated into ovocytes and
spermatocytes.
SEXUAL PHASES IN WOOD-BORING MOLLUSKS
171
By their rapid proliferation and differentiation the spermatogenic cells
encroach upon the spaces occupied by the vacuolated follicle cells and
eventually fill the entire lumen of the follicle (Fig. 1, B). In some indi-
viduals the follicle cells contain numerous fragmenting and degenerating
nuclei, representing a kind of atypical spermatogenesis, as described by
Coe and Turner (1938) for Mya.
spg
fc
fc
FIG. 1. Bankia sctacca. Development of the primary ambisexual gonad.
portion of section of young follicle, showing large vacuolated follicle cells
with a few primary gonia (/></) and a single young ovocyte (or) peripherally;
several follicle cells contain atypical, degenerate nuclei (a), derived from original
primary gonia. B, portion of follicle in early male phase, with a few remaining
vacuolated follicle cells (fc) and various stages of spermatogenesis; a, atypical de-
generate nuclei; oc, ovocyte; sp(j, spermatogonia ; spt, spermatids j sj>s, spermatozoa.
There is considerable variation in the number and size of the ovocytes
which are always present on the walls of the follicles during spermato-
genesis. Some individuals, corresponding with the so-called true males
of T. navalis, have only a few small ovocytes in each of the follicles (Fig.
3, A}, while others show a preponderance of ovocytes in some or all of
the follicles before the spermatozoa are fully ripe (Fig. 2).
Occasionally all the spermatozoa are discharged before ovulation
occurs, resulting in a distinctly female phase (Fig. 3, 5), but more
frequently a functionally hermaphroditic condition is found. Both
spermatozoa and ova may ripen at the same time and evidently both
172
WESLEY R. COE
may be discharged simultaneously. Under experimental conditions self-
fertilization occurs readily ; this is followed by the formation of the
polar bodies and cleavage, but only as far as the blastula and gastrula
stages. For the normal processes of larval development the environ-
mental conditions peculiar to the maternal gill chambers appear to be
necessary.
A
FIG. 2. Teredo diegensis. Sections of three follicles from gonad of second
male-phase individual which had branchial brood pouches distended with larvae.
A, immature follicle, principally in male phase, with relatively few ripe spermatozoa
and with only small ovocytes in basal layer of germinal cells. B, more nearly ma-
ture follicle in male phase, distended with spermatogenic cells and many ripe
spermatozoa; numerous half-grown ovocytes in basal layer of germinal cells. C,
ripe follicle in hermaphroditic male phase ; lumen filled with ripe spermatozoa and
with nearly mature ovocytes densely crowded on periphery.
During some seasons this species becomes particularly injurious by
boring in mooring ropes. A similar habit has been reported for T.
navalis (Coe, 1933). Under such conditions only dwarf individuals
are produced but many of these are nevertheless capable of forming a
small number of ripe gametes. A single change of sexual phase, from
male to female, may occur, although many individuals are killed by the
disintegration of the rope before even the primary male phase is
completed.
SEXUAL PHASES IN WOOD-BORING MOLLUSKS
SEXUAL PHASES OF BANKIA SETACEA
173
This species differs from Teredo diegensis, with which it is often
associated in the cooler waters on the coast of southern California, in
being oviparous rather than larviparous. On reaching the female phase
vast numbers of minute ova are produced and these are discharged di-
rectly into the water. Fertilization of these ova by sperm of other indi-
viduals takes place in the water. Then follows a free-swimming larval
period of perhaps four weeks before the larva is ready to settle on a
piece of wood and transform to the adult condition.
It is evident that all individuals pass through a functional male phase
a few weeks after entering the wood. The two types of males are more
FIG. 3. Teredo diegensis. A, portion of gonad of young individual in " true
male " phase, showing spermatogenesis and a single, more highly enlarged spermato-
zoon ; a single ovocyte is shown at the base of the spermatogenic cells. B, por-
tion of gonad in female phase, with ripe ova ; those ova still attached to wall of
follicle are surrounded by follicle cells with undifferentiated gonia basally.
easily distinguished than in either Teredo naval is or T. diegensis. About
half of the young, male-phase individuals are apparently hermaphroditic;
these complete spermatogenesis early and then change to the female
phase. These are evidently genetically protandric females, while an ap-
proximately equal number retain the primary male phase much longer.
The latter presumably represent the " true males " of other species (Coe,
1933fl, 1936). Fully adult individuals, measuring 20 to 50 cm. in length,
usually have the appearance of being either males or females, with little
indication of ambisexuality. Only occasionally do their gonads reveal
distinctly their essentially hermaphroditic nature. Careful examination
of the gonad in full spermatogenesis, however, usually reveals a few
ovocytes on the walls of the follicles and these may be considered as an
indication that a change of sexuality may later occur. In the female
phase likewise indifferent gonia show the potentiality of a sex change.
174 WESLEY R. COE
The sexual conditions in this species are therefore similar to those
of the oyster Ostrea virginica (Coe, 1938), which is seasonally of sepa-
rate sexes but in which the sexual phase of any season cannot be pre-
dicted from the sexual condition of the preceding season. In Bankia,
however, many of the females experience but a single change of sex.
from male to female, while the true males may retain the male phase
throughout life.
On the coast of southern California some individuals of B. sctacca
are found in spawning condition throughout the year. The majority of
individuals, however, spawn only during the autumn and spring months,
with resting periods in the winter and summer. Consequently wooden
blocks and timbers become much more quickly and more heavily infested
in the spring and autumn than at other times of the year.
Johnson and Miller (1935) found that settlement of this species in
Puget Sound occurred principally from October to December and less
abundantly from March to September. Kofoid and Miller (1927) also
observed that in San Francisco Bay settlement of this species was con-
fined to the cooler months of the year.
When removed from the body the eggs of Bankia develop rapidly to
the free-swimming larval condition after artificial fertilization by sperm
from another individual. The eggs of juvenile protandric females are
sometimes capable of self-fertilization.
CONCLUSIONS
It is evident that the sexuality of these three species of pelecypods
represents a graded series of ambisexual or hermaphroditic conditions
intermediate between such dioecious forms as Mya or Mytilus, which are
almost strictly of separate sexes, and those that are uniformly mo-
noecious, as the larviparous oysters, Ostrea cdulis or 0. lurida. In all
the dioecious pelecypods of which the sexuality has been extensively in-
vestigated hermaphroditism is found occasionally and this may include
a large or only a small portion of the gonad.
Even the monoecious species usually have some dioecious tendencies,
with some individuals ("true males") showing a preponderance of
masculine characteristics, while others are more nearly feminine. A se-
quence of functional male and female phases is of common occurrence
and in the case of long-lived species this may constitute an alternating
rhythm. The wood-boring mollusks are of this type.
Protandry is characteristic of many species. This represents a juve-
nile type of sexuality and often occurs when the individual is very young
and when the bodv has reached but a small fraction of its definitive size.
SEXUAL PHASES IN WOOD-BORING MOLLUSKS 175
As mentioned above for Bankia and as Loosanoff (1937) found in
Venus, the genetic females pass through a juvenile male phase before
adult sexuality is realized. This has been reported for other species. In
the oviparous oyster Ostrea inrginica the proportion of the genetic fe-
males which pass through a functional male phase during their first
breeding season depends both upon the particular local race concerned
and upon the environmental conditions. This juvenile male phase is
more frequently aborted or omitted under conditions favorable to rapid
growth, thereby increasing the proportion of juvenile females. This
species also shows a rhythmical tendency toward seasonal change of sex
in later life (Coe, 1938).
In all the examples mentioned and in many others belonging to the
various classes of mollusks, the sex-differentiating mechanism is so deli-
cately balanced between the two sexual tendencies that relatively slight
differences in environmental conditions may be potent in determining
which of the two contrasted aspects of sexuality shall be realized. In
some the entire population functions as male when young and as female
when fully adult. An intervening functionally hermaphroditic phase
may occur.
SUMMARY
1. The three species of wood-boring mollusks Teredo navalis, T.
diegensis and Bankia sctacea, are all protandric, with a strong tendency
toward rhythmical changes of functional male and female phases.
2. Each species differs as to the degree of ambisexuality characteristic
of the primary male phase and of the subsequent sexual phases.
3. The primary gonad in all three species develops from branching
follicles filled with large vacuolated follicle cells and having the primary
gonia scattered along walls of the follicles.
4. In each of these species the gonads of young animals indicate that
there are two types of primary male-phase individuals: (1) ambisexual
males or protandric females, characterized by many ovocytes on the
walls of the spermatic follicles, and (2) true males with few ovocytes.
In those of the former type the male phase is of short duration, while
true males retain the male phase longer or in some cases indefinitely.
5. In T. navalis the first female phase does not usually become func-
tional until nearly all the sperm of the primary male phase have been
discharged. Functional hermaphroditism is not usual, although the
gonad is histologically ambisexual during the change of sexual phase in
both directions.
6. In T. diegensis, on the other hand, functional hermaphroditism is
of usual occurrence and the sexual phases are not sharply demarcated.
176 WESLEY R. COE
7. In B. setacea functional hermaphroditism occurs only occasionally
in the primary male phase; the subsequent sexual phases are clearly dif-
ferentiated, often with a resting stage intervening between two sexual
phases. The sexual phases are of the alternative type in that any sexual
phase, after the first, may be followed by either a male or female phase
if the length of life suffices. The relatively short life of many individ-
uals, however, allows but a single change of sex, from male to female,
in the genetic females, and none at all in true males.
8. In all of the three species the eggs begin development after arti-
ficial fertilization. In Bankia the larvae may be reared to the free-
swimming veliger stage, but in the other two species the larval stages
require the peculiar environmental conditions of the maternal gill cham-
bers. Under experimental conditions self-fertilization and apparently
normal cleavage occurs readily in the two species of Teredo and occasion-
ally in Bankia.
LITERATURE CITED
COE, W. R., 1933. Destruction of mooring ropes by Teredo : growth and habits in
an unusual environment. Science, 77 : 447-449.
COE, W. R., 1933a. Sexual phases in Teredo. Biol. Bull., 65 : 283-303.
COE, W. R., 1936. Sequence of functional sexual phases in Teredo. Biol. Bull.,
71 : 122-132.
COE, W. R., 1938. Primary sexual phases in the oviparous oyster (Ostrea vir-
ginica). Biol. Bull, 74: 64-75.
COE, W. R., AND HARRY J. TURNER, JR., 1938. Development of the gonads and
gametes in the soft-shell clam (Mya arenaria). Jour. Morph., 62: 91-111.
GRAVE, B. H., 1928. Natural history of the shipworm, Teredo navalis, at Woods
Hole, Massachusetts. Biol. Bull., 55: 260-282.
GRAVE, B. H., AND JAY SMITH, 1936. Sex inversion in Teredo navalis and its
relation to sex ratios. Biol. Bull., 70 : 332-343.
JOHNSON, MARTIN W., AND ROBERT C. MILLER, 1935. The seasonal settlement of
shipworms, barnacles, and other wharf-pile organisms at Friday Harbor,
Washington. Ihiir. Wash. Publ. in Oceanography, 2: 1-18.
KOFOID, C. A., AND R. C. MILLER, 1927. Marine borers and their relation to the
marine construction on the Pacific Coast. Biological Section Final report
of the San Francisco Bay Marine Piling Committee, pp. 188-343. Pub.
by the Committee, San Francisco.
LOOSANOFF, VICTOR L., 1937. Development of the primary gonad and sexual
phases in Venus mercenaria Linnaeus. Biol. Bull., 72 : 389-405.
ROCH, FELIX, 1940. Die Terediniden des Mittelmeeres. Thalassia, 4 : 1-147.
SIEGERFOOS, C. P., 1908. Natural history, organization, and late development of
the Teredinidae. or shipworms. Bull. U. S. Bur. Fish., 27 : 191-231.
YONGE, C. M., 1926. Protandry in Teredo norvegica. Quart. Jour. Micr. Sci.,
70: 391-394.
REGENERATION OF COENOSARC FRAGMENTS REMOVED
FROM THE STEM OF TUBULARIA CROCEA
A. GOLDIN AND L. G. EARTH
(From the Department of Zoology, Columbia University, the Marine Biological
Laboratory, Woods Hole, Mass., and Queens College, New York)
The role of environmental factors in the regeneration of hydroids has
been studied extensively, the evidence all pointing to the extreme lability
of hydroid systems. The literature on this subject has recently been
reviewed by Earth (1940ft). Morgan (1903) found that, when the
cut end of a Tubularia stem was placed in sand, regeneration was in-
hibited at that end. Regeneration is inhibited also when the sectioned
ends of a Tubular ia stem are ligated. The reason for this inhibition was
made clear by experiments in which Tubularia stems were exposed to a
differential of oxygen in the sea water. Earth ( 1938a) was able to re-
verse the normal polarity by placing the distal end of the stem in a glass
capillary, and he attributed this reversal to a lack of oxygen at the
covered end. Miller (1937) reversed the polarity of Tubularia steins
by exposure of the proximal end to a higher oxygen tension. Further,
the rate of regeneration was shown to be dependent upon the oxygen
tension (Earth, 1938a). All of these experiments have been interpreted
as meaning that regeneration in Tubularia is dependent upon the avail-
ability of oxygen (Earth, 1940ft).
Experiments designed to determine the origin of polarity in regener-
ating Tubularia stems are complicated by the presence in these stems of
an already existing polarity. This polarity is evidenced by a gradient
in the rate of regeneration and a gradient of oxygen consumption in the
stems (Earth 1938ft, 1940o), and by the dominance of distal over prox-
imal levels (Earth, 1938ft). Direct exposure of the coenosarc to sea
water provides a sufficient stimulus for regeneration (Zwilling, 1939),
and since the process of regeneration involves reorganization of cells, it
was indicated that exposure of the entire coenosarc surface to sea water
might result in sufficient reorganization to obliterate the existing gradi-
ents in the stem. The coenosarc fragments could then be subjected to
carefully controlled environmental differentials in an attempt to deter-
mine the role of the environment in regeneration.
The experiments were therefore designed to ascertain : ( 1 ) the nature
of the structural changes which occur during the development of coeno-
177
178 A. GOLDIN AND L. G. EARTH
sarc fragments; (2) whether there is a gradient of oxygen consumption
in coenosarc taken from different levels of the stem; (3) the polarity
exhibited by coenosarc fragments during regeneration.
METHODS
During June and July the experiments were performed on Tubularia
crocea collected from the wharf piles at the Marine Biological Labora-
tory. From September through December colonies were collected from
floats in the Far Rockaway channel in New York City. Uniform, clean
stems 5 to 8 cm. in length were chosen for the experiments. Segments
10 mm. long were used, the hydranth plus the first 5 mm. being discarded.
The cuts were made with iridectomy scissors. Holding the perisarc at
one end of the stem segment with a jeweler's forceps, a needle was passed
gently over the perisarc, and the coenosarc expressed at the opposite end.
During the summer most satisfactory survival and regeneration were
obtained when the coenosarc fragments were kept in running sea water
which had been filtered through absorbent cotton. The coenosarc frag-
ments were placed on agar (2 per cent agar in sea water) in Syracuse
dishes, and the latter transferred to a large glass aquarium through which
the filtered sea water constantly flowed. The fragments were kept one-
half inch from the surface of the water by elevating the Syracuse dishes
in the aquarium. This was done to insure the availability of oxygen.
The coenosarc fragments were moved around in the dish every few hours
to insure uniform healing. Twenty-four hours after removal, they were
transferred to Syracuse dishes which contained no agar. The coenosarc
fragments removed from stems collected at Far Rockaway during the
fall and winter were very hardy, satisfactory viability and regeneration
being obtained using filtered sea water. Agar and continuous circulation
of the sea water were not necessary. The operations and observations
EXPLANATION OF PLATE I 1
FIGS. 1-3, and 8-11 (X 15) ; Figs. 4 and 6 (X 160) ; Figs. 5 and 7 (X 950).
1. Coenosarc fragment (above) and empty perisarc (below) immediately after
expression of the coenosarc.
2 and 3. Coenosarc fragment two hours (Fig. 2) and twenty-four hours (Fig.
3) after removal from the perisarc.
4 and 5. Section of coenosarc fragment immediately after removal.
6 and 7. Section of coenosarc fragment two hours after removal.
8-11. The types of regenerants obtained from expressed coenosarc fragments.
Unipolar (Fig. 8) ; bipolar (Fig. 9) ; bipolar-unipolar (Fig. 10) ; multipolar (Fig.
11).
1 The authors wish to thank Mr. Jack Godrich for his assistance in the prepara-
tion of the photomicrographs.
REGENERATION OF TUBULARIA COENOSARC
179
11
PLATE I
180 A. GOLDIN AND L. G. EARTH
were made with the aid of a binocular microscope. The coenosarc frag-
ments were fixed in Bouin's picro-formol-acetic fixative. They were
sectioned at five microns, and stained with Delafield's haematoxylin.
THE DEVELOPMENT OF COENOSARC FRAGMENTS
When coenosarc is removed from the perisarc, the tissues undergo a
series of structural changes resulting, finally, in regeneration of hy-
dranths. For convenience of description, the process may he divided
into six stages based on characteristic morphological relationships.
Stage 1. The coenosarc has just been removed from the perisarc.
There has been some morphological disturbance due to the mechanics of
the operation. Plate I, Fig. 1, shows the condition of the coenosarc,
above, and the empty perisarc, below. When examined histologically
(PI. I, 4), it may be noted that the ectodermal and endodermal layers are
well defined, although the coelenteron has been obliterated. The nuclei
of the ectodermal cells are centrally located and there is no trace of peri-
sarc present (PI. I, 5).
Stage 2. Two hours after removal the fragment has begun to con-
tract along the original distal-proximal axis (PI. I, 2), and the interior
has become somewhat vacuolated (PI. I, 6). The ectoderm is still well
defined, but the nuclei of the ectodermal cells are located peripherally and
the cells are somewhat elongated and swollen (PI. I, 7). No perisarc is
present. The atypical appearance of the ectodermal cells may be an indi-
cation of cellular degeneration, which is followed by a sloughing off of
the original ectodermal cells. This loss of cells from the coenosarc may
be observed, with the aid of a binocular microscope, from the time the
perisarc is removed until a new perisarc is formed.
Stage 3. Twenty-four hours after removal, the coenosarc has under-
gone further contraction and is now somewhat spherical (PI. I, 3). The
outer layer is not well defined, and the inside of the spherical mass con-
sists of numerous, closely packed cells (PI. II, 12, 13). Traces of new
perisarc may be noted around the periphery of the tissue mass (PL II,
13).
Stage 4. Thirty-six hours after removal, the center of the tissue
EXPLANATION OF PLATE II
FIGS. 12, 14, and 16 (X 160) ; Figs. 13, 15, and 17 (X 950).
12 and 13. Section of coenosarc twenty-four hours after removal from the
perisarc.
14 and 15. Section of coenosarc thirty-six hours after expression.
16 and 17. Section of coenosarc sixty hours after expression.
REGENERATION OF TUBULARIA COENOSARC
181
•
12
14
/
„ w
*\ vr i.
*•- t-M*
v> A' - ^ •
*^-» ^
• %'/'
Ji':*
15
16
..J«
17
PLATE II
182 A. GOLDIN AND L. G. EARTH
mass is less solidly packed with cells, and ectodermal cells have started
to become arranged around the periphery (PI. II, 14, 15).
Stage 5. Sixty hours after removal, the center of the mass of tissue
is hollow. Well-defined ectodermal and endodermal layers have been
formed around the periphery (PI. II, 16, 17). The ectodermal cells are
smaller than the ones in Stage 2 (PI. I, 7).
Stage 6. Seventy-two to ninety-six hours after removal of the
coenosarc fragments from the perisarc, regeneration occurs. This stage
is characterized by the formation and emergence of new hydranths (PI.
I, 8-11). Hydranth formation is preceded by an aggregation of cells
and later of pigment at the point of regeneration. Some of the frag-
ments, although health)- in appearance, do not regenerate. They develop
as far as Stage 5 (PI. II, 16), at which time a heavy perisarc is secreted
and development stops.
The changes which occur after removal of the coenosarc from the
perisarc result in a general morphological dedifrerentiation, namely, the
formation of a spherical mass of coenosarc tissue in which the original
ectoderm and encloclerm are no longer clearly defined. Cellular dedif-
ferentiation was not observed, no evidence being found that the cells
return to an embryonic condition. The morphological dedifferentiation
is followed by a redifferentiation, involving the reorganization of ecto-
derm and endoderm, formation of new perisarc, and subsequent regen-
eration. It is of interest to note that the time required for regeneration
of the fragments is longer (72-96 hours) than the time required for in-
tact stem segments (approximately 36 hours) at the same temperature.
The additional time required for regeneration of the fragments is under-
standable when the time required for the initial dedifferentiation and
early reorganization is taken into account.
THE RATE OF OXYGEN CONSUMPTION OF COENOSARC REMOVED FROM
DIFFERENT LEVELS OF THE STEM
In these experiments young unbranched stems were selected. The
segments of stem used were 10 mm. long. The distal segments were
taken from the region extending from 5 to 15 mm. proximal to the hy-
dranth, and the proximal segments 20 to 30 mm. proximal to the hy-
dranth. The coenosarc fragments were removed from these stem seg-
ments and kept in running filtered sea water until ready to be placed in
the Warburg manometers. The fragments were placed in the manom-
eters from 17 to 24 hours after removal from the perisarc. At this
time the fragments have reached their greatest morphological dediffer-
entiation (Stage 3, PI. II, 12). They have become spherical and are
REGENERATION OF TUBULARIA COENOSARC
183
protected from the mechanical shaking of the Warburg manometers by
a thin, newly secreted perisarc. The rate of oxygen consumption was
calculated on the basis of mm.3O2 used per hour per ten mg. of dry
weight. The results are summarized in Table I. In most of the ex-
TABLE I
Oxygen consumption of distal and proximal coenosarc fragments removed from
the perisarc. Rate = mm.3 O»/hr./10 mg. dry weight. The temperature of the sea
water during the experiments was 18.5±.02°C.
Description of coenosarc fragments
Oxygen consumption
Exp.
No.
Region
0-
Time
Dry
weight
Rate
mm.3
hours
mg.
RE 1
19
distal
48.5
10.75
0.494
91.0
19
proximal
37.2
10.75
0.391
88.5
RE 2
15
distal
19.8
9
0.46
48.0
15
proximal
23.4
9
0.69
37.6
RE 3
4
distal
6.6
22
0.224
13.4
14
proximal
41.2
22
1.294
14.4
RE 4
18
distal
10.5
9
0.720
16.2
17
proximal
10.8
9
0.735
16.3
RE 5
14
distal
13.1
8
0.378
43.5
15
proximal
15.2
8
0.482
39.5
RE 6
19
distal
24.3
7
0.729
47.7
19
proximal
19.6
7
0.694
40.5
RE 7
15
distal
18.2
11.5
0.764
20.7
15
proximal
17.0
11.5
0.721
20.4
periments the distal rates are approximately the same as the proximal
rates. The averages of the distal and proximal rates for the seven ex-
periments are 40.1 and 36.7 respectively. The distal and proximal re-
gions of stem segments with perisarc, however, show a distal-proximal
gradient in rate of oxygen consumption (Earth, 1940o). This differ-
ence in rate is present after the stems are cut, and persists from 24
through 36 hours after cutting. The distal and proximal coenosarc frag-
ments, however, show only slight difference in rate 24 hours after re-
moval from the perisarc. This means that the coenosarc fragments must
have lost the differential during the first 24 hours. Thus, the reorganiza-
tional changes in the coenosarc fragments involve a dedifferentiation of
the physiological gradient present in the intact stem.
184 A. GOLDIN AND L. G. EARTH
Apparently then, exposure of the coenosarc to sea water has an effect
on the rate of regeneration. In order to clarify the nature of this effect,
the rates of distal coenosarc fragments and distal stem segments may be
compared. The average rate of oxygen consumption of distal coenosarc
fragments is 40.1 as compared with 21.8 for distal stem segments, the
latter average being calculated from data presented by Earth (1940a).
If the dry weight of the stem perisarc represents even as high as 50 per
cent of the dry weight of the stem, the rate of distal coenosarc fragments
would still be as high as the distal rate of the stems. Thus, the distal
coenosarc fragments consume oxygen at a rate at least as high as the
distal stems. Since, in addition, distal and proximal coenosarc frag-
ments respire at about the same rate, the effect of exposure of the coeno-
sarc to sea water and the resultant dedifferentiation is to increase the rate
of oxygen consumption of coenosarc from proximal levels of the stem
up to the higher rate of the distal levels.
POLARITY OF THE REGENERATED COENOSARC FRAGMENTS
The polarity relationships exhibited by the regenerated coenosarc
fragments are worthy of examination for comparison with stems, where
only unipolar and bipolar forms regenerate after cutting. Uniform ex-
posure of the coenosarc to sea water removes the possible complication
of an environmental differential created by the presence of the perisarc.
The regenerated fragments may be classified using the general termi-
nology employed by Child (1927) for Corymorpha, the groupings being
based upon the axial pattern developed by the fragments. The regen-
erated coenosarc fragments may be classified as unipolar, bipolar, bipolar-
unipolar, multipolar, and apolar. The unipolar forms have regenerated
a single hydranth on the rounded mass of tissue (PI. I, 8). Bipolar
regenerated fragments have formed hydranths at two opposite poles of
the coenosarc mass (PL I, 9). The bipolar-unipolar forms have regen-
erated two hydranths from the same region of the coenosarc (PI. I, 10).
These may be two independent hydranths or two partially fused hy-
dranths. The category " multipolar " is used to designate fragments
which have regenerated more than two hydranths. This group includes
regenerated fragments ranging from tripolar forms, to forms in which
the entire surface of the coenosarc has become covered with tentacles
(PI. I, 11). In each of these categories are included forms in which
the regenerated hydranths are not complete. Thus, a hydranth may be
lacking in the number of oral or basal tentacles or lacking in any of the
structures necessary for a complete hydranth. For the purpose of this
analysis, however, it was not deemed necessary to subdivide the various
REGENERATION OF TUBULARIA COENOSARC
185
categories with respect to these irregularities. Many of the hydranths
which form do not emerge from the new perisarc. These may be readily
classified in the above groups, so that no distinction is made between
emerged and non-emerged hydranths. The apolar forms are those frag-
ments which fail to regenerate. They develop as far as Stage 5 (PL II,
16), form a thick perisarc, and remain in that condition.
The classification of the regenerated coenosarc fragments is arranged
in Table II. The unipolar and multipolar forms make up the greatest
percentage of the regenerants (78.6 per cent). The bipolar and bipolar-
unipolar forms comprise a much smaller percentage of the total (21.4
TABLE II
Classification of the regenerated coenosarc fragments obtained after the removal
of the perisarc. The observations were made at 96 hours. Percentage regeneration
equals the number of a particular kind of regenerated coenosarc fragment divided by
the total number of fragments which formed hydranths. The temperature of the
sea water was 19±2°C.
Description of the regenerated coenosarc
fragments
No regeneration
Unipolar
Bipolar
Bipolar-
unipolar
Multi-
polar
Apolar
Dead
Number
73
26
9
56
50
166
Percentage Regen-
eration
44.5
15.9
5.5
34.1
per cent). Since exposure of the naked coenosarc to sea water is a suf-
ficient stimulus for hydranth formation (Zwilling, 1939), the formation
of hydranths should be enhanced when the entire coenosarc is naked, and
therefore in more direct contact with sea water and oxygen dissolved in
the sea water. That this may be true is demonstrated by the relatively
high percentage of multipolar regenerants (34.1 per cent) obtained after
removal of perisarc. These multipolar forms are never obtained when
the perisarc is left intact. The appearance of a high percentage of uni-
polars (44.5 per cent) suggests that in these cases the mass of tissue may
have been exposed to a uniform gradient of oxygen in the sea water, for
many of them became attached to the bottom of the dish at an early stage
and the hydranths always formed away from the attached end. This
interpretation is supported by Child's experiments with Corymorpha
(1928) in which hydranths were regenerated at the upper surface of un-
disturbed cell aggregates. Coenosarc fragments develop in the same
way, irrespective of the level of the stem from which they are removed.
186
A. GOLDIN AND L. G. EARTH
Data for the regeneration of coenosarc fragments removed from three
different levels of the stem are summarized in Table III. The coenosarc
fragments of all three levels of the stem give rise to regenerants having
similar types of polarity relationships. In addition, it may be noted that
96 hours after removal of the coenosarc from the perisarc, approximately
TABLE III
Classification of the regenerated coenosarc fragments removed from different
levels of the stem. The observations were made at 96 hours after removal of the
fragments from the perisarc.
Description of the regenerated coenosarc fragments
No regeneration
Unipolar
Bipolar
Bipolar-
unipolar
Multi- '
polar
Total
Apolar
Dead
Distal
10
4
2
12
28
14
18
Middle
12
3
1
13
29
19
12
Proximal
15
6
2
12
35
19
6
the same number of regenerants appear at all three levels. Thus, the
gradient in the rate of regeneration present in Tiibnlaria stems (Barth,
19385, 1940o) apparently disappears when the coenosarc fragments are
removed from the perisarc.
DISCUSSION
One of the chief difficulties in any attempt to analyze the role of
the environment on regeneration is the inability to work with homo-
geneous systems. That hydroids have a gradient of metabolic activity
has been well established. This gradient is developed, apparently as the
result of an environmental differential, early in the development of the
organism. Thus, Child (1925) has shown that a metabolic gradient is
probably established as a result of the nature of the orientation of the
egg during its growth. Further, once a gradient has been established, it
may maintain itself in a uniform environment, and the gradient has
therefore become a function of localized differences within the tissues
themselves. Barth (19385) demonstrated a gradient in the rate of re-
generation along the length of the stem of Tubularia, distal segments re-
generating at a higher rate than proximal segments. There is likewise
a gradient of oxygen consumption of the parts of the stem (Barth,
1940a). Barth (19385) also suggested that the dominance of distal
over proximal levels of Tubularia stems might be interpreted as a compe-
REGENERATION OF TUBULARIA COENOSARC 187
tition for substance " S," the success of which is dependent upon the
activity of enzyme " E." Thus, an organism living in a uniform ex-
ternal environment may nevertheless maintain its own heterogeneity once
this heterogeneity has become established.
In order to determine more accurately the role of environmental fac-
tors on the origin of new gradients in regeneration, it is of importance
to obliterate first any existing gradients in the animal tissues themselves.
Some attempts in this direction have been made. Child (1928) found
that cells of Coryinorplia, when dissociated mechanically, will aggregate
and establish new polarity relationships. Coryuwrpha stems, when sub-
jected to toxic agents, may lose their established polarity relationships
and form new gradients of regeneration (Child, 1927), the new gradients
being produced by a differential exposure to the environment.
The experiments with expressed Tubularia coenosarc indicate an ob-
literation of the original polarity after the coenosarc is removed from the
perisarc. This is borne out by the reorganizational changes which the
coenosarc undergoes after removal. There is, at first, a morphological
dedifferentiation, in which the mass becomes spherical and the ectoderm
and endoderm are no longer clearly defined. Cellular dedifferentiation,
however, was not observed. The morphological dedifferentiation is fol-
lowed by a redifferentiation. The ectoderm and endoderm are re-
organized and a new perisarc is formed. Subsequently, regeneration
occurs. If the polarity of an organism is dependent upon a gradient of
activity of some enzyme, as suggested by Barth (1938ft). then it is quite
likely that the activity of this enzyme is radically changed during the
process of reorganization. That the initial polarity is lost, is evidenced
also by the appearance of regenerated hydranths at the free surface of
fragments which have become attached to the bottom of the dish, the
regenerated hydranths having no necessary relation to the original po-
larity. It is further substantiated by the appearance of a high percentage
of multipolar forms in which appear numerous and apparently unrelated
polarity relationships. The appearance of these multipolar forms must
mean that the original polarity is no longer extant ; the exposure of the
coenosarc to the sea water being sufficient to stimulate regeneration at
many points on the uniform mass of tissue.
Correlated with the disappearance of the original polarity is the dis-
appearance of the difference in oxygen consumption of tissues removed
from distal and proximal levels of the stem. The sharp gradient of
oxygen consumption found along the length of the stem (Barth, 1940o)
is not exhibited by the excised fragments at the time when morphological
dedifferentiation has reached its peak (17-24 hours after removal of the
tissue). At this time the rates of oxygen consumption of dedifferenti-
188 A. GOLDIN AND L. G. EARTH
ated coenosarc fragments from both distal and proximal levels of the
stem are at least as high as the rate for distal stem segments. The loss
of the gradient of oxygen consumption is due, therefore, to a general
increase in rate. This increase in rate is probably stimulated by a high
availability of oxygen to the coenosarc fragments, made possible by their
removal from the perisarc. Thus, the removal of the coenosarc from
the perisarc results in a reorganization involving not only morphological
dedifferentiation but also a dedifferentiation of the physiological gradient.
The end product of this process is a more homogeneous mass of Tubu-
laria cells. The localized differences in the ability to regenerate, found
in stems covered with perisarc, also disappear as a result of the morpho-
logical and physiological dedifferentiation. Coenosarc fragments re-
moved from distal and proximal levels of the stem regenerate at the same
rate and develop similar types of polarity relationships.
Expressed coenosarc, therefore, if used at the time when dedifferenti-
ation is greatest (approximately twenty-four hours after removal from
the perisarc), offers good biological material for studies of the environ-
mental factors stimulating regeneration and for an investigation of the
origin of polarity in regeneration.
SUMMARY
The morphogenesis of coenosarc expressed from the perisarc of
Tubularia stems is described. A series of structural changes occurs in
the coenosarc, there being first a dedifferentiation of histological struc-
ture, followed by a redifferentiation culminating in the regeneration of
new hydranths.
The gradient of oxygen consumption present in the stem of Tubularia
disappears when the coenosarc is removed from the perisarc. This
physiological dedifferentiation represents an increase as well as an equali-
zation of oxygen consumption by coenosarc fragments from distal and
proximal levels of the stem.
Concomitant with the morphological and physiological dedifferentia-
tion, differences in the ability of distal and proximal levels of the stem to
regenerate disappear. Distal and proximal coenosarc fragments regen-
erate at the same rate and develop similar types of polarity relationships.
The different kinds of regenerants obtained are described and classi-
fied on the basis of their polarity relationships. Evidences were given
that these polarity relationships are new, and have no relation to the
original polarity in the intact stem.
The value of using expressed coenosarc to study the effect of the
environment on regeneration and on the origin of polarity in regeneration
is discussed.
REGENERATION OF TUBULARIA COENOSARC 189
LITERATURE CITED
EARTH, L. G., 1938a. Oxygen as a controlling factor in the regeneration of Tubu-
laria. Physiol. Zool., 11 : 179-186.
EARTH, L. G., 1938/>. Quantitative studies of the factors governing the rate of
regeneration in Tubularia. Biol. Bull., 74: 155-177.
EARTH, L. G., 1940«. The relation between oxygen consumption and rate of re-
generation. Biol, Bull, 78: 366-374.
EARTH, L. G., 19406. The process of regeneration in hydroids. Biol. Rev., 15:
405-420.
CHILD, C. M., 1925. The axial gradients in Hydrozoa. VI. Embryonic develop-
ment of hydroids. Biol. Bull., 48 : 19-36.
CHILD, C. M., 1927. Modification of polarity and symmetry in Corymorpha palma
by means of inhibiting conditions and differential exposure. Jour. E.vpcr.
Zool., 47 : 343-383.
CHILD, C. M., 1928. Axial development in aggregates of dissociated cells from
Corymorpha palma. Physiol. Zool., 1 : 419-461.
MILLER, J. A., 1937. Some effects of oxygen on polarity in Tubularia crocea (ab-
stract). Biol. Bull., 73: 369.
MORGAN, T. H., 1903. Some factors in the regeneration of Tubularia. Arch,
f. Entw.-mech.. 16: 125-154.
ZWILLING, E., 1939. The effect of the removal of perisarc on regeneration in Tubu-
laria crocea. Biol. Bull, 76 : 90-103.
THE ROLE OF FERTILIZIN IN THE FERTILIZATION OF
EGGS OF THE SEA-URCHIN AND OTHER ANIMALS
ALBERT TYLER
(From the William G. Kcrckhoff Laboratories of the Biological Sciences,
California Institute of Technology)
INTRODUCTION
The striking phenomenon of the specific agglutination of spermatozoa
by a substance obtained from the eggs has been described in a number
of species of marine animals (see Lillie, 1919; Just, 1930; Tyler, 1940a).
Lillie considered this substance, which he called fertilizin, to play a cen-
tral role in the fertilization process, and developed a theory of the mecha-
nism of fertilization based on the ability of fertilizin to combine with
the spermatozoon and with some substance within the egg. One of the
principal arguments for his views was the evidence that eggs of the sea-
urchin which had been deprived of fertilizin lost their capacity for ferti-
lization. In his first experiments (1914) the fertilizin was removed by
prolonged washing of the eggs (Arbacia), combined in some cases with
shaking to remove the jelly layer which he had shown (1913) to be
heavily charged with fertilizin. Loeb (1914, 1915) raised the objection
that the decrease in fertilizability was due to the aging and death of the
eggs during the washing period of one to three days. He showed, on
the other hand, that fresh eggs of Strongylocentrotus purpuratus, that
had been deprived of their jelly layer (which he considered identical with
fertilizin) by means of acidified sea water, would still give 100 per cent
fertilization. Lillie (1915) repeated the acid sea water experiments with
Arbacia and found the capacity for fertilization (per cent fertilized) to
be much reduced as a result of the treatment. He also noted that some
fertilizin could be obtained from the acid-treated eggs although the jelly
layer appeared to be completely gone. Later (1921, footnote p. 16),
with Strongylocentrotus, he found that acid-treated jellyless eggs could
still be fertilized although there could not be obtained from these eggs
sufficient fertilizin to agglutinate the spermatozoa. He interpreted that
result to mean that " an amount of fertilizin insufficient for sperm agglu-
tination is yet adequate for fertilization."
The present experiments resolve these differences as apparently being
due to differences in amount of sperm employed for insemination. The
190
FERTILIZIN AND FERTILIZATION 191
results show that jellyless, fertilizinless eggs can be fertilized but that
they must be inseminated with much higher concentrations of sperm
than are necessary for untreated eggs. The present, as well as some of
the earlier work of the author (1939, 1940), also lends support to Lillie's
view that fertilizin is concerned in the fertilization process, and some
suggestions are made here as to its role. It is further shown that the
sperm agglutinating property of fertilizin can be destroyed without alter-
ing its ability to combine with the sperm. An interpretation is offered
of the temporary nature of the agglutination reaction in the sea-urchin
and its more permanent nature in forms like the keyhole limpet. Evi-
dence is also presented that fertilizin is not merely confined to those spe-
cies of animals whose egg water causes iso-agglutination of sperm, but
is more generally distributed and may very likely be universal.
IDENTITY OF FERTILIZIN WITH THE GELATINOUS COAT OF THE EGG
It has been shown (Tyler, 1940a) in experiments with the sea-urchin
Strongylocentrotus pur pur at us and the keyhole limpet Megathura crenu-
lata that the sperm agglutinin (fertilizin) is located in the jelly layer
surrounding the egg. On the rather reasonable assumption that the ma-
terial of the jelly is a single substance, this means that fertilizin is identi-
cal with the jelly. In any event, the evidence showed that fertilizin is a
component of the jelly layer and is not secreted by the ripe eggs. Re-
moval of the jelly layer was readily accomplished by means of sea water
acidified to between pH 4.5 and 3.5 and also by means of a 1 per cent
solution of chymotrypsin in sea water. No fertilizin could be obtained
from such jellyless eggs even after prolonged standing. When ripe eggs
are allowed to stand in sea water, the jelly slowly goes into solution and
the concentration of fertilizin in the solution increases. But this, it was
shown, does not increase the total amount of fertilizin that can be ob-
tained from the eggs. In other words, extraction of freshly shed eggs
with acid sea water gives just as much fertilizin as that in the acid extract
of eggs that had stood for some time in sea water plus that in the super-
natant sea water.
Hartmann, Schartau and Wallenfels (1940) support the view that
fertilizin is identical with at least a part of the material of the jelly layer.
They found in Arbacia pustulosa that fertilizin is given off in repeated
changes of sea water as long as remains of the jelly layer are present on
the eggs. They also removed the jelly layer by means of a sperm extract
containing an antifertilizin (see Frank, 1939; Tyler, 1939a, 1940b ;
Tyler and O'Melveny, 1941) and obtained no fertilizin from the treated
eggs. Further evidence for this view is given by their finding that Ar-
192 ALBERT TYLER
bacia sperm agglutinate on the surface of the jelly layer of Astropecten
eggs but fail to do so if the eggs are first treated with the sperm extract
which forms a precipitation membrane on the surface of the jelly.
Additional evidence along this line is contained in some experiments
by Evans (1940). He found that Roentgen radiation caused an imme-
diate disappearance of the jelly from around the Arbacia egg. Using
Janus green as a test for the presence of jelly in egg water, he found
that after an irradiation of 59,000 r or more it could not be demonstrated
in the egg water. He also noted that the agglutinating power of the
egg water is greatly reduced after irradiation, and this agrees with Rich-
ards and Woodward's (1915) earlier results.
FERTILIZATION AFTER REMOVAL OF FERTILIZIN
The primary question concerning the role of fertilizin is whether or
not its complete removal in a non-injurious manner renders the eggs
non-fertilizable. This question was examined in some experiments with
eggs and sperm of the west coast sea-urchin Strongylocentrotus purpu-
ratus. Since, as the evidence shows, fertilizin is identical with, or at
least a component of the gelatinous coat of the egg, its removal involves
the dissolution of this coat. In Strongylocentrotus, the jelly is rapidly
dissolved by placing the eggs in sea water acidified to between pH 3.5
and 4.5 (Tyler and Fox, 1940). If the eggs are not allowed to remain
too long in the acid sea water, there is no visible sign of injury.
Although the jelly is colorless and transparent, its absence is readily
noted by the fact that the eggs can then be brought into contact with
one another by their surfaces (Tyler, 1940b, Fig. 1, d). When eggs of
Strongylocentrotus are deprived of their jelly coat and washed, no de-
tectable (by agglutination of sperm) amount of fertilizin can be ob-
tained either by allowing them to remain for prolonged periods in sea
water or by macerating and extracting them with various solvents (Tyler
and Fox, 1940).
Upon insemination the jellyless eggs are capable of fertilization to
the extent of 100 per cent, as Loeb (1914, 1915) and Lillie (1921) had
reported for eggs of 6". purpuratus. One typical experiment may be
cited. Two 20 cc. samples of a 0.1 per cent suspension of fresh 5\ pur-
puratus eggs in sea water were taken and one of them acidified to pH 4.0.
After 5 minutes both dishes of eggs were given a set of four washings
with a total of 100 cc. of sea water, allowing the eggs to settle and 1 cc.
of suspension to remain in the dishes between washings. The acid-
treated eggs were observed to be deprived of their jelly. The addition
of 0.05 cc. of a 1 per cent fresh sperm suspension gave 100 per cent
FERTILIZIN AND FERTILIZATION 193
membrane elevation and cleavage in both the acid-treated and control
eggs. Similar results were also obtained when the jelly was removed
with chymotrypsin.
It may be concluded from this evidence that fertilizin is not essential
for fertilization. However, such a conclusion is only valid if the ferti-
lizin has in fact been completely removed from the treated eggs. That
this may not be the case is indicated by other evidence and considerations
presented below. But, even if it be assumed for the present that ferti-
lizin is not essential for fertilization, the question may still be raised as
to whether or not it is an aid to fertilization.
FERTILIZIN AS AN AID TO FERTILIZATION
It is well known that the number of spermatozoa required for fertili-
zation is in general much greater than the number of eggs present in
the suspension, and as the number of spermatozoa employed for in-
semination is decreased, the percentage fertilization decreases. The fac-
tors responsible for this fact, that many more than one spermatozoon per
egg must in general be present in the suspension in order for fertilization
to succeed, have been examined by several investigators (Glaser, 1915;
Lillie, 1915; Cohn, 1918; Morgan, 1927, p. 27 et seq.), and will not be
discussed in any detail here. The present question is whether or not
more spermatozoa are required for fertilization when fertilizin is re-
moved from the eggs. This question was investigated with eggs and
sperm of Strongyloccntrotus purpuratus and the results do, in fact, show
a decrease in " fertilizability " (increase in amount of sperm required
for fertilization) upon removal of the jelly.
Table I lists the results of nine experiments in which the jelly was
removed by means of acidified sea water or chymotrypsin. In all cases
the eggs were washed after treatment and the pH brought back to that
of normal sea water. In the table, cleavage rather than membrane eleva-
tion is taken as an index of fertilization inasmuch as the treated eggs
often fail to form or to elevate fertilization membranes but may never-
theless cleave (see Tyler and Scheer, 1937). The amounts of sperm
added are for convenience all given on the basis of a 0.01 per cent sperm
suspension although actually the larger amounts of sperm were taken
from more concentrated suspensions. In the different experiments there
are, as the table shows, considerable variations in the amount of sperm
required to give the same percentage fertilization of the control eggs.
This may be due to variations in the condition of the sperm and eggs, in
aging of the sperm at various dilutions, in temperature, etc. For the
point in question, however, it suffices to compare simply the jellyless with
194
ALBERT TYLER
the control eggs in each horizontal line. The results show that with the
larger amounts of sperm the jellyless eggs give practically the same per-
centage fertilization as the controls. But with smaller amounts there are
considerable differences. Thus, with small amounts of sperm that give
between 75 and 100 per cent fertilization in the control eggs, only 0 to
TABLE I
Fertilization of jellyless and normal eggs of S. purpiiratus inseminated with
various amounts of sperm. The egg suspensions contain 200 to 400 eggs in 5 cc. of
sea water.
Experiment
Treatment for removal of jelly coat
Amount of
0.01 per cent
sperm sus-
pension added
Percentage cleavage
Jellyless eggs
Control eggs
1
30 min. in pH 4.5 sea water
cc.
0.1
0.5
2.0
5
35
99
95
98
99
2
30 min. in pH 4.0 sea water
0.05
0.5
5.0
0
15
95
20
90
95
3
5 min. in pH 3.5 sea water
0.2
1.0
5.0
0
55
100
25
95
100
4
10 min. in pH 3.7 sea water
0.2
10
85
5
10 min. in pH 3.9 sea water
0.1
1.0
20
100
100
100
6
30 min. in 1 per cent chymo-
trypsin in pH 8 sea water
0.05
1.0
0.2
45
90
95
7
30 min. in 1 per cent chymo-
trypsin in pH 8 sea water
0.1
2.5
0.1
100
75
100
8
10 min. in 1 per cent chymo-
trypsin in pH 6 sea water
0.25
2.5
15
100
100
100
9
10 min. in 1 per cent chymo-
trypsin in pH 6 sea water
0.5
5.0
45
95
95
95
20 per cent is obtained in the jellyless samples. To get the same per-
centage fertilization as in the controls, the amount of sperm required
for the treated eggs is roughly five to ten times greater. While the
variations in the results do not permit an exact figure to be given for
this ratio, it is clear that the differences are all in the same direction
in each experiment and are well outside the limits of error. It should
FERTILIZIN AND FERTILIZATION 195
also be noted here that, since sufficient sperm gives as much fertilization
in the treated eggs as in the controls, there is no particular injurious
action of the treatment involved.
FERTILIZIN AS A BARRIER TO FERTILIZATION
It appears then that the presence of fertilizin on the eggs is an aid
to fertilization in that smaller amounts of sperm are required than in its
absence. It might be supposed, then, that restoration of the fertilizin
would eliminate the difference and that addition of fertilizin to normal
eggs would lower the amount of sperm required for fertilization. Un-
fortunately, no way is as yet known by which the fertilizin can be re-
stored in its normal state ; that is, in the form of a gelatinous coat around
the egg. When the jelly is dissolved in acidified sea water it does not
go back on to the eggs upon neutralization of the suspension but remains
in solution. One might, however, enquire whether or not the presence
of fertilizin in solution in the egg suspension increases the fertilizing
power of the sperm. This was examined with both jellyless and normal
eggs, and it was found that, instead of increasing the fertilizing power
of the sperm, the presence of fertilizin in solution had the opposite effect.
In one experiment the fertilizin was restored in its original amount (but
in solution) and in roughly ten times that amount to suspensions of naked
eggs. Various amounts of sperm were used for insemination. The
lowest quantity of sperm that gave 100 per cent fertilization in the jelly-
less controls gave only 15 per cent in the sample with original fertilizin
content and 0 per cent in that with the ten-fold concentration. In an
experiment with normal eggs the smallest amount of sperm that gave 100
per cent fertilization, gave about 35 per cent when an amount of fertilizin
roughly equivalent to the content of the eggs was present in solution and
0 per cent when ten times that amount was present.
The presence of fertilizin in solution evidently acts as a barrier rather
than an aid to fertilization. This action, it appears, is due to increase
in amount of agglutination of sperm that occurs with increase in amount
of fertilizin present in the solution. It is not merely the temporary
locking up of the sperm in the agglutinates that causes the decrease in
fertilizing power, but, as the next section shows, it involves a permanent
effect of the fertilizin on the sperm.
Loss OF FERTILIZING POWER AS A RESULT OF AGGLUTINATION
F. R. Lillie (1913) showed that the agglutination of sea-urchin sperm
by egg water (fertilizin) is temporary. On testing the sperm after
reversal of agglutination, he found them to have about half the fertiliz-
196 ALBERT TYLER
ing power (fertilized half the percentage of eggs) of the control sperm
suspension. He also noted (1919) that after reversal of agglutination
the sperm cannot be re-agglutinated. I have confirmed these findings
with S. purpuratiis and have obtained a much greater reduction in fer-
tilizing power of the sperm.
In twelve experiments that were run, the sperm was agglutinated with
sufficiently strong egg water, so that further addition of egg water, after
reversal, gave no visible agglutination. The agglutination usually lasted
30 to 40 minutes. Insemination with amounts of sperm that were well
above the control minimum for 100 per cent fertilization gave in all tests
with the agglutinated and reversed sperm between 0 and 3 per cent fer-
tilization. To obtain the same percentage fertilization with the control
as with the treated sperm was found to require between a forty- and a
two-hundred-fold reduction in the amount of control sperm used for
insemination. The possibility was also examined that the reversed sperm
might be more capable of fertilizing jellyless eggs, but the results were
negative.
Along with this reduction in fertilizing power of the sperm there is
no visible sign of any injurious effect after reversal of agglutination, nor
is there any reduction in the activity of the sperm. In fact, the egg
water, as is well known, increases the activity of the sperm and as meas-
urements of respiratory rate showed (Tyler, 1939fr) the increase persists
long after the reversal of agglutination. The experiments show, then,
that sperm which have been agglutinated are, after spontaneous reversal,
incapable of fertilization. The small percentages of fertilization that
result when large amounts of treated sperm are used are evidently due
to the fact that some spermatozoa in the treated suspensions may escape
being agglutinated.
It may be concluded, then, that some change is produced in the sper-
matozoa, as a result of their reaction with fertilizin, which, although es-
sentially non-injurious, renders them incapable of fertilizing normal eggs.
This change might occur during the initial reaction or upon the spon-
taneous reversal of the agglutination.
THE SPONTANEOUS REVERSAL OF SPERM-AGGLUTINATION
IN SEA-URCHINS
The temporary nature of the agglutination reaction exhibited by sea-
urchin sperm in egg water is an exceptional affair. In the usual sero-
logical reactions, the agglutination of various types of cells (blood cells,
spermatozoa, bacteria, etc.) by their antisera does not spontaneously
reverse, but persists indefinitely. Natural agglutinins, too, such as the
FERTILIZIN AND FERTILIZATION 197
blood group agglutinins in humans, give permanent agglutination which
can only he reversed by special treatment. It is of interest, then, not
only in connection with fertilization, but in regard to the nature of
agglutination reactions in general, to consider the possible causes of the
spontaneous reversal.
We shall use as a basis of the present discussion the lattice or frame-
work theory of Heidelberger (1938) and Marrack (1938). This theory
postulates that in agglutination as well as precipitation reactions the anti-
gen and antibody are structurally complementary and both are multi-
valent in regard to their combining groups. Thus one molecule of anti-
gen may combine with more than one molecule of antibody which in turn
may combine with more than one molecule of antigen and so build up
large aggregates. Where both of the complementary substances are in
solution, precipitation results. Where one is present as the surface of
the cell, agglutination occurs. The following interpretations may then
be suggested for reversal of agglutination in the sea-urchin. (1) The
fertilizin molecules plus the combined antifertilizin split off from all of
the spermatozoa, leaving neutralized fertilizin in the suspension. (2)
They split off at some, rather than all, combining sites in such a way that
each (completely neutralized) fertilizin molecule remains attached to
not more than one spermatozoon. (3) The fertilizin molecules are split
by the action of the sperm leaving univalent fragments combined with
the antifertilizin on all the spermatozoa.
All three of these interpretations can account for the failure of re-
agglutination and the loss of fertilizing capacity on the part of the re-
versed sperm. Attempts were made to eliminate one or another of these
possibilities but the experiments were inconclusive and need not be de-
scribed here. However, some new findings and further consideration
of earlier work lend support to the third interpretation.
It was shown (Tyler and Fox. 1940) that fertilizin of the keyhole
limpet is much more resistant than that of the sea-urchin to inactivation
by heat and proteolytic enzymes and that this greater stability correlates
with the more permanent nature of the agglutination reaction in that
form. That the difference is not due to differences in the relative
amounts of fertilizin involved is evident by the fact that the reaction
is of long duration in the keyhole limpet even when weak fertilizin solu-
tions are employed, whereas it does not in the sea-urchin surpass a maxi-
mum of very much shorter duration when the strongest available ferti-
lizin solutions are added. This suggests then that, in the sea-urchin, the
combined fertilizin may be broken down fairly rapidly by action of the
sperm. It has also been noted that when fertilizin solutions are heated
or treated with proteolytic enzymes there is at first a small but definite
198 ALBERT TYLER
increase in activity followed by the gradual inactivation. This suggested
the possibility that the fertilizin is first split into smaller but still multi-
valent molecules. Such behavior is not unique for it has been fre-
quently noted with immune antibodies (see Marrack, 1938; Zinsser,
Enders and Fothergill, 1939; Petermann and Pappenheimer, 1941) and
the altered agglutinin is termed " agglutinoid." It seemed possible then
that, by careful inactivation of fertilizin solutions, univalent fragments
might be obtained. The " univalent "' fertilizin should be incapable of
causing agglutination, but should inhibit subsequent agglutination by un-
treated fertilizin. It should also be expected to be effective in destroying
the fertilizing power of the sperm. As will be shown in the next section,
both of these effects have been obtained with heat-treated fertilizin solu-
tions. This, then, lends support to the third interpretation of the spon-
taneous reversal of agglutination ; namely, that the fertilizin molecules
are split and the univalent fragments remain attached to the combining
groups on the sperm.
" UNIVALENT " FERTILIZIN
In five experiments concentrated solutions of S. purpuratus fertilizin
that had been purified by previously described methods (Tyler and Fox,
1940) were heated at 90° to 100° C. just up to the time at which the
agglutinating activity had practically disappeared. Sperm was then
added to samples (at room temperature) of (A) the heated solutions,
(B~) the control solutions, and (C) sea water, the relative amounts being
such that complete agglutination (no reaction to additional fertilizin after
reversal) occurred in the control solution. When unheated fertilizin
was added to samples of the sperm in A, there was either a very weak
reaction or no visible agglutination at all. After reversal of agglutina-
tion in B, normal eggs were inseminated with various amounts of the
sperm suspensions. When amounts of sperm were used that, in the
case of the sea water controls, C, were near the minimum for 100 per
cent fertilization, the A-sperm gave 0 to 5 per cent (av. 0.5 per cent)
and the 5-sperm gave 0 to 1 per cent (av. 0.2 per cent) fertilization. A
further control was run in those experiments where A -sperm showed a
weak agglutination reaction upon addition of unheated fertilizin. This
was done by diluting the control fertilizin to a concentration giving a
similar reaction and adding sperm to the dilute solution at the same time
and in the same relative amounts as employed in the other solutions.
The fertilizing capacity of the sperm in the diluted fertilizin was found
to be only slightly lower than that of the sea water control sperm. An
absorption experiment was also performed by the addition of excess
FERTILIZIN AND FERTILIZATION 199
sperm to a sample of the heated fertilizin solution and, after centrifuga-
tion, the active agent was found to have disappeared from the super-
natant solution.
The results show, then, that the agglutinating property of fertilizin
can be destroyed without altering appreciably its capacity to combine
with the sperm. The heated fertilizin is usually somewhat weaker than
the control in its ability to prevent subsequent agglutination and in its
ability to destroy the fertilizing power of the sperm. This most likely
means that a small amount of the fertilizin is more completely degraded
during the heat treatment. It is clear, however, that by careful heat
treatment a modified (non-agglutinating) fertilizin can be produced that
differs only slightly, if at all. in its ability to combine with the sperm.
Since according to the modern theory a specific agglutinating substance
is assumed to be multivalent in respect to its specific combining groups,
it is reasonable to consider the non-agglutinating substance in this case
univalent.
The formation of univalent fertilizin may be assumed to involve the
splitting of the molecule into fragments each of which contains a single
combining group or it might involve the splitting off of the combining
groups alone. In the latter instance the active agent would be expected
to be of small molecular size. Dialysis tests showed, however, that the
active agent is incapable of passing through a cellophane membrane.
The first assumption appears then to be the more likely one. Other
properties of the active agent have not as yet been studied except for a
preliminary test of its inactivation by heat. It is inactivated in about
one and one-half to three times the time required for destruction of the
agglutinating property of the original fertilizin.
FERTILIZIN IN ANIMALS NOT EXHIBITING ISO-AGGLUTINATION
OF SPERM
Lillie (1919) and Just (1930) assumed that eggs of all species of
animals possessed fertilizin, although they, themselves, had shown that
in many species there is no detectable agglutination of sperm by homolo-
gous egg water. They regarded the agglutination reaction simply as an
indicator for the presence of fertilizin, but they did not offer any evi-
dence or tests that would demonstrate an analogous substance in the ab-
sence of the clumping reaction. The present concept of univalent fer-
tilizin has led to the demonstration of specific sperm-combining sub-
stances in species in which the agglutination reaction is lacking. If, in
a particular species of animal, the fertilizin obtained in the egg water is
univalent, then it should give no agglutination of homologous sperm, but
it should destroy their fertilizing capacity.
200 ALBERT TYLER
This point was examined in the starfish Patina niiniata and in the
gephyrean worm Urechis caupo. In the starfish, concentrated egg watei
causes no agglutination of homologous sperm. In Urechis there ma}
occasionally be a weak reaction. Concentrated egg waters were pre-
pared from eggs of these two species by extraction with pH 4 sea water.
Sperm was then added to the neturalized homologous and heterologous
egg waters as well as to sea water and after a few minutes various
amounts were taken for insemination of the homologous eggs. In all
cases there was found to be a great reduction in the fertilizing capacity
of the sperm treated with homologous egg water, while that treated with
heterologous egg wTater showed approximately the same fertilizing ca-
pacity as the sea water controls. A typical experiment may be cited.
Concentrated Patina and Urechis egg waters were prepared from 10 per
cent egg suspensions. One part of a 1 per cent Patiria sperm suspension
was added to nine parts of (A) Patina egg water, (B) Urechis egg
water and (C) sea water. The same was done with a one per cent
suspension of Urechis sperm. Insemination of homologous eggs (ap-
proximately 200 eggs in 5 cc. of sea water) with 0.05 cc. of these mix-
tures gave for Patiria no fertilization with A, 100 per cent with B and
99 per cent with C. For Urechis the results were 100 per cent with A
and C and 0 per cent with B.
These results then lend support to the view of Lillie and Just that
fertilizin is of general distribution in animals. When appropriate mate-
rial is available, the investigations will be extended. For the present it
is evident in two species of animals that a specific sperm-combining sub-
stance is obtainable from the eggs and, since the substance has no ag-
glutinating action on homologous sperm, it may be termed univalent
fertilizin.
DISCUSSION
It has been shown that fertilizin, when present in the form of a gelat-
inous coat, is an aid to fertilization in the sea-urchin. It would also
appear from the experiments that fertilizin is not entirely essential to
fertilization. But this assumes that all of the fertilizin is removed upon
removal of the jelly. While no detectable fertilizin is obtainable from
the jellyless eggs, it is quite conceivable that it may be present in com-
bined form on the surface of the egg. It has been shown (Tyler, 1940&)
that there is an antifertilizin below the surface of the egg and it would
be reasonable to assume that the surface of the egg is composed of a
fertilizin-antifertilizin complex. Upon removal of the jelly, this com-
bined fertilizin would remain as a monomolecular layer with free spe-
FERTILIZIN AND FERTILIZATION 201
cific combining groups on its outer surface. In support of this view
may be cited the observation of Frank (1939) that jellyless as well as
normal sea-urchin eggs can be agglutinated by means of an antifertilizin
obtained from the sperm. The possibility may then be admitted that
fertilizin is indispensable for fertilization but further evidence along this
line would be desirable before any attempt is made to develop a theory
of fertilization with it as an essential agent.
In regard to the manner in which fertilizin may act as an aid to
fertilization there are several possibilities. In the first place it is clearly
not merely the greater volume due to the presence of the jelly that is
involved, since the spermatozoon must, in any event, reach the surface
of the egg for fertilization to ensue. It is possible that the gradient pro-
duced, as the jelly slowly goes into solution, exerts a chemotactic effect
on the sperm. There is, however, still no general agreement as to chemo-
taxis. Hartmann (1940) reports demonstrating such action of fertilizin
by means of the Pfeffer capillary method, whereas Cornman (1941)
could obtain no positive results with that method.
Another possibility is that the jelly serves as a trap for the sperm.
This appears reasonable on the basis of the fact that the spermatozoon
reacts with fertilizin in solution. One may suppose that, while most
of the fertilizin is in the form of a jelly, some of it is in solution in the
interstices ; or that even as a gel there are some free combining groups
available. The formation of a precipitation membrane on the surface
of the jelly by the action of antifertilizin (Tyler, 19405) is more readily
explainable on the basis of the latter assumption. Trap action would
help to explain how fertilizin (as a jelly) acts as an aid to fertilization,
since it would restrict the random movements of the spermatozoa to a
small volume and thereby increase the chance of fertilization. However,
other and more quantitative experiments are needed before decision can
be made as to whether or not it alone can account for greater fertiliza-
bility of the normal in comparison with the jellyless eggs.
Another possibility is that some structural property of the jelly causes
the sperm to approach so that its long axis is normal to the surface.
While observations (see Morgan, 1927; Chambers, 1933) indicate that
a radial approach is more favorable for fertilization, it has not definitely
been shown that oblique approach and contact with the surface results
in failure of sperm entry.
The possibility should also be considered that the greater fertiliza-
bility of the normal eggs is clue to the activating effect of fertilizin on
the sperm. But before decision can be made as to the value of this
factor, it would be important to know that there is no corresponding
decrease in the fertilizable life of the sperm.
202 ALBERT TYLER
In connection with these possibilities, it must be recalled that after
the sperm has reacted with f ertilizin in solution it is incapable of fertiliza-
tion and that, probably because of this, the presence of fertilizin in
solution in a suspension of eggs acts as a barrier to fertilization. Thus
excess sperm is required to take up the fertilizin in solution and leave
uncombined sperm available for fertilization. It is evident that in nor-
mal fertilization the spermatozoon must reach the surface of the egg
before the inhibiting action of the fertilizin surrounding the egg has taken
place. If, as suggested above, fertilizin in the form of a jelly has only
a few superficial combining groups available, it is quite conceivable that
they may serve as the initial trap for the sperm but would not be suf-
ficient to neutralize all of the reacting groups on the sperm before the
latter has reached the surface of the egg. The increased activity of the
sperm upon reaction with fertilizin would also aid its reaching the surface
before the fertilization-inhibiting reaction went to completion. While
this seems to be the most likely interpretation, it requires considerably
more experimental support. Also, it appears that the information so fat-
available does not warrant a detailed discussion of Lillie's theory of
fertilization, nor of the recent views of Hartmann (1940), nor of the
development of a new theory of the exact function of fertilizin and
other specific substances.
It has been shown in the present work that appropriate treatment of
sea-urchin fertilizin converts it into a non-agglutinating agent that is
still capable of reacting specifically with the sperm. On the basis of the
lattice theory of agglutination reactions this altered fertilizin may, quite
legitimately, be designated a univalent substance. It was also shown
that the egg waters of certain species of animals that do not contain spe-
cific sperm agglutinins nevertheless contain specific sperm-combining
substances which may likewise be designated univalent. The absence of
the agglutination reaction in many species of animals does not, then, mean
the lack of fertilizin, if by that term we mean simply a substance that
reacts specifically with the sperm.
This concept may also be extended to problems in general immu-
nology. It is well known that certain animals, such as the rabbit and the
horse, readily produce upon immunization, specific agglutinins and pre-
cipitins. Others, such as the mouse and the rat, produce little or none
but do form protective or neutralizing antibodies. It may be suggested,
then, that the antibodies produced in the latter species are principally
or entirely of the univalent type. This possibility can be readily tested
experimentally; — cells treated with the univalent antibodies should be
rendered incapable of being agglutinated by the specific agglutinating
antibodies obtained in the former species.
FERTILIZIN AND FERTILIZATION 203
SUMMARY
1. It has been shown in the sea-urchin that the presence of fertilizin,
in the form of the jelly coat of the egg, serves as an aid to fertilization.
In solution it acts as a harrier to fertilization.
2. Confirmation is presented of Lillie's finding that sea-urchin sperm
cannot be re-agglutinated after reversal of an initial agglutination. It
is also shown that the reversed sperm are incapable of fertilization.
3. Appropriate heat treatment converts fertilizin into a substance that
does not cause sperm agglutination but still combines with the sperm as
shown by the inability of the sperm to be subsequently agglutinated by
ordinary fertilizin and by loss of fertilizing power. In accordance with
the assumption of multivalency in the lattice theory of agglutination, the
modified fertilizin is assumed to be univalent. It is found to be non-
dialyzable.
4. In the starfish and in Urccliis the egg water is shown to contain a
specific sperm-combining substance (univalent fertilizin) that is incapable
of causing iso-agglutination of sperm.
5. Of various interpretations of the spontaneous reversal of agglu-
tination in the sea-urchin, a splitting of the fertilizin into univalent frag-
ments is considered the most likely.
6. Reasons are presented for holding open the possibility that ferti-
lizin plays an indispensible part in fertilization. Various possible expla-
nations as to the manner in which it serves as an aid to fertilization are
discussed and that involving trap action is considered the most likely.
7. It is suggested that some species of animals produce upon immu-
nization only, or principally, univalent antibodies and a method of de-
termining this point is offered.
LITERATURE CITED
CHAMBERS, R., 1933. The manner of sperm entry in various marine ova. Jour.
Exper. Biol, 10: 130-141.
COHN, E. J., 1918. Studies in the physiology of spermatozoa. Biol. Bull., 34 :
167-218.
CORNMAN, I., 1941. Sperm activation by Arbacia egg extracts, with special rela-
tion to echinochrome. Biol. Bull., 80: 202-207.
EVANS, T. C, 1940. Effects of roentgen radiation on the jelly of the Arbacia egg
(abstract). Biol. Bull., 79: 362.
FRANK, J. A., 1939. Some properties of sperm extracts and their relationship to
the fertilization reaction in Arbacia punctulata. Biol. Bull., 76: 190-216.
GLASER, O., 1915. Can a single spermatozoon initiate development in Arbacia?
Biol. Bull.. 28: 149-153.
HARTMANN, M., 1940. Die stofflichen Grundlagen der Befruchtung und Sexualitat
im Pflanzen- und Tierreich. I. Die Befruchtungsstoffe (Gamone) der
Seeigel. Nutiinviss., 51 : 807-813.
204 ALBERT TYLER
HARTMANN, M., O. SCHARTAU, AND K. WALLENFELS, 1940. Untersuchungen tiber
die Befruchtungsstoffe der Seeigel. II. Biol. Zcntralbl, 60: 398-423.
HEIDELBERGER, M., 1938. The Chemistry of the A mi no Acids and Proteins, Chap.
XVII, pp. 953-974. Charles C. Thomas, Springfield.
JUST, E. E., 1930. The present status of the fertilizin theory of fertilization.
Protoplasma, 10: 300-342.
LILLIE, F. R., 1913. Studies of fertilization. V. The behavior of the spermatozoa
of Nereis and Arbacia with special reference to egg-extractives. Jour.
Expcr. Zoo/., 14: 515-574.
LILLIE, F. R., 1914. Studies of fertilization. VI. The mechanism of fertilization
in Arbacia. Jour. Expcr. Zool., 16 : 523-590.
LILLIE, F. R., 1915. Sperm agglutination and fertilization. Biol. Bull., 28: 18-33.
LILLIE, F. R., 1919. Problems of Fertilization. University of Chicago Press,
Chicago.
LILLIE, F. R., 1921. Studies of fertilization. VIII. On the measure of specificity
in fertilization between two associated species of the sea-urchin genus
Strongylocentrotus. Biol. Bull., 40: 1-22.
LOEB, J., 1914. Cluster formation of spermatozoa caused by specific substances
from eggs. Jour. Exper. Zool.. 17: 123-140.
LOEB, J., 1915. On the nature of the conditions which determine or prevent the
entrance of the spermatozoon into the egg. Am. Nat.. 49: 257-285.
MARRACK, J. R., 1938. The Chemistry of Antigens and Antibodies. Medical Re-
search Council, Special Report Series. No. 230, London.
MORGAN, T. H., 1927. Experimental Embryology. Columbia University Press,
New York.
PETERMANN, M. L., AND A. M. PAPPENHEIMER, JR., 1941. The action of crystal-
line pepsin on horse anti-pneumococcus antibody. Science, 93 : 458.
RICHARDS, A., AND A. E. WOODWARD, 1915. Note on the effect of X-radiation on
fertilizin. Biol. Bull., 28 : 140-148.
TYLER, A., 1939</. Extraction of an egg membrane-lysin from sperm of the giant
keyhole limpet (Megathura crenulata). Proc. Nat. .-lead. Sci., 25: 317-
323.
TYLER, A., 1939/>. Crystalline echinochrome and spinochrome : their failure to
stimulate the respiration of eggs and of sperm of Strongylocentrotus.
Proc. Nat. Acad. Sci., 25 : 523-528.
TYLER, A., 1940«. Sperm agglutination in the keyhole limpet, Megathura crenulata.
Biol. Bull., 78: 159-178.
TYLER, A., 1940/>. Agglutination of sea-urchin eggs by means of a substance ex-
tracted from the eggs. Proc. Nat. Acad. Sci.. 26: 249-256.
TYLER, A., AND S. W. Fox, 1940. Evidence for the protein nature of the sperm
agglutinins of the keyhole limpet and the sea-urchin. Biol. Bull., 79:
153-165.
TYLER, A., AND K. O'MELVENY, 1941. The role of antifertilizin in fertilization.
Biol. Bull., in press.
TYLER, A., AND B. T. SCHEER, 1937. Inhibition of fertilization in eggs of marine
animals by means of acid. Jour. Expcr. Zool., 75: 179-197.
ZINSSER, J., J. F. ENDERS. AND L. D. FOTHERGILL, 1939. Immunity. Macmillan,
New York.
SPECIFICITY AND HOST-RELATIONS IN THE
TREMATODE GENUS ZOOGONUS 1
HORACE W. STUNKARD
(Front the Department of Biology, New York University, and ike Marine
Biological Laboratory, Woods Hole, Mass.)
The genus Zoogonus was erected by Looss (1901) to contain Z.
minis, (as type), and Distoiinuti viviparum Olsson, 1868. Zoogonus
mints was described from two specimens found in the intestine of
Labrus incrula at Trieste. The worms measured 1.55 mm. in length and
about 0.45 mm. in width. The following year, Odhner (1902) desig-
nated Zoogonus viviparus (Olsson, 1868) Looss, 1901 as type of a new
genus Zoogonoidcs. In this paper he redescribed Distomuni rubeUuui
Olsson, 1868, and transferred the species to Zoogonus. Like Olsson,
he found the parasites in Labrus bcrggylta (syn. L. maculatus} from the
west coast of Sweden, but the examination of twenty fishes at the
Zoological Station of Kristineberg disclosed only two infections and only
a few worms were obtained. The specimens were yellowish in color.
1.1-1.4 mm. in length and 0.45 mm. in greatest width. Zoogonus rubcl-
lus was distinguished from Z. minis on the size of suckers and length of
the miracidia. Although Looss and Odhner were two of the ablest
students of the trematodes, their observations on Zoogonus were limited
to the study of very few specimens.
The specific identity of Z. minis, questioned by competent investi-
gators, still remains an unsolved problem. Goldschmidt (1902, 1905),
after comparing specimens collected, at Trieste with the descriptions of
Z. minis and Z. rubcllus, stated that there were no morphological dif-
ferences. The dimensions of suckers and miracidia, characters used by
Odhner to separate the species, were found to be variable and hence
invalid as specific criteria. Furthermore, Goldschmidt was unwilling to
differentiate the species on the presence or absence of yellow pigment in
the tissues. Nicoll (1909), who reported Z. rubcllus as consistently
abundant in Anarhiclias lupus from St. Andrews Bay, described the
worms as pale yellow in color, 0.75-1 mm. in length and about one-half
1 The observations at the Station Zoologique de Wimereux were made while
the writer held a fellowship from the Oberlaender Trust of Philadelphia, Pa.
Grateful acknowledgment is made to the Oberlaender Trust for financial assistance
and to both Professor Maurice Caullery, Directeur, and Dr. Jean Vivien, Sous-
Directeur of the Station Zoologique, for laboratory facilities generously and gra-
ciously placed at my disposal.
205
206 HORACE W. STUNKARD
as wide as long. Referring to the descriptions of Looss, Odhner and
Goldschmidt, Nicoll stated, ' My specimens agree best with Gold-
schmidt's description." It is significant that the worms studied by
Nicoll and Odhner came from the same region, whereas Goldschmidt' s
material was collected in the Mediterranean and presumably was iden-
tical with that of Looss. Concerning specific determination, Nicoll ex-
pressed the opinion that, " Looss's Zoogonus minis is in all probability
identical with Odhner's Z. rub ell its ... at any rate, features sufficient
to distinguish them are not at present apparent."
In a systematic review of the family Zoogonidae, Odhner (1911)
maintained that the specimens of Zoogonus from the Mediterranean and
North Sea are specifically distinct. After collecting material from both
locations, he distinguished the two species on differences in size and
color, size of suckers and shape of sucker cavities, location of the
acetabulum, length of the digestive ceca, position of the cirrus sac, and
number of eggs in the uterus. Odhner stated that extended specimens
of Z. mints never exceed 0.6 mm. in length and 0.2 mm. in width,
whereas similar specimens of Z. rubcllus measure 0.9-1.2 mm. in length
and about 0.25 mm. in width. He noted that the pharynx and the
miracidia are approximately the same size in the two species, but appear
to be larger in the smaller specimens of Z. mints.
The specimens of Z. mints studied by Odhner were apparently con-
tracted and probably not entirely mature. His statement that extended
worms never exceed 0.6 mm. in length cannot be accepted, since the type
specimens described by Looss measured 1.55 mm. in length. The num-
ber of eggs in the uterus obviously is correlated with the degree of
sexual development, and the other features employed by Odhner to
differentiate the species manifest so much variation that subsequent
authors have disagreed on the identity or distinctness of the European
species of Zoogonus. It is apparent that morphology of the adult stages
is inadequate for a final solution of the problem.
Although a beginning has been made on the life history of Zoogonus,
information from this source is still too fragmentary to permit final
specific determination. In a series of papers, Timon-David (1933.
1934, 1936, 1938) described encysted metacercariae, identified as larvae
of Z. mirus, in sea-urchins collected in the Gulf of Marseilles and along
the coast of Roussillon. The metacercariae were found only in the
muscles of the lantern of Aristotle. The degree of infection was vari-
able and from one to sixty cysts were recovered from individual urchins.
The incidence of infection in Paracentrotus lividus reached 50 to 60 per
cent, a somewhat lighter infection was common in Sphacrcchinus granu-
laris, only a few cysts were recovered from Arbacia acqiiitubcrculalu,
SPECIFICITY AND HOST-RELATIONS IN ZOOGONUS 207
whereas no infection was observed in Echinus aciitus or Psanmiechinus
microtuberculatns. All the parasites apparently belonged to a single
species. The cysts increased in size with the development of the meta-
cercariae, and measured from 0.15 to 0.25 mm. in diameter. A cyst
(1934, Fig. 1), fixed in picro-formol-alcohol solution under moderate
compression, measured about 0.4 mm. in diameter, according to the
scale accompanying the drawing. The cyst wall measured 0.003 mm. in
thickness and was not surrounded by a connective tissue capsule. The
worm was bent upon itself, with the dorsal surface applied to the wall
of the cyst. Released from their cysts, the mature larvae averaged 0.6
mm. in length and 0.2 mm. in width. Specimens from P. lividits, A.
aequitubcrculata and ^. granularis (1934, Figs. 2, 3, 4), fixed in ex-
tended condition, measured 0.83, 0.93 and 0.66 mm. respectively (length
calculated from scales accompanying the figures). Timon-David (1936)
reported that metacercariae fed to Blcnmus gattoniglnac excysted and
persisted in the intestine for 45 days. Such a specimen, figured in the
report, measured 0.64 mm. in length and w-as little if any farther ad-
vanced in development than larvae freshly removed from their cysts.
The observation of Timon-David, that remains of sea-urchins were fre-
quently present in the intestine of Labrns mcrula, supports his opinion
that the metacercariae from the urchins are actually larval stages of
Z. mints. In his (1934) paper he recalled that the development of the
miracidia has been known since the accounts by Looss (1901), Gold-
schmidt (1902) and Wassermann (1913), but that the first intermediate
host remains as yet unknown. He expressed the belief that the cer-
carial stages are to be sought among the gastropods of the region.
In a report on larval trematodes from the region about Roscoff,
Finistere, Stunkard (1932) described a tailless cercaria, C. rcticulatntn
from Nassa reticulata, which shows such remarkable morphological
agreement with the metacercariae described by Timon-David that the
two must be closely related and may possibly belong to the same species.
One item in the description of Stunkard requires correction. In the
figure, the pharynx is represented as only a short distance in front of
the acetabulum, whereas notes made at the time state that the pharynx
is situated about midway between the suckers.
A single species of Zoogoinis has been recorded from the Atlantic
coast of North America. It was first described in the cercarial stage
by Leidy (1891), who named it Distoinmn lasium. The larvae develop
in Nassa obsolcta. Subsequent studies on the cercaria were reviewed by
Stunkard (1938), who completed the life cycle. The cercariae encyst in
polychaete annelids, principally Nereis vircns. Natural infections were
found in eels and sexually mature specimens were recovered after ex-
208 HORACE W. STUNKARD
perimental infection of the eel and toadfish. Comparison of adult speci-
mens with descriptions of Z. rubcllus and Z. minis provided no positive
basis for specific distinction and so notwithstanding certain differences
in hosts, life cycle, and morphological details, Stunkard regarded the
American specimens as specifically identical with Z. rubcllus and Z.
mirus.
Subsequent studies of the European species carried on at the
Station Zoologique de Wimereux in 1939 and of the American species
at the Marine Biological Laboratory in Woods Hole during 1940, have
yielded such discordant results that the question of specific identity
must be reconsidered. The results of these observations were reported
in abstract (Stunkard, 1940).
A sojourn at Wimereux, France, from July 8 to August 14, 1939,
provided an opportunity to reexamine European phases of the Zoogonus
problem. Metacercariae were found in Psammcchinus iniliaris, the
common sea-urchin of the region. Urchins were collected at different
locations from the Port de Boulogne to Ambleteuse, a stretch of some
ten kilometers. The heaviest infection appeared in specimens from the
Port de Boulogne where encysted larvae were recovered from more thai:
50 per cent of the urchins dissected. Different individuals harbored
from one to thirty-six metacercariae. Lighter incidence and degree of
infection were found in urchins taken between Boulogne and Ambleteuse.
All of the larvae appeared to belong to a single species.
The metacercariae were encysted in the muscles and connective tissue
of the lantern of Aristotle. The cysts were transparent, with no obvious
reaction of the host to the parasite. The cyst wall was thin, colorless and
very tough. The cysts measured from 0.2 to 0.28 mm. in diameter. The
larvae, freed from their cysts, measured 0.42 to 0.65 mm. in length and
0.15 to 0.22 mm. in width. The cuticula was spined, although the spines
were reduced in size and number behind the level of the testes. Larvae
fixed under cover-glass pressure measure up to 0.95 mm. in length and
a small one (Fig. 3), apparently equally mature but fixed without pres-
sure, is only 0.37 mm. in length. When the worms are fixed without
compression, the preacetabular portion bends ventrad, so that in mounted
specimens the oral sucker may appear above or below and immediately
in front of the acetabulum (Figs. 1, 2, 3). A representative specimen,
fixed under slight pressure and shown in Fig. 4 is 0.75 mm. in length.
The acetabulum, situated near the middle of the body, measures 0.08 by
0.086 mm. in diameter. The pharynx is located about midway betwreen
the suckers and measures approximately 0.06 mm. in diameter. When
the specimen was extended the pharynx tended to be longer than wide.
Its anterior end is dentate and the nuclei of the organ are concentrated
SPECIFICITY AND HOST-RELATIONS IN ZOOGONUS 209
_Ph
— cs
sv
dc
- -vit
— sr
ut
PLATE I
Abbreviations
ac acetabulum
cs cirrus sac
tie digestive cecum
gp genital pore
o-i' ovary
ph pharynx
sr
sv
ts
ut
vit
seminal receptacle
seminal vesicle
testis
uterus
vitellaria
All figures are drawn to the same scale from fixed, stained and mounted
specimens.
FIG. 1. Mature metacercaria, developed six weeks in Nereis I'irens, fed to a
toadfish and removed two days later ; Woods Hole. One of the largest speci-
mens, fixed without compression, anterior end bent ventrad ; dorsal view.
FIG. 2. Sexually immature specimen, developed six weeks in N. virens and
six weeks in an eel ; Woods Hole. Shows preacetabular ventral bending of body
in specimens fixed without compression ; lateral view from left side.
FIG. 3. Mature metacercaria from Psammcchinus miliaris; Wimereux. One
of the smallest specimens, fixed without compression, anterior end bent ventrad ;
ventral view.
FIG. 4. Mature metacercaria from P. miliaris; Wimereux. An average size
specimen, fixed under cover-glass pressure, 0.75 mm. long ; ventral view.
210 HORACE W. STUNKARD
in its posterior half. The excretory system was worked out completely
and agrees in detail with that of Cercariaeuin reticulatwn and with that
of the American species of Zoogonus.
Measurements of the metacercariae from Wimereux do not differ
greatly from those given by Timon-David for metacercariae from sea-
urchins in the Mediterranean. The suckers in my specimens are slightly
smaller than those measured by Timon-David, although his figures show
the apertures of the suckers to be wide open and the specimens may
have been more flattened. He figured the acetabulum slightly behind
the middle of the body, whereas in my material it tends to lie slightly in
front of the middle, although this feature is variable and changes with
the extension or retraction of anterior and posterior regions of the body.
Consequently, it appears likely that the specimens from Wimereux are
specifically identical with those from the Mediterranean.
After discovery of the metacercaria, attempts were made to find the
other stages in the life cycle of the European species of Zoogonus. Ex-
amination of many fishes, including several specimens of Labrus sp.,
were fruitless. In view of the possibility, expressed previously, that the
tailless larva, C. reticulatum, may represent a stage in the life history,
wide search was made for it. The host, Nassa reticulata, is abundant in
the region but the examination of more than 1200 specimens did not yield
a single infection with tailless cercariae. Over 800 of these snails were
collected from mud between rocks of the breakwater in the Port de
Boulogne, and sea-urchins taken from these rocks were heavily infected
with metacercariae. Other mollusks examined for Zoogonus larvae,
with negative results, included 240 Mytilus cditlis, 146 Barnea Candida,
28 Tapes pullastra, 10 Ensis sp., 240 Patella vulgaris, 35 Purpura lapil-
lus, 34 Littorina obtusa, 86 L. nidis, 56 L. littorca. and 45 Gibbula
cineraria.
To determine whether annelids as well as echinoderms harbor meta-
cercariae of the European species of Zoogonus, worms were carefully
dissected under a binocular microscope. The examination of 14 Eu-
nereis longissima, 12 Nereis errorata and representatives of other un-
identified polychaetes from the Port de Boulogne did not disclose any
metacercariae.
In view of the failure to discover other stages in the life cycle of
Zoogonus in the Wimereux area, the origin of the infection in the sea-
urchins and subsequent fate of the larvae are entirely problematical.
The completion of the life history by trematode parasites would be diffi-
cult in this region since the tides have an amplitude of eight to ten meters
and the collecting grounds, exposed at low tide, are covered by an enor-
mous volume of water six hours later.
SPECIFICITY AND HOST-RELATIONS IN ZOOGONUS 211
Comparison of the larval stages of Zoogonus found along the north
coast of Europe and the eastern coast of Xorth America shows slight
but apparently significant differences. Cercariae from Nassa reticulata
at Roscoff average slightly larger than those from N. obsoleta at Woods
Hole and the suckers are also larger. In the European form the ranges
of size are: acetabulum, 0.068-0.076; oral sucker. 0.076-0.085; and
pharynx, 0.03-0.04 mm., whereas measurements of the corresponding
structures in the American form are : 0.062-0.075 ; 0.043-0.055 ; and
0.022-0.028 mm. Moreover, in the European form the prepharynx is
relatively shorter and the pharynx is about midway between the suckers,
whereas in the American form the pharynx is farther posteriad and fre-
quently overlaps the acetabulum. The metacercariae from Wimereux
and from Woods Hole show the same differences as the cercariae from
the two regions. Cysts of the European form are slightly larger, the me-
tacercariae are larger (compare Figs. 1, 2, and 3) and the relative sizes of
suckers persists. Average measurements of ten specimens from the two
localities give the following sizes (dimensions of Wimereux specimens
first, of Woods Hole specimens second): acetabulum 0.085 vs. 0.075;
oral sucker 0.09 vs. 0.065 ; pharynx, 0.06 vs. 0.042 mm.
To determine whether the American species of Zoogonus may occur
in sea-urchins as well as polychaete annelids, attempts were made at
Woods Hole in the summer of 1940 to infect urchins with Distomum
lasium (=-C. linfoni). Many freshly dredged urchins, both Arbacia
punctulata and Strongylocentrotus drobachicnsis, were dissected with
negative results. Since enormous numbers of these animals have been
used during the past forty years for embryological and other studies with-
out the reported finding of metacercariae. natural infection with trema-
tode larvae must be absent or very slight. Portions of dissected urchins,
including the denticles and attached tissues, were placed in dishes of sea
water with scores of naturally emerged cercariae of Zoogonus. The
larvae crawled about over the tissues but did not penetrate or encyst.
They were not attracted toward intact sea-urchins or dissected portions
of them. Single urchins were exposed for several hours during the day
in finger bowls to hundreds of cercariae and maintained during the inter-
vening time in large aquaria. Dissection of the urchins later did not
disclose any metacercariae.
Although the factors concerned with infection of the secondary inter-
mediate host are virtually unknown, it is apparent that experiments de-
vised to secure experimental infection in the laboratory must approximate
natural conditions as closely as possible. Accordingly, on August 16.
1940, twenty specimens of N. obsoleta from which cercariae were emerg-
ing in large numbers were placed in each of two aquaria. Fifteen speci-
212 HORACE W. STUNKARD
mens of A. punctulata were added to one aquarium; five specimens of
A. punctulata and five specimens of S. drobachiensis to the other. After
an interval of a week, dissection of the urchins was begun. No infection
was found in 5\ drobachiensis but Zoogonus larvae were recovered from
eleven specimens of A. punctulata. These urchins, examined in the
period from August 23 to September 11, yielded 79 cysts in which the
larvae were dead, 60 cysts containing living larvae, 32 unencysted, dead
larvae, and 16 unencysted. living larvae. The dead, encysted larvae were
often partly disintegrated. Live larvae in cysts had extruded their sty-
lets, but showed no evidence of development. The gonads were no
larger than those of the cercaria and the ducts of the penetration glands
were still visible. It is evident from these results that cercariae of the
American species of Zoogonus will enter and encyst in sea-urchins and
that they may live there for a time. But no development was observed
and the finding of so many dead larvae, both free and encysted, indicates
that A. punctulata is not a suitable host. It is probable, therefore, that
these sea-urchins are not involved in the life cycle of the parasite.
The problem of specificity in host-parasite relations can be solved
only by the experimental methods developed in studies on the life cycles
of parasites. Formerly it was believed that different species of hosts
harbored different parasites. In the case of trematodes, it is now known
that a single parasitic species may infect a wide variety of hosts. Allas-
sostoma parvuni may infect frogs and turtles ; Zygocotyle lunata may in-
fect birds, rodents and ruminants ; Cryptocotyle lingua may infect birds,
rodents and carnivores ; Notocotylus urbanensis may infect ducks and
muskrats ; Fasciola hcpatica may infect cattle, pigs, rodents, the elephant,
kangaroo and man ; Echlnostoma rcvolutum may infect various species
of birds and mammals ; Psilostomum ondatrac may infect the muskrat,
duck, pigeon and canary. These examples, selected from a large list,
represent five families and show that the possibility of multiple hosts is
general. Furthermore, as a result of development in widely separated
hosts, representatives of a single trematode species may manifest mor-
phological differences which under other conditions might reasonably be
regarded as specific. Specimens of F. hcpatica from a guinea pig and
others from a cow would hardly be assigned to the same species on the
basis of morphology.
The digenetic trematodes manifest a comparable lack of specificity in
their intermediate hosts. This is true particularly in cases involving a
second intermediate host, often nothing more than a "transfer host" in
which no development occurs. The condition is similar to that in Fas-
ciola and Zygocotyle, where cercariae encyst on vegetation or other ob-
jects which are eaten by the final host. Even in the first intermediate
SPECIFICITY AND HOST-RELATIONS IN ZOOGONUS 213
host, specificity may be far from rigid. For Fasciola hepatica, which
has become cosmopolitan in distribution, snails belonging to the following
genera may serve as first intermediate hosts: Lymnaea, Galba, Bulinus,
Physopsis, Physa, Stagnicola, Fossaria, Pseudosuccinca and Ampullaria.
In any particular region, one variety of snail is selected, but in different
regions the species is different.
The data on Zoogonus are hard to interpret. In view of the lack of
specificity in the life cycles of other trematodes, it is not impossible that
a single species of Zoogonus employs different primary, secondary and
definitive hosts on the two sides of the Atlantic ocean. In such event, the
morphological variations are readily explained. On the other hand, the
bionomic and morphological differences may represent valid specific cri-
teria. This opinion is supported by cytological observations. According
to Goldschmidt (1905), Z. minis has 10 chromosomes, while Brooks
( 1930) found 12 chromosomes in the American form. At present there
is no basis for a positive distinction between species of Zoogonus from
the North Sea and the Mediterranean, but it appears probable that tin-
European and American forms are specifically distinct. If this proves
to be true, the American species is Z. lasius (Leidy, 1891) Stunkard,
1940.
SUMMARY
Encysted metacercariae of Zoogonus are reported from the sea-
urchin, Psammechinus uiilians, at Wimereux, France. Comparison with
descriptions of other larval stages found at Roscoff and Marseilles indi-
cates that all belong to the same species. Attempts to infect sea-urchins
at Woods Hole with the American form of Zoogonus were only partially
successful. Bionomic and morphological differences between the Euro-
pean and American representatives of Zoogonus are discussed. It ap-
pears probable that they belong to different species.
BIBLIOGRAPHY
BROOKS, F. G., 1930. Studies on the germ cell cycle of trematodes. Am. Jour.
Hygiene, 12 : 299-340.
GOLDSCHMIDT, RICHARD, 1902. Ueber Bau und Embryonalentwickelung von Zoo-
gonus mirus Lss. Zentr. Bakt., Parasit. u. Infekt., I, 32 : 870-876.
GOLDSCHMIDT, RICHARD, 1905. Eireifung, Befruchtung und Embryonalentvvick-
lung des Zoogonus mirus Lss. Zool. Jahr., Anat., 21 : 607-654.
LEIDY, JOSEPH. 1891. Notices of Entozoa. Proc. Acad. Nat. Sci, Philadelphia,
42: 410-418.
Looss, A., 1901. Ueber einige Distomen der Labriden des Triester Hafens.
Zcntr. Bakt.. Parasit. u. Infckt.. I, 29: 398-405, 437-442.
NICOLL, W., 1909. A contribution towards a knowledge of the Entozoa of British
marine fishes. Part II. Ann. Mag. Nat. Hist., 8 ser., 4: 1-25.
214 HORACE W. STUNKARD
ODHNER, T., 1902. Mitteilungen zur Kenntnis der Distomen. I. Ueber die Gat-
tung Zoogonus Lss. Zcntr. Bakt., Parasit. u. Infekt., I, 31 : 58-69.
ODHNER, T., 1911. Zum natiirlichen System der digenen Trematoden II. Zool.
Ans., 37 : 237-253.
STUNKARD, H. W., 1932. Some larval trematodes from the coast in the region of
Roscoff, Finistere. Parasitol, 24: 321-343.
STUNKARD, H. W., 1938. Distomum lasium Leidy, 1891 (Syn. Cercariaeum lintoni
Miller and Northup, 1926), the larval stage of Zoogonus rubellus (Olsson,
1868) (Syn. Z. minis Looss, 1901). Biol Bull, 75: 308-334.
STUNKARD, H. W., 1940. Life history studies and specific determination in the
trematode genus Zoogonus. Jour. Parasit., 26 (Suppl.) : 33-34.
TIMON-DAVID, J., 1933. Contribution a 1'etude du cycle evolutif des Zoogonides
(Trematodes). Comfit. Rend. Acad. Sci., 196: 1923-1924.
TIMON-DAVID, J., 1934. Recherches sur les Trematodes parasites des Oursins en
Mediterranee. Bull. lust. Occanogr. Monaco, No. 652, 16 pp.
TIMON-DAVID, J., 1936. Sur 1'evolution experimentale des metacercaires de Zoo-
gonus mirus Looss, 1901 (Trematodes, Famille des Zoogonidae). Comfit.
Rend, dc I'Assoc. Franc,. Avanc. des Sciences, Marseille, 1936, pp. 274—276.
TIMON-DAVID, J., 1938. On parasitic trematodes in Echinoderms. Livro Jubilar,
L. Travassos, Rio de Janeiro, Brasil, pp. 467-473.
WASSERMANN, F., 1913. Die Oogenese des Zoogonus mirus Lss. Arch. f. mikr.
Anat., 83 (Abt. II) : 1-140.
FACTORS INFLUENCING MOULTING IN THE
CRUSTACEAN, CRANGON ARMILLATUS
WALTER N. HESS
(From the Biological Laboratory of Hamilton College and the Tortugas Laboratory
of the Carnegie Institution)
INTRODUCTION
According to Darby (1938), C rang on armillatus exhibits diurnal
moulting in which most moultings occur during the early afternoon.
Although this is the warmest period of the day, he did not consider that
there was any correlation with temperature. This investigation was
undertaken in order to determine whether light or temperature, or both
of these factors, are concerned with the diurnal moulting of this animal.
Crangon of different sizes were collected and placed in individual
finger bowl culture dishes in the laboratory. The animals were fed
abundantly on algae and the flesh of the spiny lobster, Panulims argus.
Some were kept on the table in the laboratory while others were placed
in constant temperature incubators.
DIURNAL MOULTING
One hundred and thirty-six specimens of Crangon armillatus, vary-
ing in length from 10 to 39.5 mm., were selected so that there were ap-
proximately the same number in each size group, as shown in Table III.
They were kept in individual finger bowls on the laboratory table where
at midday the average light intensity was approximately 75 foot-candles.
The experiment was begun on June 19 and continued until each animal
had moulted twice with the exception of eight fatalities, for which other
specimens were substituted. The data obtained in this study are shown
in Table I.
This study confirms the existence of diurnal moulting in this animal
under the conditions of this experiment. The great majority of the
animals moulted between the hours of 10:00 A.M. and 5:00 P.M., with
the largest number moulting between the hours of 1 :00 and 2 :00 P.M.
No animals moulted between the hours of 9:00 P.M. and 7:00 A.M.
There is a diurnal rise and fall of air temperature at Tortugas which
corresponds very closely with the diurnal moulting of Crangon armillatus.
During the period of this experiment the average temperature in the
215
216
WALTER N. HESS
laboratory at 8:00 A.M. was 28.6° C, at 1 :00 P.M. it was 31.9° C. and
at 5 :00 P.M. it was 29.4° C. The temperature in the laboratory at night
fell to an average of approximately 27.8° C. Since the animals that
were used in this experiment were kept in small dishes with a compara-
tively small amount of water they were, to a very large degree, subject
to these temperature changes.
Under the conditions of this experiment the animals did not begin
moulting until the temperature had risen to approximately 29° C. in the
morning and ceased moulting when it fell to approximately this same
temperature in the late afternoon or evening.
Crangon annillatus lives near low tide in small bays at Tortugas that
are protected from strong wave action. At night and at high tide the
temperature at this season is approximately 28° C. At low tide, at
midday, the temperature in these bays often rises to 39° C., although the
TABLE I
Number of Crangon armillatus that moulted between the hours indicated when kept
in individual culture dishes on the laboratory table.
7-8
A M.
8-9
A.M.
9-10
A.M.
10-11
A.M.
11-12
A.M.
12-1
PM.
1-2
P.M.
2-3
P.M.
3-4
P.M.
4-5
P.M.
5-6
P.M.
6-7
P.M.
7-8
P.M.
8-9
P.M.
1
6
6
21
26
27
55
33
31
25
18
16
5
2
average is four or rive degrees lower. Thus in nature these animals are
subject to much the same temperature changes as in the laboratory.
EFFECT OF KEEPING ANIMALS AT A CONSTANT TEMPERATURE
This study was undertaken to ascertain if possible the effect of re-
versing the effect of daylight from daytime to night. Since it was dis-
covered that the midday heat affected the incubators by raising the tem-
perature above 30° C., when they were set at this temperature, a higher
temperature was used.
Two incubators with thermostat control, which were set at 33° C.,
were used for this study. The inside of one of the incubators was illu-
minated from 8:00 P.M. to 8:00 A.M. by a Mazda lamp which cast a
light of approximately 75 foot-candles on the animals, thereby reversing
the relation between light and daytime. The inside of a second incubator
was kept in total darkness both day and night in order to determine
whether the light used in the first incubator had any effect on moulting.
Sixty-two Crangon, each in separate culture dishes, were kept in the
illuminated incubator, and 57 were kept in the non-illuminated incubator.
MOULTING IN CRANGON ARMILLATUS
217
TABLE II
Percentage of Crangon armillatus that moulted from 8:00 A.M. to 8:00 P.M., and
from 8:00 P.M. to 8:00 A.M. when kept at 33° C. One group was kept
in total darkness while the other was illuminated only at night.
In total
darkness
Illuminated with
75 f.c. at night
Moulted from 8:00 A.M. to 8:00 P.M 54.4
Moulted from 8:00 P.M. to 8:00' A.M.. . 45.6
51.6
48.4
The number of moulted individuals were counted twice daily ; at 8 :00
A.M. to determine the number of individuals that moulted during the
night and at 8:00 P.M. to determine the number that moulted during
the daytime. The results obtained are shown in Table II.
Although the results above show that a few more animals moulted
during the daytime than at night, moulting is not restricted to the daytime
when Crangon are kept at a constant temperature. It further shows
TABLE III
Comparison of the average number of days between moults of Crangon that were kept
in the laboratory at an average daily temperature of approximately 29.5° C.
with those that were kept at a constant temperature of 33° C.
Size
10-15
mm.
1 5-20
mm.
20-25
mm.
25-30
mm.
30-35
mm.
35-40
mm.
Average
days
At lab. temp, of
upprox 29 5° C
7
9 7
11 1
12 5
14 7
18
12 2
At 33° C. . .
5
7.8
9.2
9 6
11 5
14 1
9 5
that light of 75 foot-candles has little if any effect on the moulting of
these animals. Crangon that are kept in culture dishes on the laboratory
table moult only in the daytime or early evening whereas at a constant
temperature they moult practically as often at night as in the daytime.
EFFECT OF AGE AND TEMPERATURE ON MOULTING RATE
Animals which were used in the preceding studies included 136 that
were kept in the laboratory at laboratory temperature, and 119 which
were kept at a constant temperature of 33° C. These were selected so
that there were approximately the same number of animals in each of the
six age groups shown in Table III. The average number of days be-
tween moulting periods for each size group is shown in the table. In
this study, size was taken as a general criterion of age.
As shown in Table III the length of the period between moults was
shortened, on an average, 2.7 days. By raising the average temperature
218
WALTER N. HESS
3.5° C. the moulting interval was decreased by 22.1 per cent. This is
in agreement with Smith (1940), who showed that the length of the
intermoult in young crayfish is directly dependent on temperature.
MOULTING RATE OF NON-SEEDED FEMALES, SEEDED FEMALES AND
SEEDED FEMALES FROM WHICH EMBRYOS WERE REMOVED
For this study, 30 non-seeded females, 30 seeded females, and 30
seeded females from which the embryos were removed were placed in
separate finger bowls on the laboratory table. All of the seeded females
selected including those from which the embryos were removed were
carrying very young embryos. Numbers were equally distributed among
the three size groups shown in Table IV.
TABLE IV
Comparison of the average moulting interval in days of non-seeded females of
different sizes with the interval between time of collecting and the next moulting
period of seeded females bearing very young embryos, and with that of seeded
females from which very young embryos were removed.
Size
20-25 mm.
25-30 mm.
30-35 mm.
Non-seeded females
11.4
12.1
14.3
Seeded temales
16.1
15.7
18.4
Seeded females from which em-
bryos were removed
11.2
10.3
12.9
In the above study it is impossible to state how much time elapsed
between the last moulting period and ovulation in the case of the seeded
females. The embryos that were attached to the seeded females were
from one to three days old when the experiment began. If these seeded
females moulted three or more days before ovulation, which seems
probable, the period between moults of the seeded females would be
approximately twice as long as that of non-seeded females. Moreover,
when the embryos were removed from the seeded females the period
between moults of these females was materially shortened. This indi-
cates that there is something which inhibits moulting in seeded females.
In no instance did a seeded female moult while she was carrying
embryos. However, all seeded females moulted within five days aftei
shedding their embryos and twelve moulted within one day.
MOULTING IN CRANGON ARMILLATUS 219
DISCUSSION
The data presented in this paper indicate quite clearly that light of
the intensity of 75 foot-candles has very little if any effect on moulting
in Crane/on anuillatus and hence cannot he considered as an important
factor in causing the diurnal moulting. On the other hand, the daily
rise and fall in temperature is a very important factor in causing the
diurnal moulting. Increase in temperature sets in operation the factors
causing moulting, while a fall in temperature checks them.
From the above data it seems probable that at least two factors are
concerned with moulting in Crangon annillatus. One, which causes
moulting, manifests itself when the temperature in which the animal lives
rises to or above approximately 29° C. The other, which inhibits
moulting in seeded females, is apparently dependent upon the attach-
ment of the embryos to the female. Moulting in insects, as shown by
\Vigglesworth (1934) and others is apparently caused by hormones.
Brown and Cunningham (1939). Hanstrom (1939), Abramowitz and
Abramowitz (1940), and Smith (1940) have shown the importance of
a moult-inhibiting substance produced in the eye-stalk of crayfish and
certain other crustacea.
CONCLUSIONS
1. Crangon annillatus exhibits diurnal moulting which begins in
mid-forenoon, reaches its height at about 1 :30 P.M. and ceases in later
afternoon or early evening.
2. Light of 75 foot-candles has very little if any effect on moulting
in these animals.
3. Temperature changes are very important in causing the diurnal
moulting. Increase in temperature sets in operation the factors causing
moulting while a fall in temperature checks them.
4. Animals kept at a constant temperature fail to exhibit diurnal
moulting.
5. By raising the average daily temperature approximately 3.5° C.
the moulting interval was decreased by 22.1 per cent.
6. Females carrying embryos do not moult even though the period
of carrying embryos exceeds the normal period between moults.
7. At least two factors appear to be concerned with moulting.
One, \vhich is greatly influenced by temperature changes, causes moult-
ing. The other, which inhibits moulting in seeded females, appears to
be dependent upon the attachment of the embryos to the female.
220 WALTER N. HESS
LITERATURE CITED
ABRAMOWITZ, R. K., AND A. A. ABRAMOWITZ, 1940. Moulting, growth, and sur-
vival after eyestalk removal in Uca pugilator. Biol. Bull., 78 : 179-188.
BROWN, F. A., JR., AND O. CUNNINGHAM, 1939. Influence of the sinusgland of
crustaceans on normal viability and ecdysis. Biol. Bull., 77: 104-114.
DARBY, HUGH H., 1938. Moulting in the crustacean, Crangon armillatus. Anal.
Rcc., 72 : Suppl., p. 78, No. 98.
HAN STROM, B., 1939. Hormones in Invertebrates. Oxford Press.
SMITH, RALPH I., 1940. Studies on the effects of eyestalk removal upon young
crayfish (Cambarus clarkii Girard). Biol. Bull. 79: 145-152.
WIGGLESWORTH, V. B., 1934. The physiology of ecdysis in Rhodnius prolixus
(Hemiptera). II. Factors controlling moulting and 'metamorphosis.'
Quart. Jour. Mic. Set.. 77: 191-222.
FEEDING MECHANISMS AND NUTRITION IN THREE
SPECIES OF BRESSLAUA
C. LLOYD CLAFF, VIRGINIA C. DEWEY AND G. W. KIDDER
(From the Arnold Biolin/ical Laboratory, Brou'ii [University, and the Marine
Biological Laboratory, Woods Hole, Massachusetts)
The question of food taking by protozoa has attracted considerable
attention in the past and there have appeared numerous accounts of the
various mechanisms employed, the type of food taken and the conditions
of acidity and alkalinity during the digestive process. Regarding the
last-mentioned observations there seems to be general agreement that,
in the bacteria-feeding species at least, there is an acid-alkaline cycle
from the time the food is ingested until the residue is defecated. Prac-
tically all of the observers employed some type or combination of types
of indicator dyes, watching for the color changes which occur as the
food is ingested, digested and the residue defecated. The most fre-
quently used indicator has been neutral red because of the ease with
which most protozoa take up this dye. Unfortunately, this indicator is
useful only to detect shifts in hydrogen ion concentrations through a
relatively small range.
We have examined the problem of feeding and acid-alkaline reac-
tions in three species of the genus Bresslaua. These ciliates are car-
nivorous members of the family Colpoclidae and one species, due to its
peculiar feeding habits, offers exceptional opportunities for direct ob-
servations for long, uninterrupted periods of time.
In the ensuing account we will give a short description of the experi-
mental organisms, an account of their feeding habits, some evidence for
an acid-alkaline cycle during digestion and a brief account of food
selectivity.
MATERIAL AND METHODS
The carnivorous ciliates were obtained from dry hay collected in
Stuart, Florida. The same procedures of excystation and isolation were
employed as were previously used in the case of most of the Colpoda
material reported from this laboratory (Kidder and Gaff, 1938; Kidder
and Stuart, 1939; Burt, 1940).
For studies on the hydrogen ion concentration within the vacuoles
221
CLAFF, DEWEY AND KIDDER
and the protoplasm various indicator dyes were used. These will be
described in a later section. It was found expedient first to stain the
food organisms (usually Colpoda steinii) and then to add a few Brcsslaua
to the culture. The culture was then placed in a moist chamber until
the food organisms had all been ingested. As in the case of Woodruffia
metabolica (Johnson and Evans, 1939; 1940), these carnivores formed
resistant cysts after the food had become depleted. These were caused
to excyst by the addition of fresh hay infusion. Food organisms were
then added and the feeding process studied under a water immersion
lens (X40). The dye brought into the protoplasm of the carnivores
during the previous period of feeding was sufficient to allow us to gain
an idea of the changes in acidity and alkalinity which took place during
feeding, digestion and the subsequent defecation of residue.
For the study of food selectivity bacteria-free ciliates were neces-
sary. The Bresslaua were freed of their associated microorganisms by
the employment of our modification of the Parpart method of direct
washing, using Syracuse watch glasses enclosed in cellophane bags
(Kidder, Lilly and Claff, 1940). Because of the structural peculiarities
of these ciliates it was found necessary to allow them to encyst and
divide after the tenth wash in sterile hay infusion. Close watch was
kept of the dividing ciliate so that the washing could be continued imme-
diately after the emergence of the daughter organisms. Each of the
two, four or more daughters was then washed individually through five
or more changes of sterile medium and placed in tubes containing the
food organism to be tested. Adequate bacteriological tests showed that
the majority of the carnivores so treated were free of bacteria.
The food organisms tested will be discussed in a later section. They
have all been mentioned in previous accounts from this laboratory.
Description of Brcsslaua Kahl
The three members of this genus which we have studied resemble
the various species of Colpoda in their general structure, mode of divi-
sion within a cyst and permanent cyst formation. They all possess a
macronucleus of the Colpoda citcullus type (Kidder and Claff, 1938;
Burt, 1940) and a single micronucleus. The chief differences are
found in the structure of the mouth, which has become modified and
extended for the carnivorous mode of life. The following brief de-
scriptions are given to add to the account of Kahl (1931) of B. vora.v
and to establish two new species.
Bresslaua vora.v Kahl (Fig. 1, A}. — This species is evenly rounded
posteriorly, but the anterior end is compressed laterally. The left an-
FEEDING AND NUTRITION IN BRESSLAUA 223
terior side is depressed in such a way that the whole anterior end is
twisted. This twisted appearance is seen best in an organism imme-
diately after excystment. The size varies greatly depending upon the
amount and kind of food taken. Freshly excysted ciliates range in
length from 40 p, to 90 p, and in width from 25 //. to 50 p.. Ciliates which
have fed on relatively large prey (such as Glaucoma scintillans or Col-
poda cucullus) attain a size of 180 /x X 100 /A or even larger.
The ciliary pattern, as seen after the silver technique of Klein or
when treated with opal blue or nigrosin, resembles that of other members
of the family. The peripheral cilia arise in pairs, as is true of most of
the cilia among the members of the genus Colpoda (Taylor and Furga-
son, 1938; Burt, 1940). This is in contrast to the condition in Wood-
ruffiia metabolica (Johnson and Larson, 1938) where the cilia are single.
The cilia are relatively short and delicate. The ciliary rows originate
from a short keel and extend over the general body surface as well as
the right interior of the cytostomal cavity, converging in a field at the
posterior end of the body.
The mouth is a large, cilia-lined cavity, open toward the ventral
surface and the left side. On the roof of the mouth are folds or
" rugae," roughly resembling those on the hard palate of mammals. On
the floor of the mouth, which is somewhat raised, there is a row of
membranelle-like structures, 40 to 45 in number. These beat in such a
way as to create a strong current out of the mouth. At the back of the
mouth there is a rather short, broad gullet directed posteriorly. It is
on the brink of this gullet that the membranelles are located.
Bresslaua vorax exhibits activity when not actually feeding. It tends
to remain on or near the bottom of the culture and to move in small
circles. It comes in contact with the bottom so that the left side of the
body, and therefore the mouth-opening, is up. Prey are swept into the
mouth by strong currents. During the time the live prey is in the mouth
until it has entered the food vacuole at the base of the gullet the move-
ment of the carnivore is much reduced. This is due to a change in the
beating of all the peripheral cilia and will be described in greater detail
in the case of one of the other species. After the prey has been success-
fully trapped in the posterior food vacuole, movement is resumed.
Bresslaua insidiatrix sp. nov, (Fig. 1, B}. — The general departures
from the Colpoda-\ike structure which were described for B. vorax are
accentuated in this species. The mouth opening is more extensive in
relation to the size of the body and the twisting of the anterior end is
somewhat greater. No " rugae " are present in the mouth. This spe-
cies varies in size from 40 ^ X 25 p. when starved to 120 p X 90 p, when
224
CLAFF, DEWEY AND KIDDER
;
A
* ^
B
«
I W.-M
FIG. 1. All drawings were taken from life. X 460. A. Bresslaua vorax.
The food inclusions are Glaucoma scintillans. B. Bresslaua insidiatrix sp. nov.
during early stages of feeding on Glaucoma scintillans. C. Bresslaua sicaria sp.
nov. after ingesting a number of Colpoda stcinii.
FEEDING AND NUTRITION IN BRESSLAUA 225
ready to divide after active feeding. The general pattern of the periph-
eral ciliary lines is similar to that in B. vorax. The cilia originate in
pairs and are very long and stiff. They are easily visible in life and
stand out at nearly right angles to the body while the organism is at
rest. There are 10 to 15 membranelle-like structures in the mouth lo-
cated in the same relative position as those in B. vorax.
One of the most characteristic things about Brcsslaua insidiatrix is
its mode of feeding. It normally rests on the bottom of the culture
dish with its right anterior end in contact with the substratum. It will
remain for two to three hours in one spot, only occasionally pivoting
slightly. During this time there is a strong current being directed into
the very large mouth-opening and all small objects are drawn in. In-
animate objects are rapidly whirled toward the posterior border of the
mouth and shot out by means of the out-going current created by the
membranelles. Moving ciliates or flagellates, on the other hand, receive
different treatment. Some mechanism within the mouth seems to be
stimulated by ciliated or flagellated organisms and this appears to affect
the whole neuromotor system. The peripheral cilia immediately lose
their stiff, vibratile appearance and move slowly in waves (Fig. 2, B, C).
The current going into the mouth slackens or disappears as does the out-
going current along the posterior border. The mouth-opening is con-
tracted, forming an efficient barrier against the escape of the prey. The
prey moves about freely in the mouth for from one to two minutes and
gradually the posterior border of the mouth begins to form the prospec-
tive food vacuole. This vacuole forms well ahead of the prey and not
under direct impact of it. The prey may partially enter the forming
food vacuole and draw back into the mouth a number of times before
it is finally trapped. Once well within the vacuole it begins to rotate
and the vacuole closes off. The closure is effected by what appears to be
a thin sheet of protoplasm originating from the region just posterior to
the zone of membranelles and flowing across the vacuole opening from
ventral to dorsal. At the instant the sheet of protoplasm fuses with the
opposite side the prey is killed. This phenomenon will be discussed later
in the section on hydrogen ion concentrations. The closure of the vacu-
ole also sets off another reaction which immediately causes the peripheral
cilia to resume their stiff, vibratile condition (Fig. 2, D).
Bresslaua insidiatrix appears to be the most highly specialized for a
carnivorous habit of the three species observed by us. It feeds only on
living ciliates and flagellates. Other bodies (cysts, amoebae, algae, yeast
and detritus) do not evoke the " swallowing " response. That this
evocation is largely physical is indicated by the following fact. In an
excysting culture of Colpoda steinii it is common to see these small
226 CLAFF, DEWEY AND KIDDER
ciliates rotating rapidly within the thin endocyst. These ciliates may
be drawn into and swept out of the mouth of B. insidiatrix a number of
times while the endocyst is still intact, but immediately the Colpoda es-
capes its cyst wall and is drawn into the mouth, it evokes the general
responses noted above. In contrast to this, both Bresslaua vorax and
the third species, yet to be described, are able to ingest certain types of
non-moving microorganisms, but not all organisms ingested are adequate
as food.
Bresslaua sicaria sf>. nov. (Fig. 1, C). — This species shows a closer
resemblance to the typical Colpoda-form than either of the above-
mentioned species. The mouth opening is confined to the ventral surface
and does not extend to the left side. The interior of the mouth cavity
is similar in structure and relative size to that of B. vorax, but lacks
" rugae." The zone of membranelle-like structures is composed of from
20 to 25 components and occupies the same general position as that in
the preceding species. A well-formed gullet is present running pos-
teriorly a short distance into the cell.
Bresslaua sicaria varies from 35 ju, to 110 /A in length and from 23 /A
to 92 ^ in width depending upon its state of nutrition. The peripheral
ciliary lines are less numerous than those of the other two species, but
the general patterns are very similar. The cilia are long and wavy and
originate in pairs.
Bresslaua sicaria, unlike the other two species, rarely comes to rest.
It swims in a characteristic looping fashion and draws its prey into the
mouth while swimming. There is a change in the ciliary motion during
the act of swallowing resulting in general and violent movement of the
whole organism. Immediately a food organism is caught the Bresslaua
starts rotating rapidly on its lateral axis and continues the rotation until
the prey enters the vacuole, when it resumes its swimming motion. The
feeding reactions of this species are very difficult to observe because of
its extreme activity.
The feeding habits of the three species described above are so charac-
teristic that it is possible to identify each of them under very low magni-
fications. Bresslaua vorax and B. insldiatrix take their prey while they
are in contact with the solid substratum, while B. sicaria feeds while
swimming free in the medium. Of the first two, only Bresslaua in-
sidiatrix remains motionless while waiting for its prey. Because of this
characteristic, B. insidiatrix is an ideal carnivore to use in experiments
and observations on feeding mechanisms.
The establishment of two new species of the gen,us Bresslaua seems
to us to be justified because of the characteristics noted above (number of
FEEDING AND NUTRITION IN BRESSLAUA 227
ciliary rows, length and characteristics of cilia, shape and extent of cyto-
stomal opening, feeding habits and food selectivity).
FOOD VACUOLES AND HYDROGEN ION CONCENTRATION
After Bresslaua insidiatrix has fed on Colpoda steinii previously
stained with a 1 : 12 million dilution of neutral red, it becomes highly
colored by virtue of its food inclusions. After the food has been ex-
hausted the carnivores form protective cysts. Many red food balls are
still present in the encysted organisms. These food balls are defecated
during or shortly following excystment (Fig. 2, A, B), leaving the cili-
ates nearly colorless. Under the water immersion lens it is possible to
detect a number of neutral red stained granules in the endoplasm. Ex-
cystment with alkaline hay infusion imparts a slight yellowish tinge to
the medium, but does not change the color of the endoplasmic granules.
The small freshly excysted ciliates settle to the bottom of the culture dish
and immediately begin feeding when numbers of Colpoda steinii are
added with the excysting fluid (Fig. 2, B). The clearest observations
are made during the capturing and killing of the first several ciliates.
As the prospective food vacuole forms its fluid contents become
slightly pink (Fig. 2, C] . This coloration deepens as the prey enters,
but there appears to be no change in the motions of the prey at this time.
At the instant that the food vacuole is closed off by the protoplasmic
sheet there suddenly appear a large number of brilliant red granules or
droplets in the protoplasm surrounding the vacuole (Fig. 2, D). The
fluid surrounding the prey then becomes more deeply colored and simul-
taneously the prey is killed. The prey becomes motionless and the cilia
stand out from the body. The fluid rapidly disappears from the vacuole
and its lining comes to lie very close to the prey. The red granules in the
cytoplasm rapidly fade out. There appears to be no indication that they
enter the vacuole as has been described by Nirenstein (1905) for Para-
mecium. This is the first color-change to be noted. The reaction with
neutral red shows that an acid condition is suddenly attained and that
the hydrogen ion concentration is equal to or less than a pH of 6.8.
The above observations were repeated a number of times using a
number of indicator dyes. None of them was quite as spectacular as the
neutral red, either because they did not penetrate or because the colors
were more difficult to see. Methyl red, methyl orange, brom cresol
green, brom phenol blue, brom phenol purple, chlor phenol red, para-
dimethyl-amino-azobenzene (Topfer's reagent), Congo red and benzene-
azo-alpha-naphthylamine were used and of these methyl red was by far
the best. Although not as brilliant as the neutral red reaction, all of
CLAFF, DEWEY AND KIDDER
the phases appeared with this dye. The appearance of bright red gran-
ules with methyl red indicates that their acidity must be in the neighbor-
hood of pH 4.2 or lower. Failure of blue coloration with Congo red
indicates that the hydrogen ion concentration is probably not higher than
pH 3.0.
It appears likely that the sudden death of the prey is the result of
the release of an acid from the protoplasm of the carnivore into the
vactiole. Topfer's reagent failed to give positive results in this organ-
ism, although the dye penetrated well. Nirenstein (1905; 1925) had
reported using this dye to detect the presence of mineral acid in the
vacuole of Paramecium, as indicated by the appearance of a red color.
No red coloration was obtained in Bresslaua. Just what type of acid is
released is obscure.
Separate experiments show that the acidity indicated by the color
changes with methyl red are compatible with the death points of the
various types of prey. Thus, Colpoda steinii is killed almost instantly
in a phosphate buffer of pH 3.8, while the Bresslaua is still alive after
one hour at pH 3.4. C. stcinii died only after long periods at pH 4.5
and above this value no death was observed. This experiment simply
shows that Bresslaua is more resistant to high acidity than is Colpoda
and lends support to the idea that the killing within the vacuole is a
result of the release of acid. A similar conclusion regarding the func-
tion of acid was reached recently by Mast and Bowen (1940) in the case
of Vorticella. Other food organisms which were tested were more re-
sistant than Colpoda. Euglena yracilis and Astasia klebsii survived for
a long time at pH 3.8 and this checks writh the reactions of these two
flagellates within the food vacuole of Bresslaua. After the protoplasmic
sheet has closed over either of these organisms there elapses from two
to ten minutes before euglenoid movement ceases.
Following the killing process the body of the Colpoda begins to move
anteriorly due to the general cyclosis of the protoplasm of the Bresslaua.
When the prey contains an indicator dye, such as neutral red, it is pos-
sible to follow the color changes occurring during the hour or two re-
quired for digestion. At first the prey is nearly colorless, but it rapidly
becomes yellowish. This indicates a faintly alkaline reaction and corre-
sponds to the situation found in Paramecium (Nirenstein, 1925) except
that the vacuole has never been observed to swell. The yellow color re-
mains for from 15 to 20 minutes and then gradually changes through
orange to a bright cherry red (Fig. 2, £). By the time the prey has
reached the red condition its general outline is lost and it has become a
compact ball. A number of these balls later fuse and form the fecal
mass which is extruded during or following the next encystment, either
B
C
.'••"
• '*. •
FIG. 2. Brcsslaua insidicitri.r showing the color changes with neutral red dur-
ing feeding. A. Freshly excysted ciliate with old residue. Note the position of
the cilia. B. Trapping of prey, Colpoda stcinii, and defecation of residue. The
cilia are bent and move in slow waves during this stage. C. Prey entering the pros-
pective food vacuole, the fluid content of which is a faint pink. D. Food vacuole
closed off from the mouth. At this stage the prey is instantly killed. Note the
appearance of the cherry red granules in the cytoplasm surrounding the vacuole.
The peripheral cilia of the carnivore have again assumed a stiff appearance. E.
Carnivore after having ingested a number of ciliates. Note the color changes in
the bodies of the prey as digestion proceeds.
230 CLAFF, DEWEY AND KIDDER
from a division cyst or a protective cyst. Once released into the sur-
rounding medium the fecal masses rapidly lose their red color, become
pale yellow and disintegrate.
As mentioned previously, these observations are best made with
Bresslaita insidiatri.r, because of its feeding habits. As far as could be
detected, the same general phenomena take place in the other two species.
Certainly the color changes during digestion and defecation are the same,
but the color changes accompanying killing, being of such short duration,
could never be definitely established due to the constant movement of the
carnivores.
FOOD SELECTIVITY
The following account of the food selectivity is based on our observa-
tions of the three species of Bresslaua in the presence of a mixed flora
of bacteria and in bacteria-free culture. \Yhile these observations do
not represent a complete survey of the possibilities, they are presented in
order to indicate the differences between the species and the possibilities
for future work. In Table I we have listed the various food organisms
which were used and have summarized the pertinent observations. It
will be noted that ingestion does not invariably mean that the organism
in question represents adequate food for growth. The various species
of Colpoda supported growth in all three species of Bresslaua and these
ciliates probably represent their natural food. The very nature of their
protective cyst formation makes this assumption plausible. When dry
hay is placed in spring water the various species of Colpoda excyst first,
feed and multiply lief ore the Bresslaua excyst. This means that in na-
ture there would usually be a source of Colpoda at the right time.
Glaucoma seintillans was ingested by Bresslaua vora.v and B. insi-
diatri.r, while in the case of B. sic aria this was never observed. Thriv-
ing bacteria-free cultures of B. I'ora.r and B. insidiatri.r were maintained
for a number of months with G. seintillans as food. In neither case,
however, were normal protective cysts formed. After the food or-
ganisms had all been ingested the carnivores continued in the trophic
condition for many days, getting smaller and smaller. Occasionally, in
the case of B. Tora.r, they would round up and form temporary cysts
(Johnson and Evans. 1940) from which they would spontaneously ex-
cyst within a few hours. This process might be repeated for days until
finally all of the carnivores were dead. Serial transplants were always
made while some food was still present. Eventually the various series
declined in division rate and failed in transfer. The causes associated
with this decline must receive further investigation.
FEEDING AND NUTRITION IN BRESSLAUA
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CLAFF, DEWEY AND KIDDER
Tetrahymena yclcii was ingested by all three species but did not
support continued growth in any case, although a few divisions of
Bresslaua vorax usually resulted. Tetrahymena appears to be toxic to
B. insidiatrix, for after a single organism had been ingested the car-
nivore would leave the bottom of the dish, swim rapidly for a few
minutes and then round up and encyst. These cysts were never viable.
Bresslaua sicaria behaved in a similar manner.
One other item worthy of note in these investigations on nutrition
is the case of Stichococcus bacillaris. Bresslaua vorax readily ingests
this alga and flourishing cultures result. Normal protective cysts are
formed and may be collected, dried and stored for future use. By use
of the glass plunger-sponge method (Kidcler, 1941) any number of
sterile carnivores may be kept on hand.
DISCUSSION
While the work on the bacteria-free cultures has not progressed to
a point where the nutritional requirements of the members of the genus
Bresslaua can be stated definitely, a number of points of interest have
come to light. One of the most interesting observations is the great
difference in the food organisms as evidenced by the differences in nutri-
tional quality between Glaucoma sclntdlans, Tetrahymena geleii and the
various species of Colpoda for Bresslaua. Colpoda was utilized by all
three species of Bresslaua. Glaucoma was utilized by Bresslaua vorax
and B. insidiatrix, while Tetrahymena was utilized by only B. vorax and
then the growth was poor and not transplantable. This is exactly the
reverse of the situation with the carnivorous hypotrichs, Stylonychia
and Plcurotricha. Lilly (1942) has shown that these ciliates will feed
and reproduce on Tetrahymena but not on Glaucoma and Colpoda. It
is becoming apparent that the exact nutritional requirements of car-
nivorous ciliates are delicately adjusted and this fact may be of use in
the future for comparisons between food organisms.
Regarding our observations on the hydrogen ion changes during swal-
lowing, killing, digestion and defecation, a few comparisons with reports
of other workers may be noted. Prowazek (1898) described neutral
red granules around the periphery of food vacuoles of Paramecium, He
supposed that these granules might be the carriers of the digestive
enzymes. An alkaline stage during the digestive process was not de-
scribed. The work of Nirenstein (1905; 1925) was the most complete
on this subject. He describes an initial acid phase in the newly-formed
food vacuoles of Paramecium, the pH being equal to that of a 0.3 per
cent solution of HC1. These vacuoles were much more acid than com-
FEEDING AND NUTRITION IN BRESSLAUA
parable ones in a number of other ciliates. After the initial acid stage
the food vacuoles increased in volume and became alkaline. Nirenstein
believed that digestion occurs only at this stage, the digestive enzymes
being trypsin-like in nature. It had earlier been proposed by Hemmeter
(1896) that the appearance of an acid phase was the response to living
prey and that the acid served as a killing agent. This contention was
denied by Metalnikow (1912) because he was unable to demonstrate any
regularity in the acid production even in the event that living prey were
ingested. In our work on Bresslaua insidiatrix the conclusion was
reached that the initial acid production around the vacuole was stimu-
lated by the closure of the vacuole and that it was probably this acid
which caused the death of the prey. The prey did not become acid in
its reaction, however, which may have been due to the combination of
the acid with its proteins. Later, enough alkaline material was taken up
to cause the protoplasm of the prey to give an alkaline reaction. This
alkaline phase appears to be the phase of active digestion, indicating,
therefore, that the enzymes involved are catheptic in nature. Before
defecation the residue becomes acid, possibly due to the acidic properties
of some of the products of digestion. The appearance of a final acid
stage in the food vacuole in Bresslaua seems to differ from the condition
in Paraiiicciuiu. In the latter organism the residue remains alkaline
(Shapiro, 1927).
Up to the present time most of the observations on the hydrogen ion
concentration of food vacuoles have been confined to bacteria-feeding
ciliates. It will be interesting to see if the conditions described above
will be found in other carnivorous types.
SUMMARY
1. Three species of Bresslaua,, B. vorax Kahl, B. insidiatrix sp. nov.
and B. sic aria sp. nov. are described.
2. These ciliates are carnivorous and feed on other small ciliates,
members of the genus Colpoda being especially favorable as food.
3. Using indicator dyes it was found that the prey is killed simul-
taneously with a sudden release of an acid into the newly-formed food
vacuole. The hydrogen ion concentration of the vacuole fluid was esti-
mated to be between pH 3.0 and pH 4.2. This range includes the death
point of various species of Colpoda. During digestion the protoplasm
of the prey becomes alkaline. The undigested residue becomes acid
before defecation.
4. Bacteria-free Bresslaua were tested with a number of food or-
ganisms and a preliminary survey of their food requirements recorded.
234 CLAFF, DEWEY AND KIDDER
LITERATURE CITED
BURT, R. L., 1940. Specific analysis of the genus Colpoda with special reference to
the standardization of experimental material. Trans. Am. Mic. Soc., 59:
414-432.
HEMMETER, J. C., 1896. On the role of acid in the digestion of certain rhizopods.
Am. Nat., 30 : 619-625.
JOHNSON, W. H., AND F. R. EVANS, 1939. A study of encystment in the ciliate,
Woodruffia metabolica. Arch. f. Protist., 92: 91-116.
JOHNSON, W. H., AND F. R. EVANS, 1940. Environmental factors affecting cyst-
ment in Woodruffia metabolica. Physiol. Zool., 13: 102-121.
JOHNSON, W. H., AND ENID LARSON, 1938. Studies on the morphology and life
history of Woodruffia metabolica, nov. sp. Arch. f. Protist., 90: 383-392.
KAHL, A., 1931. Urtiere oder Protozoa. I. Wimpertiere oder Ciliata (Infusoria).
In: Dahl's Tierwelt Deutschlands, Teil 21. Fischer, Jena.
KIDDER, G. W., 1941. The technique and significance of control in protozoan cul-
ture. In: Protozoa in Biological Research. Ed. G. N. Calkins and F. M.
Summers. Columbia University Press, New York.
KIDDER, G. W., AND C. L. CLAFF, 1938. Cytological investigations of Colpoda
cucullus. Biol. Bull., 74: 178-197.
KIDDER, G. W., D. M. LILLY AND C. L. CLAFF, 1940. Growth studies on ciliates.
IV. The influence of food on the structure and growth of Glaucoma vorax
sp. nov. Biol. Bull, 78: 9-23.
KIDDER, G. W., AND C. A. STUART, 1939. Growth studies on ciliates. I. The role
of bacteria in the growth and reproduction of Colpoda. Physiol. Zool.,
12: 329-340.
LILLY, D. M., 1942. Nutritional and supplementary factors in the growth of
carnivorous ciliates. Physiol. Zool. (in press).
MAST, S. O., AND W. J. BOWEN, 1940. The hydrogen ion and the osmotic con-
centrations of the cytoplasm in Vorticella similis (Stokes) as indicated
by observations on the food vacuoles. Biol. Bull., 79: 351 (Abstract).
METALNIKOW, S., 1912. Contributions a 1'etude de la digestion intracellulaire chez
les protozoaires. Arch. Zool. exper. et gen., 5e ser., 9 : 373-499.
NIRENSTEIN, E., 1905. Beitrage zur Ernahrungsphysiologie der Protisten. Zcit-
schr. f. allg. Physiol., 5 : 435-510.
NIRENSTEIN, E., 1925. Uber die Natur und Starke der Saureabscheidung in den
Nahrungsvacuolen von Paramecium caudatum. Zeitschr. f. zsrisscns. Zool.,
125: 513-518.
PROWAZEK, S., 1898. Vitalfarbungen mil Neutralroth an Protozoen. Zeitschr. f.
wissens. Zool, 63 : 187-194.
SHAPIRO, N. H., 1927. The cycle of hydrogen-ion concentration in the food
vacuoles of Paramecium, Vorticella and Stylonychia. Trans. Am. Mic.
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TAYLOR, C. V., AND W. H. FURGASON, 1938. Structural analysis of Colpoda duo-
denaria sp. nov. Arch. f. Protist., 90 : 320-339.
SECRETION 1 OF INULIN, XYLOSE AND DYES AND ITS
BEARING ON THE MANNER OF URINE-FORMATION
BY THE KIDNEY OF THE CRAYFISH
N. S. RUSTUM MALUF-
(From the Department of Zoology, Tlic Johns Hopkins Unircrsity, ami the
Department of Tropical Medicine, The Tnlane University)
INTRODUCTION
The following data are the outcome of an attempt to find whether
filtration occurs through the nephron of the crayfish. While they do not
conclusively exclude filtration, they are sufficiently interesting to warrant
presentation.
In contrast to the glomerular kidney of vertehrates, the aglomerular
vertebrate kidney cannot eliminate glucose, even during hyperglycaemia
and phloridzination (Marshall, 1930), and cannot eliminate xylose (Jol-
liffe, 1930). or inulin (Richards, Westfall, and Bott, 1934). Further-
more, there is little or no doubt that inulin is not secreted by nor passively
resorbed through the vertebrate nephron (see Smith, 1937). Accord-
ingly, it was presumed that the presence or absence of inulin in the urine
of the crayfish (a classical freshwater invertebrate), after its injection
into the haemocoele, would demonstrate whether filtration occurs.
It is here shown that although inulin and xylose do appear in the urine
of the crayfish, they are, at least in part, actually secreted. It is therefore
unnecessary to invoke filtration to explain the excretion of these carbo-
hydrates. The ability of all parts of the nephron of this animal to se-
crete or accumulate one dye or another (see below) and of the coelomosac
to secrete calcium (Maluf, 1941a) indicates that this nephron is mainly,
if not entirely, a secretory organ.
The subject is Cainbants clarkii, which frequents the freshwater
swamps of southern Louisiana.
1 Throughout this paper, secretion implies the transport of a substance from a
region of lower to one of higher diffusion potential for that substance. Excretion
refers merely to the outward elimination of undesirable material, regardless of
whether the latter is secreted or filtered.
This work was begun in the Department of Zoology, The Johns Hopkins Uni-
versity, while the author was Johnston Research Scholar. Many thanks are due
to Prof. S. O. Mast for numerous kindnesses and appreciative criticism.
2 Now of the Department of Physiology, School of Medicine, Georgetown
University, Washington, D. C.
235
236 N. S. RUSTUM MALUF
METHODS
Inulin
Analysis. — The concentration of inulin in the blood and urine was
measured, after acid-hydrolysis, by the Shaffer-Hartmann-Somogyi
method (see Shaffer and Somogyi, 1933) using Shafrer-Hartmann re-
agent " 50 " and Somogyi 's (1931) procedure for deproteinization of the
serum. The technique was adapted to the small quantities used in this
work as follows. Blood was taken by amputating a leg at the femur
and allowing about 0.15 cc. to run into a small test-tube (9 X 75 mm.).
Bleeding was instantly and permanently stopped by compressing the
stump with the hot tips of a blunt forceps. The test-tube was stop-
pered and heated at 80° C. for about a minute or until the blood became
opaque and was then cooled rapidly. The resulting solid was broken up
with a fine glass-rod and the tube centrifuged for a few minutes. A
sample of the supernatant liquid, usually about 75 cti.mm. was drawn
into a fine calibrated pipette of the constricted type (Fig. I, A). It was
deproteinized by adding an equal volume of 7 per cent CuSO4-5H2O and
another equal volume of 10 per cent Na2WO4-2H.,O. Distilled water
was added according to the required dilution (5 to 16 times), the same
pipette being used in adding the water and reagents as that in taking the
sample. After stoppering, shaking, and permitting to stand for at least
20 minutes, the tubes were centrifuged and 80 cu.mm. aliquots drawn for
analysis. About 150 cu.mm. distilled water were added to increase the
volume and then about 70 cu.mm. N H2SO4 for hydrolysis. The tubes
were capped with glass-bulbs and heated in rapidly boiling water for 15
minutes. After cooling, a small drop of phenolphthalein was added.
The solutions were neutralized with N KOH. If the color became too
intense it was brought to pink with 0.1 N H2SO4 ; 0.161 cu.mm. of the
Shaffer-Hartmann reagent was added and then a few drops of distilled
water to augment the volume to about 1 cc. The test-tubes were shaken,
capped with glass-bulbs, and heated without agitation in rapidly boiling
water for 15 minutes. After cooling, the cap was removed only just
before the contents of that tube were to be titrated and about 250 cu.mm.
N H2SO4 introduced. The solid was completely dissolved with a glass-
rod without undue agitation and the contents titrated with 0.01 N
Na2S2O3 until the color, due to the free L, became a very light yellow.
About 35 cu.mm. of a 1 per cent aqueous solution of starch were added
and the titration continued to the end-point. Titration was from a
Linderstrp'm-Lang-Keys microburette of 250 cu.mm. capacity, divided
into cubic millimeters, and of uniform bore as shown by measurements
of the length of a drop of mercury at all levels. The concentration of
URINE-FORMATION IN CRAYFISH KIDNEY
237
inulin was ascertained by interpolation in a graph established from
aqueous solutions containing a known quantity of the inulin (Pfanstiehl
inulin, c.p.). Blanks and standards were run frequently. To obtain
values with respect to the plasma, 5 per cent was deducted from the
ascertained value, this being the approximate quantity of total solids in
the whole blood of the crayfish and presumably close to the quantity
which fell out by heating the blood at 80° C. The accuracy was within 5
per cent of the amount present.
FIG. 1. A. Volumetric micro-pipette of the constricted type with mouth-piece
and rubber tubing, c, cotton-plug. B. Apparatus for the collection of urine and
emptying of bladders. Arrow indicates direction of suction.
The urine was treated in the same manner as the blood with the ex-
ceptions that heating and deproteinization were unnecessary, the urine
being protein-free, and that, in the first 26 experiments, a correction for
evaporation had to be applied (see below).
The preparation of inulin contained some impurity which, without
being hydrolyzed, reduced Benedict's qualitative and which could not be
N. S. RUSTUM MALUF
removed by yeast. The Pfanstiehl Company advised us that they had
not been able to eliminate the impurity by repeated crystallization. The
impurity was, however, negligible because within less than an hour after
the injection of inulin, in the quantities used in this work (see Table II),
the blood (whole or protein-free) was non-reducing unless subjected to
acid-hydrolysis. Evidently the tissues removed the reducing substance
rapidly. At no time could the urine reduce Benedict's reagent without
preliminary acid-hydrolysis. Yeast-adsorption was therefore unneces-
sary in the analysis of inulin. Possibly because the animals were starved
for a few days, the blood (whole or protein-free) of unsubjected animals
was non-reducing even with preliminary acid-hydrolysis ; the urine of
these animals was invariably non-reducing. Crayfish can endure, with-
out appreciable injury, starvation for four months at least (Brunow,
1911).
Inulin-clearance. — The renal clearance of a substance has a definite
physiological meaning, being the virtual volume of blood cleared of that
substance per unit time by the kidneys. It is expressed by C - UV/P,
in which C is the clearance, P the concentration of the substance in the
plasma, and V the rate of urinary flow. It is necessary to know the
average concentration of that substance in the blood throughout the
time that the urine to be analyzed is being formed.
Before an experiment, the crayfish was kept overnight fully sub-
merged in running aerated freshwater. In measuring the inulin-clear-
ance, the integument and branchial chambers were drained of moisture,
the animal was weighed, and a fraction of a cc. of crayfish-saline 3 con-
taining a given quantity of inulin injected slowly through the proximal
abdominal venter and the wound cauterized. The amount of inulin
injected was adjusted mainly by altering the concentration of the dis-
solved inulin in the saline because it was not desired to augment the
blood-pressure by injecting a relatively large quantity of liquid (Table
II). By thorough bleeding, the total quantity of blood in an average-
sized Cambarus clorkii was found to be 6.6 per cent of the wet weight,
which corresponds closely to the 6.7 of Herrmann (1931), who used
the same method with Potamobius astacus.
After about 45 minutes the animal, including its branchial chambers,
was drained of moisture and the anterior margins of the latter plugged
with cotton- wool. The bladders were emptied by suction (about 12 mm.
Hg) applied at the nephropores through the arrangement in Fig. 1, B.
3 The saline was based on the most acceptable data on the concentration of
inorganic electrolytes in the blood of the crayfish (see Maluf, 1940, for references)
and was as follows (g./l.) : NaCl, 7.81; CaCL, 1.31; MgCl,, 0.82; KC1, 0.70;
buffered at pH 7.6 by 0.5 cc. M/5 Na2HPO4/NaH2PO4. This assumes a A of
about 0.66° C. (see Lienemann, 1938, and Schlatter, 1941).
URINE-FORMATION IN CRAYFISH KIDNEY
239
\Yhen no urine could be obtained, firm bilateral digital pressure was
applied to the integument lateral to the bladders (Maluf, 1941 a) and
suction again used until no further urine issued. The first sample of
blood was taken immediately after and at what was considered zero
time. The concentrations of inulin in blood from the pericardial sinus
and from a leg were practically identical at that time, thus showing uni-
form distribution of the foreign material. The nephropores were cau-
terized to ensure a dry surface. "Ames Temporary [dental] Cement:
FIG. 2. A. Manner of handling the crayfish while the bladders are emptied
and the nephropores are being sealed, c, cotton-plug in anterior margin of right
branchial chamber ; .s-, seal on the basal segment of the left antenna. B. Dorsal as-
pect of a crayfish with dorsal part of carapace and crop-gizzard removed, showing
both bladders, bl, distended. C. Same as B but with viscera moved posteriorly and
with wad of cotton, r, between viscera and bladders, bl.
hydraulic, non-irritant " was applied to the excretory eminence and basal
segment of the antenna in two layers, under a magnification of 10.5 X,
by means of a forceps. The animal was handled as shown in Fig. 2, A.
The cement should not extend beyond the basal antennal segment because
movement of the antenna would be likelv to crack the dried seal. Hard-
240 N. S. RUSTUM MALUF
ening, which is due to the formation of zinc phosphate from zinc oxide
and phosphoric acid, is rapid. Other cements were tried but were
incomparably inferior. Ten minutes after completing the application
the animal was fully immersed in freshwater and kept undisturbed.
Three or four blood-samples were taken through the experiment,
which lasted 8 to 15 hours. Immediately after the last sample, the
ventral nerve-cord was transected at the proximal level of the abdomen
so as to prevent abrupt abdominal flexion, the chelipeds were amputated
basally, and the dorsal surface of the carapace and the crop-gizzard,
which is wedged over and between the bladders, were carefully removed.
The distended bladders presented themselves conspicuously (Figs. 2. B
and C, bl ) . The urine contained in the translucent bladders was crystal-
clear and the kidneys could be seen beneath (Fig. 2, C). The viscera
were then pushed back and a wad of absorbent cotton (Fig. 2, C, r)
was applied over them to keep any fluid trom flowing near the bladders.
The urine was rapidly and completely collected by applying suction (about
12 cm. Hg) through the orifice of the arrangement shown in Fig. 1, B
to the surface of each bladder. The animal was tipped on the side of
collection with the head downward while this was done. The rate of
urinary flow was thus accurately measured. The rapid collection ob-
viated a correction for loss by evaporation. In the very few instances
in which both bladders were not equally distended, the kidney corre-
sponding to the lower rate of urinary flow was diminutive in size.
In the first 26 experiments on inulin-clearance the nephropores were
not sealed because it was assumed that undisturbed animals with emptied
bladders would not urinate appreciably during the interval. Urine, in
these instances, was collected by suction from the nephropores and a
correction applied for the fraction of water lost by evaporation. This
is quite appreciable, and because previous investigators have not taken
it into account the writer has no doubt that their values for the concen-
tration of solutes in the urine of the crayfish are higher than the actual.
The necessary corrections were obtained by aspirating, with the same
pressure, a known quantity of distilled water, from the tip of a fine
pipette of the constricted type (Fig. 1, A), into a test-tube of the same
dimensions as that used for the collection of urine (Fig. 1, B). The
resultant quantity of water, after light centrifugation of the test-tube
for a few seconds, was measured by drawing it into a calibrated glass-
tube. The loss in collecting 0.148 cc. in two minutes was 16.4 per cent
and that in collecting 0.0739 cc. in five minutes was 35 per cent. The
first correction was the one generally applied, as it was the writer's
policy to collect the maximal quantity of urine in the minimum time with
the above pressure. Generally an amount of 0.15 to 0.2 cc. could be
URINE-FORMATION IN CRAYFISH KIDNEY 241
readily collected within two minutes. A successful and rapid collection
depends to a great extent upon the aspirating tip. This should not have
sharp edges but should be blunt and regular ; its diameter should not be
so large as to cover the entire excretory eminence. There is no doubt
that the water lost was due entirely to evaporation. Scrupulous care
was taken to prevent water-contamination of the urine, by draining the
animal thoroughly, sucking water from the branchial chambers and
rostral region, and plugging the anterior margins of the chambers with
cotton-wool. The urine \vas not contaminated with blood, as was shown
by negative biuret-, heat-, and H2SO4-tests, by an uninjured operculum,
and by the fact that the concentration of inulin and dyes in the urine
was considerably greater than in the blood (see below).
The rate of urinary flow was not measured in the first 26 experi-
ments. It was assumed to be constant from one individual to another
per unit weight, being determined, in a fully submerged animal, by the
rate of diffusion of water into the body (Herrmann, 1931). Otherwise
the rate of flow was measured by the above technique which necessitates
sacrificing the animal at the end of the experiment. In ten experiments
with inulin (Table I), the rate averaged 5.0 cc. per 100 grams per 24
hours. This is quite close to the average (==5.2) of Lienemann
(1938), who collected the urine by aspiration from nephropores which
had been sealed, and was taken as the rate of flow for the animals in
the first 26 experiments. The rate of urinary flow7 in the crayfish is
low as compared with the frog (Forster, 1940) and freshwater turtle
(Friedlich, Holman, and Forster, 1940), and even relative to that in
birds and a terrestrial reptile (Marshall, 1932). This emphasizes the
low permeability of the gills of the crayfish to water.
The inulin-clearances and U/P's obtained through the use of direct
measurements of urinary flow with sealed nephropores (Figs. 6 and 7,
inulin; solid circles) and from an average rate of flow with unsealed
nephropores (Figs. 6 and 7, inulin; open circles) are quite comparable.
The average concentration of inulin in the blood through the ex-
perimental period was secured by averaging the interpolated values at
the mid-period of each hour (see curves representing concentration-time,
Fig. 3). Three or four blood-samples were sufficient to establish the
shape of the curves. Furthermore, it was undesirable to take more blood
than necessary.
Xylose
The analysis of xylose was identical with that of inulin except that
acid-hydrolysis was omitted.
The nephropores were sealed and the rate of urinary flow measured
directly (see above and Table I).
242
N. S. RUSTUM MALUF
FIG. 3. The concentration of inulin in the blood in mg. per cent (ordinate) as
a function of the time in hours (abscissa) during measurements of inulin-clearance.
URINE-FORMATION IN CRAYFISH KIDNEY
243
Three blood-samples were sufficient (Fig. 4). The average concen-
tration of xylose in the blood through the experimental period was calcu-
lated in the same way as for inulin.
Creatinine
Deproteinization of the serum was unnecessary because of the large
dilution (about 26 )<). To 80 cu.mm. of serum or urine were added 2
cc. of distilled water. The tubes were capped and shaken and 1 cc. of the
alkaline picrate was added to each. A Dubosque-type colorimeter with
TABLE I
Urinary flow in cc. per 100 grams per 24 hours.
No.
Male
Female
Material injected
1
3.4
0.3 cc. 10% inulin in crayfish-saline.
2
4.5
0.4 cc. 10% " "
3
3.3
1 cc. 20% " "
4
4.4
0.4 cc. 5% " "
5
6.1
0.4 cc. 5% " "
6
5.4
0.6 cc. 10% " "
7
6.4
0.6 cc. 10% " "
8
8.8
0.2 cc. 5% " "
9
4.4
0.2 cc. 5% " "
10
3.7
0.2 cc. 5% " "
11
5.4
1 cc. 30% xylose in % crayfish-saline.
12
7.2
1 cc. 30% " " "
13
7.9
0.3 cc. 10% xylose in dist. water.
14
8.85
0.2 cc. 10% " " "
15
7.1
0.5 cc. 10% " " "
16
4.6
0.5 cc. 10% xylose in crayfish-saline.
17
2.9
0.5 cc. 5% creatinine in cravfish-saline.
18
4.6
0.5 cc. 5% " " ''
19
5.9
0.2 cc. 5%
20
5.7
0.2 cc. 5%
21
7.6
0.5 cc. 10% creatinine in dist. water.
22
8.9
0.5 cc. 10% " " "
23
2.9
0.5 cc. 15% creatinine (somewhat toxic) in dist. water.
24
3.1
0.5 cc. 15%
1-cc. cups was used. The light was passed through a green filter.
Standards were made each time as expected. There was never as
much as a 50 per cent difference between the samples and the standards.
The blood of unsubjected animals did not give a positive Jaffe reaction.
The blood-curves were almost straight lines (Fig. 5). The average
concentration of creatinine in the blood through the experimental period
was calculated in the same way as for inulin.
The nephropores were sealed and the rate of urinary flow measured
directly (see above and Table I).
244
N. S. RUSTUM MALUF
1600,
woo-
200-
FIG. 4. The concentration of xylose in the blood in mg. per cent (ordinate) as
a function of the time in hours (abscissa) during the measurements of xylose-
clearance.
URINE-FORMATION IN CRAYFISH KIDNEY
245
RESULTS
Excretion of Inulin
In all the preliminary experiments, the injection of 0.2 cc. 5 per
cent inulin in crayfish-saline resulted in a renal output of inulin. Thus,
the urine, which was collected by suction from the nephropores, gave
a positive result with Benedict's qualitative only after acid-hydrolysis.
Before the introduction of inulin the urine was non-reducing even with
acid-hydrolysis.
900
700-
FIG. 5. The concentration of creatinine in the blood in mg. per cent (ordinate)
as a function of the time in hours (abscissa) during the measurements of creatinine-
clearance.
Similar results followed the injection of xylose or glucose. To
guard against possible glucose-contamination in the sample of xylose,
the urine was shaken for a few minutes with an equal quantity of a
20 per cent suspension of washed yeast in distilled water and centri-
fuged. Blanks, with only the yeast-centrifugate, were non-reducing.
The glucose was given in high concentration (0.6 cc. 70 per cent per
40 grams) because the tissues tended to remove it from the blood.
246
N. S. RUSTUM MALUF
Because glucose, xylose, and inulin are excreted by the kidney of
the crayfish and not by the vertebrate aglomerular kidney, it at first
seemed that filtration occurs in the former. This might imply that the
hypotonicity of crayfish-urine is produced as in the Amphibia, namely,
by the formation of a protein-free filtrate at the proximal end of the
nephron and by subsequent resorption of relatively more salts than
water by the tubule. On the other hand, other important data (see
Discussion) centra-indicate filtration.
Because inulin is neither secreted by nor passively resorbed through
the vertebrate nephron, the inulin-clearance in this phylum is an unvary-
ing function of the concentration of inulin in the plasma. This is true
o
o°
20O 3OO 40O 5OO 60O TOO BOO 90O COO IIOO COO OOO MOO 60O BCD TOO BX>
FIG. 6. The renal clearance of inulin, xylose, and creatinine in cc. per hour
(ordinate) as a function of the concentration of these compounds in the plasma in
mg. per cent (abscissa). Solid circles, inulin-clearances with direct measurement
of urinary flow and nephropores sealed ; open circles, inulin-clearances without di-
rect measurement of urinary flow and nephropores not sealed ; triangles, xylose-
clearances with direct measurement of urinary flow and nephropores sealed ; crosses,
creatinine-clearances with direct measurement of urinary flow and nephropores
sealed. Each point stands for a single separate animal.
even at the low plasma-concentrations (Miller, Alving, and Rubin,
1940). To find whether secretion can account for the marked occur-
rence of inulin in the urine of the crayfish, a study was made of the
inulin-clearance at various levels of inulin in the plasma. Variation of
the renal clearance with the plasma-concentration would demonstrate
secretion. Parenthetically, even if the renal clearance of a substance
does not vary with its plasma-concentration, secretion is not theoretically
excluded (Shannon, 1938, 1939).
The actual inulin-clearance : plasma-inulin relationship (Fig. 6, inu-
lin) demonstrates an outward secretion of inulin. The U/P : plasma-
inulin curve (Fig. 7, inulin} is similar and the U/P's were above unity.
Because the renal clearance is a product of the U/P and rate of urinary
URINE-FORMATION IN CRAYFISH KIDNEY 247
flow, the approximate identity in the curves of Figs. 6 and 7 is equivalent
to stating that the rate of urinary flow tends to be constant among differ-
ent individuals.
It should be pointed out that the wet weight of both kidneys is
normally a direct rectilinear function of the wet weight of the crayfish,
at least in animals weighing between 10 and 50 grams. The relation-
ship is expressed by y == 0.0026.r, in which y is the mass of both kidneys
and x the mass of the entire animal. Because the inulin-secreting mass
of the kidney is probably a direct function of the total mass of the
kidney, all animals should be approximately the same weight in an ideal
set of experiments. In this investigation, because the lower plasma-
concentrations were by no means confined to the larger animals (Table
II), size, within the experimental range, cannot have been a determining
factor in the inulin-clearance : plasma-inulin relationship. This is fur-
ther brought out by the fact that the variation in the inulin-clearance
with the concentration of inulin in the plasma, in crayfish which range
between average and large size, is determined practically entirely by the
U/P (Figs. 6 and 7, inulin} and not by the volume of urine excreted,
which is greater in the larger animals although fairly constant per unit
weight. In other words, the hourly differences in the absolute rate of
urinary flow among individuals of somewhat different size are relatively
small and inconsistent as compared with the variation of the U/P with
the concentration of inulin in the plasma. Assuming a constant con-
centration of inulin in the plasma, the inulin-clearance (— UV/P)
would doubtless vary with the mass of the kidney, but the U/P probably
would not because the inulin-secreting mass of the kidney may bear a
constant value with respect to the water-secreting mass. This implies
that while the large kidney would secrete more inulin than the small one,
it would also secrete proportionally more water.
The shape of the U/P : plasma-inulin curve indicates that the renal
cells asymptotically become functionally saturated with inulin as the
plasma-level of this compound rises. If filtration does not occur one
would expect that, at extremely low concentrations of inulin in the
plasma, the U/'P's would be less than unity because there would be very
little inulin available to the renal cells within a given interval of time.
Apparently because of the relatively high avidity of the renal cells for
inulin, it was not practicable to measure inulin-clearances at extremely
low plasma-levels ; sufficient urine was not formed before the blood was
freed from inulin. With xylose, U/P's below unity occur even at mod-
erate plasma-levels. It is possible that, at moderate concentrations, the
kidneys secrete relatively more inulin than water and that the reverse is
true for xylose.
248
N. S. RUSTUM MALUF
Attempts were made to locate the site of inulin-secretion in the
nephron by the colorimetric method of Alving, Rubin, and Miller (1939).
About 0.8 cc. 10 per cent inulin in crayfish-saline were injected into
TABLE II
Excretion of Inulin
No.
Wgt. in g.
and sex
Inulin
Urinary
flow
Amt. of crayfish
saline and cone,
of inulin injected
Duration of
experiment
Plasma
Urine
mg. per cent
mg. per cent
cc./hr.
hrs.
1
42.79
433
1,590
0.088
0.6 cc. 10%
12.5
2
30.09
345
1,716
0.062
0.25 cc. 10%
11.5
3
27.89
355
1,600
0.058
0.27 cc. 10%
13.75
4
42.5 9
1,810
1,970
0.088
1 cc. 20%
14.25
5
34.2d*
1,390
1,974
0.072
1 cc. 20%
14.75
6
31.0 d"
99
540
0.064
0.2 cc. 5%
13.5
7
25.7tf
234
468
0.054
0.2 cc. 5%
13.7
8
33.09
1,086
1,246
0.068
0.75 cc. 10%
12.5
9
42. 7c?
235
578
0.090
0.4 cc. 5%
13
10
44.8 d"
296
494
0.094
0.4 cc. 5%
12.6
11
33.4tf
758
883
0.070
0.5 cc. 10%
11.3
12
23.1 9
276
550
0.048
0.17 cc. 10%
11.5
13
51. Id*
304
825
0.106
0.38 cc. 10%
11
14
37.3d1
280
742
0.076
0.28 cc. 10%
13
15
51. 7d"
356
841
0.108
0.45 cc. 10%
12
16
26.09
359
825
0.054
0.26 cc. 10%
12.3
17
28.0 d"
299
858
0.058
0.28 cc. 10%
12.4
18
25.09
1,568
2,350
0.052
0.5 cc. 20%
11.6
19
23.09
1,549
2,130
0.048
0.5 cc. 20%
12
20
46.5 d1
307
494
0.096
0.65 cc. 10%
14
21
48.5 d1
425
462
0.10
0.68 cc. 10%
12.5
22
27.69
830
1,155
0.12
0.7 cc. 10%
12
23
21.79
330
718
0.094
0.2 cc. 10%
11
24
25.0d"
330
882
0.108
0.3 cc. 10%
10.6
25
20.49
300
1,090
0.088
0.4 cc. 10%
11.2
26
21.4cT
370
1,156
0.092
0.4 cc. 10%
11.2
27
28.09
670
1,180
0.040
0.3 cc. 10%
9.7
28
33.59
574
1,160
0.06
0.4 cc. 10%
9.5
29
30.3 9
1,380
2,220
0.042
1 cc. 20%
10.2
30
26.59
166
530
0.049
0.4 cc. 5%
9.8
31
29.5 cf
131
520
0.075
0.4 cc. 5%
8.9
32
47.3d1
467
720
0.106
0.6 cc. 10%
9.7
33
32.0d"
424
1,250
0.089
0.6 cc. 10%
10.6
34
23.89
60
192
0.088
0.2 cc. 5%
7.75
35
29.59
102
240
0.054
0.2 cc. 5%
9
36
42.0C?
45
180
0.065
0.2 cc. 5%
8.3
medium-sized animals. After about three hours the kidneys were re-
moved, rinsed in saline, and the coelomosac, tubule, and labyrinth teased
apart. Approximately equal amounts of coelomosac, labyrinth, and
tubule were put into separate small test-tubes. To each was added 1
URINE-FORMATION IN CRAYFISH KIDNEY
249
cc. of the freshly prepared diphenylamine reagent. The tubes were
capped and put into a boiling water bath for six minutes. The color
which developed at the end of this time was evidently maximal. Un-
aided visual examination of the intensity of color did not indicate any
differences in the amount of inulin present in the tubes. The intensity
was determined solely by the mass of tissue used.
Regardless of whether a substance is removed from the blood by
extrarenal tissues, the renal clearance of the substance will be a function
of the concentration of that substance in the blood. It was nevertheless
of interest to find if inulin can be hydrolyzed by the tissues of the cray-
- o
o
o
o o
o
o
o
FIG. 7. The U/P ratio of inulin, xylose, and creatinine (ordinate) as a func-
tion of the concentration of these compounds in the plasma (abscissa). Notations
identical with those in Fig. 6. Each point stands for a single separate animal ; the
same as those in Fig. 6.
fish. The kidneys and samples of the hepatopancreas, somatic muscles,
and blood were frozen in solid carbon dioxide, thoroughly macerated,
and extracted in a known quantity of saline. To aliquots of the cen-
trifugates were added a solution of inulin and a small drop of xylol.
The mixtures were analyzed for inulin immediately and after 13 hours
at room temperature. The controls contained only a solution of inulin
and the preservative. There was no change in the concentration of
reducing carbohydrate, with or without acid-hydrolysis, in any tube.
This indicates that, under the conditions of the experiments at least,
inulin is not hydrolyzed by the tissues of the crayfish. Similar experi-
ments showed a destruction of d-xylose in the following descending
250
N. S. RUSTUM MALUF
order : hepatopancreas, kidneys, somatic muscles, blood. This may ex-
plain how the concentration of xylose in the blood falls more rapidly
than that of inulin (Figs. 3 and 4) even though the renal xylose-
clearances (see below) are lower than the inulin-clearances. Xylose
may also diffuse out through the gills.
Excretion of d-Xylosc
The xylose-clearance varies directly with the concentration of xylose
in the plasma (Fig. 6, xylose) and at moderately low plasma-levels the
U/P's are well below unity (Fig. 7, xylose}. Assuming the occurrence
of filtration and resorption, this relationship may be explained by an
incapacity of the nephron to resorb xylose beyond a maximal rate ; as a
TABLE III
Excretion of d-Xylose
No.
wgt.
in g.
and
sex
Xylose
Urinary
flow
Remarks
Plasma
Urine
mg. per cent
mg. per cent
cc./hr.
1
29.6
988
1,370
0.067
1 cc. 30% xylose in % crayfish-saline;
9.75 hr. duration.
2
38.1
890
1,120
0.115
1 cc. 30% xylose in % crayfish-saline;
8.5 hr. duration.
3
31.5
89
10
0.10
0.3 cc. 10% xylose in dist. water; 10 hr.
duration.
4
22.5
64
10
0.083
0.2 cc. 10% xylose in dist. water; 9.5 hr.
duration.
5
33.6
210
61
0.10
0.5 cc. 10% xylose in dist. water; 8.8 hr.
duration.
6
34.5
127
19
0.066
0.5 cc. 10% xylose in crayfish-saline;
8 hr. duration.
consequence, an increasing amount would " spill over " as the plasma-
level is raised. Because at low plasma-concentrations the U/P is below
unity (Fig. 7, xylose) the resorption would presumably be active, i.e.
xylose would be inwardly secreted. There is a resemblance to the han-
dling of glucose and other threshold-substances by the mammalian
kidney.
On the other hand, the process can be readily explained, without
resort to filtration, by assuming that both xylose and water are out-
wardly secreted and that, at low plasma-levels of xylose, the rate of
secretion of water is relatively large compared with the secretion of
xylose. At moderately high plasma-levels the xylose-clearance is nearly
identical with the inulin-clearance (Fig. 6). In the experiments which
necessitated the introduction of sufficient xylose to raise the average
URINE-FORMATION IN CRAYFISH KIDNEY
251
plasma-concentration to about 1000 mg. per cent (see Table III), the
animals became torpid soon after the injection but recovered completely
within several minutes. It was therefore not considered within the
scope of a physiological experiment to measure xylose-clearances at still
higher plasma-levels. The injurious effects are probably osmotic. Inu-
lin was not toxic even at the high concentrations.
For the same reason as with inulin, the xylose-clearance : plasma-
xylose curve is practically identical with the U/P : plasma-xylose curve
(Figs. 6 and 7, .vylose).
TABLE IV
Excretion of Creatinine
No.
Wgt. in
g. and
sex
Creatinine
Urinary
flow
Remarks
Plasma
Urine
mg. per
mg. per
cc./hr.
cent
cent
1
25.59
230
460
0.031
0.5 cc. 5% creatinine in crayfish-saline; 9.8 hr.
duration.
2
26.5 d"
155
240
0.051
0.5 cc. 5% creatinine in crayfish-saline; 9 hr.
duration.
3
30.09
65
140
0.074
0.2 cc. 5% creatinine in crayfish-saline; 9.1 hr.
duration.
4
39.0 9
60
105
0.093
0.2 cc. 5% creatinine in crayfish-saline; 8.25 hr.
duration.
5
34. 7c7
280
450
0.112
0.5 cc. 10% creatinine in dist. water; 8.25 hr.
duration.
6
32.09
225
350
0.118
0.5 cc. 10% creatinine in dist. water; 8.1 hr.
duration.
1
33.8cf
537
850
0.040
0.5 cc. 15% creatinine in dist. water; 8.7 hr.
duration. (Somewhat toxic.)
8
25.59
610
1,150
0.033
0.5 cc. 15% creatinine in dist. water; 8.25 hr.
duration. (Somewhat toxic.)
Excretion of Creatinine
Because the inulin- and creatinine-clearances are identical in certain
vertebrates at all plasma-levels, it was desirable to compare the same
clearances in the crayfish. The results were not elucidating and are
presented here merely for record because it is believed that they are
accurate (Figs. 6 and 7, creatinine; Table IV). Plasma-concentrations
higher than 900 mg. per cent were definitely injurious if not fatal. The
maximal ones on record are just within the threshold of toxicity, judging
from the activity of the animal.
Excretion of Dyes
The initial objective of the experiments under this heading was to
find if the nephron of the crayfish is capable of eliminating dyes which
252 N. S. RUSTUM MALUF
the vertebrate aglomerular kidney is incapable of excreting. It was also
desirable to study the capacities of the different parts of the nephron to
secrete or accumulate various kinds of dyes.
The dyes were dissolved in crayfish-saline immediately before use.
A description of the chemical composition of most of the dyes can be
found in Conn's (1925) monograph.
Cyanol (DuPont).4 — This is an aniline dye giving an intense blue in
solution even when very dilute. Cyanol is not eliminated by the
aglomerular vertebrate kidney if given in doses of the order of several
mg. per kg. (Hober, 1930) but is slightly excreted when in quantities of
125-300 mg./kg. (Marshall and Grafflin, 1932).
Immediately after emptying the bladders, a fraction of a cc., con-
taining a dose of about 1.7 mg./kg., was injected through the proximal
abdominal venter. This colored the blood a vivid blue. Urine was
collected after five hours and had to be diluted about tenfold to bring
the intensity of color down to that of blood taken only one hour after
the injection. Within five hours the blood lost all trace of blue. The
experiment was repeated with similar results. As stated above, the
concentration of foreign material, one hour after injection, is about equal
in blood taken from the legs as in that from the pericardial sinus.
Other subjects were opened one to two hours after the injection.
The viscera were rinsed writh saline. Cyanol was not found in any
organ other than the labyrinthic epithelium. The intensity of blue in
the labyrinth not only greatly exceeded that of blood at the time but
even that of blood taken only twenty minutes after the injection. The
dye did not stain the bladder nor diffuse out from the contained urine
even at a time, five or six hours after the injection, when it was absent
from the blood.
The accumulation of cyanol in the labyrinth cannot be considered due
to a resorption of water by the labyrinth, from a filtrate conceivably
formed at the coelomosac, because : ( 1 ) The dye is greatly concentrated
in the labyrinthic cells and yet not appreciably apparent in the more
distal parts of the nephron; (2) the labyrinthic cells, even in "living"
hanging-drop preparations, indicate a marked outwardly secretory activity
as shown by the frequent presence of globules apparently being pinched
off toward the lumen : the labyrinth therefore can scarcely be considered
as a water-resorbing organ from a cytological standpoint; (3) the data
indicate that the coelomosac is a secretory organelle (Maluf, 1941o, and
below).
Fcrrocyanide. — Iron salts, such as ferric ammonium citrate and so-
dium ferrocyanide, are not excreted by the aglomerular vertebrate ne-
4 Kindly supplied me by Professor E. K. Marshall, Jr.
URINE-FORMATION IN CRAYFISH KIDNEY 253
phron (Marshall and Grafflin, 1932) but are filtered through the glo-
merular nephron of vertebrates (see Smith, 1937).
Both bladders were emptied and 0.5 to 1.2 cc. of 2.4 per cent sodium
ferrocyanide injected into animals weighing from 27 to 48 grams. The
Prussian blue color was developed by adding a known quantity of Folin's
(1929) ferric sulfate reagent to the NaoWO4-H2SO4 protein-free blood-
centrifugate. At the end of five hours a scarcely appreciable quantity
of urine could be collected, which gave a Prussian blue test. The ferric
sulfate reagent produced an intense blue throughout the teased nephron ;
the color was more intense than that of the blood taken only 0.5 hour
after the injection and seen through the same or greater depth. This
experiment indicates that the kidney is capable of accumulating ferro-
cyanide but that the cells apparently become too poisoned to secrete urine.
The hepatopancreas, muscles, and alimentary tract, rinsed free from
blood and teased apart, gave no reaction.
Phenol Red. — Phenol red is secreted by the aglomerular teleost kid-
ney (Marshall and Grafflin, 1932). The bladders of the crayfish were
emptied immediately before the injection of the dye. The dose was 1 cc.
of 34 mg. per cent phenol red into animals weighing about 30 grams.
To develop the maximal intensity of color, both urine and blood were
either exposed to NH3 or received a known quantity of NH4OH. The
urine, collected five hours after the injection, had to be diluted over ten-
fold to reduce its intensity to that of blood taken 20 minutes after. On
examining the kidneys in situ five hours after the injection, only the
posterior part of the labyrinth had a reddish tinge. On adding a drop
of 0.1 N NaOH to the nephron in crayfish-saline, the whole labyrinth
became an intense red which was even deeper than that of blood taken
as early as 0.5 hour after the injection. The labyrinth is thus capable
of secreting phenol red and the pH of its cells is evidently about 7.0.
Other tissues, including the coelomosac and nephric tubule, after being
briefly rinsed from blood, showed no trace of phenol red.
The urine, as it issued from the nephropore, was a clear orange-red,
not the purple-red of maximal intensity, and therefore has a pH of
about 7.5.
Neutral Red.— In the three animals studied (dose : 1.2-1.8 cc. 80 mg.
per cent per 30 grams) there was no indication of a concentration of
this dye in the urine. The dye penetrated the labyrinth and tubule but
the coelomosac did not show a trace of it. There seemed to be some
accumulation in the hepatopancreas as the color was more intense in this
organ (on adding a drop of acetic acid) than in the blood of equal depth.
As the urine issues from the nephropore it is a light yellow and turns
•pink on the addition of acid. This shows that its pH is greater than 7.4.
254 N. S. RUSTUM MALUF
It has already been noted that the phenol red experiments indicate a pH
of about 7.5.
" Indigo Carmine."- —Indigo carmine is composed of carmine blue
and indigo disulfonate. The sample used had been passed by The Com-
mission on Standardization of Biological Stains. It is long-known that
indigo disulfonate is outwardly secreted by the vertebrate tubule. The
dose was 0.7 cc. 80 mg. per cent per ca. 30 grams. Four hours after the
injection the dye was markedly more concentrated in the bladder-
contained urine than in blood even when collected only 25 minutes after
the injection. On examination of the kidneys, no dye was found in the
coelomosac or distal portion of the tubule. In one example concentrated
dye was seen to leave the lumen of the proximal portion of the tubule
upon application of pressure to the labyrinth, but there was no indication
that the cells of the tubule take up the stain. The dye was concentrated
in irregular patches in the labyrinth especially at the posterior end.
There was no trace of it in the hepatopancreas and other tissues.
Congo Red. — Six-tenths of a cc. of 160 mg. per cent Congo red was
injected into a 31-gram animal. Blood taken forty minutes later was a
very light pink. The kidney was examined four hours after the injec-
tion; the coelomosac was a deep pink but the dye was absent from the
rest of the nephron, and from the hepatopancreas, muscles, and gut.
"Basic Fuchsin" (aniline red; diamond fuchsin R.F.N. ; magenta;
passed by the C.S.B.S.). — The dose was 0.75 cc. 80 mg. per cent 30
grams. The animal was opened four hours after the injection. The
stain had penetrated the muscles, hepatopancreas, coelomosac, nephric
tubule, and other tissues. As compared with the blood, it was concen-
trated only in the labyrinth where it was a very intense purple. Soon
after the injection the animals lay on their side in semi-torpor but recov-
ered completely and removed all traces of dye from the blood.
Acid Fuchsin. — Eight-tenths of a cc. of 80 mg. per cent dye was in-
jected into a 40-gram animal. The kidneys were examined after about
4.5 hours, at which time the dye was more concentrated in the bladders
than in blood taken even 35 minutes after the injection. The labyrinth
was a more intense pink than the blood seen through the same depth.
The nephric tubule, coelomosac, hepatopancreas, muscles, and gut were
not stained.
Methylene Blue (Passed by the C.S.B.S.). — Five-tenths of a cc. 50
mg. per cent were injected into a 22-gram animal. A blood-sample,
taken after about two hours, was a very light blue. The animal was
opened after about five hours. Methylene blue was concentrated only
in the proximal portion of the tubule, where it existed as intracellular
granules. After fixing the fresh kidney in 20 per cent formalin, the blue
URINE-FORMATION IN CRAYFISH KIDNEY 255
concretions disappeared and the proximal portion of the tubule became
a uniform blue. Evidently the intracellular granular condition depends
upon an active process. The hepatopancreas and gills, but not the mus-
cles, were merely stained. The experiment was repeated with identical
results.
Colloidal Carbon. — Five-tenths of a cc. of " Higgins American India
Ink: waterproof, black," diluted 6X with crayfish-saline, was injected
into a 20-gram animal. This was sufficient to give a very dark brown
color to the blood. The animal was opened after four hours and the
organs rinsed in situ with saline. The colloid had not penetrated
any tissue. This is a functional demonstration of the absence of a
nephrostome.
DISCUSSION
The primary question is whether filtration occurs through the nephron
of the crayfish. The paper of Bethe, von Hoist, and Huf (1935), which
appears to furnish po'sitive evidence for filtration, should be read with
care, especially as certain investigators have taken their results at face-
value. Bethe et al. augmented the internal hydrostatic pressure of the
crab, Corcinus inacnas, by a vertical column of saline which communi-
cated with the haemocoele. The aqueous column then sank in abrupt
steps, indicating a fall in the internal pressure. They stated that this is
evidently a physiological event because raising the hydrostatic pressure
after death resulted in only a slight fall of the column which they at-
tributed to an expansion of the soft membranes of the integument.
They also pointed out that if the crab dies during the experiment the
column of saline either does not fall or sinks very slowly. The animals,
which were observed in air, were stated to have shown a loss of fluid
from three sites: (1) the gill-chambers; (2) the mouth; and (3) the
nephropores. These investigators noted that the fluid from the gill-
chambers contained protein but was cell-free ; the writer thinks that this
fluid may have issued partly from the mucus-secreting glands. Above
all, the authors explicitly remarked that, during the fall of the aqueous
column, generally no loss of liquid by way of the nephropores could be
observed. Their suggestion that the kidneys of the crab regulate the
internal hydrostatic pressure per se, i.e. even when the osmotic pressure
does not vary, is therefore unfounded. As noted above, augmentation
of the blood-volume by about one-third apparently does not increase the
rate of urinary flow.
By measuring the oncotic pressure of the blood and the haemocoelic
pressure of crayfish, Picken (1936) indicated that filtration is appar-
ently possible.
256 N. S. RUSTUM MALUF
The writer is not aware of any facts which can be taken as positive
evidence for the filtration-resorption theory or against the absence of
filtration and the outward secretion of liquid. Analogy with the verte-
brate nephron is inadequate. Furthermore, outward secretion of liquid
is known to occur in aglomerular fish (Marshall, 1930; Bieter, 1931).
The urine of the latter is, like that of the crayfish, hypotonic to the blood.
It is unknown, however, whether the hypotonicity of the urine of
aglomerular forms is due to an outward secretion of a hypotonic liquid
or to the elimination of an iso- or even hypertonic liquid, in the proximal
part of the nephron, followed by a resorption of salts. Owing to phylo-
genetical reasons (see Marshall, 1934), the latter method does not appear
probable.
There are several facts which indicate that the nephron of the crayfish
is primarily if not entirely an organ of outward secretion :
1. There is no tenuous syncytium such as the glomerular capsule of
the vertebrate nephron (Maluf, 1939, 1941c).
2. Large calcareous concretions sometimes occur in the lumen of the
coelomosac, the most proximal organelle of the nephron, thus indicating
that the coelomosac can secrete calcium (Maluf, 1941a). The coelo-
mosac is also capable of accumulating Congo red (see above).
3. Experimental cytological evidence indicates an outward secretion
of water by the distal half of the tubule (Maluf, 1941&).
4. Histologically there is no doubt that the labyrinth secretes mate-
rial outwardly (Maluf, 1939). The labyrinthic cells are capable of accu-
mulating and outwardly secreting cyanol, phenol red, indigo carmine,
basic fuchsin, and acid fuchsin (see above).
5. The cells of the proximal portion of the tubule can accumulate
methylene blue (see above). All parts of the nephron are therefore
capable of secreting or accumulating one dye or another.
6. Inulin is outwardly secreted (see above).
7. From a teleological viewpoint the coelomosac is evidently not a
filtration-organelle (Maluf, 19410).
8. Injecting into a moderate-sized crayfish 1 cc. of crayfish-saline, i.e.
a volume about one-third that of the initial blood-volume, and thus very
probably increasing the internal hydrostatic pressure, does not augment
the rate of urinary flow (Table I).
The Malpighian tubule of insects, as a result of physiological and
cytological evidence, probably should be considered as an entirely secre-
tory nephron. The beautiful live preparations of Wigglesworth (1931o,
b, c) show that the Malpighian tubule can excrete fluid, in an apparently
normal way, even under conditions when the hydrostatic pressure is zero.
URINE-FORMATION IN CRAYFISH KIDNEY 257
The ingenious experiments of Patton and Craig (1939) show that the
Malpighian tubule can absorb various isotonic salines isosmotically even
when the hydrostatic pressure must be zero (the saline rose into the capil-
lary gauge up to 10 to 15 mm. admittedly by capillarity). They also
state that hydrostatic pressure does not cause an increase in " filtration "
rate. It is not apparent to the writer why Patton and Craig assumed
that the isosmotic uptake of solution by the Malpighian tubules is due to
filtration. It is known that the alimentary epithelium of vertebrates ab-
sorbs solutions isosmotically and, at the same time, absorbs, selectively,
ions of a particular species.
Kowalevsky (1889), Cuenot (1895), and Bruntz (1904) studied the
affinity of the crustacean nephron, in situ, for ammonium carminate.
indigo carmine, and certain other dyes. They did not indicate, however,
whether the dyes were concentrated by the nephron because they made
no statements as to the relative intensity of dye in the blood and urine.
Kowalevsky and Bruntz noted that ammonium carminate and litmus
stain the coelomosac but not the rest of the nephron while indigo carmine
stains the tubule and labyrinth. Because the coelomosac stained red
with litmus, Kowalevsky concluded that this organelle has an acid reac-
tion. He also observed that the coelomosac, and not the labyrinth, has an
affinity for Congo red and methylene blue. Cuenot believed that the
labyrinth of the crayfish, lobster, and crabs has a strongly alkaline reac-
tion (italics his) because it "energetically decolorised acid f uchsin " ;
the color reappeared on macerating the kidney in acetic acid. He also
noted that alizarin violet (an alkaline dye) retains its color instead of
going into the orange-red phase. I have found, on the other hand, that
the labyrinth is capable of concentrating acid fuchsin and that treating
the nephron with acetic acid does not augment the intensity of color.
The dye was more concentrated in the urine than in the blood.
The present observations with the pH indicators, phenol red and
neutral red, show that the cytoplasmic pH of the labyrinthic cells is
about 7.0 and that the pH of the bladder-contained urine is about 7.5.
Because the labyrinth will take up an acid dye, such as indigo carmine, is
no reason to believe that its cells are basic. The uptake of dyes during
life is not equivalent to the affinity of fixed dead tissues for dyes. This
is a distinction which Kowalevsky and Cuenot did not make.
The statements of Kowalevsky and Cuenot that the labyrinth is alka-
line led the writer (1938) to suggest that the nitrogenous products of
protein-catabolism are outwardly secreted by the labyrinth. The facts
that the labyrinth is not alkaline and that the concentration of the N-P-N
is markedly lower in the urine than in the blood (see Delaunay, 1927 and
1931, for the crabs Mala sqninado and Cancer pagurus; the crayfish has
258 N. S. RUSTUM MALUF
not been studied with this regard) have greatly weakened that supposi-
tion. It should be borne in mind that M. squinado and C. pagunis are
marine crabs without nephric tubules and eliminate a urine isotonic with
their blood. It is therefore unlikely that the N-P-N is subjected to
dilution by an outward secretion of water.
Because the main nitrogenous excretory product of the Crustacea is
a highly diffusible substance, — ammonia (Delaunay, 1927, 1931), it
seems probable that this escapes largely through the gills. Although we
possess data on the over-all rate of ammonia-output by the crayfish
(Potamobius astacus; see Brunow, 1911), there is no statement in the
literature concerning the concentration of ammonia in the urine ; conse-
quently the rate of output of ammonia by the renal route is unknown.
Partly because the concentration of ammonia is practically the same in
the urine as in the blood of the above-mentioned crabs, it is possible that
the existence of ammonia in the urine is merely due to diffusion.
SUMMARY
1. The techniques of measuring the rate of urinary flow and of col-
lecting urine are described. The collection of urine from the nephro-
pores by suction is a satisfactory procedure provided a correction is ap-
plied for the water lost by evaporation.
2. The techniques of collecting blood and of measuring renal clear-
ances in the crayfish are described.
3. Raising the internal volume by one-third and therefore, presum-
ably, augmenting the internal hydrostatic pressure, by the injection of 1
cc. of crayfish-saline, does not increase the rate of urinary flow.
4. Inulin and xylose will appear in the urine after being injected into
the haemocoele. Glucose will occur in the urine provided enough is in-
jected to permit its existence in the blood for a sufficient period.
5. The inulin-clearance and the U/P ratio of inulin vary inversely
with the concentration of inulin in the blood. This demonstrates that
inulin is secreted.
6. Inulin is not hydrolyzed by the hepatopancreas, kidneys, somatic
muscles, or blood.
7. At low plasma-levels, the U/P ratios of xylose are very much be-
low unity but rise above unity at high plasma-levels. This shows that
xylose is either actively resorbed from a filtrate or is outwardly secreted
but, with the low plasma-levels, at a relatively low rate compared with
the secretion of water. The xylose-clearance : plasma-xylose curve is
practically identical in shape with the U/P : plasma-xylose curve.
8. Although the renal clearances of xylose are much lower than the
renal clearances of inulin, the plasma-concentration of the monosaccha-
URINE-FORMATION IN CRAYFISH KIDNEY 259
ride falls more rapidly than that of the polysaccharide. This may be
partly because the tissues can destroy xylose.
9. Only the labyrinthic cells can accumulate and outwardly secrete
cyanol, phenol red, indigo carmine, basic fuchsin, and acid fuchsin.
The coelomosac, but not the labyrinth or tubule, can accumulate Congo
red. These dyes cannot accumulate in, and apparently do not penetrate
into, other tissues of the body.
10. Only the cells of the proximal half of the tubule accumulate
methylene blue.
11. Colloidal carbon does not enter the kidney; this is functional
proof of the absence of a nephrostome.
12. The cytoplasmic pH of the labyrinthic cells is about 7 ; the pH
of the bladder-contained urine is about 7.5.
13. The available facts (histological, chemical, physiological, and phy-
logenetical) indicate that the nephron of the crayfish is primarily if not
entirely an organ of outward secretion.
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HOBER, R., 1930. Beweis selektiver Sekretion durch die Tubulusepithelium der
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— , 1934. The comparative physiology of the kidney in relation to theories of
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PATTON, R. L., AND R. CRAIG, 1939. The rates of excretion of certain substances
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81 : 437-457.
PICKEN, L. E. R., 1936. The mechanism of urine formation in invertebrates. I.
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SHAFFER, P. A., AND M. SOMOGYI, 1933. Copper-iodometric reagents for sugar
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Am. Jour. Physiol.. 122: 775-781.
— , 1939. Renal tubular excretion. Physiol. Rev., 19: 63-93.
SMITH, H. W., 1937. The Physiology of the Kidney. New York : Oxford Uni-
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SOMOGYI, M., 1931. The use of copper and iron salts for the deproteinization of
blood. Jour. Biol. Chcm.. 90: 725-729.
WIGGLESWORTH, V. B., 1931a. The physiology of excretion in a blood-sucking
insect, Rhodnius prolixus (Hemiptera, Reduviidae). I. Composition of the
urine. Jour. Ex per. Biol., 8: 411-427.
— , 1931/L II. Anatomy and histology of the excretory system. Ibid., 8: 428-
442.
, 1931c. III. The mechanism of uric acid excretion. Ibid., 8: 443-451.
ACTIVATION OF CUMINGIA AND ARBACIA EGGS BY
BIVALENT CATIONS *
JOSEPHINE HOLLINGSWORTH
(From the Department of Zoology, University of Pennsylvania, and the
Marine Biological Laboratory, Woods Hole, Massachusetts)
The activation of unfertilized eggs by isotonic salt solutions was first
described by R. S. Lillie (1910, 191 la, 1911ft). Since then, the activa-
tion by isotonic salt solutions of the eggs of seven marine invertebrates,
belonging to three different orders, has been reported ; Pomatoccros by
Horstadius (1923), Astcrias by Dalcq (1924a, 1924ft), Phase olion by
Pasteels (1935), Hydroidcs by Pasteels (1935), Barnca by Dalcq
(1928), Thalcsscma by Hobson (1928) and Nereis by Spek (1930).
The present work is a study of the effects of isotonic solutions of
CaCl2, BaCl2, SrCl2, MgCL, NaCl, KC1 and LiCl, singly and in varying
binary mixtures and proportions, on the eggs of Cumingia tcllinoidcs and
Arbacia puuctnlata; of the relative effectiveness of CaCU in the activa-
tion of ovary eggs and shed eggs and of shed eggs that have been washed
and shed eggs that have not been washed ; and of the relative effective-
ness of solutions of isotonic CaCL which vary in pH. While its prin-
cipal contribution is an extension of our knowledge of the effects of
various isotonic salt solutions in the activation of eggs, it is hoped that
it may illuminate further our understanding of the fundamental reaction
or series of reactions which underlie the vital response of the cell.
Many careful investigators have shown that various types of stimula-
tion cause an increase in permeability of the plasma membrane of various
kinds of living material. However, Heilbrunn (1937) points out
: There is one type of stimulation which can scarcely be conceived of as
producing an increase in permeability. This is the stimulation produced
by calcium salts. Students of permeability are quite unanimous in re-
garding the calcium ion as a permeability lowerer rather than a perme-
ability increaser. Hence the action of calcium in producing stimulation
cannot be explained on the basis of the permeability theory." Heilbrunn
and his students have developed a colloid chemical theory of stimulation
in which calcium plays the dominant role (see Heilbrunn, 1928; Heil-
1 A thesis in zoology presented to the faculty of the Graduate School of the
University of Pennsylvania in partial fulfillment of the requirements for the degree
of Doctor of Philosophy.
261
262 J. HOLLINGSWORTH
brunn and R. A. Young, 1930 ; Heilbrunn and Daugherty, 1933 ; Heil-
brunn and Mazia, 1936; Angerer, 1936; Mazia and Clark, 1936; Heil-
brunn and Wilbur, 1937; Donnellon, 1938). This theory postulates the
following series of changes : calcium is released from the cortex resulting
in a liquefaction of .the cortex ; free calcium enters the interior of the
cell ; as the concentration of free calcium increases in the cell interior, a
series of reactions is initiated which includes an initial decrease in vis-
cosity followed by a characteristic clotting reaction. This series of re-
actions constitutes the vital response of the cell. My observations on the
activating effect of bivalent cations appear to support the colloid chemical
theory of stimulation as developed by Heilbrunn and his students.2
MATERIAL AND METHODS
In most of the experiments the same general procedure was employed.
In any given comparison the eggs from one female were used. The eggs
were shed into sea water, the supernatant fluid withdrawn and two drops
of a dense suspension of eggs were quickly pipetted into dishes of experi-
mental solutions previously prepared. No attempt was made to control
the temperature of the experimental solutions. The temperature of the
air was no higher than 26° C. at any time and was usually between 21°
and 25°. In any given experiment the range was rarely more than 2° C.
Merck's C.P. chemicals were used in making the solutions. Nad,
KC1 and LiCl were made up in 0.53 M concentration and MgCL, BaCL,
CaCl2 and SrCl2 in 0.3 M concentration. These solutions are isotonic
with sea water and the eggs do not shrink or swell in them. In studying
the effects of various mixtures of isotonic salt solutions in the activation
of eggs, the pH of the various solutions was adjusted, by the addition
of 0.1 N HC1 and 0.1 N NaOH, so as to lie in the range found experi-
mentally to be most favorable for activation, i.e. pH 6.2 to 8.6 for Cu-
mingia eggs and pH 8.8 to 9.0 for Arbacia eggs.
Eggs were exposed for varying periods of time to isotonic solutions
of a single chloride or to mixtures of chlorides in varying proportions.
The eggs were not transferred to sea water as is the usual procedure in
experiments of this kind, inasmuch as a high percentage of cleavage could
be obtained in the experimental solutions. In each experiment, hundreds
of eggs were examined and 100 eggs were counted. The time factor is
very important in determining the percentage of cleavage. At the end of
2 This work was done at the Marine Biological Laboratory at Woods Hole
during the summers of 1935, 1936 and 1937.
The problem was suggested by Dr. L. V. Heilbrunn. I wish to express my
appreciation for his invaluable guidance and kind criticism during the course of
this investigation.
The complete data are on file in the Library of the University of Pennsylvania.
ACTIVATION BY BIVALENT CATIONS
263
a certain period, which is roughly three hours in the case of Cmningia eggs
and five hours in the case of Arbacia eggs, there is no further increase
in the percentage of cleavage and cell injury occurs a little later. It is
desirable to count the percentage of activation at the end of this optimum
period which varies with the solution, the pH and the temperature.
Conclusions concerning the effectiveness of a reagent in activating
eggs are based on the percentage of cleavage. Although the first visible
sign of activation of Cumingia eggs is the extrusion of polar bodies, it
is difficult to make an accurate count of the percentage of eggs with polar
bodies, for if the egg lies with the animal pole down, the polar bodies
cannot be seen. While the first sign of activation when Arbacia eggs
are inseminated is the elevation of the vitelline membrane, this reaction
cannot be employed with isotonic solutions since they do not cause mem-
brane elevation although they do cause the membrane to swell.
TABLE I
Experiments were performed to determine the relative effectiveness of barium,
calcium and strontium solutions on the eggs of 33 individuals and tables were pre-
pared of the percentage of cleavage and polar body formation and of the number of
minutes elapsing before polar body formation.4 The following results were obtained.
Activated by
Average time of pb
formation
Average percentage cl
Average percentage pb
barium
11 min. 48 sec.
5.3
3.6
calcium
11 min. 27 sec.
35.4
26.1
strontium
10 min. 54 sec.
35.8
38.2
sperm
10 min. 6 sec.
RESULTS
Cumingia
Effect of 0.3 M CaCL. — When unfertilized Cumingia eggs are placed
in 0.3 M CaCL, at any pH between 6.0 and 8.6, the first polar body is
extruded in from 5 to 12 minutes and the first cleavage is completed in
from 40 to 60 minutes. This is approximately the same as the time of
polar body formation and of cleavage in eggs activated by sperm. The
percentage of cleavage at the end of several hours varies widely among
the eggs of different individuals. In some individuals 100 per cent of
the eggs undergo apparently normal activation. They continue to cleave
for several hours, reach the 8—16 cell stage and appear to be healthy and
normal. After several hours, however, the blastomeres pinch in and fall
apart.2 Polar body formation is extremely irregular, but there appears
264
J. HOLLINGSWORTH
to be an inverse relationship between the percentage of polar body forma-
tion and the percentage of cleavage (compare Morris, 1917).
The result of experiments in which eggs were exposed to solutions
of CaCl2 which vary in pH from 3.6 to 9.0 is shown in Table I.
The various solutions from pH 6.0 to 8.6 are equally effective in inducing
activation of the eggs of most individuals. The pH of the solutions
appears to have more effect on the percentage of cleavage than on the
percentage of polar body formation, polar body formation proceeding at
pH 9.0 while the percentage of cleavage decreases above pH 8.6. 3 Both
are almost completely inhibited at pH 4.1. (See Table II.) While the
TABLE II
Effect of pH on Activation of Cumingia Eggs by Isotonic Calcium Chloride
No. of e>
Time of
expos.
Temp. °(
.p.
Hrs.
Min.
1
4
20
21.3
pb cl
2
4
55
21.3
pb cl
3
4
15
21.3
pb cl
4
6
20
20.0
pb cl
5
6
20
20.0
pb cl
6
7
15
20.0
pbcl
7
4
20
21.3
pbcl
8
4
55
21.3
pb cl
9
6
55
20.0
pbcl
10
7
15
20.0
pb cl
pH
4.1
0 0
3 1
0 0
0 0
0 0
0 0
4.6
60 5
32 0
19 0
0 0
13 0
40 0
4.9
55 2
34 18
21 12
1438
927
5.8
13 70
40 13
60 2
7 70
18 1
35 2
6.1
1345
1651
20 19
1650
6.2
298
698
494
30 5
8 10
7.1
298
688
691
25 5
2 14
7.6
2245
1063
3032
298
590
989
1655
866
56 3
3 15
8.2
1849
1459
11 54
3292
1459
465
8.3
296
692
690
52 0
626
8.6
1960
1850
2055
1387
11 88
1077
1960
1050
35 1
12 27
9.0
1733
24 22
652
3422
2767
2767
1733
1255
20 3
13 18
sw
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
strontium solution is somewhat more effective than the calcium solution,
the former causes the blastomeres to separate in less time. It is difficult
to make accurate counts of the percentage of cleavage when blastomeres
separate.
Effect of Mixtures of Calcium Chloride and Some Monovalent Ca-
tions.— The result of experiments in which eggs were exposed to mix-
tures of potassium chloride, sodium chloride or sea water and calcium
chloride in various proportions is shown in Fig. 1. The degree of acti-
vation decreases rapidly as the proportion of potassium increases up to a
ratio of 1-16 and then remains fairly constant. It may be concluded
that there is an antagonism of calcium by potassium between ratios 1--64
3 This work is not a study in artificial parthenogenesis and no attempt was
made to develop procedures for securing later stages of development.
ACTIVATION BY BIVALENT CATIONS
265
and 1-16 since the percentage of activation decreases too rapidly to be
due to the dilution of calcium ions by potassium ions. Sodium has only
a slight inhibiting influence on the activating effect of calcium from ratio
1-64 to ratio 1-4, but inhibition increases markedly above an average
ratio of 1-4. The slight decrease in the percentage of activation up to
an average ratio of 1-4 is probably due to the dilution of the calcium
Ratio
FIG. 1. Action on Ciwiingia eggs of isotonic solutions of some monovalent
cations and of CaCL singly and in various mixtures and proportions.
K/Ca — average of ten experiments.
" Na/Ca — average of sixteen experiments,
sea water/Ca — average of five experiments.
solution, but there is an antagonism of calcium by sodium above this
ratio. Sea water has an inhibiting effect on the activating effect of cal-
cium beginning in a ratio of 1—64 and increasing as the proportion of
sea water increases, with complete inhibition in most cases at a ratio of
about 1-2. The inhibiting effect of sea water is no doubt clue to the
monovalent cations.
Effect of Mixtures of Calcium Chloride and of Some Bivalent Ca-
tions.— The result of experiments in which eggs were exposed to mix-
tures of strontium, barium or magnesium and calcium in various proper-
266
J. HOLLINGSWORTH
tions is shown in Fig. 2. Strontium is somewhat more effective than
calcium in inducing cleavage in Cumingia eggs but causes the polar bodies
to be extruded far from the cell surface and the blastomeres to separate
in a short time. Strontium inhibits slightly the activating effect of cal-
cium while calcium inhibits somewhat the activating effect of strontium.
Solutions in which the Sr/Ca ratios are from 1-1 to about 64—1 are more
injurious than the single salt solutions and it is difficult to make an accu-
60-
Ratio
FIG. 2. Action on Cumingia eggs of isotonic solutions of some bivalent cations
singly and in various mixtures and proportions.
Sr/Ca — average of seventeen experiments.
Ba/Ca — average of twelve experiments.
Mg/Ca — average of thirteen experiments.
rate count of the percentage of cleavage between these ratios. Barium
has a slight activating effect on Cumingia eggs. It may be concluded
that there is an antagonism of calcium by barium as the latter produces
a marked inhibition of the activating effect of calcium, beginning in a
ratio of 1-64. The percentage of activation resulting from exposing
eggs to isotonic MgCU is negligible. Magnesium inhibits very slightly
the activating effect of calcium even in a ratio of 64-1. Inasmuch as
magnesium has no activating effect on Cumingia eggs, it might be ex-
ACTIVATION BY BIVALENT CATIONS 267
pected that as the dilution of the calcium solution by magnesium in-
creases, the percentage of activation would decrease. It is interesting
and noteworthy that this is not the case. Even in an Mg/Ca ratio of
64—1 there is a high percentage of cleavage.
In summary, monovalent cations are not effective in activating the
eggs of Cuminyia and they inhibit the activating effect of calcium, the
effect increasing as the K/Ca, Na/Ca, sea/Ca ratios increase. The bi-
valent cations, with the exception of magnesium, are able to activate Cu-
mingia eggs and in certain combinations and proportions, mutually inhibit
activation. There is considerable variation in the behavior of different
lots of eggs, but it is a variation in magnitude rather than in kind. The
results of experiments performed on the eggs of a single individual (see
original manuscript) are more interesting than the average of the results
of many experiments as presented in this paper, because the former pre-
sents a more characteristic picture of the behavior of marine eggs.
Arbacia
Effect of 0.3 M CaCl.,. — When unfertilized Arbacia eggs were placed
in 0.3 M CaCL,, at any pH between 8.0 and 8.5, a certain percentage
(rarely more than 25 per cent) of the eggs of most individuals undergo
cleavage. It is difficult to make reliable counts of the percentage of acti-
vation in Arbacia eggs inasmuch as cells that have undergone cleavage
usually occur in groups. The time required for maximum percentage of
cleavage is from 7 to 10 hours or considerably longer than the time of
cleavage in eggs activated by sperm. In a study of the eggs of 40 indi-
viduals the percentage of cytolysis was found to be high (33.5 per cent)
if the eggs were aged for about 11 hours before being placed in the
calcium solution.
A comparative study was made of the percentage of cleavage when
the eggs were obtained in various ways. In some instances a fragment
of ovary was placed directly in the solution to be tested. The exuding
eggs are called ovary eggs. If such exuded eggs were washed in sea
water they are called washed eggs. Shed eggs were obtained in the usual
manner. Experiments were performed on the eggs of about 60 indi-
viduals and tables were prepared on the comparative percentage of cleav-
age. These tables are elaborate and it was thought unwise to attempt
their publication (see original paper). The following results were
obtained :
Average percentage of cleavage
Ovary eggs 11.8
Shed eggs 22.8
Shed eggs 23.8
Washed shed eggs 30.9
268
J. HOLLINGSWORTH
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ACTIVATION BY BIVALENT CATIONS
269
There is a higher percentage of cleavage in shed eggs than in ovary eggs
and a slightly higher percentage of cleavage in washed shed eggs than
in shed eggs.
Table III shows that when the unfertilized eggs of one individual
are placed in solutions of CaCL in which the pHs vary from 3.8 to 9.6,
for from 5 to 9 hours, the percentage of cleavage is low below pH 8.0
and is only slightly higher at pH 8.5 while the highest percentage of
cleavage takes place at about pH 9.0. Cleavage takes place in the short -
100
o
0>
c.
Rotio
FIG. 3. Action on Arbacia eggs of isotonic solutions of some monovalent and
bivalent cations singly and in various mixtures and proportions.
K/Ca — average of fourteen experiments.
Na/Ca — average of nine experiments.
Li/Ca — average of eight experiments.
Na/Mg — average of three experiments.
est time at pH 9.2 but the eggs soon undergo cytolysis. High alkalinity
also seems to cause nuclear division without cytoplasmic cleavage in a
large percentage of eggs. In many cases there is little and sometimes
no cleavage in solutions of which the pH is about 8.8 while there may
be a high percentage of cleavage in solutions of which the pH is about
9.0. Arbacia eggs are not activated by alkaline sea water.
Effect of Mixtures of Calcium Chloride and Some Monovalent Ca-
tions.— The result of experiments in which eggs were exposed to solu-
tions of potassium, sodium or lithium and calcium chloride in various
270
J. HOLLINGSWORTH
proportions and of sodium and magnesium chloride in various propor-
tions is shown in Fig. 3. There is a gradual increase in the percentage
of activation as the proportion of potassium increases up to a ratio of
about 1-2 while above this ratio there is a decrease with activation ceas-
ing at a ratio of about 8-1. From ratios 16-1 to 64—1 there are many
eggs in which the nucleus has undergone several divisions and in which
Ratio
FIG. 4. Action on Arbacia eggs of isotonic solutions of some bivalent cations
singly and in various mixtures and proportions.
Mg/Ca — average of eight experiments.
Sr/Ca — average of eight experiments.
Ba/Ca — average of eleven experiments.
there has been no cytoplasmic division. There is a marked increase in
the percentage of activation as the proportion of sodium increases up to
about ratio 1-4 followed by a marked decrease, with no activation in
mixtures in which the ratio is above 16-1 or in the Na solution. Iso-
tonic LiCl solution is able to activate a small percentage of Arbacia eggs.
The degree of activation increases rapidly as the proportion of lithium
increases beginning in a ratio of about 1-16, with the highest percentage
at a ratio of about 1-2, followed by a sharp decline at a ratio of about
ACTIVATION BY BIVALENT CATIONS 271
2-1. Isotonic MgCL is able to activate a very small percentage of eggs.
There is a very great increase in the percentage of activation as the
proportion of sodium increases up to an average ratio of 1-8 followed
by an equally sudden decrease with activation ceasing at a ratio of about
8-1. This result is of interest because there is a marked increase in
the percentage of activation both in Na/Ca mixtures and Na/Mg mix-
tures between ratios of about 1-32 and 1-8.
Effect of Mixtures of Calcium Chloride and Some Bivalent Cations.
-The result of experiments in which Arbacia eggs are exposed to solu-
tions of magnesium, strontium or barium chloride and calcium chloride
in various proportions is shown in Fig. 4. While magnesium has a
slight activating effect, there is a mutual anatagonism between calcium and
magnesium. Strontium is able to activate a small percentage of eggs but
is much less effective than calcium. The degree of activation by calcium
decreases gradually as the proportion of strontium increases up to a ratio
of about 1-8 with a sharp decrease above a ratio of 1-8. It may be
concluded that there is an antagonism of calcium by strontium. Barium
alone is able to activate a small percentage of eggs. The degree of acti-
vation decreases as the proportion of barium increases with activation
ceasing at a ratio of about 2-1. It may therefore be concluded that
there is antagonism of calcium by barium. There are many eggs with
nuclear divisions without cytoplasmic cleavage in mixtures where the
Ba/Ca ratio is between 32-1 and 64-1.
In summary, Na and K are not effective in activating Arbacia eggs
while Li activates a small percentage. The monovalent cations increase
the percentage of activation by calcium when present in certain definite
proportions. The bivalent cations, Sr, Ba and Mg each have an inhibit-
ing effect on activation by Ca and the antagonism is mutual. There is
even more variation in the behavior of Arbacia eggs than in Cumingia
eggs but again it is a variation in magnitude rather than in kind (see
original paper).
DISCUSSION
The problem of the activation of unfertilized eggs by an alteration
of their chemical environment has been vigorously attacked by a number
of investigators. R. S. Lillie (1910, 191 la, 1911ft) was the first to
report activation of marine eggs by means of isotonic salt solutions. To
date, the activation by isotonic salt solutions of the eggs of seven species
of marine invertebrates has been reported. In each case, activation was
accomplished by exposing eggs to varying combinations and proportions
of the chlorides of the cations of sea water. In all, the presence of Ca
appears to be essential while there is variation in the element which it is
necessary to add.
272 J. HOLLINGSWORTH
R. S. Lillie (1910) reported the initiation of development in Arbacia
eggs when exposed to isotonic NaCl for varying periods of time fol-
lowed by return to sea water. He reported no activation of Arbacia
eggs when exposed to isotonic solutions of CaCL, SrCL and MgCL
followed by return to sea water. In the present work the opposite of
these observations is reported. The difference in results obtained with
the monovalent cations may be due to the fact that in the present work
the eggs were not returned to sea water and the difference in results ob-
tained with bivalent cations may be due to the fact that Arbacia eggs
must be exposed to isotonic solutions of CaCL, SrCl2 and MgCL for
hours in order to obtain a noteworthy percentage of activation.
The results of the experiments on the eggs of Arbacia reported in this
paper are in agreement with the results of the work on marine eggs re-
ported by Dalcq (1928) on Barnca Candida, Hobson (1928) on Thalcs-
sema ncptuni and Pasteels (1935) on Phascolion and Hydroidcs where
alkalinity and the monovalent cations K, Na and Li increase markedly
the percentage of activation of Arbacia eggs by Ca and are necessary in
order to obtain a high percentage of activation of the eggs of most indi-
viduals and where cleavage appears to be more nearly normal in favorable
binary mixtures than in isotonic CaCL alone.
However, the results on activation of the eggs of Cumingia by iso-
tonic salt solutions are not in agreement with the results of the work
reported by Dalcq, Hobson and Pasteels on activation of marine eggs by
isotonic salt solutions. The addition to the calcium solution of excess
OH ions or of the monovalent cations Na or K does not increase the
percentage of activation of Cumingia eggs but has the opposite effect.
The segmenting eggs appear more nearly normal and more healthy in
isotonic CaCL alone than in any of the binary mixtures used. Activa-
tion by isotonic CaCL with respect to time of polar body formation and
percentage of activation compares favorably with activation of eggs by
sperm. We may say that in the case of Cumingia eggs, Ca is the sole
activating agent and that no other external agent or treatment is necessary.
There are several theories to explain the activation of unfertilized
eggs. All these theories are aspects of more general theories of stimula-
tion. The oxidation theory of activation was stated by J. Loeb (1913).
It is now quite certain that not all activating agents increase the rate of
oxidation. Heilbrunn (1915) pointed out that cyanide does not prevent
the first stages of development in Arbacia eggs and (1920a) that matura-
tion in Cumingia eggs is not dependent on an increase in oxygen con-
sumption. Whitaker (1931, 1932) reported that in the eggs of Nereis
and Arbacia there is an increase in the rate of respiration following
fertilization whereas in the eggs of Chaetoptcrus and Cumingia there is
ACTIVATION BY BIVALENT CATIONS 273
a decrease following fertilization. Activation of eggs by an isotonic
solution of CaCL can scarcely be due to an increase in the rate of oxida-
tion for calcium is usually thought to decrease the rate of oxidation (see
for example, Ahlgren, 1925; Hoick, 1934; and Thunberg, 1937). It
may therefore be concluded that rate of oxygen consumption is not the
primary factor in the initiation of development of eggs.
The permeability theory of activation, founded by R. S. Lillie (1916,
1917, 1918) has been used to explain initiation of development in eggs.
That there is an increase in permeability in some marine eggs following
activation has been convincingly demonstrated by a number of careful
investigators. The work of Lillie (1916, 1917, 1918) and McCutcheon
and Lucke (1932) shows that the permeability of Arbacia eggs to water
increases after fertilization and the work of Stewart and Jacobs (1932)
shows that permeability of these eggs to ethylene glycol increases after
fertilization. However, activation of eggs by isotonic CaCL cannot be
conceived of as due to an increase in permeability. It is universally
agreed among students of permeability that bivalent cations such as mag-
nesium and calcium cause a decrease in cellular permeability and antago-
nize those reagents known to increase it. Therefore the action of cal-
cium in the activation of Cumingia eggs cannot be explained on the basis
of the permeability theory.
Dalcq (1924o, 1924/7) has developed a depolarization theory of acti-
vation. This theory depends upon the presence of charges of definite
sign upon the cortex and constituents of the egg and upon the existence
of a potential gradient on the cortex. He concluded that a disturbance
of the intraovular cations results in depolarization, that Ca is the most
effective agent in bringing about depolarization and that activation is the
result of depolarization. However, the depolarization theory of Dalcq
seems highly speculative and is difficult to understand from the electro-
chemical standpoint.
Heilbrunn (1915) favored the coagulation theory of activation. This
theory, which is now termed the colloid chemical theory is, as the
permeability theory, a broad theory of stimulation for all types of irri-
table systems. In a study of the chemical changes in the egg following
activation, Heilbrunn and his students have shown that whenever a cell
is stimulated, Ca is released from the cortex. Heilbrunn and his stu-
dents have further shown that if Ca is first removed from egg cells by
oxalate, stimulating agents are not effective but that upon the return to
sea water the usual response may be obtained (see Heilbrunn and R. A.
Young, 1930; Heilbrunn and K. Wilbur, 1937). For a full discussion
of the colloid chemical theory of stimulation see Heilbrunn's " Outline
of General Physiology," 1937. The results of the study of the activa-
274 J. HOLLINGSWORTH
tion of the eggs of Arbacia by favorable binary mixtures of bivalent and
monovalent cations and of the study of the activation of the eggs of
Cumingia by isotonic solutions of bivalent cations alone, where 100 per
cent of the eggs of some individuals undergo apparently normal cleavage
in a period of time which compares favorably with the time of activation
of eggs activated by sperm, favor the colloid chemical theory of Heil-
brunn and are directly opposed to any interpretation in terms of the
oxidation or permeability theories.
SUMMARY
1. When unfertilized Cumingia eggs are placed in 0.3 M CaCl,, 100
per cent of the eggs of some individuals undergo apparently normal cleav-
age. The time of polar body formation and of the first cleavage in eggs
activated by Ca is approximately the same as the time of polar body for-
mation and of cleavage in eggs activated by sperm.
2. Polar body formation and cleavage in Cumingia eggs proceed nor-
mally in 0.3 M CaClo at the various pHs between pH 6.2 and pH 8.6 but
are inhibited above and below this range.
3. The bivalent cations Sr, Ca and Ba are able to activate Cumingia
eggs and are named in the order of their effectiveness.
4. The time of polar body formation in Cumingia eggs activated by
isotonic solutions of SrCl2 and BaCl2 is approximately the same as the
time of polar body formation in eggs activated by sperm.
5. The monovalent cations K and Na and sea water inhibit activation
of Cumingia eggs by Ca. The percentage of activation decreases as the
K/Ca, Na/Ca and sw/Ca ratios increase. K has a greater inhibiting
effect than Na.
6. Ba inhibits the activation of Cumingia eggs by Ca, Sr inhibits very
slightly the activating effect of Ca, while Mg does not appear to have an
inhibiting effect on activation of eggs by Ca.
7. When unfertilized Arbacia eggs are placed in 0.3 M CaCU, from
40 to 60 per cent of the eggs of most individuals undergo cleavage if the
pH of the solution is between 8.8 and 9.2. No membrane is elevated in
isotonic salt solutions.
8. Below pH 8.8 the percentage of cleavage is low and above pH 9.0
the percentage of cytolysis is high in Arbacia eggs activated by isotonic
CaCl,.
9. Isotonic solutions of SrCl,, BaCU and MgCL are able to activate
a certain percentage of Arbacia eggs, but these ions are not so effective
as Ca and their action is somewhat variable.
10. The monovalent cations Na, Li and K in certain definite propor-
tions increase the percentage of cleavage induced by Ca while in other
ACTIVATION BY BIVALENT CATIONS 275
proportions they have the opposite effect. Similarly isotonic NaCl, in
certain definite proportions increases markedly the percentage of activa-
tion by the Mg solution while in other proportions Na has the opposite
effect.
11. Sr, Mg and Ba inhibit the activation of Arbacia eggs by Ca, the
inhibiting effect increasing as the Sr/Ca, Mg/Ca and Ba/Ca ratios
increase.
12. The results are brought into relation to the colloid chemical theory
of stimulation.
LITERATURE CITED
AHLGREN, G., 1925. Zur Kenntnis der tierschen Gewebsoxydation sovvie ihrer
Beeinflussung durch Insulin, Adrenalin, Tliyroxin und Hypophysepriiparate.
Skand. Arch. Physiol., 47 : Suppl, 1-266.
ANGERER, C. A., 1936. The effect of mechanical stimulation on the protoplasmic
viscosity of Amoeba protoplasm. Jour. Cell, and Coinp. Physiol., 8 :
329-345.
DALCQ, A., 1924a. Le role des prinicipaux metaux de 1'eau de mer dans 1'activation
de 1'oeuf en maturation. Bull. d'Hist. a/'/'/, a la Phys. ct a la Path., 1 :
465-485.
— , I924b. Recherches experimentales et cytologiques sur la maturation et 1'acti-
vation de 1'oeuf d'Asterias glacialis. Arch, de Biol., 34: 507-674.
— , 1928. Le role du calcium et du potassium dans 1'entree en maturation de
1'oeuf de Pholade (Barnea Candida). Proto plasma, 4: 18-44.
DONNELLON, J. A., 1938. An experimental study of clot formation in the peri-
visceral fluid of Arbacia. Physiol. Zobl, 11: 389-397.
HEILBRUNN, L. V., 1915. Studies in artificial parthenogenesis. II. Physical
changes in the egg of Arbacia. Biol. Bull, 29: 149-203.
— , 1920a. Studies in artificial parthenogenesis III. Cortical change and the
initiation of maturation in the egg of Cumingia. Biol. Bull., 38 : 317-339.
— , 1928. The Colloid Chemistry of Protoplasm. G. Borntraeger, Berlin.
— , 1937. An Outline of General Physiology. W. B. Saunders Co., Philadelphia.
HEILBRUNN, L. V., AND K. DAUGHERTY, 1933. The action of ultraviolet rays on
Amoeba protoplasm. Protoplasma, 18 : 596-619.
HEILBRUNN, L. V., AND R. A. YOUNG, 1930. The action of ultra-violet rays on
Arbacia egg protoplasm. Physiol. Zobl., 3: 330-341.
HEILBRUNN, L. V., AND D. MAZIA, 1936. The action of radiations on living proto-
plasm. Duggar's Biological Effects of Radiation, 1 : 625-676. •
HEILBRUNN, L. V., AND K. WILBUR, 1937. Stimulation and nuclear breakdown in
the Nereis egg. Biol. Bull., 73: 557-564.
HOBSON, A. D., 1928. The action of isotonic salt solutions on the unfertilized eggs
of Thalessema neptuni. Brit. Jour. E.rpcr. Biol., 6 : 65-78.
HOLCK, H. G. O., 1934. Studies on the effect of calcium, strontium and barium
chlorides on the oxidation processes in various tissues. Skand. Arch.
Physiol., 70 : 273-294.
HORSTADIUS, S., 1923. Physiologische Untersuchungen iiber die Eireifung bei
Pomatoceros triqueter L. Arch. f. mikr. Anat., 98: 1-9.
LILLIE, R. S., 1910. The physiology of cell division. II. The action of isotonic
solutions of neutral salts on unfertilized eggs of Asterias and Arbacia.
Am. Jour. Physiol., 26: 106-133.
— , 191 la. The physiology of cell division. III. The action of calcium salts in
preventing the initiation of cell division in unfertilized eggs through iso-
tonic solutions of sodium salts. Am. Jour. Physiol., 27 : 289-307.
276 J. HOLLINGSWORTH
— , \9\\b. The physiology of cell division. Jour. Morph., 22: 695-730.
— , 1916. Increase of permeability to water following normal and artificial acti-
vation in sea urchin eggs. A in. Jour. PhysioL, 40 : 249-266.
— , 1917. The conditions determining the rate of entrance of water into fertilized
and unfertilized Arbacia eggs, and the general relation of changes of
permeability to activation. Am. Jour. PhysioL, 43: 43-57.
— , 1918. The increase of permeability to water in fertilized sea-urchin eggs and
the influence of cyanide and anaesthetics upon this change. Am. Jour.
PhysioL, 45 : 406-430.
LOEB, J., 1913. Artificial Parthenogenesis and Fertilization. Chicago.
MAZIA, D., AND J. M. CLARK, 1936. Free calcium in the action of stimulating
agents on Elodea cell. Biol. Bull., 71 : 306-323.
McCuTCHEON, M., AND B. LucKEi, 1932. The effect of temperature on permeability
to water of resting and of activated cells, etc. Jour. Cell, and Com[>.
PhysioL, 2: 11-26.
MORRIS, M., 1917. A cytological study of artificial parthenogenesis in Cumingia.
Jour. E.vpcr. Zob'L, 22 : 1-35. "
PASTEELS, J. J., 1935. Recherches sur le determinisme de 1'entree en maturation
de 1'oeuf chez divers invertebres marins. Arch. Biol., 46: 229-262.
SPEK, J., 1930. Zustandsanderungen der Plasmakalloide bei Befruchtung und Ent-
wicklung des Nereis-eies. Protoplasma, 9 : 370-427.
STEWART, D. R., AND M. H. JACOBS, 1932. The effect of fertilization on the per-
meability of the eggs of Arbacia and Asterias to ethylene glycol. Jour.
Cell, and Comp. PhysioL, 1 : 83-92.
THUNBERG, T., 1937. Die hemmende Wirkung der Erdalkalien und besonders des
Kalkes auf gewisse physiologische Oxydations-prozesse. Skand. Arch.
PhysioL, 75 : 279-294.
WHITAKER, D. M., 1931. On the rate of oxygen consumption by fertilized and
unfertilized eggs. Jour. Gen. PhysioL, 15: 167-200.
— , 1933. On the rate of oxygen consumption by fertilized and unfertilized eggs.
Jour. Gen. Pli\su>l., 16: 475-525.
PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS
PRESENTED AT THE MARINE BIOLOGICAL
LABORATORY, SUMMER OF 1941
JULY 8
The source of pancreatic juice bicarbonate. Eric G. Ball.
(This paper has already appeared in full in the July, 1941 number of the
Jmtnnil of Biological Chemistry.)
The permeability and the lipid content of the erythrocytes in experi-
mental anemia. Arthur J. Dziemian.
In a series of albino rabbits the permeability of the erythrocytes to glycerol,
diethylene glycol, ammonium propionate and ammonium salicylate was studied and
the total lipid, cholesterol and phospholipid contents of the red cells, calculated per
ml. of cells, per erythrocyte and per square micron of cell surface, were determined.
The rabbits received subcutaneous injections of 50 mg. of phenylhydrazine hydro-
chloride and changes in the permeability of the red cells to the above substances
were studied. Within about nine days after injection, practically an entirely new-
population of red cells was present in the animals, as shown by a study of changes
in cell diameters. At this time the rate of penetration into the cells of diethylene
glycol and glycerol had greatly increased, while the ammonium propionate and
salicylate penetrated slower than normal. Thereafter the times of 50 per cent
hemolysis of the red cells in all the solutions used returned slowly toward the
original values. When lipid analyses on the red cells of the experimental animals
were made, no correlation was found between the changes in permeability and the
changes in the lipid content of the erythrocytes.
TJie rectifying property of the giant axon of the squid. Rita Guttman
and Kenneth S. Cole.
Measurements of the resistance of the squid giant axon have been made by
means of a direct current Wheatstone bridge between one injured end of the axon,
placed in KC1, and the other end immersed in sea water, with the inter-electrode
region hanging in oil. During the passage of small currents through the axon, the
resistance of the fiber does not depend upon the magnitude and direction of the
current. But when larger currents are used this is no longer true, i.e. if the un-
injured end is the anode, the over-all resistance of the axon is greater than that
found during the passage of small currents while at the cathode the reverse is
true. The apparent resistance of the nerve fiber, then, depends upon the magnitude
and direction of the current flowing through it. The nerve fiber thus does not
obey Ohm's Law and is an electrical rectifier which permits current to pass more
easily in one direction than the other, rather than a pure resistance.
Cocaine and veratrine cause progressive and reversible loss of rectification. As
the axon dies, excitability is lost, and the rectification and the resting potential
disappear. When the fiber is completely dead, there is no rectification and the fiber
acts as a pure resistance.
Such a rectification is to be expected if the membrane conductance is a measure
of ion permeability and this permeability is increased at a cathode and decreased at
an anode. Also, rectification has been suggested as an explanation of some elec-
trotonic and excitation phenomena.
277
278 PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS
Metabolism and fertilisation in the starfish egg. Herbert Shapiro.
The respiration of immature, mature and fertilized eggs of the starfish, Astcrlas
forbesii, was studied during May and June by means of Warburg manometers,
over a temperature range 11.5 to 27.8° C. Oxygen uptake by unfertilized eggs
may remain constant for periods as long as ten hours. Fertilized eggs show a
relatively constant rate at first, and then a slowly increasing one, as embryological
development advances. An average increase of approximately 30 to 50 per cent
was found in the rate of fertilized eggs, as compared with unfertilized, the rise
being slightly higher at the low temperatures.
Although some experiments yielded results in agreement with those of Loeb
and Wasteneys (Arch, entw.-mech. Organism., 35: 555, 1912) and of Tang (Biol.
Bull., 61: 468, 1931) in showing little or no change after fertilization, the more
extended series reported here, done on eggs showing high percentages of cleavage,
and during the optimal portion of the breeding season, demonstrate that the average
starfish egg undergoes a significant acceleration of oxidations subsequent to
fertilization.
JULY 15
Factors in the lunar cycle which may control reproduction in the Atlantic
palolo. L. B. Clark.
Of all the physiological influences attributed to the lunar cycle, the coincidence
of reproduction of certain marine polychaetes with specific phases of the moon has
been best determined. Of such animals, the palolo worms are perhaps outstanding
because of their size, striking reproductive behavior, and the apparent specific
relation between the moon's phases and time of reproduction.
A number of experiments on artificially changing the light relations of the
lunar cycle by illuminating or shading rocks containing worms were undertaken.
The results of all the experiments are consistent in that if the average duration of
light is increased, reproduction occurs before the controls, and if the average
duration of moonlight is decreased, the time of swarming occurs after the controls
or not at all. It is concluded, therefore, that this is a factor involved in reproduc-
tion and that the effectiveness of the various phases of the moon's cycle is corre-
lated with the average duration of moonlight during the cycle.
If this were the only factor involved, the effectiveness of moonlight to induce
swarming would increase to a maximum about three days after the full moon and
then decrease. But the effectiveness of moonlight is bimodal, the modes centering
about the first and last quarter moon, with the latter much more effective. Ob-
viously there must be some other factor operating in moonlight. The only other
factor varying in the desired manner is the daily difference in the rate of change
of moonlight. This reaches a maximum at the new and full moons and a minimum
at the first and third quarter. If it is postulated that the effectiveness of moon-
light in determining the time of swarming bears some correlation to the reciprocal
of the difference in the daily rate of change of moonlight, the resultant varies in a
manner similar to the incidence of swarming during the lunar cycle.
Accumulation of manganese and the sexual cycle in Ostrea virginica.
Paul S. Galtsoff.
Oysters of known age and origin, planted on an experimental bottom in Long
Island Sound near Milford, Connecticut, showed a distinct annual cycle in the
Mn-content which varied from 7.3 to 51.0 mg. per kilo d.w. During the twenty-
nine months' period of observations the high Mn content (from 30 to 50 mg.
p.k.d.w.) invariably coincided with the period of gonad development and sexual
activity of the oysters (May-August) while low Mn content (from 7 to 11 mg.
PRESENTED AT MARINE BIOLOGICAL LABORATORY 279
p.k.d.w.) occurred in winter and early spring (November-April). Ovaries were
found to be particularly rich in Mn (51.0-59.6 mg. p.k.d.w.) and testes contained
only from 4.6 to 7.2 mg. p.k.d.w. Other tissues contained the following amounts :
gills, 17 to 18 mg. p.k. in winter and from 35 to 38.6 in summer; mantle, 8.7 in
January and from 14.2 to 17.0 in September; visceral mass, 8.9 to 18.4; and ad-
ductor muscle, 4.3 to 5.2 in July and 4.1 to 9.3 in January. These results indicate
that the Mn cycle is associated with the development of a female phase of the
oyster. The physiological role of the metal in the metabolism and its relation to
the sex change in this species is not known.
Studies on the life history of Siphodcra vinaledwardsii, a trematode
parasite of tiic toadfish. R. M. Cable and A. V. Hunninen.
Experimental studies on the life history of Siphodcra vinaledwardsii (Linton)
have demonstrated that this trematode is related to the Heterophyidae as postulated
by Manter, Price and Wilhelmi on the basis of morphological and serological in-
vestigations. The definitive host in the Woods Hole region is the toadfish, Opsainis
tan, practically all of which are naturally infected. The small marine snail, Bit-
tiuin alternation, serves as the molluscan host in which the cercariae develop in
simple, elongate radiae. The cercaria is a pleurolophocercous form of an unusual
type since the tail is inserted ventrally and coiled when at rest, the fourteen pene-
tration glands have two instead of the usual four bundles of ducts in the region of
the oral sucker, and the excretory formula is 2[(2-f 2) + (2 + 2)] == 16 flame
cells. The cercariae penetrate and encyst in various species of flounders, develop-
ing into apparently infective metacercariae in approximately two weeks. Cysts
occur in the fins, body wall and even the myocardium of the flounder. Feeding
experiments thus far completed indicate that the toadfish becomes infected by eating
fish containing metacercariae. Three toadfish, isolated for four weeks, were fed
fish containing 13-day metacercariae. Two of these have been examined to date
and found to harbor large numbers of very young worms in addition to a few
mature specimens from previous natural infection.
Pathology and immunity to infection with heterophyid trematodes.
Horace W. Stunkard and Charles H. Willey.
The term heterophyid refers to a large family of digenetic trematodes which
infect fish-eating birds and mammals. Cryptocotyle lingua is a common hetero-
phyid species in the Woods Hole area; its life cycle was reported by Stunkard
(1930). The larval stages are produced in Littorina Httorca and L. rudis, while
the cercariae encyst in the dinner and other fishes.
Stunkard and Willey (1929) studied the development of C. lingua in cats and
rats. In these hosts, the worms developed to sexual maturity between the intes-
tinal villi and no intramucosal invasion was observed. Since there is evidence to
indicate that cats and rats are not favorable hosts, the studies were continued on
terns and dogs.
Young terns developed a very severe infection from the sixth to the fourteenth
day, when the number of eggs in the feces began to diminish. After the twentieth
day the feces contained very few eggs and large numbers of young worms recently
liberated from their cysts. After an initial heavy infection, gulls and terns develop
a strong resistance to superinfection and the presence of a few worms serves to
maintain a substantial immunity.
A dog, fed enormous numbers of cysts, began to pass eggs of the parasite on
the fifth day. Large numbers of immature and mature worms were present on
the surface of tine mucosa and in the crypts between the villous folds. The villi
showed acute inflammatory changes, desquamation, hyperemia, and excessive mucous
secretion. There was no invasion of the intestinal glands or tunica propria. An-
280 PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS
other dog, similarly fed for fourteen days, was in a moribund condition and autopsy
revealed the presence of thousands of sexually mature worms. Dogs were given
moderate infections and allowed to recover. Eggs began to appear in the feces
on the fifth day, were numerous for about four weeks, after which the number
began to decline. At the end of three months very few eggs could be found and
the feces were negative at the end of six months. After resistance had been estab-
lished in dogs, the feeding of large numbers of metacercariae produced no visible
ill effects and very few eggs appeared in the feces.
These experiments show that birds and dogs, if the latter survive an initia'
infection, effect a "self-cure" (as that term was defined by Stoll, 1929) and there-
after are resistant to any substantial reinfection.
JULY 22
The organisation of the melanophore system in bony fishes. G. H.
Parker.
Catfish color changes, which range from pale yellowish-green to coal-black, are
mediated in the main by three receptors, the dorsal retina, the ventral retina, and
the skin. The pale phase of this fish is excited through the dorsal retina, which is
best illuminated when the fish is on a white background lighted from above. Chro-
matic nerve tracts extend from the dorsal retina through the central nervous organs
and the autonomic system to the melanophores. The final fibers in these tracts are
adrenergic in that they discharge adrenaline which blanches the fish by causing
melanophore pigment concentration. The fiber tracts concerned with this response
may be designated as the retino-adrenergic arc. The blood of a pale catfish is
devoid of the darkening agent intermedine, a state which indicated the inhibition of
the intermediate pituitary lobe. From the dorsal retina nerve tracts presumably
extend through the central nervous organs to the pituitary gland and thus mediate
pituitary inhibition. Such tracts may be called the retino-pituitary inhibition arc.
The dark phase of the catfish is induced through the ventral retina and the
skin. When the fish is on a black background the ventral retina is excited by light
from above. From it impulses pass through the central nervous organs and the
autonomic system over whose cholinergic fibers they reach the melanophores. Here
the resultant acetylcholine excites the melanophores to disperse their pigment and
thus to darken the fish. The tracts concerned with these activities constitute the
retino-cholinergic arc.
The second receptor concerned with the dark phase of the catfish is the skin,
which can be studied best in blinded fishes. Such fishes, dark, intermediate, or
pale, if put at once into darkness retain their original tint for days, but on being
exposed to daylight quickly become coal-black. From the photoreceptors in the
skin nerve-fibers pass in tracts to the pituitary gland which is thereby excited to
discharge intermedine. This then passes by way of the blood to the melanophores
whose dispersed pigment darkens the fish. These nerve tracts and blood courses
may be called the dermo-pituitary arc. Other bony fishes whose melanophore sys-
tems are much like that in the catfish are the angelfish, eel, snakefish, Japanese
catfish and the stickleback. The killifish and the flatfishes are similar but lack
effective pituitary organs.
Some aspects of pigment deposition in feather germs of chick embryos.
Ray L. Watterson.
A study of the developmental history of melanophores in the wing skin and
feather germs of Barred Rock embryos indicates that pigment deposition is not a
function of pigment cells alone, but is definitely controlled by recipient barbule
cells. (1) Melanophores are packed with pigment granules early in development.
Nevertheless, pigment is not distributed to epidermal cells until certain of them
"PRESENTED AT MARINE BIOLOGICAL LABORATORY 281
become visibly differentiated as barbule cells. (2) Pigment granules accumulate
at the tips of pigment cell processes and become pinched off, whereupon they lie
freely among the epidermal cells. Pigment liberated in this manner is later taken
up by barbule cells. (3) Pigment is deposited in each row of barbule cells in a
definite sequence. The most peripheral barbule cells are the first to elongate and
to form keratin; only when these visible differentiation processes begin can they
receive pigment. As this wave of differentiation spreads toward the pulp, more
axial cells become capable of receiving pigment. (4) A study of the development
of down feathers with split barb-vanes indicates that pigment cell processes are
specifically attracted toward barbule cells. In their development barbule cells dif-
ferentiate in the center of a barb-vane ridge where they normally do not occur.
Melanophore processes leave their normal paths, extend toward these centrally
located cells and carry pigment to them. (5) Pigment deposition stimulates the
melanophores involved to undergo proliferation. Melanophores undergoing mitotic
division occur almost exclusively at those levels of feather germs where pigment
deposition is in progress. (6) Feather germs elongate slowly until 10 days and
18 hours, whereupon they elongate rapidly, attaining their full growth by 13 days.
The onset of pigment deposition coincides with the onset of rapid growth. Lillie
and Juhn have estimated that 90 per cent of the axial growth of regenerating
feathers is accomplished by cell elongation. Pigment deposition begins at that
phase of development when barbule cells begin to elongate rapidly.
The influence of hormones on the differentiation of melanophores in
birds. Howard L. Hamilton.
When explants of skin from embryos of birds which have red and black pig-
ments in their feathers are grown in a tissue culture medium consisting of blood
plasma and embryonic extract, black melanophores appear but red ones occur very
infrequently. If sex hormones are added to the culture medium, then many red
melanophores as well as black ones differentiate in the explant. The two kinds
of pigment cells are recognized as discrete cell types because of color, shape, and
solubility differences in the granules, viscosity differences in the cytoplasm as
shown by more rapid granule movement in red melanophores, and differences in
their reactions to various hormones. In general, sex hormones increase the number
of red melanophores which differentiate in treated explants from red breeds. Ses-
ame and olive oils also produce a stimulation (possibly due to traces of sterols).
Black melanophores are inhibited by estradiol, but estrone and testosterone favor
their differentiation.
Desoxycorticosterone, an adrenal cortical hormone, decreases the number of
melanophores in the New Hampshire Red, White Leghorn, and Barred Rock breeds
of fowl. Sex hormones produce a similar inhibition of black melanophores in the
latter breed. Young tissue (5-6 days) yields few or no melanophores when grown
in the presence of hormone; in older tissue (7-8 days) there is a reduction in num-
ber of melanophores and inhibition of feather germ formation as well. Crystalline
hormones act apparently by slowing the process of melanin synthesis, so that the
treated cells, although chronologically of the same age as the controls, are physio-
logically younger. It is concluded that genetic differences in the precursor cells
must determine whether they become red or black melanophores, but that environ-
mental factors (physiological differences in feather germs; hormones) may directly
influence which of the two kinds will predominate.
The distribution and development of the melanophore hormone in the
pituitary of the chick. Hermann Rahn.
In the chicken a structural pars intermedia is absent. The melanophore hor-
mone, however, is present in considerable quantities and is found in greatest concen-
PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS
tration in the region of the pars anterior furthest removed from the infundibular
process (Kleinholz and Rahn). Quantitative assays of the melanophore hormone
were made on the pituitary of chicks throughout the whole development. The
Anolis lizard test was used for these determinations. The first appearance of the
hormone can be detected on the fifth day of incubation, i.e. five days before differ-
entiation in the pituitary can be seen by ordinary cytological methods. During the
last half of the incubation period the melanophore hormone per unit weight of
pituitary tissue increases rapidly and reaches its greatest concentration at hatching
time. All further apparent increase in hormone per total gland after hatching time
can be accounted for by the increasing growth or weight of the pituitary gland.
JULY 29
Effect of sea water on the radio sensitivity of Arbacia spenn. T. C.
Evans and J. C. Slaughter.
The percentage fertilization resulting from inseminations with sperm irradiated
"dry" is much greater than that irradiated in sea water. The amount of injury to
the sperm increases with dilution. For example, to reduce the fertilizations to 50
per cent, sperm diluted to 1 : 100 must receive 20,000 roentgens, whereas a suspen-
sion of 1 : 1000 needs only 3,000 r.
The sperm are more susceptible immediately after the addition of sea water,
when the rate of oxygen consumption is high, than thirty minutes later when the
rate of oxygen consumption is lower.
Concentrations of as low as 0.01 per cent egg albumen greatly increase the
radioresistance of sperm in sea water. The resistance in sea water is also increased
upon the addition of sufficient amounts of dead Arbacia sperm or living sperm of
Nereis.
It therefore appears that the condition of the medium during irradiation mark-
edly affects the radiosensitivity of the sperm as measured by the decrease in per-
centage fertilization.
N
The fractionation of cellular respiration by the use of narcotics. Ken-
neth C. Fisher.
An examination has been made of new data and of data from the literature
concerning the effects of narcotics on oxygen consumption. The relation between
concentration and effect seen in all these cases possesses a feature which suggests
that the inhibitor affects independently two discrete parallel respiratory systems in
each of the preparations. In general the concentration range over which the effects
are graded is not identical for the two systems.
Inhibition of cell division in yeast, a protozoon, the sea-urchin egg and of light
production in luminous bacteria, approaches completion at narcotic concentrations
which affect oxygen consumption in the same preparation relatively much less.
Actually, inhibition of these activities parallels closely inhibition of the more sensi-
tive of the two respiratory systems.
One substance, benzoate, has been found for which the sensitivities of the two
respiratory systems in yeast are just the reverse of those shown for narcotics, i.e.,
as the concentration of benzoate is gradually increased, the "second" system is
inhibited before the "first." As would be expected ideally, this compound inhibits
oxygen consumption approximately 55 per cent before beginning to affect the rate
of cell division.
It is concluded that in many cells the oxygen consumption, at least at the site
of action of narcotics and allied substances, is composed of two independent parallel
respiratory systems. The metabolism for cell division and light production appears
to be associated with only one of these two.
These observations have been made with the collaboration of Messrs. J. R.
Stern, R. J. Henry and Richard Ormsbee.
PRESENTED AT MARINE BIOLOGICAL LABORATORY
Effect of aside on Cypridina luciferin. Aurin M. Chase.
There are a number of pieces of information now available which bear upon
the chemical nature of Cypridina luciferin. Anderson (1936) has suggested a
resemblance to certain naturally occurring hydroxy-benzene derivatives studied by
Ball and Chen (1933), on the basis of the similarity in oxidation-reduction potential.
Chase (1940) has described changes in the visible absorption spectrum during oxi-
dation of luciferin which indicate a possible quinoid structure. Giese and Chase
(1940) have postulated an aldehyde or keto group in the luciferin molecule on the
basis of an irreversible combination with cyanide. Chakravorty and Ballentine
(1941) have proposed as a partial structure for the luciferin molecule a hydro-
quinone nucleus with a keto-hydroxy side chain. The hydroquinone nucleus would
explain the reversible, non-luminescent oxidation of luciferin and the keto group
would be the point of combination with cyanide or with luciferase.
Giese and Fisher (unpublished data) have described inhibition of luminescence
in luminous bacteria by sodium azide and this observation prompted the present
study of the effects of azide on luminescence of purified Cypridina luciferin and
luciferase. At pH 6.6 luminescence is found to be reversibly inhibited at azide
concentrations from about O.OOS to about 0.1 M. At pH 5.4 these same azide con-
centrations are much more effective, indicating that the HN. may be the active
agent. The effect appears to be chiefly upon the luciferin. Plotted in terms of
the mass law equation, the data fall upon straight lines with slopes approximately
equal to one.
It is tentatively suggested that HN3 reacts with luciferin in the same way that
it has been shown by Fieser and Hartwell (1935) to react with benzo- and naphtha-
quinones. Further evidence for a quinoid group in the luciferin molecule would
therefore be indicated.
AUGUST 5
Aging phenomena, and factors influencing the longevity of Mactra eggs.
Victor Schechter.
(This paper has appeared in the Jour. Expcr. Zool., Vol. 86, No. 3, for 1941.)
Comparison of the respiratory rates of different regions of the chick
blastoderm during early stages of development. Frederick S.
Philips.
As an introductory study of the chemical processes involved in the regional
differentiation of the chick blastoderm (the area pellucida of the head-process
embryo), the rates of oxygen consumption were studied of various isolated pieces.
In addition, the respiratory rate of pieces containing most of the pellucid area was
determined at various stages of development from the unincubated blastoderm to
the 12-somite embryo. Oxygen consumption was measured in the Cartesian diver
microrespirometer. The total-nitrogen of the tissues was estimated by a modifica-
tion of the Conway micro-Kjeldahl procedure.
The pellucid area of head-process embryos was divided into pieces containing
respectively the head-process, node and anterior streak, middle streak, posterior
streak, and right and left lateral regions. The Qo2' (m./*l. O^ consumed/hour/yN)
of all these regions is essentially similar. No major differences are apparent in
the rate of oxygen consumption of the various regions studied which can be corre-
lated with their marked regional differences in developmental potency.
The Qo/ of the embryonic area increases from a value of about 33 in the
unincubated blastoderm until it reaches a value of about 75 in the 17-hour embryo,
the early definitive streak stage. The head-process, 5-6-somite, and 11-12-somite
embryos have rates of oxygen consumption similar to that of the 17-hour embryo.
284 PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS
During this period of development from the unincubated to the head-process
stage the total quantity of nitrogenous material in the whole area pellucida increases
only slightly. However, the total amount of O2 consumed/hour increases at least
threefold. Apparently, therefore, the increase in respiratory rate during the first
17 hours of development depends on the large increase in cell-number coincident
with the conversion of intracellular yolk material into active cellular constituents.
Further interpretations of the effects of CO and CN on oxidations in
living cells. Mathilda M. Brooks.
In these experiments, the rate of O2 consumption, of cleavage and length of life
of eight different stages in the development of sea-urchin and starfish eggs, as af-
fected by certain accelerators and inhibitors, was studied. The stages included un-
fertilized eggs, first cleavages, morula, blastula, early gastrula, late gastrula, early
pluteus and late pluteus. The reagents were methylene blue (.00012 M) ; KCN
(.00025 M) and CO as near 100 per cent as possible. It was found that methylene
blue accelerated O2 consumption in the early stages, decreased it or had no effect
in the middle stages and increased it again in the late stages. The decrease pro-
duced by KCN varied, so that the rate varied from 63 per cent of the normal to
about 6 per cent depending upon the stage of development ; the decrease produced
by CO varied causing a rate of 92 per cent of the normal in certain stages and a
rate as low as 20 per cent in others. Methylene blue accelerated the rate produced
by CO about 10 per cent, and either increased it when KCN was used or produced
no effect.
In the case of cleavage, KCN produced multiple aster formation without cell
division even in the presence of methylene blue at this concentration. Methylene
blue prevented cytolysis produced by CO : doubled the life of the embryo ; caused
a faster rate of development ; and increased the size of the pluteus stage of
Arbacia from 280 M (controls) to 420 M.
These experiments suggest new aspects of the respiratory enzymes and asso-
ciated redox systems. Since methylene blue poises the potential, it appears that
the optimum redox potential at which these systems function changes with devel-
opment. This can be interpreted either that the respiratory enzyme assumes a
different role or that these enzyme systems are actually different at the various
stages of development.
AUGUST 12
Studies on conditions affecting the survival in vitro of a malarial parasite
(PlasmodiwH lophurac). William Trager.
The malarial protozoa are obligate intracellular parasites which have never been
cultured in vitro. Indeed, little has thus far been discovered concerning even the most
elementary conditions which might favor their survival outside of their living host.
Accordingly, a series of experiments was conducted in which parasitized blood cells
taken from a chicken infected with P. lophwac were placed in various media and
their time of survival at 40-42° C. determined. The chief criterion of survival was
the ability of the parasites to infect baby chicks under a set of standard conditions.
It was found that survival in vitro was favored by aeration but not by a very high
oxygen tension, by a balanced salt solution of high potassium content, by certain
concentrations of glucose or glycogen, by glutathione, by red cell extract, by low
concentrations of chick embryo extract and chicken liver extract, by daily renewal
of the medium and by an optimal density of parasites per cu. mm. In the best
preparations, as judged by infectivity, at least 40 per cent of the original parasites
were alive on the third day, at least 20 per cent on the fourth day, about 1 per cent
on the fifth day and about 0.05 per cent on the sixth day. In these preparations
there was a small increase in parasite number during the first day of incubation.
PRESENTED AT MARINE BIOLOGICAL LABORATORY
The effect of dyes on the response to light in Peranema trichophorum,
Charles C. Has sett.
When stimulated by a sudden increase in the intensity of light, the flagellate
Pcrancma trichophorum responds by a shock reaction, i.e., it ceases forward motion
and bends sharply, then moves off at an angle to its original direction of movement.
The time required to produce this response was used as a measure of the photo-
dynamic effect of a number of dyes. The average reaction-time of untreated
peranemae was found to be 12.1 seconds; the optimum concentration of active dyes
(ca. 5 X 10~4 M), decreased this to ca. 1.0 second; weaker solutions of these dyes
and all solutions of less active dyes produced longer reaction-times, with 12 seconds
as the approximate maximum. The order of effectiveness of the dyes was : rose
bengal, eosin, neutral red, methylene blue, Nile blue sulfate, auramine O. Orange
G had no effect. Brilliant green increased the reaction-time to 16.2 seconds in
5 X 10~5 M solution ; weaker solutions produced shorter reaction-times down to
12.4 seconds at 1 X 10~7 M. This may have been due to the greater toxicity of
brilliant green.
These results indicate that (1) the photodynamic action of dyes can affect the
response of Pcrancma to light; (2) the fluorescence of a dye is not a measure of
its photodynamic effect; (3) there is no correlation between the wave-length of the
light absorbed by a dye and its effect on the response of Pcrancma; (4) dyes with
very different molecular structures produce similar effects.
The utilization of ammonia by Chilomonas paraniecium. John O.
Hutchens.
Using a solution containing CH.COONa, NH4C1, (NH4)2SO4, K2HPO4, CaCL,
MgCL, FeCl-, and thiamin hydrochloride in which Chilomonas attains populations
up to 10" cells/cc., sufficient quantities of cells have been obtained to permit accurate
analyses of the composition of the cells. Also growth is accompanied by sufficient
conversion of substrates to permit the compilation of balance sheets.
All data deal with cells and the conversions achieved by them during the
logarithmic phase of the growth curve. The results of the experiments are as
follows : 10" cells weigh 2.5 mg. wet and 0.61 mg. dry, i.e. they are 24 per cent solid
material ; 2.5 per cent of the wet weight is tungstic acid precipitable nitrogen, there-
fore the protein (N X 6.25) accounts for 16 per cent of the wet weight or 67 per
cent of the solids. Of this nitrogen 28 per cent is amide nitrogen, indicating a high
percentage of dicarboxylic amino acids in the protein. Ammonia utilized by the
chilomonads (up to 27 per cent of the original ammonia in the solution) is all
recoverable by Kjeldahl digestion. No significant amounts of ammonia are oxi-
dized to- nitrate or nitrite. Therefore, while ammonia is a satisfactory source of
nitrogen for Chilomonas, it is not utilized as a source of energy in the solution
containing acetate.
The possibility of thiamin synthesis by ciliatcs. Virginia C. Dewey and
G. W. Kidder.
The only ciliate so far investigated, Tctrahymcna getcii, has been reported to
require the whole thiamin molecule for growth. Using a vitamin-free casein basic
medium, in which no growth of the ciliate occurred, it was found that the addition
of thiamin permitted extremely slow, but transplantable growth. The addition of
alfalfa or timothy hay extract to the casein permits optimum growth. In an at-
tempt to make a quantitative estimate of the thiamin requirement, the alfalfa
supplement was treated to destroy the thiamin by heating at 121° C. at pH 10 or
higher for periods of 1-3 hours. The treated supplement, when added to casein,
permitted optimum growth of T. gcleli even in the absence of thiamin. This is
286 PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS
true also of T. rora.v with regard to alfalfa, but not timothy hay. Glaucoma scin-
lillans, however, requires both thiamin and the alfalfa supplement. The same ex-
periment was repeated using proteose-peptone treated to destroy the thiamin as a
basic medium with essentially the same results. Four possible explanations of this
phenomenon are offered: (1) heat treatment does not destroy thiamin, which seems
unlikely, since thiamin must be added to the medium besides the alfalfa in order to
obtain growth of Glaucoma: (2) there is some substance in plant material which,
acting catalytically, permits the resynthesis of thiamin from the fragments formed
by the heat treatment; this seems most likely at present; (3) there are present in
plant materials thiazole-like substances more resistant to heat than the thiazole of
thiamin and which can take the place of the latter in the thiamin molecule ; there is
no evidence for or against this possibility; (4) there is some entirely different sub-
stance in plant materials which can substitute for thiamin. This last possibility
seems to be excluded by the fact that Glaucoma will grow in the casein-heat treated
alfalfa medium after it has supported a population of Tctrahymcna which were
killed by heat before inoculating the Glaucoma. This indicates synthesis of thia-
min by Tetrahymena.
AUGUST 19
Electrical potential and activity of cholinc cstcrasc in nerves. David
Nachmansohn.
(Some of this material is scheduled to appear in the September, 1941 issue of
the Journal of General Physiology. Part of it has already appeared in the Journal
of Neurophysiology for July, 1941.)
Chemical composition of mitochondria and secretory granules. Albert
Claude.
(This material is scheduled to appear in Symposia on Quantitative Biology,
Cold Spring Harbor, Vol. 9, October, 1941.)
Native proteins and the structure of cytoplasm. Dorothy Wrinch.
GENERAL SCIENTIFIC MEETINGS
AUGUST 26
Further studies of metamorphosis of ascidian larvae. Caswell Grave.
The mechanism of metamorphosis in the larva of Cynthia partita and in the
larva of Amaroucium constcllatmn does not differ significantly from that in larvae
of Ascidia nigra and of species of Polyandrocarpa.
Larvae of all of these species are induced to metamorphose very soon after
hatching from the egg, or after they have been liberated from the brood pouch of
the parent, by treatment with aqueous solutions of CuCl2 in concentrations of the
order of about 5 X 10~8 molar.
Observations of large numbers of larvae of Cynthia show that the normal
duration of its free-swimming life varies between about 9 and 100 hours when
segregated in vials containing 10 cc. of sea water, but that similar groups are in-
duced to metamorphose within 2 hours when treated immediately after hatching
with a 7 X 10~" molar solution of CuCL.
The Amaroucium larva has a short free-swimming life of about 100 minutes
maximum duration. This period is reduced to 40 minutes by treatment of larvae
with a 1 X 10~4 molar solution of CuCK. Its mechanism of metamorphosis is even
PRESENTED AT MARINE BIOLOGICAL LABORATORY 287
more sensitive to an aqueous extract of the tissues of the adult Ainaroucimn
zooids. Groups of larvae treated with such an extract are induced to metamorphose
within 20 minutes or about one-fifth of the normal period of larval life. Ascidian
tissues are known to contain copper.
It is assumed that the copper salt may act as an enzyme poison, inhibiting an
enzyme system of larval metabolism, thus ending larval life and liberating the
adult action system, with its lower rate of metabolism, from the inhibition imposed
upon it by the higher metabolic rate of the larval action system.
The " eye-spot " and light-responses of tJic larva of Cynthia partita.
Caswell Grave.
The "eye-spot" of the Cynthia larva is degenerate and the responses the
larva makes to light are correspondingly deficient.
The eye consists of a single defective, opaque lens and a small number of
retinal cells that are wholly devoid of pigment granules and are not arranged to
form an optic cup.
Cynthia larvae soon after hatching swim to the water surface in negative
orientation to gravity but they show no orientation of their swimming movements
to light. There is no persistent aggregation of the actively swimming larvae either
at the most illuminated edge of the container or at the least illuminated edge. The
larvae at all times tend to take an even distribution over the water surface.
However, if a lot of larvae are placed in a rectangular container (Leitz filter
cell) enclosed in a box from which light is excluded except for a small opening at
one end, the larvae after an interval of several minutes will be found to have moved
to the least illuminated end of the cell. The same negative response to the directive
beam will be found to have occurred as often as the cell is reversed in the box.
The mechanism by which larvae of the Ammaroucium type orient their swim-
ming movements with reference to a source of light involves an eye with a rela-
tively deep, pigmented optic cup into which light is concentrated intermittently at
each rotation of the body of the actively swimming larva, by a system of three
lenses. The Cynthia hirra, lacking both optic cup and functional lenses, is deficient
in an orientation mechanism. It may be partially compensated for by placing the
larva in the path of a beam of directive rays of light.
Regeneration in tJw early sooid of Amaroucium constellatum. Lloyd
Birmingham.
Oozoids 1-8 days old were cut in one of three regions: (a) just below the
pharynx, separating pharynx, atria, and hindgut in the anterior fragment and leav-
ing stomach, gut, epicardium and heart in the posterior fragment, (b) through
middle of pharynx leaving mainly pharyngeal and atrial tissues in the anterior
fragment, and (r) just anterior to the heart leaving only the heart and part of the
epicardium in the posterior fragment. The criterion of successful regeneration
was the development of the beating heart.
Each fragment after operation (a) had about the same potentialities. Some
65 per cent of all fragments regenerated. About 70 per cent of fragments regen-
erating were members of the pair from one individual. The frequency of regen-
eration of anterior fragments was approximately the same as that of posterior.
Operation (b) showed a high frequency of regeneration in posterior fragments
and intermediate frequency in anterior fragments. Only rarely did the posterior
fragment from operation (r) regenerate; the anterior portion nearly always regen-
erated.
The time required for the development of a beating heart varied between 1 and
7 days. Average time was two days. The smaller the fragment relative to the
total size of the individual the longer the time required.
PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS
The removal of the tail or adhesive papillae of the tadpole leads not to regen-
eration but to metamorphosis. Such treatment seems to hasten the breakdown of
the larval action system allowing the adult action system to take over. Thus, re-
generation of larval tissues has not been demonstrated ; however, regeneration in
the adult is of a complex type. Both tissue and organ regeneration take place.
The latter requires the action of both totipotent cells and factors promoting proper
differentiation.
Normal asexual reproduction of zooids occurs 15-20 days after metamorphosis
of the larva, yet the capacity for perfect regeneration is already present in such
zooids the day following metamorphosis.
Characteristics of the acceleration of Arbacia egg cleavage in hypotonic
sea water. Ivor Cornman.
The acceleration, as previously reported, is a response of the egg to dilutions
of sea water down to a concentration 88 per cent that of normal sea water. Ac-
celeration of the first cleavage can be produced by beginning the treatment any
time from a few minutes after insemination until at least as late as prophase of
the first cleavage, and acceleration of the second cleavage by treating after the first
cleavage. While the acceleration can result from exposure beginning during the
mitotic cycle, a direct effect upon some phase of mitosis is not as yet demonstrated.
Rather, evidence so far obtained favors the supposition of an indirect action, perhaps
like a stimulus. (1) There is no clearly defined optimum when the acceleration is
obtained by a range of dilutions. The effect resembles an all-or-none response,
rising to a plateau in sea water diluted to 98 per cent, and continuing only slightly
diminished to 90 per cent, where it begins to drop toward a retardation. (2) The
first or second cleavage can be accelerated, but not both in the same egg. (3) Eggs
from different urchins vary, some responding well, others not at all. This varia-
tion shows no correlation with the natural concentration of the sea water. Were
the effect of hypotonicity a direct one, altering the cytoplasm so as to facilitate
some phase of mitosis, it would be consistent with a more sharply defined optimum
response, an acceleration of both cleavages, and possibly some correlation between
the concentration of sea water and responsiveness of the eggs. On the contrary,
the evidence, while not conclusive, points to a system within the egg which reacts
to the full extent of its responsiveness to any dilution not great enough to interfere
with cleavage, and which does not react in eggs which have been once stimulated,
or for some natural reason lack the necessary energy or irritability.
Maternal inheritance in echinodcnn hybrids. Ethel Browne Harvey.
Three different echinoderm hybrids have been studied, the California sea-
urchins, Strongylocentrotus purpitratus $ X S. franciscanus <$, the Woods Hole and
Maine sea-urchins, Arbacia pnnctulata ? X Strongylocentrotus drobachiensis d and
the Woods Hole sea-urchin and sand dollar, Arbacia pnnctulata % X Echinarachnius
parma cf. In all three crosses, there is a marked maternal inheritance. The rate
of development of the hybrid egg is that of the normal egg, and the size, shape,
pigmentation and skeleton structure of the hybrid pluteus are like that of the
mother with very little paternal influence. It has not been possible to obtain plutei
from the reciprocal crosses, but Matsui, working at Woods Hole, found that " a
cross between a female Echinarachnius and a male Arbacia ... is in general purely
maternal or nearly so, paternal characters in no case appearing" (1924, Jour. Coll.
Agr., Imperial Univ. Tokyo, 7: 211-236). Early larval development in these
echinoderms therefore seems to be controlled by the cytoplasm rather than the
nucleus. These experiments are preliminary to crossing the non-nucleate half-egg
of one species, obtained by centrifuging, with the sperm of the other species,
whereby conclusive evidence will be obtained as to cytoplasmic versus nuclear
inheritance.
PRESENTED AT MARINE BIOLOGICAL LABORATORY 289
Intermediary carbohydrate metabolism of eggs and sperm of Arbacia
punctulata before and after fertilisation. E. S. Guzman Barren and
J. M. Goldinger.
In 1928, Perlzweig and Barren found that the eggs of Arbacia punctulata con-
tained carbohydrates and produced lactic acid ; the lactic acid formation was in-
creased when the oxidation was inhibited by HCN. The eggs of Arbacia contain
also pyruvic acid (about 850 micrograms per gram dry weight). When lithium
pyruvate was added to a suspension of eggs, the unfertilized eggs metabolized
pyruvate at a rate of about 70 micrograms per hour per gram dry weight. The
utilization of pyruvate increased five-fold after fertilization (354 micrograms per
hour). Pyruvate metabolism is presumably catalyzed by diphosphothiamine, as in
mammalian tissues and bacteria, for it is present in both fertilized and unfertilized
eggs. The increased pyruvate metabolism after fertilization is not due to increased
concentration of diphosphothiamine, because its concentration is not altered by
fertilization (2.72 micrograms per cc. packed cells). The metabolism of pyruvate
in the eggs does not proceed through the Szent-Gyorgyi cycle, because neither
a-ketoglutarate nor succinate is oxidized. Addition of pyruvate depressed the O,
consumption of fertilized eggs. The sperm of Arbacia also utilized pyruvate. In
addition, the sperm oxidized a-ketoglutarate, succinate, and /( + )-glutamate, thus
possessing all the elements for the operation of Szent-Gyorgyi's cycle for carbo-
hydrate oxidation. The diphosphothiamine content of .sperm was about twice that
of eggs, 5.15 micrograms per cc. Whether this fivefold increase in the metabolism
of pyruvate after fertilization is responsible for the increased O, uptake of the
eggs on fertilization cannot be demonstrated by these experiments.
On metabolism of the heart of Venus mercenaries. A. E. Navez, J. D.
Crawford, D. Benedict and A. B. DuBois.
In the study of the substrate (s) underlying the contraction of the heart of
Venus mercenaria investigated by us (1940), [ the following preliminary observa-
tions were made. The excised heart will keep for a long period its characteristic
contractions when it is placed in a small quantity of aerated sea water. It may be
whole, or " cut " in 3-5 pieces or " chopped " up in a large number of small pieces :
the tissue remains highly contractile. Also for long periods this tissue (in any
form or even in completely "minced" state) will respire at a uniform rate. Com-
pared to O2 consumption of the whole heart (100 per cent) the "cut" heart ex-
ceeds it by about 10 per cent, while the " chopped " heart is lower by about 10 per
cent and the " minced " heart by about 50 per cent. But all seem to carry on the
O2 fixation with an R.Q. around 1.0. This applies to the heart unwashed in sea
water. Often the washing (repeated from 1-6 times) of the pieces with sea water
lowers the rate of this reaction. Addition of the washings brings back the rate
around its normal value (some times a little lower, occasionally a little higher).
The "minced" pulp can be centrifuged in two components, whose. respiration rates
are here given as percentage of the rate of the original "minced" pulp (100 per
cent): (1) supernatant fluid: 30-35 per cent and (2) granular part: 65-70 per
cent. Acetonic extracts and residues when reunited in water are inactive or inac-
tivated.
In the study of the respiratory system we used the cytochrome-cytochrome
oxidase-dehydrogenase system as a working hypothesis, in view of the presence of
cytochrome C and succinic dehydrogenase in the heart (Ball and Meyerhof, 1940, -
confirmed by us also). The addition of p-phenylenediamine determines a large
1 Navez, A. E., Crawford, J. D, and Benedict, D., Biol. Bull, 79: 358, 1940.
2 Ball, E. G., and Meyerhof, B., Jour. Biol. Chan., 134: 791, 1940.
290 PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS
increase in Qo., (up to 350 per cent) depending on concentration. It persists for
long periods. The poisoning of the heart by KCN (even at high concentration)
determines an inhibition of 40 per cent at the most and the same in the presence or
absence of p-phenylenediamine. The addition of succinate alone raises the Qo, by
10 per cent; succinate + methylene blue increase it to 150 per cent maximum, but
this action is also elicited by M.B. alone and to the same extent. The inhibition of
this reaction by KCN is as above 30-40 per cent. The other inhibitors tried gave
markedly small or no effects : i.e. sodium fluoride, sodium azide, sodium iodoacetate,
sodium selenite. Ethyl urethane alone has an enhancing effect (up to 20 per cent
increase at concentration 0.1 per cent by weight). Definite indications (a strong
lumiflavin reaction) point to the presence of a flavoprotein ; a weak glutathione
reaction is given also. In conclusion the simple working hypothesis does not fit
the observational facts. Other experiments not reported here confirm this rejec-
tion. Additional observations are needed and are planned for the near future.
Coordination of ciliary movement in the Modiolns gill. Alfred M.
Lucas and James Snedecor.
A study previously undertaken on this problem (Jour. Morph., 1932, 53 : 243-
276) employed moving pictures to record the waves of the lateral cilia of the
Modiolus gill, but only wave-length could be satisfactorily determined and much
time and film were used to obtain the data. The stroboscope employed in the present
study has some advantages in that frequency and rate of wave propagation could be
recorded : the wave-length was calculated. Even this method did not give suffi-
cient number of records to allow critical analyses of data and some better procedure
should be worked out for the problem.
A summary of results :
Temperature
Average frequency
Av. rate of wave
propagation
Av. wave-length
°C.
vib./sec.
It/sec.
M
10
3.5
47
13.8
15
6.2
75
11.7
20
7.4
102
14.5
25
10.6
102
10.6
30
15.4
142
10.0
35
15.8
158
10.9
Av. 11.9
The variation around the average was very great in every case so that the value of
11.9 /i for the wave-length is quite close to 13.1 M obtained with moving pictures.
Conduction of the coordination impulse in ciliated epithelium is similar to the con-
duction in nerves in that the wave-length is constant in both cases.
Preparing an animated diagram of somatic mitosis. Lorus J. Milne.
Although the factual basis for this study has been limited during the past year
to the behavior of dividing epithelial cells from 25-mm. larvae of the salamander
Ambystoma tigrinmn, a much greater variation has been found than was antici-
pated. The timing of separation of daughter cells by formation of new cell mem-
brane does not seem to be correlated with any given stage of the telophase trans-
formations of the nucleus. It may be early before any alterations can be seen
from the anaphasic condition of the chromosomes to very late, when the daughter
nuclei are reorganized almost into interphase. Much variation is present in the
dimensions of the spindle, the area of the metaphase plate and the volume changes
PRESENTED AT MARINE BIOLOGICAL LABORATORY 291
evident in the cytosome. Change in cell form has been followed in detail and
observed to be polygonal in inter- and prophase, to become progressively more
spherical in meta- and anaphase, and to recover the polygonal condition in late
telophase or early interphase.
In technique a number of advances have been made, and an animation unit has
been assembled from equipment provided by the Carnegie Corporation of N. Y.
A very smooth S-curve was found to be the haversine relation such as given in
tables of recent editions of Handbook of Chemistry and Physics (haversine 6
- (1 — cos#)/2). This smooth curve was found excellent for transitions such
as starts and stops of movements, rendering these completely free of "jump."
Difficulty in applying ink and paint to cellophane was overcome by mixing the
pigment solutions with 2 per cent honey and 8 per cent of 10 per cent Fotofoam,
90 per cent of water color. The honey keeps the pigment solution from drying out
completely, hence it does not crack or peel off. Fotofoam, apparently a bile salt
derivative, reduces the surface tension of the color and allows it to spread easily
on the shiny cellophane. A dissolving shutter with both manual and automatic
control has been developed, using the new Polaroid-H glass. The two uncrossed
plates of this glass transmit about 50 per cent of incident light ; crossing the axes
to 85° cuts the transmission to about 1/400 of the 50 per cent value, while at 90°
the transmitted light from bright sun through an F:1.5 lens is photographically
inactive to even the fastest films. The decrease in transmission is almost linear,
and until crossed more than 86° seems uniform throughout the spectrum. Beyond
that limit, the violet end is less restricted than the longer wave-lengths.
Stimulation by intense flasJies of ultra-violet light. E. Newton Harvey.
Any effective stimulus must be of sufficient intensity and also change rapidly
in intensity. In order to obtain high intensity ultra-violet light a three micro-
farad condenser discharge at 20,000 volts is passed through a quartz mercury vapor
sterilamp, according to the method of Rentschler. A single discharge, lasting a
few millionths of a second, is capable of immediately killing bacteria, disintegrating
protozoa, stopping cyclosis, ciliary and amoeboid movement, contracting myonemes
and suppressing bacterial luminescence. In Nitclla the protoplasmic rotation is
reversibly stopped, sometimes only on the side of the cell toward the ultra-violet
or only in a portion of the cell covered with quartz, not in that region covered with
glass. It was found that sometimes the ultra-violet light would start an action
potential locally and sometimes the potential was propagated over the whole cell,
showing that ultra-violet light can stimulate in the same manner as electrical stim-
uli. The stimulation of vertebrate muscle and nerve is not yet certain. None of
the above effects ever occur when the cells are shielded from the discharge by glass.
The influence of the medium on the radioscnsitivity of sperm. T. C.
Evans, J. C. Slaughter, E. P. Little, and G. Failla.
The ability of sperm to fertilize eggs is affected by roentgen radiation. How-
ever, it has been found that: (1) Sperm irradiated in the seminal fluid in concen-
trated form are very radioresistant. (2) If the seminal fluid (containing sperm)
is diluted with sea water and then irradiated, the sperm become progressively more
radiosensitive with dilution. The increase of radiosensitivity in a certain range of
dilution is inversely proportional to the concentration of the sperm. (3) Sperm in
dilute suspensions can be made more resistant again by the addition, before irradia-
tion, of small amounts of egg albumen, gelatin, Arbacia egg water, and glycylgly-
cine. (4) Sperm, in sea water, made inactive (by centrifugation) are more radio-
resistant than those actively swimming about during the exposure to the roentgen
radiation. (5) The actions possibly involve two stages: (1) some harmful agent is
momentarily produced in the water, and (2) the activity of the sperm affects the
amount of contact with the harmful agent.
292 PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS
The effect of roentgen radiation on the fertilizing capacity of the sperm may
be regarded as an indirect effect which can be altered by changing the medium. A
more direct action of the radiation (not easily modified by the medium) is the
delay in cleavage observed when eggs are inseminated with irradiated sperm.
Comparative pharmacology of myogenic and neurogenic hearts. C. Ladd
Prosser and George L. Zimmerman.
The hearts of mollusks and adult vertebrates are inhibited by acetylcholine.
These are myogenic hearts. The hearts of decapod crustaceans of insects and of
Limulus are accelerated by acetylcholine and are neurogenic.
Acetylcholine accelerates and raises the tonus of the hearts of Arcnicola cristata.
In high concentration it stops the heart in systole. The threshold is one part in
108 without eserine. Eserine potentiates and atropine antagonizes the action of
acetylcholine. The dorsal vessel is accelerated by acetylcholine but higher concen-
trations are required. Small amounts of potassium added to sea water bathing the
Arenicola heart accelerate and raise the tonus while small amounts of excess cal-
•cium slow the hearts. A pacemaker is located in the small vessel connecting the
laterogastric vessel with the heart. Adrenalin accelerates the hearts and in high
concentrations stops them in diastole. From the above results we postulate that the
heart of Arcnicola is neurogenic.
In the Linnilus embryo the heart begins its beat myogenically on the twenty-
first day. It becomes neurogenic at about the twenty-eighth to thirty-third day.
During the myogenic period this heart is insensitive to acetylcholine (1 in 104)
with or without eserine. Beginning from the thirty-first to thirty-fifth day the
hearts are accelerated by acetylcholine.
The heart of Daphnia is inhibited by acetylcholine and in the heart of Eubran-
chippus there is no effect (as in early Linnilus and in vertebrate embryos). The
heart of Talorchcstia is accelerated by acetylcholine.
It is suggested that those hearts which are accelerated by acetylcholine are
neurogenic and that those which are inhibited or unaffected are myogenic.
Structures concerned with yolk absorption in Squalus acanthias. Lois
E. TeWinkel.
In Balfour's monograph on the Development of Elasmobranch Fishes (1876)
and in a paper by Beard (1896, Anat. Anz., 12: 334) it is stated that yolk, in the
later embryonic stages of these fishes, passes bodily up the yolk stalk into an
internal sac. This sac is an outgrowth of the stalk at its point of entrance to the
intestine and yolk taken into it eventually reaches the alimentary canal, there to be
digested.
Living Squalus acanthias embryos from 110-230 mm. in length and preserved
specimens of 60 mm. have been studied. The internal yolk sac increases enormously
in size between the 60 and 230 mm. stages. It is just clearly visible in gross dis-
sections of the former, while, in the latter, the external sac has shrunk to an empty
stub only 6 mm. in length and the internal sac is very large ( approximately 45 mm.
long and 18 mm. in diameter). The method by which yolk is transported to the
internal sac has not yet been determined.
Sections show yolk platelets in the internal sac of a 60 mm. embryo but appar-
ently not any are present in the intestine, whereas, in a 150 mm. specimen, the
spiral valve region is filled with an emulsion of yolk in various stages of digestion.
Cells of the simple, low volumnar epithelium lining the external yolk sac contain
scattered fat droplets and glycogen in the 110 and 150 mm. specimens studied,
indicating that, so long as the external sac is large and well vascularized, it plays
some part in embryonic nutrition.
PRESENTED AT MARINE BIOLOGICAL LABORATORY 293
Tlic distribution of elastic tissue in the arterial pathway to the carotid
bodies in the adult dog. William H. F. Addison.
In the examination of many series of sections through the region comprising
the bifurcation of the common carotid artery, the carotid sinus and the carotid
body, there is found great uniformity in the structural tissues constituting the walls
of the arterial vessels supplying blood to the carotid body tissue. But, as is fre-
quent in the vascular system, the arrangement of the vessels may present many
variations. In the case here reported, from the right side of an adult dog, the
most striking variation is that the carotid body is not aggregated into one large
mass but is distributed along the usual arterial pathway as several separate masses.
The blood supply to the carotid body in the dog is from the occipital artery,
which in this animal is the first branch of the external carotid artery above the
bifurcation of the common carotid artery. The occipital artery arises at a variable
distance from the bifurcation and sometimes from the bifurcation itself. In the
present case there is an interval of 3 mm. between the bifurcation and the origin
of the occipital artery. The structure of the walls of the beginning is different
from that of the rest of the occipital artery, inasmuch as it is elastic-walled, non-
muscular, similar in structure to the carotid sinus. Because of its structure and its
wider diameter than the rest of the occipital artery, this beginning part of the
occipital artery may be called the occipital sinus. The further course of the path-
way to the carotid body is as follows. From the occipital sinus is given off a short
branch which is at first elastic-walled. This branch soon divides into two sub-
branches, of which one has muscular walls and the other has elastic-tissue walls.
The latter gives off the blood supply to the carotid body and then becomes muscular
in character.
In the present case this arrangement of the elastic-walled vessels is present,
but the carotid tissue is distributed at intervals alongside the carotid sinus, its
elastic-walled branch and the latter's elastic-walled sub-branch. From each of
these parts of the arterial pathway little vessels come off to supply the separate
masses of carotid tissue, and in each little vessel the wall is elastic in structure.
Thus, in this case where the carotid body tissue is divided into small portions,
each portion is still provided with blood through an elastic-walled non-muscular
series of channels, while the continuation of these vessels, except those terminating
in the carotid tissue, is always muscular, and under the control of the vasomotor
system.
Behavior of the arteriolcs in hypertensive rabbits, and in normal rabbits
following injections of angiotonin. Richard G. Abell and Irvine H.
Page.
It is well known that in hypertensive patients, and in animals made hypertensive
experimentally, there is an increase in resistance to blood flow. Although it has
been observed that the retinal arterioles of hypertensive individuals are narrower
than normal, there are no reports of measurements of their diameters before, as
well as after the development of hypertension.
In the present experiments, living arterioles in transparent moat chambers in
ears of normal rabbits were observed with the microscope, and their diameters
measured. The animals were then made hypertensive either by the method of
Goldblatt (1934) or the method of Page (1939, 1940), and the diameters of the
same arterioles measured again.
Of the 7 operated rabbits, 4 became hypertensive. The blood pressure rose to
from 1.4 to 2.1 times the normal level. The arterioles constricted from approxi-
mately 0.4 to 0.7 their control diameters when the rabbits became hypertensive.
No constriction occurred in those rabbits which did not become hypertensive.
The capillaries and venules did not constrict in any of the experiments.
294 PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS
In order to see whether the arteriolar constriction that occurred in the hyper-
tensive rabbits might have been due to angiotonin, 0.2 cc. of angiotonin was in-
jected intravenously into a normal rabbit, and the resulting arteriolar constriction
measured. After the arterioles had returned to their control diameters, the rabbit
was made hypertensive, and the resulting constriction oi the same arterioles
measured.
In both instances the arterioles constricted to approximately 0.5 their original
diameters, and the blood pressure rose to about 1.4 times its control level.
This suggests that the arteriolar constriction that occurred in these hypertensive
rabbits might have been due to angiotonin.
It should be emphasized that these studies have been made on vessels in the
ears of rabbits, which are notoriously active in dilatation and constriction; conse-
quently the extent of constriction found here should not be applied to other
peripheral arterioles without further study.
AUGUST 27
Catalysis of ionic exchanges by bicarbonates. M. H. Jacobs and Doro-
thy R. Stewart.
The acceleration of hemolysis in solutions of NH4C1 by low concentrations of
bicarbonates, first observed by 0rskov, was explained by Jacobs and Parpart as
essentially a catalysis of diffusion involving entrance of CO2 and NH3 into the cell,
conversion of CO2 into HCO-f, exchange of HCO3~ for Cl~, reconversion of HCO3~
into CO;,, and so on. This principle can be extended to other ionic exchanges in the
erythrocyte in which ammonium salts are not concerned. In general, as long as
the necessary pH differences exist between the anion-permeable cell and its sur-
roundings, and anions such as Cl" are available for exchange, a reaction which for
brevity may be represented as HCO/^ CO2 + OH~ may take place in opposite
directions on the two sides of the membrane, the resulting cycle leading to the final
equilibrium distribution of ions far more rapidly than is possible in the absence of
bicarbonates. This catalysis-like effect is illustrated by the volume changes of
erythrocytes that occur on changing the reaction of the surrounding medium or on
suspending the cells in a solution of a salt with a bivalent anion such as SO4~~.
With certain limitations and qualifications, changes in the amounts of bicarbonates
in the solution affect only the rate of the process and not the position of the final
equilibrium.
The role of carbonic anhydrase in the catalysis of ionic exchanges by
bicarbonates. Dorothy R. Stewart and M. H. Jacobs.
The theory suggested in the preceding abstract for the catalytic effect of
bicarbonates on the attainment of certain ionic equilibria involves the reversible
reactions
CO, + H2O ^ H2CO3 ^ H+ + HCO-.
The first of these reactions is known to be strongly accelerated in both directions
by the enzyme carbonic anhydrase. Considerable support is therefore given to the
theory by the observation that the catalysis-like effect of bicarbonates on ionic
exchanges in the erythrocyte is in turn strikingly influenced by this enzyme. The
importance of carbonic anhydrase in such processes can be shown either by adding
it to the medium in which the cells are suspended or by inhibiting its action within
the cells by means of sulfanilamide or cyanides. In general, under the conditions
of these experiments, the enzyme is more effective inside than outside the cells, but
under certain circumstances its external effect may also be very striking. Inhibition
of the enzyme within the cell by cyanides occurs almost instantly, that by sulfanilam-
ide reaches its maximum only after several minutes. Both effects may readily be
reversed by washing the cells.
PRESENTED AT MARINE BIOLOGICAL LABORATORY 295
Some effects of desoxycortico-sterone and related compounds on the
mammalian red cell. Martin G. Netsky and M. H. Jacobs.
The sodium salt of the phosphate ester of desoxycortico-sterone (Na DOC
PO4) was found to produce sphering of human erythrocytes in concentrations as
low as 1 mg. per cent or 2 X 1(T5 mols per liter. The sodium salt of the phosphate
ester of di-desoxycortico-sterone (Na di DOC POJ produced sphering at 0.4 mg.
per cent or 5 X 1(T7 mols per liter; 21-sodium hydrogen phosphate of 3-acetoxy-A5-
pregnene-21-ol-20-on (Na AcO pregnene PO4) also produced sphering, but sodium
glucuronidate pregnanediol did not. Neither compound E, nor free desoxycortico-
sterone, nor Kendall's amorphous fraction gave sphering. Sphering ability seems
to be associated with molecules of polar : non-polar structure, although a special
form of non-polarity is required. Sphering can be reversed or inhibited by the
addition of serum protein, the amount of protein necessary being the same in either
case. The reaction is quantitative, 1 mg. of either Na DOC PO4 or Na di DOC
PO4 being equivalent to approximately 10 mg. of serum protein. In higher concen-
trations, those substances which produce sphering are directly hemolytic. Direct
hemolysis is also prevented by the addition of serum protein, and apparently the
same type of chemical structure is required for it as for sphering. The effect of
some of these substances on permeability to ammonium chloride and to glycerol was
studied by the hemolysis method. In the case of ammonium chloride, the hydroxyl-
chloride ion exchange was inhibited and hemolysis slowed markedly, both in human
and beef cells. The effect on glycerol hemolysis of beef cells was a decrease in the
time of hemolysis at all pH levels. For human cells this was also true at pH levels
more acid than about 6.8, but at higher pH values, the effect was a more complicated
one, low concentrations increasing the time, higher ones decreasing it below the
level of the control. For the three Na PO4 salts, the order of activity was : Na di
DOC PO4 > Na DOC PO4 > Na AcO pregnene PO4. Similar effects on glycerol
permeability, obtained with compound E and Kendall's amorphous fraction, indicate
that an extremely polar : non-polar structure is not required for permeability
changes.
Permeability of the Arbacia egg to potassium.1 Herbert Shapiro and
Hugh Davson.
The permeability of the Arbacia punctulata egg to ions, and the problem of the
maintenance of concentration gradients in this cell, have hitherto not been investi-
gated. Chemical analyses were made of the potassium content of the egg and of
sea water. The eggs were found to have approximately twenty times as much
potassium as the sea water. Fertilized eggs contained very nearly the same amount
of potassium as unfertilized. Suspensions of eggs were placed in a shallow layer
on the bottom of Erlenmeyer flasks, and oxygenated by passing moistened oxygen
over the suspensions. The flasks were immersed in a thermostat maintained at
25.6° C. Samples of suspension were taken at regular intervals, centrifuged, and
the supernatant fluid analyzed chemically for potassium content. Runs were made
over periods varying from two to seven hours. Total cell volume was determined
by measurement of cell diameters, and of cell concentration by a dilution method.
Both unfertilized and fertilized eggs lost potassium on coming in contact with sea
water, though at a slow rate; from about 1.5 to 8 per cent of the cellular potassium
diffused out in a two-hour period. Eggs placed in nitrogen also lost potassium,
though at a rate not markedly different from that of eggs in oxygen. Eggs in
artificial sea water with five times the normal potassium content accumulated
potassium, and did this against a gradient. When placed in artificial sea water
1 This investigation has been aided by a grant from the Penrose Fund of The
American Philosophical Society.
296 PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS
containing four times the normal amount of calcium, fertilized eggs lost potassium
more rapidly than those in normal sea water. When the excess calcium sea water
was replaced by normal sea water, potassium began to reenter the cell, once more
against a gradient.
Lipo-protein complexes in the egg of Arbacia. A. K. Parpart.
Determinations of the total lipid were made on " lyophylled " eggs of Arbacia.
Values of total lipid were 5.4 per cent of the whole egg or 26.9 per cent of the
solids of the egg. By methods found applicable to erythrocytes it was found that
77 per cent of the total lipid behaves as though it were bound to protein.
Eggs exposed to a 30 parts NH4 Cl, 70 parts sea water mixture for four to
ten hours showed no decrease in the amount of lipid bound to protein. Eggs under-
going development for five hours did not change in total lipid or in lipid bound to
protein.
These data suggest that the major portion of the lipid acts as a, structural
component of the egg cell more than as a metabolic component.
The relation between protoplasmic streaming and the action potential in
Nitclla and Chara. Samuel E. Hill.
Much past work has shown that protoplasmic streaming is profoundly affected
by passage of electrical currents through the cell, or by passage of action currents
along the cell. Does presence or absence of streaming bear any relation to elec-
trical irritability?
Under certain conditions, for example, soaking for three days in distilled water,
*Nitella cells lose their ability to give an electrical response to stimulation, yet the
protoplasmic streaming continues. Streaming is thus not a sign of irritability.
The streaming may be stopped by very weak electrical stimulation. If the
applied voltages are small enough, the streaming slows down and gradually comes
to a stop, no action current appearing. If, however, a larger stimulating voltage is
applied, an action current appears and the streaming stops abruptly. After 30 to
60 seconds the streaming begins anew, starting and stopping abruptly several times
as if pulling against high viscosity. After a few seconds of this the streaming
again becomes regular. These abrupt starts and stops are accompanied by no
electrical changes. During the time while the protoplasm is at a standstill, it is
possible to provoke an action current by electrical stimulation. This can be re-
peated at intervals (about 25 seconds) for 30 minutes or more, every stimulation
being followed by an action current and the protoplasm at a standstill all the while.
After a rest of a few minutes the streaming begins again.
The streaming appears to have no antecedent relation to the action current,
since presence of streaming does not indicate electrical irritability, nor absence of
streaming indicate failure of electrical response.
Observations on luminescence in Mnemiopsis. Aurin M. Chase.
Harvey and Korr (/. Cell. Comp. Physiol., 1938) found that extracts or frag-
ments of the photogenic cells of Mnemiopsis Icidyi can luminesce even in the com-
plete absence of oxygen. Under such conditions continuous luminescence occurs
rather than the brief flashes characteristic of the living organism ; an indication
that nervous control of the process has disappeared.
The present experiments concern luminescence of the intact, living animal.
Upon electrical stimulation through the sea water flashing occurs along the rows
of swimming plates. After complete de-aeration (thirty to forty minutes flushing
with purified hydrogen ) no flashing can be elicited by electrical or mechanical stim-
ulation. Three to five minutes after re-admitting air the animal again responds.
PRESENTED AT MARINE BIOLOGICAL LABORATORY 297
The cycle of de-aeration and re-aeration can be repeated as many as three times
before the animal dies. Movement of the swimming plates stops at about the same
time that luminescence on electrical stimulation ceases. As the animal begins to
disintegrate, either in an atmosphere of air or of hydrogen, a dim, continuous
luminescence gradually appears along the rows of swimming plates. This lasts for
about an hour, and undoubtedly represents the basic luminescent reaction as studied
by Harvey and Korr, freed from its normal nervous control.
Mncmiopsis in sea water (air present) loses its ability to luminesce on elec-
trical stimulation within 15 seconds after addition of 0.0001 M KCN, although the
swimming plates continue to move for ten to twenty minutes. Eserine (1:2,000)
increases the sensitivity to luminesce on mechanical stimulation and also increases
the duration of the luminescent flashes. Addition of acetylcholine (1 : 3,000) en-
hances this effect. Returning the animals to plain sea water gradually restores
the normal response. Adrenaline (1:100,000) apparently decreases the sensitivity
but the effect is less clear-cut than the increased sensitivity caused by eserin and
acetylcholine.
PAPERS READ BY TITLE
Photodynamic studies on Arbacia eggs. Fred W. Alsup.
Rose bengal in concentrations of 1 part dye to 20,000 or more parts sea water
and eosin in a concentration of 1 : 2,000 have no effect, in the dark, on the relative
viscosity of the inner protoplasm of the unfertilized eggs of Arbacia punctulata.
However, when the eggs are exposed in either of these dye solutions to light from
a 1000-watt electric bulb, i.e., exposed to photodynamic action, the viscosity is
markedly increased. On the average, the viscosity of eggs exposed to photo-
dynamic action is about 40 per cent higher than that of unexposed eggs. In-
creases in viscosity can be detected within 5 seconds after the exposures and reach
a maximum in about 1-5 minutes after the exposures.
The unfertilized eggs of Arbacia become activated when exposed to photo-
dynamic action. Most of them show marked amoeboid movement and do not
cleave when left in the dye solutions or removed to sea water. A very low per-
centage of these eggs divide in an irregular fashion. If eggs are removed to
mixtures of sea water and isotonic calcium chloride soon after they are exposed to
photodynamic action, a much higher percentage cleave irregularly. Apparently
calcium strengthens the cortex of the eggs, which has been liquefied by photo-
dynamic action.
Disruption of mitosis in Colchicum by means of colchicine. Ivor Corn-
man.
Colchicum, the commercial source of colchicine, contains a concentration of the
alkaloid sufficient to block mitosis in other plants. By growing excised roots of
Colchicum conns as temporary cultures in small vials, it has been possible to test
a wide range of conditions upon uniform material. During the 8% hours of the
experiments, mitosis continued normal in tap water, in 1 per cent colchicine, and
in sucrose isomolar with the effective colchicine concentrations. Mitosis was
blocked in C. byzantiniim by 5 per cent colchicine and in C. aiitiiinnalc by 10 per
cent. The cytological picture in C. bysantinum is typical of colchicine effects :
disappearance of the spindle structure and then on the spindle material ; accumula-
tion of blocked metaphases ; inhibition of the cell plate ; appearance of tetraploid
and binucleate cells. Upon removal from the colchicine, the cells recovered and
continued dividing. They appeared normal except for the abnormal orientation of
the spindles. Onion roots cultured in the same way showed typical colchicine
effects in 0.01 per cent colchicine. It is concluded that the spindle mechanism in
298 PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS
Colchicum is the same as in other angiosperms with regard to the colchicine effect,
and that the immunity of Colchicum to its own alkaloid resides in some extra-
mitotic protective mechanism.
The effect of roentgen radiation on the jelly of the Nereis zygote. T.
C. Evans.
Several investigators have reported that irradiation of Nereis ova results in
an extreme swelling of the fertilization membrane following insemination. The
swelling of the fertilization membrane is related to the amount of jelly retained
within the membrane. Within a few minutes after insemination, unirradiated eggs
will exude the jelly rapidly and it will pass through the membrane to surround the
zygote with a thick viscous layer.
In this investigation zygotes have been irradiated after the jelly has been
exuded, and it has been found that the radiation disperses the jelly immediately.
The dosage required is about 44,000 roentgens.
The action of the roentgen radiation on the jelly, after it has been produced
externally, is similar to that previously reported for the jelly of Arbacia and
Asterias eggs.
Tests of nudcoli and cytoplasmic granules in marine eggs. R. Ruggles
Gates.
In testing the nucleoli of the eggs of Asterias, Arbacia, Mactra and Chactop-
tcrus for phospholipids the absence of lipoids and phospholipids from these bodies
was shown by negative tests with Sudan III, osmic acid, Scharlach R and Feulgen
without hydrolysis. In Mactra eggs treated with chloroform the nucleolus was
unchanged. Observation of fresh eggs of these genera in sea water under an
immersion lens showed that the nucleolus consists of two parts, like immiscible
fluids, one enclosed within the other. The outer part is more quickly soluble in tap
water than the inner portion.
While the nucleus is unaffected when Feulgen is added without previous hy-
drolysis, the cytoplasm in all these eggs unexpectedly showed a gradually deepening
magenta color, indicating the presence of substances which have a free aldehyde
group. The same reaction was obtained with the muscles of Chactoptcrns. The
cytoplasmic substances are relatively insoluble in water but more soluble in alcohol.
They appear to belong to the acetalphosphatids of Feulgen and Voit. Fucus eggs
and oogonia showed no color change at ordinary temperatures, so these substances
may be characteristic of animal cells ; but on exposure to air or rise of temperature
a pink color develops in certain tissues of the FUCKS thallus in Feulgen.
When the cytoplasm of Chactoptenis eggs is examined under an immersion
lens after the Feulgen reaction, many of the granules, both large and small, are
magenta in color. In crushed eggs, some granules are seen to be deep magenta,
some pale, some uncolored, and there is a diffuse pink in the cytoplasm. The
smallest granules are most intensely colored, some granules of all sizes remaining
uncolored.
Sex-linkage of stubby (sb) in Habrobracon. Russell P. Hager.
Linkage of fused (fu) with the sex alleles has been demonstrated to occur with
crossing-over in 8.6 per cent (Speicher) to 17.6 per cent (Bostian) of the cases
(cf. Whiting, P. W., /. Morph., 66: 323-355). Since stubby (sb) was known to
be linked with fused, tests were made to determine the percentages of crossing-over
between stubby and fused, and stubby and the sex alleles so that the order of the
factors could be mapped. Females heterozygous for stubby and fused (sb/fu)
yielded 479 sb, 507 fu, 136 sb fu, and 179 + haploid males. Crossing-over between
PRESENTED AT MARINE BIOLOGICAL LABORATORY 299
stubby and fused is therefore about 24.2 per cent as data collected previously by
others have indicated.
Orange-eyed females heterozygous for stubby were mated to stubby males :—
o sb xa/o xb X sb xa or sb xb. The crosses with sb xa yielded 370 (linked)
heterozygous and 228 (cross-over) stubby females; 10 (cross-over) heterozygous
and 32 (linked) stubby diploid males. The crosses with sb xb yielded 642 (cross-
over) heterozygous and 1132 (linked) stubby females; 70 (linked) heterozygous
and 57 (cross-over) stubby diploid males. The cross-over class among the bi-
parental males is of the type opposite to the cross-over class among the females.
The cross-over percentage was calculated by extracting the square root of the
products of the comparable classes. V642 X 228/( V642 X 228 + V1132 X 370).
Crossing-over between stubby and the sex factor was accordingly 37.5 per cent
among the females and 33.6 per cent among the diploid males.
The order of factors is therefore :
sex — (8.6 per cent to 17.6 per cent) — fused — (22 per cent to 24.2 per cent) stubby:
sex ( 33.6 per cent to 37.5 per cent) stubby.
The elasmobranch interrcnal; a preliminary note. The interrcnal body
of Alopias vulpinus (Bonnaterre). E. R. Hayes.
This description is based upon the examination of a thirteen-foot " thresher "
shark, A. vulpinus. The interrenal is an unpaired, elongate, yellow body lying be-
tween the caudal portions of the two kidneys and immediately dorsal to the posterior
cardinal sinus. Slightly asymmetric in position, the gland is more closely applied
to the left kidney. Beginning 5 cm. from the posterior limit of the kidney, it
extends craniad 30 cm. and is discontinuous at one point in its anterior half. In
cross-section, the gland is roughly oval, the greatest diameter ranging from 8-10
mm. posteriorly and tapering to 2-3 mm. anteriorly.
Microscopically, a rather thin capsule is seen covering a strikingly uniform
parenchyma which shows no tabulation. It consists of cords of lipid-laden cells
interlacing with blood sinuses and clothed by the endothelium lining the sinuses.
It is possible to distinguish only one type of cell in the parenchyma. This type
closely resembles the " spongiocytes " of the mammalian adrenal cortex. The cells
are heavily packed with droplets of Sudanophil lipid which also blacken with OsO4
(labile in xylol ) and with the Schultz test give a strongly positive reaction for
cholesterol. Paralleling the uniformity of these reactions is the even distribution
throughout the gland of a considerable amount of birefringent material. Certain
cells show evidence of nuclear pycnosis although in other respects they are indis-
tinguishable from neighboring cells. The life history of the cells of this gland
remains to be worked out.
The cytology o/( Amoeba verrucosa. Dwight L. Hopkins.
Amoeba vcrrucosa is a fan-shaped form typified by longitudinal folds and
grooves which are formed continuously on its superior surface. It feeds on a
variety of plant and animal organisms including bacteria and rotifers. In feeding
it flows over the prey, trapping it in a groove, or depression in the under-surface.
Once the prey is trapped, the sides of the groove are extended downward and under
until they meet and the victim is enclosed in an irregular tube. The folds or sides
of the groove, when they meet, form closely approximating, irregular lines, which
remain visible for some time. The tube is not closed completely. When the prey
is rejected as food it is squeezed out from the posterior end of the tube; when
accepted, only the water is squeezed out and the food is drawn into the interior.
The food vacuole thus formed contains little or no fluid.
300 PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS
Once the food vacuole is inside, small neutral red staining vacuoles which are
very numerous in the cytoplasm are attracted to its walls and enter by coalescence.
This process renders the recently formed food vacuole stainable with neutral red.
As digestion proceeds the color is first red, then yellow, and finally colorless, just
before the vacuole is expelled. Due to the addition of neutral red vacuoles, food
vacuoles during digestion may contain considerable fluid material, but most of this
is absorbed by the cytoplasm before the residues are expelled.
Contractile vacuoles arise by coalescence and swelling of smaller vacuoles.
The cytoplasm contains numerous small rod-shaped and spherical bodies which
come to the region where contractile vacuoles form through the plasmagel and
when the gel transforms into sol, they cluster around the old contractile vacuole.
These small bodies, as well as the membrane of the contractile vacuole, take Janus
Green B faintly. The small bodies which stain finally with Janus Green B form
small contractile vacuoles by coalescence and swelling.
Observations on the melanophore control of the cunner Tautogolabnis
adspersus (Walbaum). George W. Hunter, III, and Edward Was-
serman.
Background responses under a constant source of illumination were studied in
the cunner, Tautogolabnis adspersus. Black-adapted fish responded to a white
background in 15 seconds, reaching a maximum in 50 minutes, while white-adapted
fish placed in a black background required an average of about ten seconds for the
first phase of the reaction and 50 minutes to complete the response. Responses of
white- and black-adapted fish to yellow and blue backgrounds were intermediate to
the controls on white and black backgrounds. Placing cunners in complete dark-
ness produced paler fish but darker than the white-adapted ones, while enucleated
cunners gave no response to background changes. Both experiments indicate the
importance of the eye in normal responses to background changes.
Cutting of caudal fin rays and the accompanying nerves of white-adapted fish
produced dark bands due to expanded melanophores in 40-45 seconds and a maxi-
mum response in 45-60 minutes. Windows gave similar results. Cuts proximal
to the band produced darkening of the freshly cut area distally as far as the
original cut. Electrical stimulation of the medulla and roof of the mouth at the
lowest frequency possible on a Harvard inductorium using 6 volts, produced a
temporary darkening of white-adapted fish lasting about a minute, while the highest
frequency gave a partial but distinct blanching of black-adapted fish.
Observations on recutting of fading bands and the fading time of single and
multiple fin ray cuts are still being carried on. Experiments on hypophysectomized
fish as well as the effects of drugs and salts on melanophores are in progress and
will be reported elsewhere.
The evidence accumulated thus far suggests that this northern representative
of the wrasses, the cunner, has a melanophore system controlled by adrenergic and
cholinergic sets of nerve fibers. While the pituitary has given a positive test for
intermedin when injected into a blanched frog, its role in the normal control of
melanophores has not yet been determined.
The control of melanophores in the cunner is being studied as one phase of the
problem dealing with pigment production and its control about the cysts of the
trematode metacercaria, Cryptocotylc lingua, which occur on the scales of the
cunner.
The influence of temperature on reconstitution in Tubularia. -Florence
Moog.
The fact that the body size of Metazoa is generally greater at lower tempera-
tures has been noted frequently. In Tubularia the size of the reconstituted
PRESENTED AT MARINE BIOLOGICAL LABORATORY 301
hydranths is similarly affected by low temperature. The table shows the length of
the reconstituents and the time for their formation in one experiment consisting of
batches of 25 sections of stems, each 6 mm. long, kept in 200 cc. of sea water.
Time to constriction Length of
Temperature of primordium primordium
0 C. hours micra
22.2 44.7 854
19.0 55.1 918
14.0 59.5 1050
In three experiments the reconstituents at 14° averaged 15.2 per cent longer
than at 22°, and their time of formation was 39.3 per cent longer.
Some authors have pointed out, on the basis of studies on embryonic and adult
vertebrates, that the larger body size might be accounted for by the fact that less
food is needed to maintain tissues at low temperatures, so that more can be used
in building new protoplasm. But since an external food supply is not a factor in
reconstitution of Tubularia, it seems likely that the increased size is due at least
in part to modification of chemical equilibria. Low temperature would most likely
slow the chemical processes involved in the conversion of tissue, without markedly
affecting diffusion, so that the change in size might result from the deeper pene-
tration of oxygen.
It is interesting to note that low temperature increases the size of the recon-
stituted hydranth while decreasing its rate of formation. Other agents, such as
low oxygen concentration, low pH, cyanide, azide, and urethanes, which decrease
the rate, decrease the size also. Evidently the processes underlying reconstitution
velocity and determination of size of the primordium are independent to a consid-
erable degree.
Factors influencing the pigmentation of regenerating scales on the ven-
tral surface of the summer flounder. Clinton M. Osborn.
From summer flounders which had been black- or white-adapted, scales were
plucked in a definite pattern from the naturally white lower surface and the fishes
returned to their original tanks. It was apparent within two weeks that the regen-
erating scales on the white-adapted flounders were white, while those on black-
adapted fishes developed melanophores. When black-adapted flounders were blinded
by enucleation, the regenerated scales were pigmented regardless of the shade of
the background, indicating that intact eyes were not essential to pigment production.
To test the direct effect of light, flounders prepared in three different ways
were illuminated strongly underneath through special glass-bottomed aquaria. In
one experiment white-adapted fishes were illuminated ventrally. The regenerated
scales were ivhite. In the next experiment the fishes were black-adapted (black
walls and ceiling ) while ventrally illuminated and grew melaninated scales.
Einally, blinded fishes (in the dark phase, but not maximally black) regenerated
pigmented scales when ventrally illuminated. The results from these three ex-
periments were qualitatively similar to the original observations, indicating that
bright illumination had little influence on the color of the regenerating scales.
This was further substantiated in experiments where the regenerated scales were
melaninated on black-adapted fishes in tanks dimly lighted during the day and
totally dark at night.
It is concluded that the color of scales regenerating on the naturally white
lower surface of the summer flounder is influenced primarily by the physiological
factors (nervous and endocrine) which cause the upper surface of the fish to
assume the pale or the dark phase. Factors which produce the dark phase favor
302 PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS
the development of melanophores in regenerating ventral scales while physiological
agents causing the pale phase allow white scales to regenerate. Light appears to
have little direct effect on the color of regenerating scales although melanination
is somewhat accelerated by light in physiological conditions which favor pigmenta-
tion.
Hypersensitization of catfish melanophores to adrenaline by denervation.
G. H. Parker.
The melanophores in a denervated caudal band of a catfish of intermediate tint
will concentrate their pigment after the fish has received 0.008 or even 0.004 of a
milligram of adrenaline per 100 grams of fish. These dosages do not induce
noticeable pigment changes in the innervated melanophores of the rest of the fish.
Weaker doses of adrenaline have no obvious effect on either denervated or inner-
vated melanophores. Stronger doses induce pigment concentration in both dener-
vated and innervated melanophores. The greater sensitivity for adrenaline thus
shown by the denervated melanophores as compared with the innervated ones may
be due to the fact that, after the color nerves have been cut, not only the adrenergic
but the cholinergic fibers degenerate. Consequently the injected adrenaline does
not find in the denervated bands the local dispersing agent acetyl choline for an
opponent as it does in the innervated regions. Hence in denervated bands adrenaline
is able to act efficiently at lower concentrations than in innervated areas. There is
no reason to suppose that denervation alters the melanophores themselves. Dener-
vation in the catfish appears merely to remove an adrenaline opponent and thus to
give this agent more effective sway. This explanation of melanophore hypersensi-
tization to adrenaline may not apply to other instances of the special sensitization
of effectors, but it appears to meet the requirements in the melanophores of the
catfish.
Implants consisting of young buds, formed in anterior regeneration in
Clymenella, plus the nerve cord of the adjacent old part. Leonard
P. Sayles.
Worms were cut to regenerate anteriorly. After 2 to 9 days, any newly-
formed material plus the adjacent anterior nerve cord was inserted into the thir-
teenth segments of hosts. In some cases the original buds dropped off. Then
either (1) no material formed at the implant or (2) small new buds, each terminat-
ing in the dorsal half of an anal segment, developed. All of the latter terminated
ventrally in truncate regions. These results were similar to those obtained when
pieces of anterior nerve cord from non-regenerates were inserted at posterior levels
of hosts.
When the original bud was retained, the results were modified. When only a
small blastema had regenerated, the implant gave rise, in many cases, to a bud
terminating in a partial anal segment dorsally and either a large, conical mass or
a weakly developed peristomium ventrally. When a small, 3-day type head was
present, this bud might continue to develop. Frequently, however, a partial anal
segment appeared on the dorsal side of the bud. Then the portion of the original
bud beyond this anal segment regressed until it was only a small ball of pigmented
material. This ball then dropped off, leaving a growing, incomplete tail bud in
place of the original head. When the implant included a well-developed head of
the type produced after 5 to 9 days, no anal segment elements appeared. In some
cases the bud continued to grow and organize, in others it regressed somewhat.
A young head bud, therefore, was not able to maintain itself against the more
powerful tail-forming influence of the host's posterior segments, although this bud
did modify the induced bud to some extent. Older buds, however, were retained
without the host producing any additional structures.
PRESENTED AT MARINE BIOLOGICAL LABORATORY 303
Chaos nobilis Penard in permanent culture. A. A. Schaeffer.
From all published accounts of amoebas probably belonging to the genus Chaos
Linnaeus, six well-attested species emerge. Three of these : difflitcns, nitida, ncos,
Icschcri, are uninucleate and two: chaos and nobilis are multinucleate. Excepting
nobilis, all these species are readily cultured. Nobilis has been reported in the
past (Vonwillier, Penard) as dying out in cultures. A few nobilis found in a
ditch near Willow Grove, Pennsylvania, on April 20, 1941, maintained themselves in
laboratory culture until now (August 20) and are slowly increasing in number.
The rate of multiplication is much slower than in the other five species, for from
4 to 10 days elapse between divisions. This slow rate may be due to improper
culturing methods, although from their appearance, one would judge these amoebas
to be normal in every respect.
Von Stein (1867) was probably the first to see a nobilis. Next Butschli (1876)
studied it and counted and measured nuclei in a wild culture. Penard studied it
in 1902 and regarded it as a distinct species. Vonwillier later inclined to the view
that the multinucleate amoebas which he found were like those of Calkins and
Penard, but not like those of Butschli, Schubotz and Gruber. Lucy Carter de-
scribed a multinucleate amoeba similar to Butschli's. The Willow Grove amoeba
is similar to Penard's amoeba, but does not show the variability in number and
nuclear size of Butschli's amoeba.
The two multinucleate species, cliaos and nobilis, are distinctly different in size
range, in nuclear structure, size, and number, and in rate of reproduction. Thus,
the largest nobilis are about 250 M in diameter and have from 80 to 90 ovoidal nuclei
measuring 16 M X 12 M; the smallest are from 100 M to 115 M in diameter with 10
to 15 nuclei of about the same size and shape as those of the larger amoebas. The
Willow Grove amoeba therefore corresponds very closely with Penard's nobilis,
with Lucy Carter's, and with Butschli's multinucleate amoeba, but differs in nuclear
number from Vonwillier's.
Further studies on Mactra egg cells. Victor Schechter.
The study of the problem of longevity in unfertilized Mactra egg cells was
continued this summer along two directions. It was found that a non-dialyzable
factor detrimental to the life of the eggs gradually develops in sea water consid-
erably before the eggs show structural or functional deterioration. With regard
to beneficial factors, dextrose down to the surprisingly low concentration of 0.001
per cent was found to be effective.
The effect of centrifugation upon the oxygen consumption of Arbacia
eggs* Sidney F. Velick.
When unfertilized eggs of Arbacia punctilio ta are centrifuged in the cushioned
medium of E. B. Harvey at a speed sufficient to stratify the cellular elements and
stretch the cells to an axial ratio of about 1.5 to 1, the oxygen consumption is
increased 60 to 120 per cent over that of the unstratified controls from the same
egg suspension. The increment usually persists for several hours in the respirom-
eter and does not result in membrane formation or cleavage. Upon fertilization,
the oxygen consumption of the stratified eggs increases by the same order of
magnitude as do the unstratified eggs. Return of the unfertilized stratified eggs to
the spherical form is not accompanied by a decline in respiration, but a decline to
the original level does occur after the spontaneous redispersion of the stratified
granules has proceeded to a sufficient extent in a stationary vessel. The fat globules
remain aggregated in a cluster long after the decline has occurred. As in the
* This work was aided by a grant from the Jane Coffin Childs Memorial Fund
for Medical Research.
304 PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS
case of the unstratified egg, the oxygen consumption increases progressively upon
too rapid shaking, an effect which has been observed by others on unstratified eggs
and attributed to damage of the membrane. These experiments, undertaken at the
suggestion of Dr. Kurt G. Stern, are being continued.
Ectodermisation of the larva of Arbacia. Allyn Waterman.
A reinvestigation of previous work on Arbacia development has been attempted
by exposing cleavage stages, swimming blastulae and early gastrulae to 2,4-dinitro-
phenol, 3,5-dinitro-o-cresol, pyocyanin, methylene blue, neutralized iodoacetic acid
and to modifications of salt proportions in an artificial sea water formula which
supported typical development through the pluteus stage. The reactions of these
various stages differ little and then only in degree which is apparently correlated
with the stage of development. Strong concentrations of respiratory affectors
retarded or inhibited differentiation and gastrulation. Among the different ab-
normal types were found ectodennized embryos and undifferentiated exogastrulae.
Weak concentrations accelerated development by several hours, without any differ-
ential effect upon the germ layer derivatives, and these larvae died earlier than the
controls or abnormal types. Iodoacetic acid provoked ectodermization, indicating
a shift of the control of development to the material at the animal pole. Omission
of SO4, MgCL, CaCL, or an excess of MgSO, in the artificial medium caused
ectodermization. In all a great variety of abnormalities occurred which differed
little from those provoked by many other methods. Undifferentiated exogastrulae
cannot be considered a type of endodermization. During early development, at
least, Arbacia embryos show a wide tolerance to most ionic variations in their
environment.
The degree of ectodermization was variable within the same culture, showing
a differential susceptibility between individuals. Completely ectodermized indi-
viduals often attained giant proportions, developed cilia and an exaggerated apical
tuft, and survived as long as other types. Less completely ectodermized larvae
often possessed some skeletal material and an undifferentiated gut.
While these results may be explained by the ectodermal-endodermal gradient
hypothesis of Runnstrom, they appear to furnish no support to the suggestion of
Lindahl that carbohydrate metabolism predominates at the animal pole (Needham
and Needham) .
Studies on Zoochlorclla-frcc Paramecium bursaria. Ralph Wichterman.
Isolation cultures were made of 40 specimens of P. bursaria. One strain dis-
closed P. bursaria to be completely free of the alga Zoochlorella but to have in-
stead great numbers of optically active crystals. These crystals were especially
abundant in the posterior region of the ciliates. Unlike green P. bursaria, which
generally settle to the bottom of the culture dish and congregate toward the strong-
est source of light, the " white " ones swim actively throughout the culture medium
which consisted of desiccated lettuce infusion. White ones showed the mating
reaction with green individuals of certain other clones. The mean daily fission
rate of green specimens which mated with the white ones was 1.1 divisions while
white P. bursaria showed a faster division rate, namely 1.5.
Since green P. bnrsaria contained no crystals and zoochlorella-free individuals
contained many, green individuals were kept in complete darkness for varying
periods of time up to 25 days in order to find out whether the zoochlorellae dis-
appeared and crystals appeared. It was found that green individuals kept in
darkness even for 25 days lost some but not all of their zoochlorellae. On the
other hand, optically active crystals appeared in such specimens and darkness did
not prevent the mating reaction from taking place. However, large clumps of
individuals, so characteristic in early stages of the mating reaction in the same
PRESENTED AT MARINE BIOLOGICAL LABORATORY 305
strains when subjected to light, did not form when they were kept in darkness for
a considerable length of time.
Zoochlorella suspensions from green individuals of the mating type opposite
from the white P. bursaria were made. When a white specimen was placed in
such a suspension of zoochlorellae, the ciliate ingested the algae rapidly. Food
vacuoles contained from one to five zoochlorellae. However, in less than a day,
the white P. bursaria became darkly granular, sluggish, then died. Yet when
zoochlorellae from another strain which also mated with the white ones were
placed with them, no such lethal effect took place. Both of the above-mentioned
clones of green P. bursaria showed the mating reaction with each other.
. •///. experimental study of intracellular pH in tlic Arbacia egg. Floyd
J. Wiercinski.
The problem of determining the exact pH of the living cell by the microinjec-
tion of indicators presents numerous difficulties. In addition to the salt and protein
errors of the indicators these difficulties include membrane formation, granular
breakdown, and most important of all, the uptake of indicators by the granules.
In order to avoid factors due to the presence of granules, indicators were injected
into the hyaline region of the centrifuged Arbacia egg.
Ten sulfonephthalein indicators and certain mixed indicators were used. A
mixed indicator lias the advantage of a sharp color transformation point at a
given pH. Varied results were obtained with the indicators under different con-
ditions of experimentation.
Tests with phenol red show a pH < 7.0, brom thymol blue < 6.8 and > 6.0,
brom cresol purple + brom thymol blue < 6.6 and > 6.0, brom cresol purple < 6.8
and > 6.0, brom cresol green + chlor phenol red > 6.2 and < 5.8, chlor phenol
red < 6.6 and > 6.0, chlor phenol red + aniline blue > 5.8, brom cresol green + Na
Alizarine S > 5.8, methyl red > 5.8, and brom cresol green > 5.6. When eggs
were immersed in 0.29 M CaCL at pH 6.1 somewhat lower values were obtained.
This may be due to an augmentation of the injury reaction.
The results indicate a pH somewhere in the neighborhood of 6.2 for the hyaline
protoplasm of the Arbacia egg.
Heat produced by respiring whole blood of Tautoga onitis and Must el us
canis. E. Alfred Wolf, Maryon Dytche, John D. O'Neal and Milton
Schaffel.
The colorimeter vessel was a triple-walled, silver-mirrored Dewar flask of
about 100-cc. capacity. Temperature rise was measured with a Beckmann ther-
mometer. Oxygen was supplied by bubbling. A method was found to prevent
foaming which did not injure the cells. The fish used for the investigation were
selected for their availability in Woods Hole waters. The constant temperature
water bath was kept at 15.3° C.
The gas used for oxygen supply for Tautoya was oxygen and oxygen plus
5 per cent carbon dioxide ; for Mustclus, because of lack of time, oxygen only was
used. This high percentage of carbon dioxide was selected in order to have a
basis of comparison for further work with higher vertebrates. The fish used in
this investigation are normally not exposed to such high pressures of carbon dioxide
and could not survive such conditions. At such pressures the blood of these fish
could not be saturated with .oxygen and the fish would suffocate in spite of the rich
supply of oxygen (R. W. Root, E. A. Wolf and others). Our present work
strikingly verifies these findings : in pure oxygen the rate of heat production re-
mained constant for hours ; in oxygen plus 5 per cent carbon dioxide this rate
decreased after about one hour of exposure; all curves changed from straight lines
306 PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS
to curves of decreasing slope. We interpret this change as signs of approaching
suffocation in an abundance of oxygen.
The rate of heat production of blood is of significant magnitude in the bio-
economics of fish. This rate in oxygen was 82 calories per kg. of blood per hour
for Tautoga and 51 cal. for Mustelus. In oxygen plus 5 per cent carbon dioxide
the rate decreased to 40 cal. for Tautoga. A fish of comparable size at 15° C.
would produce about 360 cal. per kg. of body weight per hour.
Effect of differences between stages of donor and host upon induction of
auditory vesicle from foreign ectoderm in' tlie salamander embryo.
C. L. Yntema.
Operations were performed on the embryo of Amblystoina punctatum. Foreign
ectoderm from embryos at stages from 9 (early gastrula) to 28 (late head-process)
was placed in the ear region of host embryos at stages from 12 (late gastrula) to
35 (onset of circulation). Prospective body ectoderm was used in experiments in
which the donor was an early or middle gastrula. At older stages prospective gill
ectoderm was transplanted. The animals were preserved at stage 46 (beginning of
feeding). The contribution of the grafts was determined by retaining the Nile-
blue sulfate stain of the grafts in sections.
The ability to induce a small vesicle was retained by a host as advanced as
stage 35. The ability to induce was greatest, as measured by older grafts, during
the neural groove stage (13) and during completion of neuralation (stages 19 and
20).
The response of the prospective body ectoderm from early and middle gastrulae
was masked by induction of neural tissue from the grafts. Prospective gill ecto-
derm from stages 12 and 13 formed auditory vesicles if the hosts were neurulae.
When older hosts were used, this potency was not realized. The most normal
vesicles induced by hosts at stage 35 were from ectoderm taken from donors at
stage 22 (early head process). Ectoderm from stage 28 formed small vesicles
when placed in the ear region of stage 20.
The following are some general conclusions. If an embryo can no longer
regenerate a labyrinth, its gill ectoderm possesses under certain conditions the
capacity to form an auditory vesicle, and the ear region may still induce a vesicle.
Ectoderm which is competent to form an auditory vesicle in the ear region of some
stages is not competent to do so at certain other stages. Both the stage of the
foreign ectoderm and the stage of the host are factors which determine the
response to form a vesicle.
Vol. LXXXI, No. 3 December, 1941
THE
BIOLOGICAL BULLETIN
PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY
CS x"oiT77
THE EQUILIBRIUM BETWEEN HEMOGLOBIN AND
OXYGEN IN WHOLE AND HEMOLYZED BLOOD
OF THE TAUTOG, WITH A THEORY OF THE
HALDANE EFFECT
R. W. ROOT AND LAURENCE IRVING1
/
(From the College of the City of New York; Swarthmore College; and the
Marine Biological Laboratory, Woods Hole)
The combination of oxygen with the blood of certain fishes occurs
far less readily when the CO2 tension is raised, but in a number of these
same fishes hemolysis largely eliminates the sensitivity of the hemo-
globin toward CO2 (Black and Irving, 1938; Root, Irving, and Black,
1939). The peculiar effect of hemolysis indicates that the properties
of the whole blood, as regards O2-combination in the presence of CO2,
do not parallel the properties of the hemoglobin when released from
the cell. One cannot, therefore, infer the properties of fish hemoglobin
in their entirety from a study of whole blood alone. It has been
further shown that the whole and hemolyzed blood of the tautog not
only differed with respect to O2-combination in the presence of CO2,
but the reciprocal effect of oxygenation on CO2-combination showed
significant differences (Root and Irving, 1940). In hemolyzed blood
-ABHCO3 .
the ratio - „ - is apparently constant tor any given CO2 tension,
whereas this is not the case in whole blood. The behavior of the whole
blood is apparently exceptional, since it is commonly considered that
_
for any given hemoglobin - „ ' is constant (Henderson, 1928;
Redfield, 1933a).
The material to be presented in this paper is in part an amplification
of the work done by us on the blood of the tautog, Tautoga onitis
(Linn.). The equilibrium between hemoglobin and oxygen in both
whole and hemolyzed blood has been examined in detail over a wide
range of CO2 tensions. From the study a clearer picture has developed
1 The authors are indebted to the U. S. Bureau of Fisheries at Woods Hole for
the provision of laboratory space and facilities during the course of this investigation.
307
308
R. W. ROOT AND L. IRVING
of the contrast in behavior of whole and hemolyzed fish blood. It has
enabled us to describe theoretically not only the equilibrium that exists
between hemoglobin and oxygen in the two conditions of the blood, but
also to give an interpretation of the effect of oxygenation on CO2-
combination (Haldane effect) as observed in the blood of this fish.
Throughout this investigation the methods of handling the blood,
equilibrating it, and analyzing the gas phases were the same as those
described in the paper by Root and Irving (1940).
200
JOO
Po,
400
mm.
soo
600
7OO
FIG. 1. Oxygen dissociation curves of whole tautog blood at 15° C. and
constant COo-tensions. The curves have been drawn according to the equation indi-
cated in the text, using the following constants:
PCO2
mm. Hg.
at
at
•
«4
ao
Ki
K2 X 102
K< X 109
0-1
0
1.0
0
0
.51
10
.75
0
.25
0
.0286
.25
25
.60
0
.15
.25
.0286
.16
50
.56
0
0
.44
.0286
100
.52
0
0
.48
.0286
COMPARISON OF THE OS-DISSOCIATION CURVES
OF WHOLE AND HEMOLYZED BLOOD
The Os-dissociation curves for both whole and hemolyzed blood of
the tautog have been established at 10, 25, 50, and 100 mm. CO2
HB-CX EQUILIBRIUM IN BLOOD OF TAUTOG
309
tension. The former are shown in Fig. 1 along with a curve established
in the virtual absence of CO2 (data of Root, Irving and Black, 1939).
Those for hemolyzed blood are presented in Fig. 2. Each curve repre-
sents the data from a single large sample of blood, with the exception
of the one at 100 mm. CO2 where two lots of blood were used. The
curves at each CC>2 tension have been drawn from an equation which
seemed best to fit the data, the constants used being shown in the table
loo
140
160
mm
. Wg.
FIG. 2. Oxygen dissociation curves of hemolyzed tautog blood at 15° C. and
constant CO2-tensions. The curves have been drawn according to the equation
given in the text, using the following constants:
PCO2
mm. Hg.
«i
02
ao
Ki
Ki X 102
10
0
1.0
0
2.0
25
0
1.0
0
.59
50
.2
.8
0
.10
.11
100
.5
.5
0
.05
.033
beneath the figures. Reference to this equation and its implications
will be made later.
If one compares in a general way the family of curves obtained for
each type of blood the following major differences become evident: (1)
the hemolyzed blood has a greater affinity for oxygen at comparable
CC>2 tensions than has the whole blood; (2) the dissociation curves for
hemolyzed blood at comparable CC>2 tensions are different in shape from
those of whole blood; (3) the hemolyzed blood, up to 100 mm. CC>2
310 R. W. ROOT AND L. IRVING
tension, shows no evidence of hemoglobin inactivation, whereas whole
blood does; and (4) the reduction in affinity for oxygen with rise in
CC>2 tension is different in magnitude in the two types of blood.
In order to further the comparison of the behavior of the two types
of blood it is desirable to study as closely as possible the shape of the
O2-dissociation curves in each case. For our purpose this has con-
sisted in an attempt to fit certain existing equations describing the
equilibrium between hemoglobin and oxygen to the data for the curves.
Once a suitable fit is obtained the theoretical implications of the par-
ticular equation employed offer some insight into the behavior of the
hemoglobin at any particular CC>2 tension. Any attempt to fit existing
equations to dissociation curves established at constant CO2 tension,
instead of constant pH, is perhaps open to criticism, but we have
reason to believe that the results are not so totally different that the
general conclusions drawn from such an analysis would be invalidated
when the pH is kept constant.
It is obvious from examination of the data that the classical equa-
tion of Hill (1910) is too simple an expression adequately to describe
all of the dissociation curves. We have therefore resorted to the equa-
tion suggested by Redfield (19336) and used by Green and Root (1933)
in describing the equilibrium between hemoglobin and oxygen in cer-
tain fish bloods. This equation is based on the theory that there are
different components of the respiratory protein which act independ-
ently of each other in compliance with Hill's equation but with dif-
ferent values of n. If the Oo-dissociation constants of the components
having values of n of 1.0, 2.0, 3.0, 4.0 etc., are designated by KI, K2, K%,
Kt, etc. and the fraction of the total Oo bound by each of these com-
ponents as ai, a2, «3, «4, etc., the fraction of the total respiratory pro-
tein present in the oxygenated form, Y, at any particular O2-tension,
X< is given by the equation:
I ^^ i-*- *• £• A. I ^^ o-*- •*• o/V i
It is necessary in tautog whole blood to introduce the term a0 to
take into account the fraction of hemoglobin inactivated at high CC>2
tensions. The sum of a0 plus the other fractions will equal 1. For-
tunately it has not been necessary for us to use more than two terms of
the general equation in describing the more complicated dissociation
curves obtained with the blood of this fish. The simple curves require
but one term and could as well be described with Hill's equation, pro-
viding we take into account the fraction of hemoglobin inactivated in
whole blood at high CC>2 tensions.
HB-O, EQUILIBRIUM IN BLOOD OF TAUTOG 311
The general results of our analysis of the Oo-dissociation curves,
using the above equation, can be obtained by reference to the table of
constants beneath Figs. 1 and 2, and to the curves drawn according to
the equation, using these constants. It is clear that in whole tautog
blood the Oo-dissociation curve in the virtual absences of COo is
sigmoid and is characterized by a value of n - - 2, i.e. there is a single
component of the hemoglobin uniting with two molecules of oxygen at
a time. As CO2 is added the dissociation curve not only moves to the
right but becomes more complicated. Components with different
values of n and dissociation constants sufficiently different to produce
definite undulations in the curve come into view. Further addition of
COo brings about inactivation of some of the hemoglobin and finally
simplifies the Oo-dissociation curve to the form of a rectangular hyper-
bola, i.e. there is now a single component of the hemoglobin combining
with one molecule of oxygen at a time. We see, then, that COo, in
addition to inactivating a portion of the hemoglobin, completely
changes the Os-dissociation curve of whole blood from a second to a
first power curve, and that the intermediate stages in the conversion
apparently produce different components that unite with different
amounts of oxygen at a time, thus complicating the dissociation curves
in this region.
The picture presented is essentially that obtained earlier by Green
and Root (1933) on the same blood at 25° C. It differs in that their
intermediate curves did not show the marked inflections that ours show.
However, a too rigorous comparison of the intermediate curves is not
justified since ours were established at a constant COo tension instead of
constant pH, as theirs were, and at 15° C. instead of 25° C.
In hemolyzed blood, as the table beneath Fig. 2 will indicate, not
only are the dissociation constants for the curves much larger than
those for whole blood at comparable CO2 tensions, but those com-
ponents characterized by a value of n greater than 1 persist at COo
pressures at which they have definitely disappeared in whole blood.
Furthermore, it can be seen that there is no necessity for assuming that
any of the hemoglobin has become inactive, as was the case in whole
blood. There is this similarity, however, between whole and hemo-
lyzed blood: added COo decreases the magnitude of the dissociation
constants (Bohr effect) and changes the behavior of the hemoglobin
in the direction of components which react with only a single molecule
of oxygen at a time (n - • 1). The latter process requires a much higher
COo tension in the hemolyzed blood, not being completed even at
100 mm. COo tension.
By way of summary, the study of the Oo-dissociation curves of
312 R. W. ROOT AND L. IRVING
whole and hemolyzed blood, both comparatively and individually, has
yielded sufficient information to enable us to visualize, at least par-
tially, what the addition of CO2 does to the hemoglobin of this fish.
In whole blood it changes the dissociation constants, modifies the be-
havior of the components combining with oxygen, and effects con-
siderable inactivation of the hemoglobin. In hemolyzed blood, at
least up to 100 mm. CO2, we obtain the first two effects, but not the
latter. However, the dissociation constants are all much larger in
magnitude, and the change in the components of the hemoglobin re-
quires a much higher tension of CO2. We are confronted then with
the apparent fact that liberation of the hemoglobin from the cell, in
the presence of CO2, in some way decreases the dissociation of oxygen
from the hemoglobin, prolongs the existence of those components of the
hemoglobin which act as if they were combining with more than one
molecule of oxygen at a time, and abolishes, or greatly postpones, any
inactivation of the (^-combining groups. The reason for this is yet
to be elucidated.
THEORETICAL INTERPRETATION OF THE EFFECT OF OXYGENATION ON
THE CO2 BOUND BY THE BLOOD (HALDANE EFFECT)
In a previous paper (Root and Irving, 1940) evidence was presented
to show that the effect of oxygenation on CO2 transport in tautog blood
was different from that in the blood of mammals. It was tentatively
suggested that a part of the difference might be explained on the basis
of the theory that there were several Go-combining components of the
hemoglobin behaving differently with respect to Oo-combination and
CO2 sensitivity. At the time we could not see how such an interpreta-
tion could apply to hemolyzed blood, since the Haldane effect here was
quite typical, and suggested that perhaps there were fundamental
changes in the properties of hemoglobin upon hemolysis. With the
combined picture we now have of the effect of CO2 on the O2-combining
power and the reciprocal effect of oxygenation on the CO2-combining
power, we are in a position to give a more adequate interpretation of
the Haldane effect as observed in this blood.
The primary fact to be explained is the inconstant - - ratio
found at 10 and 25 mm. CO2 pressure for whole blood. To those
familiar with mammalian blood it is well known that these ratios are
considered to be constant for any single hemoglobin, and it is usually
believed that they are constant, though of different magnitude, for the
hemoglobin of any species (Redfield, 1933a). An inconstant ratio,
then, would be considered atypical as compared with the usual constant
ratios.
HB-O2 EQUILIBRIUM IN BLOOD OF TAUTOG 313
Our interpretation rests on the fundamental postulate that the
hemoglobin consists of (^-combining components which can combine
either with a single molecule of oxygen at a time, or more, depending
upon how many prosthetic groups the components contain. On this
basis let the following assumptions be made:
1. For any single O2-combining component the - - ratio
A(J2
is constant. Let this constant be called R.
2. Different Oo-combining components have different values for R.
This is not a groundless assumption for R varies among hemoglobins of
different species (Redfield, 1933a).
3. Hence, if the proportions of the several Oz-combining com-
-ABHCO3 ,
ponents change, the ratio - „ - tor the combined components of
the whole hemoglobin may be inconstant.
We are now in a position to apply these assumptions. Let the
components of the hemoglobin be designated as a\, a?, a3, an etc. in
accordance with the previous treatment (see page 310), and the cor-
-ABHCO3
responding -- — ^— - ratios be written as follows:
1 _ -ABHOV2
BI; "2 : -;
-ABHC03*3 -ABHCO,««
' «<
Any given increment of oxygenation of the whole hemoglobin,
o", will be equal to the sum of the increments for each of the com-
ponents, i.e.
. (2)
The base correspondingly released by the whole hemoglobin,
-ABHCOs", will be equal to the sum of that released by each of the
components, i.e.
-ABHCO3U = -ABHCXV1 + -ABHCXV2
+ -ABHCO3*3+ -ABHOV4. (3)
Combining equations (2) and (3) we have
-ABHOV
AO2U
-ABHCO3al+ -AHBCO3g2+ -ABHCO3a3 + -ABHCO3a4
AO2ai
(4)
314
R. W. ROOT AND L. IRVING
It is evident from (1) that equation (4) can be rewritten in the following
form :
-ABHCCV
AO2"
AO2a2
A(V3
AO2a4
(5)
By the use of this fundamental equation curves can be drawn which
relate the total CO2 to the degree of oxygenation of the hemoglobin.
Since the effect of oxygenation upon CO2-combination is called the
Haldane effect, the curves which describe the effect of change in com-
bined oxygen (AO2) upon the combined CO2(ABHCO3) will be called
Haldane curves. These curves have been constructed from data calcu-
TABLE I
Data for construction of Haldane curve for tautog whole blood at 10 mm. CO*.
Rai = .05; Ra^ = .135; AO2U == 10 per cent HbO2.
HbOs
AO2«J
AO2a4
-ABHCOs"!
-ABHCO3a4
-ABHCOs"
Total [CO?)
per cent
per cent
per cent
vol. per cent
vol. per cent
vol. per cent
vol. per cent
0
0
0
0
0
0
20.50
10
10
0
.50
0
.50
20.00
20
10
0
.50
0
.50
19.50
30
10
0
.50
0
.50
19.00
40
10
0
.50
0
.50
18.50
50
10
0
.50
0
.50
18.00
60
8.5
1.5
.43
.20
.63
17.37
70
5.0
5.0
.25
.68
.93
16.44
80
2.5
7.5
.13
1.01
1.14
15.30
90
2.0
8.0
.10
1.08
1.18
14.12
lated according to the principle of equation (5) and presented in Tables
I and II. The change in oxygenation is expressed as an increment of
the percentage saturation of the whole hemoglobin, since the original
O2-dissociation curves are drawn in that manner; furthermore we have
given AOo"1 the arbitrary value of 10% HbO2. This is a sufficiently
small increment to provide an adequate number of points on a theoreti-
cal Haldane curve. It is to be understood that in assigning a value of
10% HbO2 to ACV it means that the fully reduced hemoglobin is
oxygenated in steps of 10 per cent and for each step the BHCO3U
released is calculated according to equation (5). The value obtained
when subtracted from the total CO2 remaining in the preceding step
of oxygenation will provide a point on the Haldane curve.
By breaking down the O2-dissociation curve at any given CO2
pressure into its components (see Figs. 3 and 5) the values for the
HB-CX EQUILIBRIUM IN BLOOD OF TAUTOG
315
AO2 of the components can readily be determined for any value of
AOif. In addition to knowing these values, the R values for each of
the components must be known in order to calculate the BHCOs"
released on oxygenation of the hemoglobin. These can be determined
from the slope of the experimentally established Haldane curve pro-
viding there is any part of it where the slope is due to only one com-
ponent acting. The latter can be determined by consulting the cor-
responding Oo-dissociation curves for the components. Fortunately
we have had to deal with only two components at any one time and
this has simplified the work of calculating the R values. Once the
value for one component is known, the other can be readily determined.
As an example of the determination of the R values, we refer to the
whole blood of the tautog at 10 mm. CO2 pressure where there are two
TABLE II
Data for construction of Haldane curve for tautog hemolyzed blood at 100 mm. CO->.
Rai == .04; Ra.2 = .06;'AO2") == 10 per cent HbO2.
Hbd
AO:«i
AO2ff2
-ABHCO3"i
-ABHCO3a2
-ABHCOii"
Total [CO->]
per cent
per cent
per cent
vol. per cent
vol. per cent
vol. per cent
vol. per cent
0
0
0
0
0
0
35.80
10
10
0
.40
0
.40
35.40
20
8.0
2.0
.32
.12
.44
34.96
30
5.0
5.0
.20
.30
.50
34.46
40
4.0
6.0
.16
.36
.52
33.94
50
4.0
6.0
.16
.36
.52
33.42
60
3.5
6.5
.14
.39
.53
32.89
70
3.0
7.0
.12
.42
.54
32.35
80
3.0
7.0
.12
.42
.54
31.81
90
3.0
7.0
.12
.42
.54
31.27
components, «i and ou acting. By examining the Os-dissociation
curves for these components, as shown in Fig. 3, it becomes evident
that below 50 per cent O2-saturation of the whole hemoglobin the 0:4
component is contributing nothing to the O2-saturation of the hemo-
globin. Therefore the slope of the corresponding Haldane curve in
this region is due solely to the «i component, i.e.:
-ABHCCV -ABHOV1
AO2"
ACV1
= Ro
The value for Rai when AO2al is put on a percentage Oo-saturation
basis, turns out to be equal to 0.05 at this particular CO2 tension. To
calculate Ra4 one may go to a position of the Haldane curve where both
components are clearly contributing to the slope of the curve. Be-
tween 60 per cent and 90 per cent CVsaturation there is such a region.
316
R. W. ROOT AND L. IRVING
Again, by consulting the Oo-dissociation curves for the components,
one can find just how much of this 30 per cent increment of 02-satura-
tion is due to each of them. It happens that approximately one-third
(10 per cent HbO2) is contributed by the a\ component, and the rest
(20 per cent HbO2) by the ou component. Rat may now be found as
follows:
-ABHCOs"1 = 3.2 vol. per cent (from experimental Haldane curve)
-ABHOV1 == 7?Ql-AO2al or
-ABHCCV1 = 0.05 X 10 == 0.5 vol. per cent
-ABHCCV4 = -ABHCCV -ABHCCV1 or
-ABHCCV4 = 3.2 - 0.5 = 2.7 vol. per cent
-ABHCQ;
ACV<
- R°< = I? - -135-
100
composite curve
100
200
300
Po
400
mm.
SCO
600
700
FIG. 3. Oxygen dissociation curves for the components of whole blood hemo-
globin at 10 mm. CO2-pressure. The upper curve represents the O2-dissociation
curve for the entire hemoglobin, obtained by adding the component curves together.
When there is no appreciable region where one component alone is
contributing to the slope of the Haldane curve, an accurate determina-
tion of the R values is difficult or impossible and one must be satisfied
with assumed values which will yield a theoretical curve closely fitting
the experimental.
Having the R values, one is now in a position to construct a Haldane
curve on the basis of the foregoing theory. With the hemoglobin fully
HB-O, EQUILIBRIUM IN BLOOD OF TAUTOG
317
reduced, and the total CC>2 known under these conditions by extra-
polating the experimental Haldane curve to 0 per cent O2-saturation,
one oxygenates the blood in steps of 10 per cent, determining for each
step the amount of BHCO3 released by each of the Oo-combining
components. The total base released, BHCOs", subtracted from the
30
9
d4
2o
<
o
Hemolyzed blood.
lOOmm.COz
Whole blood
fO mm. COZ
20
40
60
100
%HbO;
FIG. 4. Theoretical Haldane curves for whole blood at 10 mm. CDs-pressure
and hemolyzed blood at 100 mm. CO2-pressure, drawn according to the theory dis-
cussed in the text. The points on the curves are those obtained by experiment.
total CO2 present at the end of the preceding step in oxygenation will
give a point on the Haldane curve for each increment of oxygenation.
In Table I the data, derived in such a fashion, for the construction of a
theoretical Haldane curve for whole tautog blood at 10 mm. COo
pressure are presented. The values in the first and last columns of this
table have been used to plot the curve presented in Fig. 4, and the
318 R. W. ROOT AND L. IRVING
points on the theoretical curve are those actually obtained by experi-
ment. It is obvious that there is good agreement between theory and
fact.
In the basic assumption for the interpretation of the anomalous
_ ARHCO
Haldane curve for tautog blood it was pointed out that the -
AU2
ratio for the entire hemoglobin may be inconstant. It has been demon-
strated that such is the case for tautog whole blood at 10 mm. CO2
pressure. However, it does not follow that the underlying theory can
apply only to inconstant ratios, i.e. that the ratios must be inconstant
at all times. A moment's consideration of equation (5) will make it
clear that there could be such a set of R and AO2 values for the com-
ponents as to provide a practically constant release of base from the
hemoglobin for each step in oxygenation. Should it so happen, for
example, that the R values for the components are not too different,
— ABHCOs
one might readily conclude experimentally that the - M - ratio
for the whole hemoglobin is constant — at least one would be tempted
to draw a straight line through the experimental points. Or, what is
more important, if it should so happen that the AO2 values for each of
the components remains practically constant over an extended range
when the hemoglobin is oxygenated by equal steps, then it would fol-
low, no matter what the R values, that the - - ratio would be
u
practically constant in this same range. Whether such a circumstance
would occur or not would be determined both by the values of the
Oo-dissociation constants for the components and the shape of the
Oo-dissociation curves they yield.
To illustrate the possibilities outlined above, we will consider the
Haldane curve for hemolyzed tautog blood at 100 mm. CO2 pressure.
— s . . ,
At this C O2 tension experiment shows that the - ~ a - ratio is best
represented as constant, yet analysis of the O2-dissociation curve indi-
cates that one is dealing with two components, on and o:2. It is diffi-
cult to determine the exact values for Rai and Ra2 from the experi-
mental Haldane curve since there happens to be no appreciable part of
it where one component alone is acting. Such will be made clear by
consulting the O2-dissociation curves for the components presented in
Fig. 5. It is evident that one must reduce the hemoglobin below
10 per cent O2-saturation before there is any significant separation of
the components. Since there is no apparent inflection in the Haldane
curve even in this region, one must conclude that the R values are not
HB-O, EQUILIBRIUM IN BLOOD OF TAUTOG
319
too different. The values we have finally taken are indicated in
Table II. It is clear, furthermore, from Table II that the AO2 values
for the components remain about the same from 30 per cent C>2-satura-
tion to 90 per cent for each 10 per cent step in oxygenation of the
hemoglobin. Such a combination of factors can only mean that the
theoretical Haldane curve will be practically a straight line, i.e. the
-ABHCCV
- ratio is apparently constant, r igure 4 shows the tneoreti-
AO2a>
cal curve for hemolyzed blood at 100 mm. CO2-pressure drawn from the
data of Table II. The points on the curve are those actually obtained
100
80
f
"
4o
20
composite curve
ZO
40
60
80
100
120
160
mm.
. Wg.
FIG. 5. Oxygen-dissociation curves for the components of hemolyzed blood
hemoglobin at 100 mm. CO2-pressure. The upper curve represents the O^-dissocia-
tion curve for the entire hemoglobin, obtained by adding the component curves
together.
by experiment. It must be concluded that under the right circum-
_ ARHCO "
stances it is possible to have what appear to be constant - „ u
ratios even though more than one CVcombining component is con-
tributing to the oxygenation of the hemoglobin and the release of base.
Such a state of affairs does not necessarily constitute an exception to
the theory we have presented, but merely a special case.
Considering both the Haldane effect in whole blood at 10 mm. CO2
-ABHCCV
pressure, where there is obviously an inconstant - — - - - ratio, and
the same effect in hemolyzed blood at 100 mm. CO 2 pressure, where
320
R. W. ROOT AND L. IRVING
the ratio appears constant, it is clear that one must set forth certain
qualifications concerning the type of ratio one might expect. If the
values of R are quite different for each of the components and their
equilibrium with oxygen is such as to yield widely varying AOa values
for each step in the oxygenation of the hemoglobin (this would be
dependent not only on the value of n but especially on the value of the
Oo-dissociation constants for the components, which would have to be
quite different in magnitude) then there should be no difficulty in
-ABHCO3U
demonstrating inconstant - — — ~r - -ratios. It, on the contrary, the
""
R values for the components are closely similar, or especially if the
components have such an equilibrium with oxygen as to provide nearly
20 40 60 so too
%Hb02
FIG. 6. Haldane curve for toadfish blood at pH 7.2. Data of Green and Root (1933)
constant AC>2 values for each step in the oxygenation of the hemoglobin
(again this would be dependent on the value of n for the components,
and their (^-dissociation constants, which in this case would have to be
-ABHCOs"
more nearly alike) then the - „ - ratios would be practically
W
constant, and experimentally would probably not show otherwise.
The inconstant - - ratio shown in whole tautog blood led
us to re-examine some of the data of Green and Root (1933) on the
blood of the toadfish. This blood is characterized by anomalous
inflections in the O2-dissociation curves adequately explained by the
theory of components. At pH 7.2, for example, the O2-dissociation
HB-O™ EQUILIBRIUM IN BLOOD OF TAUTOG 321
curve is satisfactorily described by assuming two (^-combining com-
ponents with widely different dissociation constants. Clearly, if the
equilibrium with oxygen of these components at this pH is such as to
provide anomalous inflections in the (^-dissociation curve, then, if our
theory is correct, the corresponding Haldane curve should present in-
flections, i.e. the - „ - ratio should be inconstant. We have
plotted the values of Green and Root for total CC>2 against the per-
centage of O2-saturation and obtained the curve presented in Fig. 6.
Although the slope of the curve is enhanced due to the fact that the CO2
pressure was not kept constant (constant pH instead), it is evident from
the inflection that the type of curve obtained is similar to that for
whole tautog blood at 10 mm. CC>2 pressure, substantiating the theory
we have presented.
There is a further matter of interest regarding the Haldane effect
in the whole blood of the tautog. In our previous paper (Root and
Irving, 1940), it was pointed out that when the CO2-tension is raised
sufficiently the Haldane effect tends to disappear, i.e. the ratio
- approaches zero value. This happens when the hemo-
globin has been partially inactivated and the remainder has been
modified to a point wrhere there is but a single CVcombining component
with a value of n equivalent to 1. The explanation probably is that if
— ABHCOs"
one decreased the prl sufficiently, the - - ratio would diminish
for a particular hemoglobin, even if the components themselves did not
change their behavior, for one might eventually reach a point where the
titration curves for the reduced and oxygenated forms of the hemo-
globin are converging toward each other. In other words, it is not safe
to assume in any situation where CC>2 modifies the behavior of hemo-
globin that the change in slope of the Haldane curve is entirely due to
this effect of CO2. It will hold only as long as the pH of the blood
remains in the region where the titration curves for reduced and
oxygenated hemoglobin parallel each other. Outside these limits the
-ABHCCV
- ratio will change in a manner quite independent or any
modification of the hemoglobin or its (^-combining components.
It can be seen from the major part of the foregoing discussion that
the argument for the peculiarities of the Haldane effect in tautog blood
rests primarily on the theory that the hemoglobin is made up of dif-
ferent O2-combining components. One might turn the argument
around and say that the peculiarities of the Haldane effect, offer strong
R. W. ROOT AND L. IRVING
support for the theory of components; for it is difficult to see how one
could get inflected Haldane curves, such as we have found in whole
blood, without having different O2-combining components present,
each of which independently affects the CO2-combining power of blood
on oxygenation. As the situation now stands it has been shown that a
single scheme can be used to describe both the effect of CO2 on the
oxygenation of hemoglobin and the reciprocal effect of oxygenation on
COs-combining power.
The authors wish to acknowledge their indebtedness to Virginia
Safford and Henry Brown for technical assistance, and to Dr. Paul S.
Galtsoff, Director, and Mr. Robert Goffin, Superintendent of the U. S.
Bureau of Fisheries at Woods Hole for their cooperation during this
investigation. They also wish to express to Professor A. C. Redfield
their appreciation for his suggestions and criticisms in the preparation
of the manuscript.
SUMMARY
1. A detailed study has been made of the effect of CO2 on the
equilibrium between hemoglobin and oxygen in whole and hemolyzed
blood of the tautog.
2. The study of the O2-dissociation curves of whole blood has shown
that the addition of CO2 up to 100 mm. pressure changes the shape of
the curves from sigmoid to rectangular hyperbolae with approximately
50 per cent of the hemoglobin inactivated. The intermediate stages
in the transformation produce complex dissociation curves which can
be described by assuming that fish hemoglobin is made up of different
O2-combining components acting independently of each other and
combining with different amounts of oxygen at a time.
3. Hemolysis renders the hemoglobin less sensitive to CO2 as evi-
denced by the fact that the O2-dissociation curves move far to the left of
those for whole blood; that the O2-combining components which com-
bine with more than one molecule of O2 at a time show greater stability
than they do in whole blood as the CO2 tension is raised ; and that there
is no hemoglobin inactivation up to at least 100 mm. CO2. There is
still a prominent Bohr effect, however, and the Oo-combining com-
ponents still gradually change their behavior as the CO2 tension is
raised.
4. Based primarily upon the characteristics of the equilibrium be-
tween hemoglobin and oxygen, a theory is offered to explain certain
peculiarities of the effect of oxygenation on the CO2-combining power
of the blood (Haldane effect). The theory offered provides a common
HB-CX EQUILIBRIUM IN BLOOD OF TAUTOG 323
explanation for the anomalies in the effect of CO2 on oxygenation of the
hemoglobin and in the reciprocal effect of oxygenation on the CO 2-
combining power of the blood.
LITERATURE CITED
BLACK, E. C., AND L. IRVING, 1938. The effect of hemolysis upon the affinity of fish
blood for oxygen. Jour. Cell, and Comp. Physiol., 12: 255-262.
GREEN, A. A., AND R. W. ROOT, 1933. The equilibrium between hemoglobin and
oxygen in the blood of certain fishes. Biol. Bull., 64: 383-404.
HENDERSON, L. J., 1928. Blood, a Study in General Physiology. New Haven.
HILL, A. V., 1910. The possible effects of the aggregation of the molecules of hemo-
globin on its dissociation curves. Jour. Physiol., 40: iv-vii.
REDFIELD, A. C., 1933a. The evolution of the respiratory function of the blood.
Quart. Rev. Biol., 8: 31-57.
REDFIELD, A. C., AND E. N. INGALLS, 19336. The oxygen dissociation curves of some
bloods containing hemocyanin. Jour. Cell, and Comp. Physiol., 3: 169-202.
ROOT, R. W., L. IRVING, AND E. C. BLACK, 1939. The effect of hemolysis upon the
combination of oxygen with the blood of some marine fishes. Jour. Cell, and
Comp. Physiol., 13: 303-313.
ROOT, R. W., AND L. IRVING, 1940. The influence of oxygenation upon the transport
of CO2 by the blood of the marine fish, Tautoga onitis. Jour. Cell, and Comp.
Physiol., 16: 85-96.
OBSERVATIONS ON THE FOOD HABITS OF ENTAMOEBA
MURIS AND ENTAMOEBA RANARUM
D. H. WENRICH
(From the Zoological Laboratory, University of Pennsylvania, and the Marine
Biological Laboratory, Woods Hole, Mass.)
I. INTRODUCTION
While studying nuclear structure and nuclear division in Entamoeba
murls (Wenrich, 1940), casual observations indicated that there were
food preferences among different individuals and among different popu-
lations of these amoebae. Further investigation showed that these
amoebae often developed temporarily differentiated ingestion tubes which
stain intensely with Heidenhain's haematoxylin. A preliminary report
on these observations was made at the Marine Biological Laboratory and
an abstract published (Wenrich, 1939). Subsequent studies extended
the survey to other sets of slides showing E. muris from both rats and
mice and similar conditions were then discovered for E. rananiin from
frogs and toads. The present more extended and illustrated report
covers the entire set of observations.
These studies have been made partly at the Marine Biological Lab-
oratory and partly at the University of Pennsylvania. They have been
made entirely on fixed and stained slides. A variety of fixing and
staining agents have been employed in preparing the slides from caecal
and rectal contents but the majority of the smears have been fixed in
Schaudinn's sublimate-alcohol-acetic and stained with Heidenhain's
haematoxylin.
The rats and mice from which slides were made were secured from
a variety of sources but a good many rats were obtained from the
Wistar Institute and to The Institute, and especially to Doctor Helen
Dean King, grateful acknowledgment is made. Acknowledgment is
also made to the staff of the Department of Zoology of the University of
California at Berkeley, for aid in securing frogs and tadpoles and for
facilities for their examination. Most of the observations on Entamocba
ranarum were made on material from Rana pipicns examined at the
University of Pennsylvania.
324
FOOD HABITS OF ENTAMOEBA 325
OBSERVATIONS
Entainocba muris
Entamocba muris lives in the caecum of rats and mice. Of the more
than 500 rats and more than 100 mice that have been examined during
the past twenty-five years, relatively few have had amoebae in sufficient
numbers to warrant detailed study.
The more striking results of this study of the food habits of E. inuris
are: (1) that there is a great diversity in the kinds of objects selected as
food; (2) that some individuals may limit themselves, for a time at least,
to a single type of food with which they may engorge themselves, while
others may engulf a considerable variety of food objects; (3) that cer-
tain populations of amoebae, that is, 'those from a single host rat or
mouse, may show strong tendencies to select one kind of food material,
while in other populations, such tendencies are not manifested ; and (4)
that differentiated ingestion tubes are developed for the ingestion of
some kinds of food.
The food objects fall into two natural divisions or groups ; those of
a plant nature and those of an animal nature. The former group is
represented by a great variety of bacterial organisms, a few of which
are illustrated by Figs. 1 to 4 and 28, yeasts (Fig. 5), Blastocystis
(Fig. 12), plant filaments (Figs. 6, 24-27), all of which are apparently
resident within the caecum; and starch grains (Figs. 7, 13, 15-18)
from the host's diet. Animals are represented by the other Protozoa
resident in the host's caecum and small intestine and by various types
of cells derived from the host. Among the Protozoa are the tricho-
monads (Figs. 11, 14, 29-32), Chilomastix and Hcxamitus pulcher from
the caecum; and Giardia (Figs. 10, 13) and Hexamitus muris which
come down from the small intestine. Host cells found ingested were
erythrocytes (Fig. 8), leucocytes (Figs. 9, 19), and epithelial cells.
Diversity of food preferences among individuals of a population as
well as instances of specialization by individuals are illustrated by Figs.
3, 6, 7, and 10, all from the caecum of one mouse. Figures 15, 17, 18,
20-27 are also from the same population. Preferences by individuals
are illustrated on Plate I, where each amoeba has filled its cytoplasm
with one kind of food. Figures 1 to 7 show ingested plant materials
so that these individuals might be considered to have been " herbivorous,"
at least temporarily. Figures 8 to 11 illustrate individuals which were
" carnivorous " at the time that they were killed, and the amoebae in
Figs. 13 and 14 could be called " omnivorous " since they contain food
objects of both plant and animal nature.
Population food preferences are occasionally noteworthy. The fusi-
326 D. H. WENRICH
form bacillus shown in Fig. 1 is probably the most common food object
seen and many populations show a high percentage of their members
containing this organism. The colonial species seen in Fig. 2 is some-
times given preference by a population. On the set of slides from which
this figure was drawn, about 65 per cent of the amoebae had ingested
one or more of these colonies, most of which, however, were much
smaller than the one shown in Fig. 2 (cf. Fig. 14). Two quite different
types of diplococcoid species are shown in Figs. 3 and 4. These are not
uncommon food inclusions, but it is unusual to see so many of either
kind in any single individual. Many other types of bacteria are found
in the amoebae, but they have not been identified or drawn.
Yeasts are not uncommon food objects, but specialization on yeasts,
as shown in Fig. 5, is uncommon. Several populations were found in
which ingested filaments were more than occasionally seen, although the
proportion of individuals enclosing filaments in any one population was
never more than 2 or 3 per cent. Starch grains were not very commonly
seen, although a number of populations included individuals which had
ingested such grains.
Populations with ingested host cells were uncommon. Epithelial
cells inside amoebae were seen only on a few occasions. Erythrocytes
taken as food were noted in only two populations which were from mice.
In one the number of individuals showing erythrocytes was greater than
in the other, but in both there was a tendency for the same individual to
ingest several red cells, as illustrated by Fig. 8. Ingestion of leucocytes
was also uncommon and the one population in which a number of
PLATE I
Figs. 1-11 showing examples of specialization by individual amoebae. Figs.
3. 6, 7, and 10 are from the same mouse. Figures 8 and 9 are also from mice.
Figures 1, 2, 4, 5, and 11 are from rats.
FIGS. 1-7. Examples of " herbivorous " food preferences.
FIG. 1. Amoeba filled with fusiform bacillus — the most common type of food.
FIG. 2. Amoeba containing a large colonial organism.
FIG. 3. Amoeba containing many small diplococcoid bacteria.
FIG. 4. Amoeba with large diplococcoid species.
FIG. 5. Amoeba with a dozen yeast cells.
FIG. 6. Amoeba with long coiled filament.
FIG. 7. Amoeba containing six starch grains. Note deeply-stained granules
on side of two of them.
FIGS. 8-11. Examples showing "carnivorous" habits.
FIG. 8. Amoeba showing five erythrocytes ; one more was under the nucleus.
FIG. 9. Amoeba with four leucocytes.
FIG. 10. Amoeba with three specimens of Giardia.
FIG. 11. Amoeba with eight specimens of Trichoinonas initris.
FOOD HABITS OF ENTAMOEBA
327
PLATE I
All figures are from fixed and stained slides. They have been drawn with the
aid of a camera lucida at an initial magnification of X 3000 and reduced about one-
third in printing. Figures 1-33 are of Entainoeba tniiris and Figs. 34-42 are of
E. ranarmn.
D. H. WENRICH
amoebae were found with ingested leucocytes was in a mouse. Indi-
vidual preference is illustrated in Fig. 9.
Among the ingested Protozoa, Trichomonas uniris was the most
common. In many populations it was rarely seen as a food object,
while in others it was the population preference. In one count from a
slide from a rat about 80 per cent showed one or more trichomonads in
various stages of digestion. Individual preferences for this flagellate
to the exclusion of other food objects were common in such populations.
Chilomastix- bettencourti, Hcxamiius pulcher and Hexauiitns initris were
seen within the amoebae on only a few occasions. Giardla was seen
more frequently but was not commonly observed. In the population
from a mouse, from which Fig. 10 was taken, between 3 and 4 per cent
showed one to three individuals of this flagellate. In this population
only the trophic stages of Glardia were ingested, although the cysts were
available.
Ingestion Methods. — Entatnoeba imirls apparently adopts somewhat
different methods for the intake of food, depending upon the nature of
the material to be ingested. In the case of starch, it appears from Figs.
15 to 18 that food cups are formed which are just big enough to take
in the granules with no vacuolar space between the food body and the
cytoplasm. The absence of a vacuole around starch grains is also indi-
cated in Figs. 7 and 13. In some cases (Figs. 7 and 13), deeply-stained
bodies are seen in the cytoplasm which is in contact with the starch grain.
These bodies are absent in other cases (Figs. 15-18) and in Fig. 7 only
PLATE II
Figs. 12, 14, from rat. Figs. 13, 15, 17, 18, 20 and 21-33 from one mouse.
Figs. 16 and 19 from another mouse.
FIG. 12. Amoeba with specimen of Blastocystis.
FIG. 13. Amoeba containing two starch grains, a specimen of Giardia and
several bacteria.
FIG. 14. Amoeba containing T. innris, six colonial organisms and two bacilli.
FIG. 15. Amoeba with starch grain half ingested. Note that edge of food
cup and cytoplasmic layer in contact with starch are deeply stained.
FIG. 16. Amoeba containing large starch grain.
FIGS. 17 AND 18. Show ingestion of starch grain almost completed. Note
deeply-stained edges of closing-in pseudopodia.
FIG. 19. Amoeba with two partly ingested leucocytes. Note constriction of
leucocytes.
FIG. 20. Amoeba with empty food cup. Wall of cup composed of denser
cytoplasm but not deeply stained.
FIG. 21. Amoeba with empty food cup. Wall of cup deeply stained.
FIG. 22. Amoeba with food cup turned " wrong-side-out."
FIG. 23. Amoeba with food cup with partly ingested food object and deeply-
stained walls.
FOOD HABITS OF ENTAMOEBA
329
PLATE II
330 D. H. WENRICH
two of the six grains show them. Their nature is problematical, but it
is assumed that they are related to digestion. In a number of cases the
edges of the advancing pseudopodia which were closing in on a starch
grain were deeply stained (Fig. 15) and more especially during the later
stages of the enclosing process (Figs. 17 and 18).
In a number of instances, empty food cups were seen (Figs. 20 and
21) and the walls of these cups were obviously composed of denser
cytoplasm which might (Fig. 21) or might not (Fig. 20) stain in-
tensely. The condition shown in Fig. 22 is interpreted to be a food cup
turned " wrong-side-out." In Fig. 23 a food cup is shown with a par-
tially ingested object and with deeply-stained walls. The middle part
of this tube-like cup is more deeply stained than the rest, suggesting
greater thickness or greater density.
Figure 19 shows a small specimen of E. muris which was fixed while
ingesting simultaneously two leucocytes, one at each side. Constriction
of the leucocytes is indicated, but the edges of the two food cups are
not deeply stained.
Ingestion of filaments was in some cases (Figs. 24 and 27), but not
in others (Figs. 25 and 26), accompanied by the formation of deeply-
stained ingestion tubes in connection with the ingestion cones. In Fig.
25 a filament has been surrounded at a region away from either end.
Ingestion cones were formed and were advancing along the filament in
both directions. In Fig. 27, an especially long, deeply-stained food tube
is shown. From the left end of this tube and proceeding toward the
right, there are three thickenings in the wall of the tube on alternate
sides, suggesting a spiral band of more intensely staining material.
A definite " mouth " or ingestion cone and deeply staining " pharynx "
PLATE III
Figs. 24—27 from same mouse. Fig. 30 from another mouse. Figs. 28, 29,
31-33 from rats.
FIG. 24. Amoeba with long filament partly coiled inside. Note deeply-stained
ingestion cone and " pharynx."
FIGS. 25 AND 26. Amoeba with partly ingested filaments ; ingestion cones not
deeply stained.
FIG. 27. Amoeba with partly ingested filament ; long, deeply-stained " pharynx."
FIG. 28. Differentiated " mouth " and " pharynx " with partly ingested bacillus.
FIG. 29. Amoeba with ingestion of T. muris almost completed. Note deeply-
stained " mouth " followed by undiff erentiated food cavity with deeply-stained con-
striction farther in.
FIG. 30. Amoeba ingesting T. muris through differentiated " pharynx."
FIGS. 31 AND 32. Amoebae with apparently broken ingestion tubes, due to
traumatism. In Fig. 32 the lower tube is all inside and is possibly a constriction
tube.
FIG. 33. Amoeba with internal constriction tube.
FOOD HABITS OF ENTAMOEBA
331
PLATE III
332 D. H. WENRICH
for the ingestion of rod-shaped bacteria are shown in Fig. 28. A num-
ber of narrow tubes of this type were seen containing partly ingested
bacterial rods.
Trichoiiionas is apparently ingested by differentiated tubes. A typi-
cal case is illustrated in Fig. 30 and many variations of this picture have
been seen. In one case the axostyle had been drawn into such a tube
while the remainder of the victim remained outside. In another case
the anterior flagella had been taken in and the prey had descended a
short distance " head first." In still another case the posterior flagellum
had been ingested ahead of the rest of the animal. Apparently the
amoeba is able to " seize " the flagellate at any point on the latter's
surface. In one instance two converging tubes were attached to one
trichomonad. The ingestion tube varies in length, up to more than
half the width of the amoeba. In some cases the diameter varies in
different regions (Figs. 29 and 30). In Figs. 31 and 32 traumatic
fragmentation of ingestion tubes is indicated. The amoeba illustrated
by Fig. 31 showed definite signs of injury. In a few cases trichomonads
were found partly incased in broad food cups, such as shown in Figs. 21
and 23. It is possible that early stages of ingestion may involve such
food cups, to be followed by the gradual development of the differen-
tiated tubes such as seen in Fig. 30.
There is evidence that similar tubes are employed to break up food
masses, as illustrated in Fig. 33. It is possible that in Fig. 32 a com-
bination of an ingestion tube and a constriction tube is indicated.
Entamoeba ranarum
The finding of the conditions just described for Entamoeba muris
led to an examination of smears made from the rectum of frogs and
PLATE IV
All figures of Entamoebae from frogs. Figs. 34, 35, 36, 38, 39, and 40 of
E. rananim from Rana pificns. Figs. 37, 41, 42, of possibly different species, from
California frog, Rana draytonii.
FIG. 34. Amoeba with a dozen specimens of Hexamitus.
FIG. 35. Amoeba with a specimen of Trichomonas augusta.
FIG. 36. Amoeba with four specimens of Cliilomasti.r.
FIG. 37. Amoeba with five host cells.
FIG. 38. Amoeba with partly ingested filament ; two deeply-stajned " pharyn-
geal " regions.
FIG. 39. Amoeba with partly ingested short filaments ; not deeply-stained
" pharynx."
FIG. 40. Amoeba containing one host cell nucleus and a partly ingested second
host cell nucleus.
FIG. 41. Amoeba with empty food cup. (cf. Figs. 20, 21.)
FIG. 42. Amoeba showing constriction of food inside cytoplasm.
FOOD HABITS OF ENTAMOEBA
333
x • V.X. V
PLATE IV
334 D. H. WENRICH
toads. Here similar conditions were observed for E. ranarum. Some
of these conditions are illustrated in Figs. 34-42. Individual speciali-
zation is indicated by Figs. 34—37. The amoeba in Fig. 34 contains
twelve specimens of Hexamitus. The population in this case tended to
favor Hexamitus as a diet, since 17 per cent had ingested one or more
individuals. On the same slide 3 per cent of the amoebae contained
trichomonads. In a count on a slide showing another population, 50
per cent of the amoebae contained Hexamitus as food. The population
on this other slide showed diversity of choice, however, as indicated by
the ingestion of filaments (Figs. 38 and 39) and host cell nuclei (Fig.
40). In another frog, Chilomastix was favored by a considerable num-
ber of the amoebae (Fig. 36).
On slides from the California frog, Rana draytonii, a large majority
of the amoebae contained host cells, apparently leucocytes, although some
may have been erythrocytes (Fig. 37). As many as twelve such cells
were counted in a single amoeba. A more balanced diet was represented
by an amoeba with four host cells and four individuals of the plant,
Blastocystis. (The amoebae from this species of frog have a nuclear
structure considerably different from that typical of E. ranarum, and may
therefore be a different species.) In a similar amoeba from a California
tadpole, a starch grain was seen.
The methods of ingestion employed by these amoebae from frogs
and toads are apparently the same as those employed by E. muris. In
the ingestion of filaments, the formation of ingestion tubes with deeply-
stained annular thickenings is shown in Fig. 38. A short differentiated
" pharynx " is shown in Fig. 39. Figure 41 shows an empty food cup
similar to that seen in E. muris (cf. Figs. 20, 21). Constrictions for
the breaking up of food masses are shown in Fig. 42, where there are
two constrictions being applied simultaneously to a single food body.
An internal constriction tube similar to that shown in Fig. 33 for E.
muris was also seen on the same slide as that from which Fig. 42 was
taken. Altogether, the food habits of E. ranarum are quite parallel to
those of E. muris.
DISCUSSION
Most of the extensive literature dealing with the feeding activities of
amoebae is concerned with free-living species, there being relatively few
reports on the food habits of those that are parasitic. Since the present
study has been limited to fixed and stained specimens, the behavior
aspects must be inferred, and an extensive discussion of amoeboid nutri-
tion would be inappropriate. However, some interesting interpretations
FOOD HABITS OF ENTAMOEBA 335
can be made and their relation to existing literature can be noted. The
following items seem worthy of attention here: (1) the diversity of food
objects ingested; (2) preferences of individuals and populations for
certain kinds of food; (3) methods of ingestion ; (4) the breaking up
of food bodies after their ingestion; and (5) the appearance of secretion
bodies in contact with ingested starch grains.
Diversity of Food Materials Ingested. — Most of those who have
studied Entamoeba muris have remarked upon the variety of food ob-
jects in the cytoplasm of the amoebae. This diversity has been compared
to that frequently mentioned for E. coli from man. In amoebae from
the caecum of mice, Wenyon (1907) noted bacteria of various kinds,
Trichomonas, Giardia, Hexamitus and its cysts, yeast cells, and epithelial
cells. Kessel (1924) noted the inclusion of ChilornastLv and smaller
amoebae besides different kinds of bacteria, and Wang and Nie (1934)
state that ingested food consists mainly of starch grains, intestinal bac-
teria and plant debris. To these lists the present study adds Blasto-
cystis, long plant filaments, and host erythrocytes and leucocytes.
A similar diversity of food inclusions in E. ranarum was noted in
the present study. Dobell (1909) made few comments on the food of
this species but remarked that when blood corpuscles and broken-up
epithelial cells were available in the large intestine the amoebae readily
ingested them. In the present study host cells, not clearly identifiable,
but possibly including both erythrocytes and leucocytes, were conspicuous
food objects in the amoebae from Rana draytonli.
Individual and Population Food Preferences. — The tendency for a
single amoeba to ingest repeatedly the same kind of food object is well
known for E. histolytica, individuals of which may contain as many as
thirty to forty erythrocytes at one time ; and, in cultures, these amoebae
may engorge themselves with starch grains. Frye and Meleney (1936)
noted that in cultures this species varied considerably in its tendency to
ingest erythrocytes, depending upon the conditions in the medium with
which the amoebae were surrounded. Pavlova (1938) has confirmed
some of these results and states further that the capacity of E. histolytica
to ingest red cells depends upon the pH of the medium, the capacity
being greatest at pH values between 5.6 and 6.5. Semenoff (1938) re-
ported that ingestion of erythrocytes did not take place unless the latter
adhered to the surface of the amoebae. It is reasonable to suppose that
E. muris and E. ranarum capture bacteria and active flagellates by an
initial adhesion of the latter to the surface of the amoebae, and that this
adhesion would, in turn, be controlled by various external and internal
factors. One wonders if such factors would be sufficiently limited or
specific in their effects to explain repeated ingestion of one kind of food
336 D. H. WENRICH
body by an individual amoeba. If this were so, then the preference of
a large percentage of some populations for one kind of food might be
similarly explained. However, the divergence of choice commonly
exhibited within a population would indicate that individuals tend to
vary among themselves as to their physiological state. It is doubtful if
population preferences represent racial, that is, genetic, differences, al-
though such a possibility cannot be ignored.
Methods of Ingestion. — Much has been written about the methods by
which amoebae take food into their bodies, but Ivanic (1933) was ap-
parently the first to call particular attention to the formation of a
" cytostome " and accompanying tube sufficiently differentiated to stain
deeply with iron hematoxylin. He first noted such structures in
Amoeba vespertilio, Amoeba iuvenalis and an unnamed species of
Hartmanclla, but extended the observations to Amoeba entzl (1936),
Hartuianclla maasi (1936a) and H. blattac (1937). I have also seen
a deeply-stained (iron hematoxylin) ingestion apparatus in two dif-
ferent small free-living amoebae of the Harlmanclla type, where the
" cytostome " was funnel-shaped, the funnel opening outwardly. On
one slide showing these amoebae nearly every individual displayed from
one to a dozen of these funnels at various points on the periphery.
In E. muris, ingestion methods seem to be much more diversified,
apparently adapted to different kinds of food, but they include the
formation of specialized tubes which stain intensely with iron hema-
toxylin.
In many of his illustrations Ivanic shows, proximal to the " cyto-
stome," capacious vacuoles and speaks of food bodies as being drawn
into them. Some of the conditions seen in the present study would lend
support to this interpretation. In Fig. 19 the concentration of the more
fluid cytoplasm at the inner ends of the partly ingested leucocytes to-
gether with the constriction at the " mouth " suggests suction. Suction
is also suggested in the ingestion of Trichomonas by E. muris; here
various portions, anterior flagella, posterior flagellum, etc., can be iden-
tified as having gone down the " pharynx " in advance of other portions,
and often a rounded globule of trichomonas protoplasm occupies an
internal vacuole while other portions of the flagellate remain outside and
the two parts remain connected through the tube-like " pharynx " (Figs.
29, 30). The enlarged vacuoles into which the short filaments are
entering in Figs. 28 and 39 suggest the same thing. Semenoff (1937,
1938) found that E. histolytica frequently extracted the nuclei from
frog and chick erythrocytes although sometimes ingested fragments
might include some cytoplasm. It is difficult to understand how suction
FOOD HABITS OF ENTAMOEBA 337
can be developed within an amoeboid cell, but the evidence at hand
favors that interpretation.
The ingestion of starch appears to take place by simple extension of
pseudopodia over the food object and in contact with it (Figs. 15, 17,
18). This method resembles that frequently reported for free-living
amoebae during the act of ingesting starch or other solid bodies. Brug
(1928) saw a living specimen of E. histolytica enter a group of starch
grains and emerge two or three minutes later with four larger and two
smaller grains in its cytoplasm, but he did not see the method of intake.
E. inuris and E. ranarum apparently ingest filaments in a manner
similar to that described for free-living species in such classical papers
as those of Leidy (1879) and Rhumbler (1898); and more recently
Comandon and Fonbrune H936), have recorded their observations
with motion pictures. Ivanic (1933) showed that ingestion of filaments
by A. vcspcrtilio is accompanied by deeply stainable thickenings along
the ingestion tube and the present study reveals similar conditions for
E. inuris and E. ranarum (Figs. 24, 27, 38). That a differentiated
tube, such as shown in Fig. 27, is fairly stable — for a time at least-
is indicated by the finding of a similar tube attached to a bent filament
but with the remainder of the amoeba missing — probably having been
torn off during the smearing process. It is probable that Fig. 25 repre-
sents an early stage in the bending of the filament, a process which
might well result in the condition seen in Fig. 27. It is interesting that
Figs. 25 and 26 do not show the deeply-stained walls of the ingestion
tube that are seen in Figs. 24 and 27. It is doubtful if these differences
are the result of variations in the destaining process, since Figs. 24 and
26 were drawn from the same slide. Stainability seems to vary with
density of protoplasm and the density is doubtless correlated with degree
of contraction.
Peristaltic contractions may be indicated by the successive thickenings
on alternate sides of the " pharynx " shown in Fig. 27. Comandon and
Fonbrune (1936), employing motion pictures, record the observation of
waves of contraction along the ingestion cone surrounding a filament in
A. verrucosa. Peristaltic action during ingestion of trichomonads by
E. muris may also be indicated by the differences in diameter of the
" pharynx " shown in Figs. 29 and 30.
The Breaking Up of Food Bodies After Their Ingestion. — The abil-
ity of amoebae to break up food masses into smaller units has been
noted by a number of observers, for example by Leidy (1879) and
Penard (1912). More recently Entz (1925) has provided a good
description of successive constrictions of food objects as seen in Amoeba
vcspcrt ilio ; and, in a later paper (1932) he reviewed the literature show-
D. H. WENRICH
ing instances of the breaking up of food masses in both amoebae and
ciliates, and also in the flagellate, Collodictyon. Ivanic (1936) described
the constriction of ingested food masses, sometimes several such con-
strictions taking place simultaneously; and Mast (1938) reported the
breaking up of ingested Colpidiwn in the cytoplasm of Amoeba proteus.
The present record seems to be the first for the breaking up of food
in a species of Entamoeba and none of the observers referred to above
have reported the presence of deeply-stained constriction tubes, such as
shown in Figs. 33 and 42. It is possible that the deeply-stained tube in
Fig. 33 represents a " pharynx " which has persisted after the prey was
ingested, although the vacuole at each end does not suggest that inter-
pretation ; and such an interpretation would not be applicable to the
condition seen in Fig. 42.
Wenyon (1907) speaks of seeing several specimens of Trichomonas
in a single vacuole in E. muris (see his Fig. 1). On the slides used in
the present study, flagellates, or their fragments, were almost always in
segregated vacuoles. However, large vacuoles, each containing many
bacteria, were sometimes seen and one wonders if fusion of vacuoles
may take place as well as their subdivision. Ivanic, however, believed
that a succession of objects would be taken in through a single
" cytostome."
Digest ire Granules in Contact -with Ingested Starch Grains. — Figure
13 shows a specimen of Entamoeba muris containing two starch grains,
each of which has deeply-stained masses at its periphery. Figure 7
showrs an amoeba with six starch grains and similar stained bodies are
seen at the sides of two of them. That it takes some time for such
bodies to appear is indicated by their absence in Figs. 15 to 18 where
starch grains are being ingested, and also their absence from four of the
six grains in Fig. 7. It seems reasonable to assume that these bodies in
contact with food represent secreted material having a digestive func-
tion. Very similar bodies are shown by MacLennan (1936) for food
bodies in Ichthyophthirius and he identifies them as elements of the
vacuome since they react positively to neutral red and to the Kolatchev-
Nassanov method for impregnation of Golgi material. Volkonsky
(1934) shows similar neutral red staining bodies outside starch grains
ingested by a large granulocyte of Phascolosoma, and also by a choano-
cyte of Clathrina coriacea. In his general review of cytoplasmic inclu-
sions in Protozoa, MacLennan (1941) refers to such bodies as digestive
granules.
Various observers have denied to free-living amoebae the capacity to
digest starch. However, the avidity with which E. liistolytica and other
endamoebae ingest this form of carbohydrate is well established. It
FOOD HABITS OF ENTAMOEBA
should not be surprising therefore, if, as in the other cells referred to,
digestive secretions should be elaborated by such amoebae for the
digestion of starch.
SUMMARY
On the basis of observations on fixed and stained slides showing
Entawoeba nniris and E. ranarum, the following observations and inter-
pretations have been made.
In general, these species of En t amoeba show great diversity in the
kinds of food ingested. E. nntris more commonly feeds on a fusiform
bacillus but its diet includes many other types of bacteria, Blastocystis,
yeasts, plant filaments, starch grains, Trichornonas, Chilomastix, Hexa-
mitus, and host erythrocytes, leucocytes and epithelial cells. E. ranarum
shows a similar range of food objects.
Individuals often select for a time, at least, — a single kind of food,
with which they may engorge themselves. Others are more omnivorous
in their selection.
Populations from a single host may show decided preferences for
one type of food ; for example, about 80 per cent of one population of
E. muris contained one or more specimens of Trichomonas.
A diversity of methods of ingestion is indicated. Starch grains are
surrounded by enveloping pseudopodia without the formation of a
fluid-containing vacuole around them. Trichomonads appear to be
drawn through an ingestion tube with walls sufficiently differentiated to
stain heavily with iron hematoxylin. Plant filaments are taken in
through similar tubes some of which show the deeply-stained walls.
There is evidence that differentiated tubes are employed to constrict food
bodies into smaller units.
Bodies which stain with iron hematoxylin have been seen in contact
with ingested starch grains in E. muris. These are interpreted as diges-
tive granules in the sense that this term is used in the review by
MacLennan (1941).
LITERATURE CITED
BRUG, S. L., 1928. Observations on a culture of Entamoeba histolytica. Med.
Dicnst. Volksgcz. Ned.-Indic., 17: 225-233.
COMANDOX, J., AXD P. DE FOXBRUXE, 1936. Mecanisme de 1'ingestion d'Oscillaires
par des Amibes. Enregistrement cinematographique. Cotnpt. Rend. Soc.
BioL, 123: 1170-1172.
DOBELL, C. C., 1909. Researches on the intestinal Protozoa of frogs and toads.
Quart. Jour. Mic. Sci., 53: 201-277.
EXTZ, G., 1925. Uber Xahrungszerkleinerung im Plasma einer Amoebe. (Amoeba
vespertilio Penard). Zool. Anz., 63: 332-336.
340 D. H. WENRICH
, 1932. Bemerkungen iiber Nahrungszerkleinerung im Plasma einiger Proto-
zoen. Arch. Zoo/. Ital, 16: 967-977.
FRYE, W. W., AND H. E. MELENEY, 1936. The effect of various suspending media
on the pathogenic and phagocytic activity of Endamoeba histolytica.
Am. Jour. Hyg., 24: 414-422.
IVANIC, M., 1933. Uber die bei den Nahrungsaufnahme einiger Siisswasser-
amoben vorkommende Bildung cytostomahnlicher Gebilde. Arch. Protist.,
79: 200-233.
— , 1936. (iber die mittels cytostomahnlicher Gebilde vorkommende Gefangen-
nahme und Einverleibung der Nahrung und deren Zerkleinerung bei einer
Siisswasseramoebe (Amoeba entzi sp. nov.). La Cellule 44: 369-386.
, 1936a. Recherches nouvelles sur 1'ingestion des aliments au moyen de
cytostomes chez quelques amibes d'eau douce. (Amoeba vesperitilio
Penard et Hartmanella maasi Ivanic). La Cellule, 45: 179-206.
— , 1937. Korperbau, Ernahrung und Vermehrung einer im Enddarme der
Kiichenschabe [Blatta (Periplaneta, Stylopj'ga) orientalis L.] lebenden
Hartmanella Art (Hartmanella blattae spec. nov.). Arch. Protist., 88:
339-352.
KESSEL, ]. F., 1924. The distinguishing characteristics of the parasitic amoebae of
culture rats and mice. Univ. of Calif. Pnhl. in Zoo/., 20 : 489-544.
LEIDY, ]., 1879. Freshwater rhizopods of North America. Washington, D. C.
MACLENNAN, R. F., 1936. Dedifferentiation and ^differentiation in Ichthyoph-
thirius. II. The origin and function of cytoplasmic granules. Arch.
Protist., 86 : 404-426.
— , 1941. Cytoplasmic inclusions. Chapter III in: Protozoa in Biological Re-
search. Columbia University Press. New York.
MAST, S. O., 1938. Digestion of fat in Amoeba proteus. Biol. Bull., 75: 389-394.
PAVLOVA, E. A., 1938. A propos de quelques facteurs agissant sur la phagocytose
des erythrocytes de 1'Entamoeba histolytica en Culture. (Russian with
French summary.) Med. Parasitol. et Parasit. Dis. Moscow., 7: 119-122.
PENARD, E., 1912. Nouvelles Recherches sur les Amibes du groupe Terricola.
Arch. Protist., 28 : 78-140.
RHUMBLER, L., 1898. Physikalische Analyse von Lebenserscheinungen der Zelle.
Arch. Entii'.-mcch., 7: 103-350.
SEMENOFF, W. E., 1937. Phases of phagocytosis in Entamoeba histolytica. Bull.
Biol, Med. E.rp. URSS., 4 : 192-194.
— . 1938. Further contribution to the study of phagocytosis in Entamoeba histo-
lytica (Schaudinn 1903). Bull. Biol., Med. E.rp. URSS., 5: 186-188.
VOLKONSKY, M., 1934. L'aspect cytologique de la digestion intracellulaire. Arch.
exp. ZcUforsch., 15: 355-372.
WANG, C. C., AND D. NIE, 1934. Notes on Entamoeba muris (Grassi) and Tri-
chomonas caviae Davaine. Proc. Fifth Pan-Pacific Sci. Cong., 4 : 2991-
2993.
WENRICH, D. H., 1939. Food habits of Entamoeba muris. Biol. Bull., 77 : 313-
314.
— , 1940. Nuclear structure and nuclear division in the trophic stages of Ent-
amoeba muris (Protozoa, Sarcodina). Jour. Morph., 66: 215-239.
WENYON, C. M., 1907. Observations on the Protozoa in the intestine of mice.
Arch. Protist., Sup pi, 1: 169-201.
STUDIES ON THE GROWTH OF INTEGUMENTARY
PIGMENT IN THE LOWER VERTEBRATES
I. THE ORIGIN OF ARTIFICIALLY DEVELOPED MELANOPHORES ON THE
NORMALLY UNPIGMENTED VENTRAL SURFACE OF THE
SUMMER FLOUNDER (PARALICHTHYS DENTATUS) l
CLINTON M. OSBORN
(From the Department of Anatomy, the Ohio State University, and the Woods
Hole Oceanographic Institution, Woods Hole, Mass.)
Considerable evidence has accumulated to indicate that melanophores
may be grown experimentally on certain fishes and amphibians in areas
where these cells fail to develop naturally. Cunningham (1891, 1893,
and 1895), working with several species of flatfishes; von Frisch (1911 ),
using Eso.v and Ncmadiilus; and Osborn (1940a, b, and c), studying the
summer flounder (ParalicJitliys dcntatus) and the common bullhead
(Ameiurus melas) have all reported success in growing melanophores
on the normally unpigmented ventral 2 surfaces of these teleosts. Ex-
perimenting with the urodele, Salamandra maculosa, Herbst and Ascher
(1927) were able to develop abnormal amounts of pigment ventrally.
In spite of these observations the origin of the newly developed melano-
phores has remained an open question. Alternative possibilities are
obvious : either they differentiate in situ or they migrate in from other
areas.
This paper brings together the results of experiments referred to in
an earlier report (Osborn, 1940a) designed to gain more information
concerning the source of experimentally developed melanophores.
MATERIALS AND METHODS
A freshly caught stock of adult flounders 15 to 18 inches long was
maintained to avoid abnormal conditions in pigmentation which some-
times arise from prolonged sojourns in unnatural laboratory surround-
ings. The desired amount of pigmentation was developed ventrally in
1 Contribution No. 296 of the Woods Hole Oceanographic Institution, whose
research facilities and financial aid provided for this investigation are genuinely
appreciated.
2 The term " ventral " will be used to refer to the lower normally unpigmented
surface of the animal. In the flatfishes the unpigmented side is more strictly the
right or the left side, depending upon the species. In the summer flounder the
right side is white.
341
LI
342 CLINTON M. OSBORN
an apparatus similar to that previously described (Osborn, 1940a) with
minor improvements. When a fish was sacrificed, at least 40 scales
plucked from widely separated areas on the ventral surface were fixed
in 5 per cent neutral formalin. Of these, ten were dehydrated in
alcohol, cleared in xylol and mounted in Clarite ; ten were mounted in
glycerine jelly directly following fixation ; and 20 were treated according
to Laidlaw's modification (1932a) of Bloch's (1917) " Dopa " 3 reaction.
These preparations were finally mounted in balsam. All scales were
studied by both transmitted and reflected light and photographic records
made. Appropriate control preparations were reserved for each condi-
tion. The ventral surface was carefully examined for pigmentation
with a dissecting microscope before each animal was sacrificed and daily
observations were made during longer experiments.
EXPERIMENTAL
In connection with studies previously reported (Osborn, 1940a), it
was observed that experimentally developed melanophores appeared in
random positions and patterns on the ventral surface. Pigmented spots
of macroscopic size and irregular in shape, differing in intensity from
gray to black, appeared here and there over any part of the ventral
surface. The only position where melanophores developed with con-
siderable regularity was at the base of the tail. In this area normal
control fishes also usually possess some pigment, probably because con-
siderable light reaches this narrow region where the surface is somewhat
rounded and unprotected by fins.
The " Dopa " Reaction
This reaction first described by Bloch (1917) and later modified by
Laidlaw (1932a) has been used for the identification of melanoblasts.
Although these cells contain no melanin pigment, Bloch observed the
formation of a black substance which he called " dopa-melanin " when
treated with " Dopa." He believed this was due to an oxidizing fer-
ment, dopa-oxidase, in the cell which reacted with the " Dopa." Thus,
many investigators have interpreted the ' Dopa-positive " cell as a
potential melanophore even though it had not yet differentiated.
In order to test ventral scales for the presence of melanoblasts by
the "Dopa" technique, three groups of flounders (12 fishes in each
group) were chosen. The first group was black-adapted, then totally
blinded and finally illuminated ventrally to insure optimum conditions
3 Throughout this paper the term " Dopa " will be used to refer to 3-4 dihy-
droxyphenylalanin (levorotatory) ..
ORIGIN OF MELANOPHORES 343
for rapid growth of melanophores (Osborn, 1939, 1940a). This treat-
ment was continued until considerable ventral pigment had developed
(Fig. 6). When a random sampling of ventral scales from such a fish
was studied it became apparent that all degrees of pigmentation (melano-
genesis) were represented by the various scales (Figs. 8, 9, and 10).
In some scales there were no melanophores, in others the small number
of melanophores had only scattered melanin granules while still others
were melaninated so heavily as to be indistinguishable from scales plucked
from the dorsal surface. Such scales arranged in a progressive series
show all stages in the acquisition of a full complement of melanin in
melanophores, suggesting that the process of pigmentation occurs in the
cells in situ as they differentiate on a particular scale. Furthermore,
there appears to be no tendency for scales adjacent to naturally pig-
mented areas (the edges of the fins etc.) to become pigmented first with
subsequent spreading from originally pigmented surfaces. Rather,
melanophores suddenly appear containing a few pigment granules quite
independently of neighboring cells. In an attempt to obtain more than
circumstantial evidence on this point some scales possessing no melanin-
containing cells (microscopic examination — Fig. 2) and others con-
taining but few young melanophores (the exact number and their posi-
tion on the scale recorded in each instance) were subjected to the
" Dopa " treatment. An average of 14 out of 20 scales from each of
the 12 fishes gave a positive "Dopa" test (Fig. 3).4 In some ventral
scales positive cells were as numerous as the melanophores on dorsal
scales while in other instances only scattered cells responded positively.
An entirely satisfactory explanation for the failure of some scales to
react positively cannot be given. Two possibilities are suggested : either
these scales possessed no melanoblasts, as is apparently the case in
scales occasionally found on the dorsal surface, or the technique may
not be entirely dependable even though precautions were taken that
the solutions were fresh and the incubation temperature accurately con-
trolled.
The flounders in group 2 were illuminated ventrally for a shorter
period (4 to 10 days), only until the first appearance (macroscopic)
of partially pigmented scales here and there over the surface. For the
" Dopa " test scales were chosen which possessed no melanin-containing
cells or but few melanophores (again carefully recorded). In this group
an average of 16 out of 20 scales per animal responded positively. The
range of variation was wide as in the first group.
4 It is of interest to note that these " Dopa " positive cells appear similar to
the round melanoblasts pictured by Laidlaw (1932/7; Fig. 5, Plate 84; and Fig. 8,
Plate 85) in human skin.
344 CLINTON M. OSBORN
The third set of flounders was not subjected to ventral illumination
or any other laboratory conditions. They were used immediately with-
out allowing time for adaptation to any unnatural background. Ade-
quate scales were plucked from the ventral surface for each of the three
types of preparations previously listed and routine " Dopa " tests were
run. None of the cells of the scales used possessed microscopically
detectable melanin granules. An average of 13 out of 20 scales from
each fish reacted positively to " Dopa." Again the range of variation
in the number of positive cells from scale to scale was wide. It is of
considerable interest, however, that flounders taken directly from nature
should possess numerous potential melanophores on a surface so free
from melanin.
Observations Using Transmitted Light
A brief summary follows for the microscopic observations of ventral
scales studied by transmitted light. Some were mounted in glycerine
jelly to preserve the alcohol-soluble pigments ; others were mounted in
Clarite following dehydration and xylol clearing.
In glycerine mounts the numerous leucophores appear slightly opaque
(Fig. 2) because of their content of guanin crystals and may be easily
recognized by their relative numbers, irregular (dendritic) shape and
their size. Other cells, less numerous, flattened, and smoother in con-
tour, almost round (in fact having the same shape as those which reacted
positively to "Dopa"), could be seen scattered among the leucophores.
They are believed to be melanoblasts and are best seen when the iris
diaphragm is partly closed. Young melanophores, containing few mel-
anin granules, are of much the same appearance but usually are given
a slightly more irregular form by the extensions of simple processes.
In studying scales arranged in series progressing from those having no
melanophores to scales possessing numerous melanin-containing cells
there appears to be a direct correlation between the increase of melanin
contained in the cell and the complexity of the processes. A coincidence
observed so regularly that it should not be overlooked was that wherever
several melanophores were growing in a group the absence of leuco-
phores in that spot was strikingly obvious (Figs. 9 and 10). Viewed
with reflected light this was even more easily seen. This suggests that
in some way a substitution of melanophores for leucophores may take
place or that conditions in the tissues favoring the generation of new
melanophores may also be responsible for the degeneration of leuco-
phores. Can it be that leucophores change into melanophores? The
very existence on the dorsal surface of structures which apparently
ORIGIN OF MELANOPHORES 345
contain both melanin and reflecting material (probably guanin) is evi-
dence supporting the idea that two pigments may occur within a single
cell (melanoleucophore — Figs. 4 and 5).
It was noted also that in scales possessing many melanophores the
cells appeared to be larger and more complex with more numerous,
irregular processes in contrast with other scales which had perhaps a
half dozen or less melanophores usually of uniform small size and
simple pattern, apparently less highly differentiated. To a certain ex-
tent the melanophores on a particular scale tend to differentiate more
or less synchronously. The way in which experimental pigmentation
first appears on the ventral surface of the flounder seems to be in har-
mony with this and with the evidence gained in the " Dopa " tests. In
addition, occasional cells containing some yellow pigment (xantho-
phores) were seen.
Clarite mounts showed essentially the same picture except that no
xanthophores were detected. The leucophores were much more trans-
parent but could be recognized by reducing the light. The smaller
round cells were also visible.
Observations with Reflected Light
Glycerine mounts viewed with reflected light showed the leucophores
in clear relief but to the disadvantage of the other cells present. How-
ever, in cases where some melanophores had developed among the leuco-
phores, the negative outline of the melanin-containing cells could be
followed, aided somewhat by the absence of leucophores at that site (see
previous page and Figs. 9 and 10). Now and then xanthophores were
observed by reflected light.
The scales cleared and mounted in Clarite were less instructive when
viewed with reflected light. Because their relative transparency reduced
the clarity of the reflected image, they supplied little additional informa-
tion.
Observations Concerning Regenerating Scales
In areas of injury on the ventral surface where scales had been
scraped away, the newly regenerated ones appeared darkly melaninated
if the fish was maintained in a physiological and experimental condition
favorable to the development of ventral pigment. Such scales are black
with melanophores as they appear (Fig. 7). However, if the injured
flounders are white-adapted or on a pale natural background with nor-
mally alternating night and day (not excessive illumination), the re-
generating ventral scales will be white with leucophores and possess no
346 CLINTON M. OSBORN
PLATE I
EXPLANATION OF FIGURES
FIG. 1. White ventral surface of a freshly caught summer flounder. Note
that the scales are normally covered with leucophores (containing guanin) but
that melanophores fail to develop on this surface. About % natural size.
FIG. 2. Photomicrograph of a scale (mounted in glycerine) plucked from the
ventral surface of a normal summer flounder. This scale possessed no melano-
phores. The numerous gray-appearing cells are leucophores which appear slightly
opaque when photographed with transmitted light. About 20 X.
FIG. 3. Photomicrograph of a scale which reacted positively to the " Dopa "
treatment. The densely opaque cells which have deposited dopa-melanin are inter-
preted to be melanoblasts. Before treatment this scale appeared similar to that
in Fig. 2. About 20 X .
FIG. 4. Photomicrograph (transmitted light) of a small area of the tip of a
scale plucked from the center of a white " excitation spot " on the dorsal surface
of a black -adapted flounder. The melanophores are numerous even in this white
area but are only slightly dispersed and well concealed by guanin crystals, as will
be seen in Fig. 5 taken with reflected light. Cell " x " is the same structure marked
for purposes of orientation in Figs. 4 and 5. About 100 X.
FIG. 5. The same area as shown in Fig. 4. This photograph was made with
reflected light, however, Note that the total area appears relatively white as it
would on the fish in reflected light even though the scale is heavily melaninated.
The reflecting guanin appears to be within the bounds of the melanophores because
the cells retain a constant size and shape when viewed by reflected and transmitted
light. Such a structure is referred to as a " melanoleucophore." Compare Figs.
4 and 5 cell for cell. About 100 X.
FIG. 6. Ventral view of summer flounder blinded immediately after capture
and continuously illuminated ventrally (direct light). Although this fish was
illuminated only 18 days, its melanination is nearly as extensive as on the flounder
shown in Fig. 7. This is due to the greater efficiency of direct illumination. One-
sixth natural size.
FIG. 7. An area of ventral surface adjacent to the pectoral fin. The flounder
was black-adapted, blinded, and illuminated continuously 74 days in a white tank.
Widespread melanophore formation has occurred but pigmentation is blackest where
regenerated scales have grown in an injured area from which the scales had been
scraped. One-third natural size.
FIG. 8. Photomicrograph of the tip of a dorsal scale plucked from one of the
white " excitation spots " of a black-adapted flounder. The numerous melano-
phores are only slightly distended during excitation despite the fact that the fish
had been black-adapted several days. Scales possessing comparable melanination
are commonly found in the darker pigmented areas ventrally. About 50 X.
FIG. 9. Photomicrograph of part of a scale plucked from a lightly melaninated
area of the ventral surface of a summer flounder black-adapted 7 days, blinded and
placed in a white tank constantly illuminated from overhead for 12 days. The
photograph was taken with transmitted light. The few young melanophores pres-
ent appear in distinct contrast to the numerous leucophores which cover nearly the
entire scale surface. Note that the leucophores are absent from the newly mel-
aninated area. Melanophore " a " serves as a point of reference and orientation in
Figs. 9 and 10. About 40 X.
FIG. 10. Same area shown in Fig. 9. Photographed with reflected light, the
numerous leucophores appear white as on the lower surface of the normal flounder.
They contain no melanin and are true leucophores. The melanophores are visible
only because they reflect the least light and appear black in contrast with the rest
of the scale surface. Compare Figs. 9 and 10. About 40 X.
ORIGIN OF MELANOPHORES
347
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PLATE I
348 CLINTON M. OSBORN
melanophores. On the other hand, if the dark flounders are injured,
then placed immediately in the apparatus providing ventral illumination,
the scales which regenerate appear as some of the darkest on the entire-
ventral surface. Although the reason for this is not definitely known,
it may be that the numerous leucophores on the white scales tend to
mask the developing melanophores during their early stages. The
prompt appearance of melanin-containing cells in areas of repair far
removed from normally pigmented regions lends further support to the
/';/ situ origin of ventral melanophores and makes the possibility of mi-
gration from previously pigmented areas seem very improbable.
DISCUSSION
That melanophores may be grown under proper experimental con-
ditions on surfaces naturally white and non-melaninated is now well
established. The conditions favoring such growth are also known (Os-
born, 1939, 1940(/, b, and c) although many details await further inves-
tigations. The experimental requirements are two-fold: (1) That the
surface in question must receive light either directly or by reflection and
(2) that the physiological condition (nervous and hormonal factors) of
the fish must be such that the internal environment of the normally pig-
mented cells favors dispersion r> of the melanin granules (physiological
darkening). Odiorne (1937) concluded that this condition also favored
the slower morphological darkening in Fiindiilus. Similar findings have
been reported in the lower vertebrates by Vilter (1931), Sumner and
Wells (1933). Sumner and Fox (1935), Sumner and Doudoroff (1937),
Sumner (1939, 1940</ and b), Osborn (1939 and 1940<-), and Dawes
(1941).
The possible source of experimentally developed melanophores at-
tracted the attention of Cunningham (1893), who saw no pigment mi-
grating from the upper surface and so from negative evidence concluded
the cells developed in situ.
The positive evidence presented here although partly circumstantial
supports the view that these melanophores develop where they are first
observed. The evidence gained from the direct observation (using both
transmitted and reflected light) of small cells whose appearance and
distribution agree in general with the picture seen in positive " Dopa "
preparations requires no further comment. Likewise the observations
concerned with the melanination of regenerated scales strongly favor
5 That the degree of dispersion need not be maximal is shown by the fact that
considerable ventral pigment may be grown on an animal whose dorsal surface is
intermediate in shade. It is important to emphasize that the fish should not be in
the pale phase.
ORIGIN OF MELANOPHORES 349
the in situ origin of these melanophores. Furthermore, the fact that
evidence gained in using the " Dopa " reaction as an indicator fits well
into the other findings suggests that in this instance the reaction is
significant and reliable. It is fully appreciated, however, that a positive
" Dopa " reaction because of its non-specificity may in itself be of
limited value or may prove to be misleading unless supported by evidence
from other sources. In these experiments the " Dopa " reaction was
used only to test for the presence of positive cells, thus avoiding the
necessity of interpreting the implications of the chemical reaction. Be-
cause this problem is highly controversial at present some further dis-
cussion may be appropriate. That the positive " Dopa " test need not
necessarily indicate the presence of a specific oxidizing enzyme (" dopa "
oxidase) in the cell has been suggested by Figge (1940), who found
that "" Dopa " would react with other substances under test tube condi-
tions to produce a black deposit. However, a characteristic feature of
a melanoblast is that it contains some substance which will cause " Dopa "
to react, forming a black material. This may be formed by a substance
which oxidizes the " Dopa " or which acts as a redox substance to
accelerate the auto-oxidation of "Dopa" in the absence of an enzyme.
This latter possibility seems rather unlikely.
Another possibility is that the cells believed to be melanoblasts on
the ventral scales may contain tyrosinase which for some reason has
failed to react with tyrosine to produce pigmentation. Figge (1940)
suggested that a positive " Dopa " reaction might indicate the presence
in a cell of tyrosinase whose oxidizing action was inhibited by a gluta-
thione-like substance. Such substances are known to inhibit the action
of tyrosinase on tyrosine but do not inhibit the action of tyrosinase on
" Dopa " (Figge, 1940). Tyrosinase actually blackens "Dopa" faster
than tyrosine. It is seen then, that the observations made can be
explained on a theoretical basis, although we do not, of course, know
precisely what happens in the cell.
The question may be asked: "If the melanoblasts are present on the
ventral surface, why do they not finish differentiation normally by
manufacturing pigment ? ' It has been demonstrated that they do
differentiate to true melanin-containing melanophores when the proper
conditions are supplied. One might argue that the internal environment
is the same in the ventral cells as in the dorsal cells and that the external
environment differs only with regard to the amount of light which nor-
mally reaches the upper and lower surfaces of the flounder respectively.
If this assumption is true, the following speculation may be offered.
Is it possible that in the potential melanophores of the ventral scales a
350 t CLINTON M. OSBORN
tyrosine-tyrosinase reaction is inhibited by a glutathione-like reducing
agent in tbe absence of light and that exposure to light (experimentally)
might remove the inhibiting effect of the reducing agent and allow the
enzyme to oxidize the color substrate? That the above assumption is
not entirely true, however, is suggested by other observations. The in-
ternal environments for the cells of the dorsal and ventral scales are
presumably alike in their hormonal constituents but not necessarily so
in regard to their respective innervations. This is not known. Pouchet
(1876) suggested, however, that a partial atrophy of the sympathetic
system may accompany the migration of the corresponding eye during
metamorphosis. In view of the present findings concerning ventral
pigmentation, further experiments designed to provide new information
on the possibility of the degeneration of sympathetic fibers to the ventral
surface are needed.
SUMMARY
Melanophores differentiate on the normally non-melaninated ventral
surface of summer flounders when two conditions are satisfied. (1)
The surface must be exposed to some light source when (2) the animal
is in a physiological condition favoring darkening as witnessed by the
behavior of the dorsal melanophores.
The melanophores develop " in situ " from potential melanophores
(melanoblasts) whose presence is evidenced by the positive " Dopa "
reaction, by direct observations of various stages of differentiation using
direct and reflected light, by studies on regenerating scales, and by
additional physiological data.
Theoretical considerations of the possible reactions involved in the
experimental development of ventral melanophores and speculations as
to why they are normally absent from the ventral surface are presented.
LITERATURE CITED
BLOCK, B., 1917. Chemische Untersuchungen iiber das spezifische pigmentbildende
Ferment der Haut, die Dopaoxydase. Zcitschr. f. physiol. Chem., 98 :
226-254.
CUNNINGHAM, J. T., 1891. An experiment concerning the absence of color from
the lower sides of flat-fishes. Zool. Anzeigcr, 14: 27-32.
CUNNINGHAM, J. T., 1893. Researches on the coloration of the skins of flat fishes.
Jour. Mar. Biol. Assoc., 3 (N.S.) : 111-118.
CUNNINGHAM, J. T., 1895. Additional evidence on the influence of light in pro-
ducing pigments on the lower sides of flat fishes. Jour. Mar. Biol. Assoc.,
4: 53-59.
DAWES, B., 1941. The melanin content of the skin of Rana temporaria under
normal conditions and after prolonged light- and dark-adaptation. A pho-
tometric study. Jour. Expcr. Biol., 18 : 26-49.
ORIGIN OF MELANOPHORES 351
FIGGE, F. H. J., 1940. The significance of the dopa reaction in pigment metabolism
studies. Anat. Rec., Suppl, 78: 80 (abstract 91).
VON FRISCH, K., 1911. Beitrage zur Physiologic der Pigmentzellen in der Fisch-
haut. Arch. gcs. Physiol., 138: 319-387.
HERBST, C., AND F. ASCHER, 1927. Beitrage zur Entwicklungsphysiologie der
Farbung und Zeichnung der Tiere. III. Der Einfluss der Beleuchtung
von unten auf das Farbkleid des Feuersalamanders. Arch. Entiv.-mech.
Organ., 112: 1-59.
LAIDLAW, G. F., 1932a. The dopa reaction in normal histology. Anat. Rec., 53:
399-413.
LAIDLAW, G. F., 19326. Melanoma studies. I. The dopa reaction in general
pathology. Am. Jour. Path., 8: 477-491.
ODIORNE, J. M., 1937. Morphological color changes in fishes. Jour. Expcr. Zool.,
76 : 441^65.
OSBORN, C. M., 1939. The physiology of color change in flatfishes. Jour. Expcr.
Zool., 81 : 479-515.
OSBORN, C. M., 1940a. The experimental production of melanin pigment on the
lower surface of summer flounders (Paralichthys dentatus). Proc. Nat.
Acad. Set., 26: 155-161.
OSBORN, C. M., 19406. Studies on the origin and behavior of melanophores experi-
mentally grown on the ventral surface of the summer flounder (Para-
lichthys dentatus). Anat. Rec., Suppl., 78: 70 (abstract 69).
OSBORN, C. M., 1940c. The growth of melanophores on the normally unpigmented
surface of the black catfish, Ameiurus melas. Anat. Rec., Suppl., 78: 167
(abstract 301).
POUCHET, G., 1876. Des changements de coloration sous 1'influence des nerfs.
Jour. Anat. et Physiol., 12: 1-90, 113-165.
SUMNER, F. B., 1939. Quantitative effects of visual stimuli upon pigmentation.
Am. Nat., 73: 219-234.
SUMNER, F. B., 1940a. Further experiments on the relations between optic stimuli
and the increase or decrease of pigment in fishes. Jour. Exper. Zool., 83 :
327-343.
SUMNER, F. B., 19406. Quantitative changes in pigmentation, resulting from visual
stimuli in fishes and amphibia. Biol. Rev., 15 : 351-375.
SUMNER, F. B., AND P. DOUDOROFF, 1937. Some quantitative relations between
visual stimuli and the production or destruction of melanin in fishes.
Proc. Nat. Acad. Set., 23: 211-219.
SUMNER, F. B., AND D. L. Fox, 1935. Studies of carotenoid pigments in fishes.
II. Investigations of the effects of colored backgrounds and of ingested
carotenoids on the xanthophyll content of Girella nigricans. Jour. Exper.
Zool, 71 : 101-123.
SUMNER, F. B., AND N. A. WELLS, 1933. The effects of optic stimuli upon the
formation and destruction of melanin pigment in fishes. Jour. Expcr.
Zool., 64 : 377-403.
VILTER, V., 1931. Modifications du systeme melanique chez les Axolotls soumis a
1'action de fonds blancs ou noirs. Compt. Rend. Soc. Biol., 108: 774-778.
STUDIES ON THE GROWTH OF INTEGUMENTARY
PIGMENT IN THE LOWER VERTEBRATES
II. THE ROLE OF THE HYPOPHYSIS IN MELANOGENESIS IN THE
COMMON CATFISH (AMEIURUS MELAS) x
CLINTON M. OSBORN
(From the Department of Anatomy, The Ohio State University}
In previous communications (Osborn, 1940a, b, and r) it was shown
that smooth-skinned as well as scaly teleosts responded to experimental
procedures by developing melanophores on their normally unpigmented
surface. These findings confirm and extend the earlier works of Cun-
ningham (1891, 1893) ; von Frisch (1911) ; Herbst and Ascher (1927)
and others more completely listed in another paper (Osborn, 1941, in
press).
In several experiments concerned with the experimental growth of
melanophores it has been observed consistently that these cells developed
most rapidly and abundantly when the fish was in a physiological condi-
tion which caused pigment granules in the normally existing melano-
phores to be maximally dispersed (Osborn, 1940a and b and 1941 in
press). Odiorne (1937) suggested that " Any condition leading to the
dispersion of pigment throughout the cell will, if maintained, promote the
development of melanophores or insure their continued existence " ; and
further wrote that " The neurohumors which are instrumental in bring-
ing about the pigmentary migrations in Fundulus also exert trophic
influences upon the melanophores."
In the catfish it has been found that melanophores can be grown
experimentally on the naturally white belly of the animal by directing
light upon it while the pigmented dorsal surface is in the dark phase
(Osborn, 1940c). If the fish is white-adapted, however, ventral il-
lumination does not result in melanophore formation. It was previously
shown (Osborn, 1938) that the melanophore-dispersing principle of the
hypophysis plays a major role in producing the dark phase (physiological
darkening) in the natural color changes of the animal. By removing the
source of this secretion but maintaining constant all other conditions
favoring the growth of melanophores on the white belly surface, it
1 It is a pleasure to acknowledge that this investigation was aided in part by a
grant from the Elizabeth Thompson Science Fund.
352
ROLE OF HYPOPHYSIS IN MELANOGENESIS 353
should be possible to determine whether a substance necessary in physio-
logical darkening was also essential in the production of melanophores
(morphological darkening). In the experiments to be reported here the
fact that melanophores failed, to grow experimentally in hypophysec-
tomized catfishes indicates that a substance necessary to produce the
dark phase is also indispensable to the experimental development of
melanophores. This strongly suggests that morphological color change
is not the result of physiological color change but rather that both are
the product of a common underlying mechanism which effects the former
change more slowly than the latter.
MATERIALS AND METHODS
Common catfishes (Ameiurus melas) six to eight inches in length
were kindly furnished me by Dr. T. H. Langlois, director of the Franz
Theodore Stone Laboratory, Put-In-Bay, Ohio.2 The laboratory stock
was kept in muddy water at 12 to 18° C. in large gray tanks in an animal
room where the illumination was of low intensity and darkness was
maintained at night. Under such circumstances fishes have been kept
over a year in excellent condition and have maintained normal pigmenta-
tion. The experimental fishes were kept in water at 10 to 12° C. during
the first post-operative week and henceforth the temperature was main-
tained between 14 and 18° C. At this temperature they took food
regularly : rolled oats daily and bits of liver or ground beef about once
a \veek.
Illumination was directed to the ventral surface of the experimental
fishes either by specially constructed glass-bottomed tubs with ceiling and
sides black or white (Osborn, 1940a) or by reflection from white tubs
brightly illuminated from above. Both of these light sources have been
used successfully in growing ventral melanophores. In our apparatus
illumination by reflection grows pigment less rapidly, however, because
of the lower intensity of the light actually falling upon the lower
surface of the fish.
The fishes, after having been lightly anesthetized in a dilute chlore-
tone solution or stupefied by chilling, were totally blinded by enucleation
and were hypophysectomized by the oral approach. Hypophysectomies
were checked for completeness by reconstructions at the time of operat-
ing, by observing the post-operative color changes displayed by each fish
and by examination at autopsy. When for any reason the operation
was considered imperfect the data for that animal were discarded.
Some of the fishes were sacrificed at convenient intervals for micro-
scopic study, others for chemical determinations. In almost all cases
2 Courtesy of Mr. John Sullivan, Ohio Conservation Department.
354 CLINTON M. OSBORN
TABLE I
Animals alive 30 days after beginning
of experiment
Number
Percentage of
original
Group A
Group B
Group C
Group D
43
29
16
15
71.7
97.0
72.7
100.0
photographs were taken of living fishes but in certain instances additional
records of preserved animals were made.
EXPERIMENTAL
In these experiments over a hundred catfishes were used representing
four different physiological or operative conditions as follows :
Group A — 60 fishes — totally blinded ; hypophysectomized 12 hrs. later.
Group B — 30 fishes — totally blinded.
Group C — 22 fishes — hypophysectomized only.
Group D — 15 fishes — unoperated controls.
Animals from each of the above groups were placed in each of five
experimental tubs : four providing continuous direct ventral illumination
(apparatus only slightly modified from that previously described, Osborn
1940a) and one having a white bottom which reflected light to the
belly of the fishes. By having representative fishes from each of the
groups in every tub, any possible effects of slight differences in tempera-
ture, light intensity, feeding, etc. were automatically ruled out. The
PLATE I
EXPLANATION OF FIGURES
Figures 1 and 2 are ventral views of two fishes described below (about %
natural size). Figures 3 and 4 are lateral views of the same two fishes.
FIGS. 1 AND 3. A common catfish (Ameiurus melas) blinded and continuously
illuminated ventrally by light reflected from the white bottom of the tub in which
this experimental fish was kept for 125 days. Note dark shade and excessive
ventral melanination.
FIGS. 2 AND 4. A catfish blinded, 12 hours later hypophysectomized and main-
tained 125 days in the white tub described above continuously illuminated. Note
the pale shade and relative loss of pigment compared with blinded control Fig. 8.
The fishes in Figs. 1 and 2; 3 and 4 were photographed and printed together.
ROLE OF HYPOPHYSIS IN MELANOGENESIS
355
-
^
356 CLINTON M. OSBOKN
animals were checked twice daily for mortalities and dead or dying
fishes were placed in fixative immediately and observations recorded.
Because at least a month of continuous illumination (at the intensity
used) was required to develop more than a slight amount of ventral
pigment in the catfish, data on fishes surviving less than 30 days were
discarded. It is important, therefore, to list animals surviving the
operation by 30 days. About 80 per cent of the original fishes were
alive and distributed as shown in Table I.
After the first month the mortality rate decreased, presumably be-
cause the less vigorous animals succumbed earliest. After that, occa-
sional deaths, combined with the intentional sacrifice of an animal now
and then, reduced the number of experimental fishes considerably so
that at the end of 180 days 51 animals (about 40 per cent) surviving in
good condition were distributed in the four groups as shown in Table II.
TABLE II
Animals alive at end of experiment — 180 days
Number
Percentage of
original
Group A
Group B
Group C
Group D
11
23
6
11
18.3
76.7
27.3
73.3
At the end of the experiment (180 days) representative animals
from each group were reserved for chemical and other quantitative
PLATE II
EXPLANATION OF FIGURES
All ventral views — about % natural size.
FIG. 5. A catfish blinded and subjected to direct ventral illumination for 148
days. Note bow the normally unpigmented white vest has become almost com-
pletely blackened with melanophores. Direct illumination grows the pigment faster
than weaker reflected light. Compare with Fig. 1.
FIG. 6. A catfish (eyes intact) ventrally illuminated with direct light for 55
days. The animal remained somewhat dark-adapted to the black sides and ceiling
of the tub. Note that some ventral pigment has grown, especially at the base of
the anal fin. Compare with Fig. 8.
FIG. 7. A catfish blinded and ventrally illuminated (direct light) 79 days.
The pigmentation is somewhat less extensive than in Fig. 5.
FIG. 8. An animal blinded and kept with the stock fishes in an unlighted tank
of neutral shade 76 days. Note the dark shade resulting from blinding, but exces-
sive pigmentation has not occurred. This fish serves as an appropriate control for
some of the other animals illustrated.
ROLE OF HYPOPHYSIS IN MELANOGENESIS 357
PLATE II
CLINTON M. OSBOKX
determinations, the results of which will he assembled in a later com-
munication. Only qualitative results will lie reported here.
RESULTS
After prolonged treatment under the conditions described, the ani-
mals from all four experimental groups could he divided into only two
categories on the basis of general macroscopic appearance.
On the one hand fishes of group B which had been blinded only
(hypophysis intact ) had become coal black dorsally and laterally and had
developed dense ventral pigment especially around the cloacal aperture
and posteriorly around the anal fin in addition to numerous spots of
melanophores scattered over the normally white vest of the 1 telly surface
(Figs. 1, 3, 5 and 7). The deposition of melanin in these fishes was
strikingly excessive as evidenced by the amount of black pigment which
came off on one's hands when the fishes were handled for observations."
This never occurred in handling fishes of any of the three other experi-
mental groups.
On the other hand, fishes of groups A (blinded and hvpophysecto-
mized), C (hypophysectomized — eyes intact) and D (unoperated) were
all pale in shade (except those in group D in black-walled tubs) and in
general not easily distinguished by original groups although in some
instances those in group A seemed slightly darker than fishes in groups
C and D. \Yhether this was a significant difference may be decided
from future quantitative determinations. Of interest in our present
findings is the fact that hypophysectomized animals of group A were
unable to grow melanophores (Figs. 2 and 4) while those in group B,
alike in all experimental details except that the hypophysis was func-
tionally intact, grew abundant melanophores. All fishes in both groups
had been totally blinded, an operation which in normal catfishes results
in the pronounced darkening of the integument (Fig. 8) due to maximal
dispersion of the melanin granules within the melanophores (Parker,
1934 and 1939 ; Abramowitz, 1936 ; Osborn, 1938 ). When animals thus
blinded are hypophysectomized, however, the integument pales consid-
erably with corresponding concentration of the melanin granules (Os-
born, 1938). It appears, then, that when the pigmentary system is sub-
jected to these two opposing influences, melanogenesis is not accelerated
even though the external environment (illumination, etc.) strongly
favors the growth of melanophores. Furthermore, the melanophores
are not maintained normally but rather undergo gradual degeneration.
•"This is in all probability the result of large numbers of superficial melano-
phores being cast off through the epidermis, a condition invariably found in catfish
integuments where melanin production is going on at an accelerated rate.
ROLE OF HYPOPHYSIS IN MELANOGENESIS 359
Animals in groups C and D placed in white tubs remained very pale
and no evidence of accelerated melanogenesis was observed although
ventral illumination was continuously provided. Animals in group D
were actually white-adapted normal animals (tub walls and ceiling white)
while those in group C not only were white-adapted but were deprived
of the hypophysis, the source of the chief melanin-dispersing factor in
the normal chromatology of the catfish. Of the other fishes in groups
C and D in tubs with black walls and ceiling, those in group C were very
slightly darker qualitatively than corresponding fishes in white tubs
while group D catfishes were rather black-adapted with a noticeable
increase in pigmentation (Fig. 6).
It was noticed that the animals of groups A, C and some in D
(those in white tubs) not only failed to show evidence of accelerated
melanogenesis but actually appeared less heavily pigmented at the end
of the experiment than stock controls.
DISCUSSION
Two types of color change have been recognized for several years,
rapid and gradual. The thesis that there is a causal relation between
the phenomena of transitory and of quantitative color change referred
to as " Babak's Law " recognizes as separate features the rapid color
changes and those of a very gradual, less temporary type. Odiorne
(1937) speaks of these as " physiological " and "morphological" color
changes respectively. He found that the pigmentation of Fundulus
majalis, F. heteroclitus, and Ameiunis nebulosits is " reduced through
the degeneration of melanophores when these fishes are kept on white
backgrounds, but tends to increase when they are kept upon black back-
grounds." He also reported that " The development of pigmentation in
young fishes (Macropodus and Gambusia) is retarded if they are kept
on white backgrounds, but on black backgrounds the fishes become very
dark." Odiorne concluded that " Morphological color changes (altera-
tions in pigmentation) and physiological color changes (arising from
pigmentary movements) are phenomena resulting from a common
cause."
Other investigators (von Frisch, 1911; Vilter, 1931; Sumner and
Wells, 1933; Sumner and Doudoroff, 1937; Sumner, 1939 and 1940a
and b; and Dawes, 1941) have reported experiments concerning an in-
crease or decrease in integumentary melanin. So far as the writer is
aware, every case of melanin increase was associated with a condition
favoring melanin dispersion in the cells, whereas decreases in melanin
regularly occurred in animals maintained in the pale phase over extended
360 CLINTON M. OSBORN
periods. These observations are in total agreement with the conclusions
of Odiorne, but direct evidence to indicate that a substance active in
physiological color change is also necessary for the formation of new
melanophores has hitherto been lacking. The results recorded here
indicate that the melanophore-dispersing substance of the pituitary
gland, so important in producing the dark phase of the catfish in its
normal physiology (Osborn, 1938), is also necessary for the develop-
ment of new integumentary melanophores and for the maintenance of
those already formed. When this substance is absent from the blood
(in hypophysectomized fishes), new melanophores are not developed even
when otherwise optimum conditions for their growth are maintained.
This is most clearly seen in the white normally non-melaninated vest of
the fish, which will become pigmented with melanophores under the con-
ditions described in group B (Figs. 1, 3, 5 and 7), using ventral
illumination. Not only did such pigmentation fail to occur in catfishes
whose pituitaries had been removed, but many of the melanophores
present previous to the operation underwent degeneration.
These findings suggest that the melanophore-dispersing substance
circulated in the blood stream of the normal fish provides a favorable
medium (internal environment) in which melanogenesis may go on. We
do not yet know, of course, wrhether this pituitary fraction itself enters
actively into the chemistry of melanin formation or whether it acts as
a catalyst in some way. In this connection it is of interest to note that
Fostvedt (1940) has reported that some pituitary fractions especially
high in melanophore-hormone content produced marked acceleration
of the oxidase system in the tyrosine-tyrosinase reaction. This was
shown in hypophysectomized frogs whose legs, isolated, were incubated
for specified periods of time following injection with the extract. • Al-
though this is somewhat removed from catfish chromatology it suggests,
at least, how the melanophore hormone may enter into melanin forma-
tion naturally, especially in animals whose normal color change mecha-
nism is so highly dependent upon this pituitary secretion.
Incidental to other observations, Rahn (1941) noticed in the rattle-
snake that following hypophysectomy little, if any, melanin was de-
posited into the cells of the shedding stratum corneum. This probably
indicates a failure of the melanophores to produce normal amounts of
pigment in the absence of the hypophysis. Recent clinical reports by
Fournier, Cervino and Conti (1941) indicate success with local injec-
tions of melanophore hormone in the treatment of vitiligo in man.
Their illustrations show clearly that pigment-free patches become re-
pigmented under administration of the hormone. This finding, together
with earlier reports by With (1920), Buschke (1907) and others who
ROLE OF HYPOPHYSIS IN MELANOGENESIS 361
treated vitiligo successfully by stimulating the growth of pigment with
light baths indicates that in the human being dual factors (light exter-
nally and a hormone internally) may cooperate in the growth and main-
tenance of pigment. It is interesting that similar agents are shown
here to control pigment production and maintenance in a teleost.
Because facts in this field are just beginning to accumulate, anything
more than speculation would be quite premature. Is it not conceivable,
however, that the intermedin abundant in the mammalian hypophysis
might be concerned in maintaining the degree of pigmentation peculiar
to the individual and that an imbalance of this factor might be corre-
lated with certain pathologies where active melanogenesis is charac-
teristic ?
SUMMARY
The common catfish (Amciunts inclas) possesses naturally a white
vest ventrally in which melanophores are only rarely found. In appro-
priate apparatus it is possible to grow melanophores abundantly over
this naturally unpigmented area and increase the amount of pigment in
other areas if the dorsal aspect (normally pigmented surfaces) of the
fish is maintained in the dark phase. It is convenient, though not
necessary, to continue the dark phase permanently by blinding the fish
totally, a fact which " per se " indicates that the eyes are not necessary
in active melanogenesis.
If the pituitary gland is removed, however, melanogenesis does not
continue. In fact, melanophore degeneration sets in with the end result
that the experimental fish is paler and less heavily melaninated than
stock controls. This indicates that the melanophore-dispersing hormone
of the pituitary gland so important in the normal color change physiology
of the catfish is also, indispensable to the development and maintenance
of melanin in melanophores. Interpreted in another way, it suggests
that morphological color change is not produced by physiological color
change but rather that both are the result of a common underlying
mechanism.
A possible way in which the melanophore-dispersing fraction of the
pituitary may be involved in the production of melanin is discussed.
It is suggested that the melanophore-dispersing hormone (intermedin)
in the human hypophysis may be concerned in the production and main-
tenance of normal pigmentation in man.
362 CLINTON M. OSBORN
LITERATURE CITED
ABRAMOWITZ, A. A., 1936. Physiology of the melanophore system in the catfish,
Ameiurus. Biol. Bull., 71: 259-281.
BUSCHKE, A., 1907. Notiz zur Behandlung des Vitiligo mit Licht. Mcd. Klin.,
3: 983-984.
CUNNINGHAM, J. T., 1891. An experiment concerning the absence of color from
the lower sides of flat-fishes. Zool. Anzciger, 14 : 27-32.
CUNNINGHAM, J. T., 1893. Researches on the coloration of the skins of flat-
fishes. Jour. Mar. Biol. Assoc., 3 (N.S.) : 111-118.
DAWES, B., 1941. The melanin content of the skin of Rana temporaria under
normal conditions and after prolonged light- and dark-adaptation. A
photometric study. Jour. Exper. Biol., 18 : 26-49.
FOSTVEDT, G. A., 1940. Effect of high melanophore hormone fractions of tyrosine
and dopa oxidation. Endocrinology, 27 : 100-109.
FOURNIER, J. C. M., J. M. CERVINO, AND O. CONTI, 1941. The treatment of vitiligo
by local injections of melanophore hormone. Endocrinology, 28: 513-515.
VON FRISCH, K., 1911. Beitrage zur Physiologic der Pigmentzellen in der Fisch-
haut. Arch. gcs. Physiol, 138: 319-387.
HERBST, C., AND F. ASCHER, 1927. Beitrage zur Entwicklungsphysiologie der
Farbung und Zeichuung der Tiere. III. Der Einfluss der Beleuchtung
von unten auf das Farbkleid des Feuersalamanders. Roux Arch. Entiv.-
mcch. Organ., 112: 1-59.
ODIORNE, J. M., 1937. Morphological color changes in fishes. Jour. E.rper. Zool.,
76 : 441-465.
OSBORN, C. M., 1938. The role of the melanophore-dispersing principle of the
pituitary in the color change of the catfish. J our. Exper. Zool., 79 :
309-330.
OSBORN, C. M., 1940a. The experimental production of melanin pigment on the
lower surface of summer flounders (Paralichthys dentatus). Proc. Nat.
Acad. Set., 26: 155-161.
OSBORN, C. M., 19406. Studies on the origin and behavior of melanophores experi-
mentally grown on the ventral surface of the summer flounder (Para-
lichthys dentatus). Anat. Rec., SuppL, 78: 70 (abstract 69).
OSBORX, C. M., 1940c. The growth of melanophores on the normally unpigmented
surface of the black catfish, Ameiurus melas. Anat. Rec., SuppL, 78 :
167 (abstract 301).
OSBORN, C. M., 1941. Studies on the growth of integumentary pigment in the
lower vertebrates. I. The origin of artificially developed melano-
phores on the normally unpigmented ventral surface of the summer
flounder (Paralichthys dentatus). Biol. Bull., 81: 341.
PARKER, G. H., 1934. Color changes in the catfish Ameiurus in relation to neuro-
humors. Jour. Expcr. Zool., 69 : 199-233.
PARKER, G. H., 1939. The relation of the eyes to the integumentary color changes
in the catfish Ameiurus. Proc. Nat. Acad. Sci., 25 : 499-502.
RAHN, H., 1941. The pituitary regulation of melanophores in the rattlesnake.
Biol. Bull, 80: 228-237.
SUMNER, F. B., 1939. Quantitative effects of visual stimuli upon pigmentation.
Am. Nat., 73 : 219-234.
SUMXER, F. B., 1940a. Further experiments on the relations between optic stimuli
and the increase or decrease of pigment in fishes. Jour. Exper. Zool., 83 :
327-343.
SUMNER, F. B., 1940£>. Quantitative changes in pigmentation, resulting from visual
stimuli in fishes and amphibia. Biol. Rev., 15: 351-375.
ROLE OF HYPOPHYSIS IN MELANOGENESIS 363
SUMNER, F. B., AND P. DouDOROFF, 1937. Some quantitative relations between
visual stimuli and the production or destruction of melanin in fishes.
Proc. Nat. Acad. Sci, 23: 211-219.
SUMNER, F. B., AND N. A. WELLS, 1933. The effects of optic stimuli upon the
formation and destruction of melanin pigment in fishes. Jour. Expcr.
Zool, 64 : 377-403.
VILTER, V., 1931. Modifications du systeme melanique chez les Axolotls soumis
a 1'action de fonds blancs ou noirs. Coinpt. Rend. Soc. Biol., 108 : 774-
778.
WITH, C, 1920. Studies on the effect of light on vitiligo. Brit. Jour. Dermal.,
32: 145-155.
THE ROLE OF ANTIFERTILIZIN IN THE FERTILIZATION
OF SEA-URCHIN EGGS
ALBERT TYLER AND KATHLEEN O'MELVENY
(From the William G. Kcrckhoff Laboratories of the Biological Sciences,
California Institute of Technology)
INTRODUCTION
In recent years several investigators (Frank, 1939; Tyler, 1939o,
1940; Southwick, 1939; Hartmann, Schartau and Wallenfels, 1940) have
obtained from sperm of sea urchins and of mollusks, a substance that
reacts with the fertilizin obtained from eggs, and which is therefore
termed antifertilizin. The reaction is manifested by the following ef-
fects:— (1) When it is added to a solution of fertilizin, the sperm-
agglutinating property is proportionately destroyed; (2) under appro-
priate conditions it forms a precipitate with fertilizin; (3) it agglu-
tinates eggs of the same or closely related species ; (4) it produces a
precipitation membrane on the surface of the egg jelly. These four
effects are evidently due to the same substance which is obtained as a
sea- water extract of moderately heated or of frozen and thawed sperm.
Several other effects of sperm extracts have been described. In the
keyhole limpet and in the abalone the extracts contain a lytic agent
(Tyler, 1939a) which has the property of dissolving the membrane
normally present on the unfertilized eggs of these species and which is
much more heat-labile than the antifertilizin. The evidence does not
as yet enable a decision to be made as to whether it is a distinct sub-
stance or a complex that is only active in combination with antifertilizin
or a higher " polymer " of antifertilizin. A somewhat similar lytic
action of macerated sperm on the egg membrane was reported in am-
phibia (Hibbard, 1928; Wintrebert, 1933) and of a sperm extract on
the cell mass and membrane surrounding the egg of the rabbit (Yamane,
1935).
An agent that inhibited the activity of the spermatozoa was obtained
by Southwick (1939) in the supernatant from centrifuging concentrated
but not dilute sperm suspensions of the sea urchin. Identity of this
agent with antifertilizin has not been established nor has the possibility
been excluded that the effect is due to some simple agent such as in-
creased acidity, CO2, etc. A similar activity-inhibiting action has been
364
ANTIFERTILIZIN AND FERTILIZATION 365
reported in sea urchins by Hartmann, Schartau and Wallenfels (1940)
for a methyl alcohol extract of sperm that does not contain antifertilizin
(the agglutinin-neutralizing agent). They also find that the extract
neutralizes the stimulating effect of egg water on sperm activity and the
similar action of echinochrome which they had earlier reported to be the
sperm-activating agent in egg water. Since their findings with echino-
chrome have not been duplicated in other species (Tyler, 19396; Corn-
man, 1941), and since they have not as yet disposed of the possibility
suggested by Cornman that rise in pH might be responsible for their
results, it would be desirable to have further evidence before the effect
of their methanol sperm extract may be accepted without reserve.
Another effect reported by Hartmann, Schartau and Wallenfels
(1940) in the sea urchin is the dissolving of the jelly coat of the egg
by the action of sperm extract. They find in Arbacia pustulosa that
addition of concentrated sperm extract or of live sperm causes the dis-
appearance of the egg jelly and we have been able to confirm this in
Sti'ongyloceiitrotus fiitrpnratns. But, according to our observations, this
disappearance does not appear to be due to solution of the jelly. When
sperm extract is added to a suspension of eggs there is formed on the
surface of the jelly a precipitation membrane which, in concentrated
extract, gradually increases in thickness and contracts until it reaches the
surface of the egg. This precipitation membrane is evidently formed by
interaction of the antifertilizin in the sperm extract with the jelly. The
disappearance of the latter in concentrated extracts is most simply at-
tributable to its incorporation in the precipitation membrane and to the
considerably smaller volume it occupies in precipitated rather than in gel
form. As the precipitation membrane contracts the egg may, particu-
larly when disturbed, break out of it. When undisturbed it may con-
tract to the surface of the egg from which it is then not readily distin-
guishable. The disappearance of the jelly under the influence of
concentrated suspensions of live sperm is likewise attributable to com-
bination with the antifertilizin on the sperm. There does not, then,
appear to be, as yet, any necessity for the assumption of a jelly-dissolving
agent in the sperm extract.
In the present work the term antifertilizin is applied to that sub-
stance derived from sperm that produces the effects listed in the first
paragraph. A similar antifertilizin has been obtained from eggs (Tyler,
1940), but it will not enter into the present account. The principal
question at issue here is whether or not the antifertilizin of sperm is
concerned in the fertilization reaction. Several facts strongly favor the
presumption that it is intimately involved. In the first place it is tissue
366 A. TYLER AND K. O'MELVENY
specific, being obtainable from no other tissues (Frank, 1939). It is,
however, not very highly species-specific, since cross-reactions are ob-
tained between species that do not cross-fertilize (Hartmann, et al.,
1940). This would mean that it is not primarily responsible for the
species-specificity of fertilization, but this does not exclude the possi-
bility that it is an integral part of the fertilization process. Another
fact favoring its involvement is that it is evidently present on the surface
of the spermatozoon. Since, in solution, it reacts with f ertilizin, it most
likely is the substance on the spermatozoon that reacts in the agglutina-
tion of the sperm and therefore must form at least a part of the surface.
Furthermore, fertilizin has been shown (Tyler, 1941) to serve as an
aid to fertilization and may possibly be an essential agent in the process.
Antifertilizin, since it reacts with it, would then be expected to have a
complementary role.
For a direct test of the significance of antifertilizin, it would be
desirable to remove it completely or partially from the sperm by some
non-injurious method and to examine the fertilizing capacity of the
treated sperm. We have been able, in the experiments reported here,
to remove antifertilizin partially without appreciable damage to the
sperm. This, as the results show, causes a considerable impairment
in the fertilizing capacity of the sperm.
MATERIALS AND METHODS
Two species of sea urchins, Strongylocentrotus piirpuratus and
Lytechinus anamesus, were employed in these experiments. Sperm and
egg suspensions were prepared by removing the gonads to sea water and
straining the shed sex cells through bolting cloth of appropriate mesh.
The concentration of the sperm suspension was usually determined from
the increase in volume after removal of the remains of the testes and is
expressed as the percentage content of " dry " sperm.
The antifertilizin concentrations in the extracts were determined
roughly by the intensity of the egg agglutination reaction and more
accurately by the amount required to neutralize one unit (as defined by
Tyler and Fox, 1940) of fertilizin (sperm agglutinin). In all the tests
the pH of the solutions was checked and adjusted where necessary by
means of the glass electrode.
The respiratory rate of the sperm was employed as an index of the
extent of damage produced by the various treatments. The measure-
ments were made in the Barcroft- Warburg apparatus with the cylindrical
type of vessel previously described (Tyler and Humason, 1937). To
ANTIFERTILIZIN AND FERTILIZATION 367
avoid possible effects of CO2 and variation in pH, glycylglycine (Tyler
and Horowitz, 1937) was used as a buffer in carbonate-free sea water.
REMOVAL OF ANTIFERTILIZIN
We found that antifertilizin could be removed from the sperm by
slight acidification of the suspension and also by mild warming. The
antifertilizin is obtained in the supernatant after centrifugation of an
acidified sperm suspension but not in that of the control. When highly
concentrated control sperm suspensions are centrifuged, particularly
after aging, some antifertilizin may be obtained in the supernatant, as
Southwick (1939) reported. This may mean that antifertilizin nor-
mally goes slowly into solution or that centrifugation of the concentrated
suspensions involves some damage and consequent liberation of anti-
fertilizin.
Antifertilizin is obtained from sperm suspensions acidified to pH 6
or lower. The more acid suspensions yield the more concentrated solu-
tions. One experiment with Strongylocentrotus sperm may be cited.
Samples of a 10 per cent suspension were acidified to pH 6, 5.6, 5.1, 4.5
and 3.5. After one hour the suspensions were brought back to the
control pH (7.9) and centrifuged. The control supernatant was clear
while those from the acidified suspensions were increasingly opalescent.
Tested on eggs the control showed no reaction while the supernatants
from the acidified samples gave precipitation membranes and agglutina-
tion which increased with increase in the degree of acidity to which the
samples had been exposed. Tests of their ability to neutralize fertilizin
gave the following approximate titres for the antifertilizin concentration
in the supernatants of the acidified samples: %, Y>, 1, 4 and 32 units re-
spectively. The spermatozoa were all immotile in the sample that had
been exposed to pH 3.5 and partly so in the pH 4.5 sample, while those
exposed to the higher pH's showed considerable activity.
These results restricted then the investigation of the treatment re-
quired for the impairment of fertilizing capacity to the range between
pH 5 and pH 6. A number of tests were run at various pH's within
this range and with various times of exposure. All of these showed a
considerable reduction in the fertilizing capacity of the treated sperm.
Similar results were obtained by heating the sperm at 30° to 33° C. for
5 to 10 minutes. The data need not be presented here since only that
part which was obtained along with the respiration measurements is of
particular significance. In practically all of these tests the treated sperm
were found to be quite active, although in general not as active as the
controls. However, differences in activity of spermatozoa are hard to
368 A. TYLER AND K. O'MELVENY
estimate by direct observation. A more objective and quantitative
method consists in measurement of the respiratory rate.
FERTILIZING CAPACITY AND RESPIRATORY RATE OF
ANTIFERTILIZIN-POOR SPERM
Determinations were made, therefore, of the rate of oxygen uptake
of the treated and control sperm along with tests of their respective
fertilizing capacities. The results of five experiments are presented in
Table I. Heat treatment was employed in one of these and acidification
in the rest. The measurements were made in duplicate in each experi-
ment, and both treated and control sperm were samples of the same
original suspension. The control oxygen consumption values vary rather
considerably in the different experiments. This variation is probably
due to a number of factors such as error in initial determination of sperm
concentration, variation in original condition of sperm, in its aging, etc.
For the present purposes, however, this variation is of no particular
significance, since comparison of treated and control sperm is made in
each experiment. The duplicate runs in each experiment are in close
agreement, which is to be expected since sperm suspensions can be quite
accurately sampled and since the spermatozoa respire at a sufficiently
high rate to make the instrumental errors relatively small.
In none of the experiments listed in Table I was the respiration of
the treated sperm equal to that of the control. The highest values were
80 per cent of the control in experiments 1 and 5 and the lowest value
was 25 per cent of the control in experiment 4. The treatment is, then,
not entirely non-injurious to the sperm. However, a considerably
greater impairment of fertilizing power results from the treatment.
The fertilizing capacity of the treated sperm is listed in the last column
of the table in terms of the amount required to give the same percentage
fertilization, between 1 and 99 per cent, as is given by one part of the
control sperm. These values are obtained from the results of inseminat-
ing samples of the same batch of eggs with serial dilutions of the control
and treated sperm taken from the manometer vessels. The two figures
for each experiment cover the range of variation. Thus, in the first
experiment, the amount of treated sperm required to give the same per-
centage fertilization as the control is four to eight times the amount of
the control sperm. For comparison, the next to the last column of the
table gives the calculated amount of treated sperm that would have the
same respiratory rate as one part of control sperm. This value is, in
each experiment, considerably less than the value for the amount of
sperm having a fertilizing capacity equal to one part of control sperm.
ANTIFERTILIZIN AND FERTILIZATION
369
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370 A. TYLER AND K. O'MELVENY
In other words, there is, as a result of the treatment in each case, a very
much greater reduction in the fertilizing power than in the respiratory
rate.
It is evident, too, from the data that a considerable impairment of
fertilizing power would be obtained following a treatment that resulted
in no reduction in respiratory rate. That we have not, as yet, suc-
ceeded in finding the proper treatment which would give that result is
not surprising in view of the variability of the sperm in the different
experiments and the fact that the difference is rather small between
treatments giving no effect and those giving a definite reduction in fer-
tilizing capacity. The present results, however, suffice to show that an
impairment of fertilizing power can be obtained that is disproportion-
ately great when compared with the respiration of the sperm. This im-
pairment cannot, then, be accounted for by a decrease in activity of the
spermatozoa. It might possibly be interpreted in a rather complicated
manner by the supposition that a corresponding fraction of the sperm
are rendered non-respiring and non-fertilizing while the remainder have
an increased respiratory rate. This would mean that the effect on the
individual spermatozoa would be all or none and that mild treatment
would have a stimulating effect on the respiration of the suspension.
There is no evidence for this. The most reasonable interpretation is
that the impairment of fertilizing capacity is correlated with the loss of
antifertilizin which was shown to result from the treatment.
In the experiments described here antifertilizin is present in solution
in the treated sperm suspension. To determine whether its presence
might affect the results, antifertilizin was added to untreated sperm in
the same or slightly greater amounts. This was found to have no
effect on the fertilizing capacity of the sperm. On the other hand,
when concentrated antifertilizin solutions are employed an inhibition of
fertilization can be obtained, as Frank (1939) and Hartmann, Schartau
and Wallenf els ( 1940) have shown. This inhibition occurs more readily
when the eggs are first treated and is evidently due to the presence of
the precipitation membrane that forms on the surface of the jelly.
When this membrane is incomplete or torn the egg can be fertilized,
as was previously reported in the case of treatment with the antiferti-
lizin obtained from eggs (Tyler, 1940).
ANTIGENICITY OF ANTIFERTILIZIN AND ACTION OF ANTISERA
In order to obtain further information on the location of antiferti-
lizin and on its role in fertilization, attempts were made to produce anti-
bodies to it. Preliminary immunization experiments showed that high
ANTIFERTILIZIN AND FERTILIZATION 371
titer agglutinins could be obtained in rabbits by the injection of whole
sperm of the sea-urchin. Immunization with antifertilizin solutions
likewise was found to induce the formation of specific agglutinins for
the whole sperm as well as precipitins for the antigen in solution.
Antisera were produced against Strongylocentrotus and Lyt echinus
antifertilizin. The procedure and results in one experiment with Ly-
techinus follow. A solution of antifertilizin was prepared by extrac-
tion of a 25 per cent sperm suspension at pH 4.5 for two hours. The
content of organic solid was determined on a sample that had been
dialyzed against distilled water and was found to be between 15 and
20 mg. per cent. The rabbit was given seven intravenous injections
totaling 23 cc. within a period of two weeks and was bled two weeks
after the last injection. The antiserum showed by the ring test a pre-
cipitin titer of 8. Tested on a one per sperm suspension it showed an
agglutinin titer of 512.
The production of agglutinins by injection of antifertilizin means
not only that the substance is antigenic but is probably a surface antigen
of the sperm. An examination of the agglutinates shows that the sper-
matozoa are stuck by their tails as well as by their heads. The anti-
fertilizin, therefore, does not appear to be restricted to a particular
location on the surface of the spermatozoon. It should also be noted
here that extraction at pH 4.5 removes only a small part of the anti-
fertilizin from the sperm since subsequent freezing and thawing or brief
heating of the residue yields at least ten times the amount obtained in
the acid extract. Also the acid-treated sperm are still agglutinable by
antisera and by egg water.
The antigenicity of antifertilizin supports the view that it is a
protein. Other evidence (to be presented in detail later) consists in
its non-dialyzability, precipitation with (NH4)oSO4, inactivation by heat
and acidity, and the fact that it gives the common (xanthoproteic,
Millon's and biuret) color tests.
The effect of the antiserum on fertilization was examined by insem-
ination of eggs in its presence. Controls were run with normal rabbit
serum. The sera were adjusted to sea- water salinity by the addition
of an equal volume of concentrated (1.73 X) sea water, and equal vol-
umes of egg and sperm suspensions wrere added. In all cases where the
sperm was diluted to the minimum for 100 per cent fertilization in the
controls, no fertilization was obtained in the antiserum. With the dilu-
tions of sperm employed, agglutination is greatly retarded and may even
fail to occur- in the antiserum. The spermatozoa have not then, to any
great extent, been rendered inaccessible to the eggs by incorporation in
A. TYLER AND K. O'MELVENY
agglutinates. The inhibition of fertilization may therefore be consid-
ered to be due to the neutralization of antifertilizin on the sperm by its
antibody in the antiserum.
DISCUSSION
The results presented here show that antifertilizin is involved in the
fertilization process. In order to decide whether or not it has an indis-
pensable role, one would like to have some more direct evidence such as
the complete and reversible removal of antifertilizin might supply. But
complete extraction without destruction of the sperm has not as yet been
accomplished. From the present evidence it is reasonable to regard
antifertilizin as involved in an initial step that facilitates fertilization
but which may or may not be an essential part of the process. This
initial step is evidently the reaction with fertilizin. In a previous article
(Tyler, 1941), it has been shown that the presence of fertilizin on the
egg serves as an aid to fertilization. Antifertilizin may, then, be con-
sidered to have a similar role in the case of the spermatozoon. For this
purpose it is not effective when present in solution but only on the
spermatozoon. Partial removal of the antifertilizin or its neutralization
by means of an antiserum or by means of fertilizin results in a decrease
or even complete suppression of the fertilizing power of the sperm. As
an interpretation for the fertilization-facilitating action of fertilizin
(Tyler, 1941) it was suggested that, in the form of a gel around the
egg, it has a few superficial combining groups available which serve as
the initial trap for the sperm but which do not neutralize all of the
reacting groups (antifertilizin) on the sperm before the latter has
reached the surface of the egg. On this basis the decrease in fertilizing
power of the treated sperm may be interpreted to mean that, with fewer
reacting groups available, there is more likelihood that they will all be
neutralized before the spermatozoa reach the egg surface.
SUMMARY
1. Acidification of sea-urchin sperm suspensions to below pH 6 or
brief heating above 30° C. liberates into the solution the substance termed
antifertilizin which is defined by four manifestations of its reaction with
fertilizin; (a) neutralization and (b) precipitation of the latter, (c)
agglutination of eggs, (d) formation of precipitation membrane on egg
jelly.
2. The treatment results in a marked decrease in the fertilizing
power even when the time and intensity of exposure are not sufficient
to immobilize the sperm.
ANTIFERTILIZIN AND FERTILIZATION 373
3. The rate of oxygen consumption of sperm, that had been exposed
to mild acid- or heat-treatment, was found to be very little affected in
comparison with the effect on the fertilizing power. Short extrapolation
permits the conclusion to be drawn that a considerable reduction in
fertilizing capacity can be obtained with no reduction of activity of the
spermatozoa.
4. Injection of antifertilizin solutions into rabbits results in the
production of an agglutinin for the intact sperm. This shows that the
substance is a complete antigen and supports the views that it is a
protein and a component of the surface of the spermatozoon.
5. Fertilization is inhibited by antisera to antifertilizin.
6. Antifertilizin is considered to be concerned in an initial (perhaps
essential) step in the union of the gametes whereby the spermatozoon
is entrapped by the complementary, specific reacting substance, fertilizin,
on the egg ; and the above inhibition experiments are interpreted on the
basis of a decrease in the number of reacting groups available on the
spermatozoon.
LITERATURE CITED
CORNMAN, I., 1941. Sperm activation by Arbacia egg extracts, with special rela-
tion to echinochrome. Blol. Bull, 80: 202-207.
FRANK, J. A., 1939. Some properties of sperm extracts and their relationship to
the fertilization reaction in Arbacia punctulata. Blol. Bull., 76 : 190-216.
HARTMANN, M., 1940. Die Stofflichen Grundlagen der Befruchtung and Sexuali-
tat im Pflanzen- und Tierreich. I. Die Befruchtungsstoffe (Gamone) der
Seeigel. Natunviss., 51 : 807-813.
HARTMANN, M., O. SCHARTAU AND K. WALLENFELS, 1940. Untersuchungen iiber
die Befruchtungsstoffe der Seeigel. II. Blol. Zcntralbl., 60: 398-423.
HIBBARD, H., 1928. Contribution a 1'etude de 1'Ovogenese, de la Fecondation, et de
1'Histogenese chez Discoglossus pictus Atth. Arch, de Biol., 38: 249-326.
SOUTH WICK, W. E., 1939. Activity-preventing and egg-sea-water neutralizing
substances from spermatozoa of Echinometra subangularis. Blol. Bull.,
77: 147-156.
TYLER. A., 1939a. Extraction of an egg membrane-lysin from sperm of the giant
keyhole limpet (Megathura crenulata). Proc. Nat. Acad. Sci., 25: 317-
323.
TYLER, A., 1939ft. Crystalline echinochrome and spinochrome : their failure to
stimulate the respiration of eggs and of sperm of Strongylocentrotus.
Proc. Nat. Acad. Sci., 25 : 523-528.
TYLER, A., 1940. Agglutination of sea-urchin eggs by means of a substance ex-
tracted from the eggs. Proc. Nat. Acad. Sci., 26: 249-256.
TYLER, A., 1941. The role of fertilizin in the fertilization of eggs of the sea-
urchin and other animals. Biol. Bull., 81 : 190.
TYLER, A., AND S. W. Fox, 1940. Evidence for the protein nature of the sperm
agglutinins of the keyhole limpet and the sea-urchin. Biol. Bull., 79: 153-
165.
TYLER, A., AND N. H. HOROWITZ, 1937. Glycylglycine as a sea water buffer.
Science, 86 : 85-86.
374 A. TYLER AND K. O'MELVENY
TYLER, A., AND W. D. HUMASON, 1937. On the energetics of differentiation, VI.
Biol Bull., 73 : 261-279.
WINTREBERT, P., 1933. La fonction enzymatique de 1'acrosome spermien du Disco-
glosse. Compt. Rend. Soc. Biol., 112: 1636-1640.
YAMANE, J., 1935. Kausal-analytische Studien iiber die Befruchtung des Kanin-
cheneies. II. Die Isolierung der auf das Eizytoplasma auflosend wirken-
den Substanzen aus den Spermatozoen. Cytologia, 6 : 474-483.
ENZYMES IN ONTOGENESIS (ORTHOPTERA)
XVIII. ESTERASES IN THE GRASSHOPPER EGG x
LOREN D. CARLSON
(From the Zoological Laboratories, State University of loiva)
The respiratory quotients obtained for the grasshopper egg during
all except the first few clays of its development are characteristically
those of an organism metabolizing fat (Bodine, 1929; Boell, 1935).
Slifer (1930) has shown that the amount of fat in the egg measured in
terms of fatty acid after saponification decreases during the phases of
active development. The determination of the amounts of " lipoidal "
substance that form the centripetal layer when a saline extract of these
eggs is centrifuged also demonstrates a decrease in volume during em-
bryonic growth (Bodine et al., 1939). Further, the potency of this
" lipoidal " layer to activate the proenzyme, protyrosinase, varies in a
different fashion than its change in volume (Bodine et al., 1939).
These facts add an interest to the study of the types and activities of
lipolytic enzymes present in the egg of the grasshopper (Melanoplus
differentialis) during its embryogeny.
MATERIALS AND METHODS
Grasshopper eggs were collected daily and kept at 25° C. either in
the pods or separated upon damp sand within covered glass dishes.
Under these conditions the eggs go into diapause within a month
(Slifer, 1931). This block in development was interrupted by keeping
the eggs at 5° C. for three months and then transferring them to 25° C.,
at which temperature they hatched in 18 days. The eggs for experi-
ments were washed, sorted, and sterilized with 70 per cent alcohol for
ten minutes (eggs 0 and 5 days of age were not treated with alcohol),
rinsed and ground in a glass mortar. The ground eggs were made up
to designated volume in a glycine-NaOH buffer mixture. This egg
brei was centrifuged and the lipoidal or centripetal layer removed, as
were the shell fragments (A and C layer of Bodine and Allen, Fig. 1,
1938). The remainder was made up to volume with the buffer mixture.
Removal of the A and C layers did not alter the enzyme activity.
1 Aided by a grant from the Rockefeller Foundation for research in cellular
physiology.
375
376 LOREN D. CARLSON
Amounts of the extract were added to a 50 cc. Erlenmeyer flask contain-
ing substrate and allowed to stand at 25° C. unless otherwise noted.
Varying concentrations of enzyme and substrate were used, the total
volume of the reaction mixture being 6 cc. After a period of time, the
reaction was stopped with 25 cc. of a 2 per cent phenol solution and
the mixture titrated with 0.05 N HC1 until methyl red, used as an
indicator, turned pink. Although the H ion concentration changed dur-
ing the experiments, the addition of the phenol in buffer brought the
pH back to the alkaline side and the HC1 titre then was a measure of
the NaOH neutralized during the reaction and was equivalent to the acid
formed. The amount of acid thus formed is considered a measure of
the rate of hydrolysis and an index of the amount of enzyme present.
The equivalents of acid produced are not strictly rate values in the case
of tributyrinase (Bodansky, 1937). Because of the difficulty in deter-
mining how much of the substrate was properly emulsified, a more
accurate measure of rate was not practicable. Controls were duplicates
of the experimental with the exception that the enzyme extract was
heated at 100° C. for 5 minutes. Reaction mixtures containing no
substrate or enzyme were also tested and gave values equal to those of
the control. Shaking the flasks during the reaction period did not
change the rate of the hydrolysis.
The buffer mixture contained 0.1 N glycine and 0.1 N NaOH in the
ratio of 9 to 1, 15 per cent glycerol, and enough NaCl to make the
solution 0.17 M with respect to NaCl. The addition of salt was neces-
sary to prevent precipitation of proteins in the extract. The phenol
was dissolved in the glycine-NaOH buffer and was never used after it
had acquired a brownish tinge. The methyl butyrate (Eastman Kodak),
2 per cent by weight, and the tributyrin (Eastman Kodak) and olive oil,
4 per cent by weight, were made up in the buffer containing glycerol and
NaCl. Previous workers have experienced difficulty in making up tri-
butyrin solutions which gave consistent results. Seventy milligrams of
a commercial dispersing agent (Daxad No. 11) - per 100 cc. of solution
will stabilize a tributyrin emulsion.
The method was checked using known amounts of butyric acid in
place of the lipid in the protocol and the probable errors of the means
of ten determinations at four concentrations between 0 and 2 X 10~4 M
butyric acid were less than 2 per cent of the mean in every instance.
The amount of hydrolysis of an excess of methyl butyrate (3 cc. of 2
per cent) increases linearly with extracts of one to thirty diapause eggs.
The amount of acid produced by one cc. of an extract (20 eggs per cc.)
was linear with time for 4 hours. When tributyrin was used as a sub-
- Furnished by Devvey and Almy Chemical Co.
ESTERASES IN THE GRASSHOPPER EGG 377
strata, an extract of 2 eggs (diapause) would produce as much acid in
2 hours as an extract of 30 eggs would produce from methyl butyrate.
The amount of acid produced from 3 cc. of 4 per cent tributyrin was
proportional to the concentration between 1 to 5 eggs per cc. The
reaction on tributyrin was linear with time only for the first hour. In
making the following determinations, 1 cc. of an extract containing 20
eggs per cc. with 3 cc. of 2 per cent methyl butyrate in a reaction
period of 2 hours and 1 cc. of an extract containing 2 eggs per cc. with
3 cc. of 4 per cent tributyrin in a reaction period of one hour at 25° C.
were used as test reactions.
In a number of experiments direct titrations of reaction mixtures
were made with 0.05 N NaOH to determine the extent of hydrolysis.
In these the pH was first adjusted by the addition of acid or alkali and
titrations made to maintain this H-ion concentration. A Leeds and
Northrop pH meter with a glass electrode was used in these titrations.
The time course of the reactions under these conditions at steady pH
values between 4.5 and 8.0 was similar to that when the method de-
scribed above was used.
EXPERIMENTAL
Enzymes are present in the grasshopper egg which will catalyze the
hydrolysis of methyl butyrate and tributyrin but not olive oil. The
enzymes are designated as methyl butyrinase and tributyrinase respec-
tively in the following discussion although other substrates may be
attacked by these enzymes. According to the nomenclature of Oppen-
heimer (1936), both are esterases ; the one more specifically a lipase
since it splits a glycerol ester of the fatty acid. The amounts of the
two lipolytic enzymes vary independently during the development of the
grasshopper egg (Fig. I).3 The methyl butyrinase activity is high at
the start of development and then drops markedly between the tenth
and fifteenth day. A steady level is then maintained during the diapause
or inactive stage. Upon resumption of development a slow decline in
activity takes place. Tributyrinase action, however, remains at the same
level during prediapause and diapause and then drops rapidly in post-
diapause development. An extract from a single grinding was used on
both substrates in each of the ten determinations represented by the
averages in the figure.
Two types of experiments were used to determine the relative
amounts of the lipolytic enzymes being studied in the embryo and yolk
3 In preliminary work (Carlson, 1940) the enzyme extracts used were so con-
centrated that the changes in activity were obscured.
378
LOREN D. CARLSON
constituents of the egg. Early prediapause (6-day) eggs were irradiated
at 1000 roentgens, which is known to prevent the embryo from devel-
oping but to have no visible effect on other constituents of the egg
(Evans,. 1936). 4 The oxygen consumption of eggs treated in this
manner decreases until the time of diapause. The O2 uptake is low
during diapause, and when the diapause is broken the oxygen uptake
increases for the first two days and then remains constant (Bodine,
15
O
I
o
6 0-5
10
20
30 40
DAYS
50 0
10
FIG. 1. Average esterase activity of five lots of eggs at each of the develop-
mental ages shown. Ordinate, the equivalents of acid produced by hydrolysis of
the esters in cc. of 0.05 N HC1 ; abscissa, time in days at 25° C. since laying fol-
lowed by the time in days at 25° C. after termination of the diapause by exposure
to 5° C. for three months. Open circles, the activity of an extract of 20 eggs in
two hours with methyl butyrate ; closed circles, that of an extract of two eggs in
one hour with tributyrin as a substrate.
Carlson, and Ray, 1940). No significant difference could be shown
between the enzyme content of the irradiated eggs and that of the
controls. Determinations were carried out for 30 days after the
irradiation.
In postdiapause, the embryos could be dissected from the egg and
determinations made of the lipase content of the embryo and other egg
constituents. The dissections were carried out in the buffer mixture.
The embryos were freed from as much adhering yolk as possible and
transferred with a minimum of fluid to a mortar and ground with a
4 Dr. T. C. Evans irradiated the eggs for the author.
ESTERASES IN THE GRASSHOPPER EGG 379
small amount of sand. The remaining yolk and shells were also ground
and used with the dissection fluid in the determinations. There was
some difficulty in freeing the embryos of yolk, but in all cases this was
done as completely as possible. The amounts of enzyme in embryo plus
yolk, etc. were always less than those of the whole eggs. The lipolytic
enzymes seem to be associated writh the yolk or its derivatives until just
previous to hatching (Table I). The yolk removed in later stages of
development usually included parts of the gut that could not be ade-
quately separated. The presence of some esterase in the 5-day post-
diapause embryos is attributed to the fact that the yolk and embryos
were especially hard to separate at this stage.
Although the time course of reactions was similar at different pH
values, the extent of hydrolysis of tributyrin was markedly affected.
TABLE I
Days Post-
diapause
Embryo
Yolk etc.
Substrate
Per cent
Per cent
0
0
100
Tributvrin
5
19.3
80.7
10
0
100
15
0
100
18
100
0
0
0
100
Methvl butvrate
5
20.3
79.7
10
8.7
91.3
15
0
100
18
100
0
Reactions were carried out at pH 4.5, 5.0, 6.0, 7.0, and 7.5 by titrating
frequently with 0.05 N NaOH. In Fig. 2, curve A shows the total
amount of alkali used in this procedure over a one-hour period with
the enzyme from two eggs reacting with 3 cc. of 4 per cent tributyrin.
Similarly, curve B shows the result of experiments using the extract of
20 eggs with 3 cc. of 2 per cent methyl butyrate. The pH for maximum
tributyrinase activity is at 6 while the H-ion concentration affects the
methyl butyrinase activity to a lesser degree. This dissimilarity in the
effect of pH on the activity of the enzymes studied is one of several
differences noted. No explanation of the difference in the values for
the rate of methyl butyrate hydrolysis when the pH is kept constant
and when it is allowed to change is at hand.
The effect of heat treatment on the enzyme extract as well as its
effect on the amount of hydrolysis was determined. In the former case
380
LOREN D. CARLSON
the lipolytic activity of the extract was affected differently for the two
substrates. The activities of extracts were tested at 25° C. after ten-
minute exposures to temperatures between 25 and 85° C. The ability
to split methyl butyrate was diminished by temperatures higher than 55°
C. while tributyrinase activity was unchanged after exposures to 65° C.
(Fig. 3).
When the reaction mixtures were kept at temperatures varying from
0° to 45° C., the amount of hydrolysis of the two substrates differed in
2-4
2-0
16
I
O
Z '"2
Z
goe
u
U04
PH
FIG. 2. The amount of hydrolysis at various H-ion concentrations. The re-
actions were kept at the pH noted by continuous titrations with 0.05 N NaOH.
O, the hydrolysis of tributyrin by an extract from two eggs over a period of one
hour; D, methyl butyrate split by an extract of 20 eggs at the end of 2 hours.
Solid symbols represent amount of acid formed when the mixture was allowed to
react over the total time. Four to seven experiments averaged in each point.
Reactions at room temperature.
a striking manner. The hydrolysis of methyl butyrate increased with
temperatures up to 45° C. (Fig. 4/4) and between 0° and 35° a p, value
of 5700 calories was obtained (Fig. 45). The Q10 over the correspond-
ing range averaged 1.4. The tributyrinase activity showed a maximum
at 25° C. with a decrease on either side of this temperature (Fig. 4-A).
The Q10 value between 5 and 15° C. is 1.97; between 15 and 25°, 1.47,
using the amount of acid produced per unit time as a rate value. The
values shown in the figure were obtained using an extract made in the
ESTERASES IN THE GRASSHOPPER EGG
381
following manner : the eggs were ground and diluted to a volume so
that the concentration was 40 eggs per cc. When this was allowed to
stand a considerable precipitate was formed. This was centrifuged off
and the supernatant fluid diluted to a volume corresponding to 20 eggs
per cc. This resulting extract still retained its tributyrinase activity,
but the methyl butyrinase reaction was reduced to one-sixth to one-fifth
of that of an extract prepared in the usual manner. Falk and Sugiura
(1915) were able to separate esterase and lipase materials in the castor
bean, the one soluble in distilled water, the other in NaCl solution. The
temperature relationship to activity of the enzyme is similar to that
reported by Fiessinger and Gajdos (1936) working with an extract of
the larva of Gallcria nicllonclla. Their extract showed maximum activ-
i-o
O
6
0-5
u
u
25 35 45 55 65 75
DEGREES CENTIGRADE
85
FIG. 3. The effect of temperature on the activity of the enzyme extract. Or-
dinate, equivalents of acid produced in cc. of 0.05 N HC1 in one hour for tributyri-
nase and two hours for methyl butyrinase at 25° C. ; abscissa, temperature in °C.
to which the extract was exposed for 10 minutes. Closed circles, the hydrolysis of
methyl butyrate ; open circles, the hydrolysis of tributyrin. Methyl butyrinase from
20 eggs ; tributyrinase from 2 eggs.
ity between 18° and 25° C. and declined at temperatures above or below
this range.
Most observations concerning esterase activity indicate that the cal-
cium ion, sodium oleate and albumin accelerate the activity of the en-
zymes. This is not found to be the case in extracts of the grasshopper
egg. Sodium oleate reduces the lipolytic activity of the preparations
used in these experiments. Calcium chloride has no effect on the
enzyme but counteracts in part the effect of sodium oleate (Table II).
Neither of these substances has any effect in stabilizing the pH. Al-
bumin was not used since the extract was rich in protein. Attempts to
show hydrolysis of olive oil with addition of sodium oleate and the
calcium ion to the egg extracts at 25° C. and 35° C. were without success.
Various esterases are affected differently by such compounds as
382
LOREN D. CARLSON
phenol, quinine, atoxyl and sodium fluoride (Falk, 1924; Oppenheimer,
1936). Curiously, extracts from pancreas, liver and kidney are inhib-
ited in their action on tributyrin in a diverse manner by quinine and
atoxyl (Falk, 1924). The effect of 0.5 per cent phenol, NaF and
quinine were tested on the esterases obtained from the grasshopper egg.
1-4
1-2
i-o
z
if)
pO-6
6
0-2
B
45 35 25
15
I
5 °C.
0 10 20 30 40
DEGREES CENTIGRADE
50
32
34
36
I/T -x 10
FIG. 4. The amount of hydrolysis of methyl butyrate and tributyrin at dif-
ferent temperatures. In A, the ordinate gives the equivalents in cc. of 0.05 N
HC1; the abscissa, the temperature at which the reaction took place. O, tributyri-
nase reaction using an extract of 2 eggs with 4 per cent tributyrin for 1 hour;
•, methyl butyrinase from 20 eggs reacting with 3 cc. of 2 per cent methyl butyrate
for 2 hours. B shows the data for methyl butyrinase plotted as log concentration
of HC1, ordinate, versus the reciprocal of the absolute temperature X 104, abscissa.
The points are average values of ten determinations at each temperature. The
straight line in B is fitted by the method of least squares. The M value between
0 and 35° C. is approximately 5700 calories. For further description see text.
The results are summarized in Table III. Both methyl butyrinase and
tributyrinase are inhibited by quinine and NaF. Only tributyrinase is
inhibited by the 0.5 per cent phenol ; methyl butyrinase activity is stimu-
lated. Two per cent phenol will completely block both reactions. Fies-
singer and Gajclos (1936), in studies on the esterase obtained from the
larva of Gallcria uicllonclla, found their enzyme extract unaffected by
ESTERASES IN THE GRASSHOPPER EGG
phenol and quinine and strongly inhibited by NaF in the same concen-
trations as noted above with tributyrin as a substrate.
DISCUSSION
The expectation that the grasshopper egg contains an enzyme capable
of hydrolyzing triglycerides of higher fatty acids was perhaps based
on a fortuitous assumption. The presence of such an enzyme in an
animal metabolizing fat as the R.Q. indicates (Bodine, 1929; Boell,
1935) and consuming 60.3 per cent of its initial store of fats during
development (Slifer, 1930) seemed highly probable. No evidence for
this enzyme could be elicited using the methods described. The activity
TABLE II
0.05N HC1
(in cc.)
Control
Control
Control
NaOl
0.2 cc. -0.4%
NaOl
CaCh
0.4 cc. -2%
Control
CaCh
Substrate
1.19
0.52
0.03
0.06
0.21
0.23
0.74
0.33
Tributyrin
Methyl butyrate
TABLE III
0.05 N HC1
(in cc.)
Control 0.5% Phenol 0.5% NaF 0.5% Quinine HC1 Substrate
1.06 0.53 0.50 0.19 Tributyrin
0.77 1.64 0.19 0.42 Methyl butyrate
on the esters of the lower fatty acid (butyric) was, however, quite high
during early stages of development. A summary of the data concern-
ing the lipids of the grasshopper eggs is of interest. The fat in the
egg of Melanoplus differcntialis is liquid at room temperature (fusion
point, 26.2° C.) ; in Clwrtophaga viridifasciata the fat is solid (fusion
point, 39.4° C.) (Slifer, 1930). The former insect spends the winter
as an egg, the latter as a nymph. The iodine number of the fats is the
same in both animals (135 to 140) (Slifer, 1932). The low melting
point in the winter eggs may possibly be due to the higher proportion
of short chain fatty acids. This is the explanation of the liquid fat of
the aphid, Pemphigus, which contains glycerides of butyric, caprylic and
lauric acids (Timon-David, 1927-28). The presence of monoesters
384 LOREN D. CARLSON
rather than glycerol triesters might give similar results. The data con-
cerning the enzymes present in the grasshopper egg lend credence to the
assumption that the lower fatty acids are present in the egg. Slifer
(1930) has shown that the total fat (measured after saponification by a
method for higher fatty acids) decreases only slightly during prediapause
(9.7 per cent), yet the volume of the lipoidal layer as measured by
Bodine et al. (1939) decreases 32.5 per cent in the same period. Slifer
(1930) found a loss of 50 per cent in postdiapause, the volume deter-
minations, 42.5 per cent. The amount of fatty acids in a diapause egg
is approximately 8 per cent of the wet weight of the egg (Slifer, 1930)
as compared to an amount of lipid equal to 3% per cent of the wet
weight of the egg obtained by the centrifuge separation. The fat
obtained by this latter method is a mixture of esters (probably glycerol)
which contains C12 to C1S fatty acids (Allen, T. H., personal communi-
cation). Experiments to determine the hydrolysis of the lipid sep-
arated by centrifuging and also lipids extracted from the egg brei with
petrol ether showed demonstrable amounts of hydrolysis after a 24-hour
period only in the case of the latter. This might well be due to the
existence of esters and acids in equilibrium.
The relative amounts of hydrolysis in these two enzymatic reactions
cannot be quantitatively compared with the activity of esterases from
other sources. In general it seems evident that the enzymes are rela-
tively concentrated in the grasshopper egg, since experiments described
with other esterases involve periods of four hours and upwards at 37° C.
to produce enough acid to be measured. Fiessinger and Gajdos (1936)
found that the tributyr-inase from the larva of Galleria mellonclla was
much more active than that from human blood serum (ca. 10 times).
They also could demonstrate no reaction with olive oil as a substrate.
The two lipolytic enzymes possess strikingly different physical and
chemical properties as evidenced by the independent change in potency
during development, the inactivation by heat, the effect of temperature
on the rate of hydrolysis, the possibility of separating the two enzymes,
and the difference in effect of the inhibitors used. Curiously, the tribu-
tyrinase, per sc, is less sensitive to heat treatment than methyl butyrinase
yet more susceptible to temperature in the presence of its substrate.
This may be due to a reversal of the heat inactivation in the former case.
The evidence indicates the lipolytic enzymes in the grasshopper are
present in greatest quantities at the time the egg is laid. From these
high levels at the time of least differentiation in the egg the enzymes
decrease in amount during development or differentiation (Fig. 1). A
change in the amounts of esterase in the egg of the trout (Salmo fario)
was observed by Falk and co-workers in a careful and detailed study of
ESTERASES IN THE GRASSHOPPER EGG 385
this material. Methyl butyrate was not hydrolyzed by the esterase from
the unfertilized egg, but the hydrolysis was accomplished by eggs 35 or
more days after fertilization. Methyl and ethyl acetates were easily
hydrolyzed by the egg but steadily less so as development proceeded ;
ethyl butyrate showed a reverse effect. The value of esterase action
generally was high in immature eggs, small in mature eggs, increasing
with development (see Needham, 1931, for summary). In the work
of Falk et al. cited here no data are given for the esters of the long
chain fatty acids. In the grasshopper egg the decline in the activity of
the monobutyrinase after the tenth day of prediapause development oc-
curs somewhat later than the decline in potency of the natural activator
(presumably a lipid) of protyrosinase (Bodine et al., 1939). It is
possible that some of the substances serving as activators are mono-
esters of fatty acids and that these are utilized rapidly in early devel-
opment. Subsequent to this period the amount of monobutyrinase falls.
However, the explanation of this effect suggested by Bodine and Carlson
(1940) seems more tenable. The decline in the amounts of both en-
zymes studied during post-diapause development seems correlated with
the rapid disappearance of yolk. The possibility that these enzymes may
be found in the serosa has not been excluded in these experiments, yet
the major part seems to be contained in the yolk and probably is incor-
porated into the midgut after its absorption. This conforms to the
evidence of Stuart (1935) that the yolk cells become part of the midgut
just previous to hatching. The cells of the intestinal tract then " in-
herit " these enzymes from the yolk. Other hydrolytic enzymes may
come to be in the gut of the adult in a similar manner.
SUMMARY
1. Glycerol extracts of the grasshopper egg (Mclanoplus diffcrcn-
tialis) have been tested for hydrolytic activity on methyl butyrate,
tributyrin and olive oil during various stages in the development of the
egg. The ability to hydrolyze methyl butyrate is high when the egg
is laid ; this value declines between the tenth and fifteenth day of devel-
opment, remains constant during diapause and slowly declines again
during the post-diapause period. The action of extracts on tributyrin
is much stronger, remains constant from the time of laying until the
cessation of the diapause and then declines markedly. No action on
olive oil could be demonstrated.
2. Optimum activity in hydrolysis of tributyrin is at pH 6; the
activity of the enzyme reacting with methyl butyrate is only slightly
affected by changes in the H-ion concentration.
386 LOREN D. CARLSON
3. Temperature affected the methyl butyrinase and tributyrinase ac-
tivity in a different manner. Exposure to temperatures above 55° C.
depressed the activity of the former while the activity of the latter per-
sisted to 65° C.
4. The hydrolytic action on tributyrin increased with temperature
between 5° and 25° C. and declined at higher temperatures. Methyl
butyrinase activity increased with temperature between 0° and 45° C.
5. The esterases seemed to be associated with the yolk until just
before hatching.
6. The effect of sodium oleate, calcium ion and various inhibitors of
lipolytic enzymes on the extracts used were determined.
The author wishes to express his appreciation to Professor J. H.
Bodine for his helpful advice and criticism.
LITERATURE CITED
BODANSKY, O., 1937. The use of different measures of reaction velocity in the
study of the kinetics of biochemical reactions. Jour. Biol. Clicin., 120:
555-574.
BODINE, J. H., 1929. Factors influencing the rate of respiratory metabolism of a
developing egg (Orthoptera). Physiol. Zoo!., 2: 459-482.
BODINE, J. H., AND T. H. ALLEN, 1938. Enzymes in Ontogenesis (Orthoptera).
IV. Natural and artificial conditions governing the action of tyrosinase.
Jour. Cell, and Comp. Physiol., 11: 409-423.
BODINE, J. H., AND L. D. CARLSON, 1940. Enzymes in ontogenesis (Orthoptera).
X. The effects of temperature on the activity of the naturally occurring
and other activators of protyrosinase. Jour. Cell, and Comp. Physiol., 16 :
71-83.
BODINE, J. H., L. D. CARLSON, AND O. M. RAY, 1940. Enzymes in ontogenesis
(Orthoptera). XII. Some physiological changes in eggs the embryos of
\vhich have been destroyed by X-irradiation. Biol. Bull., 78: 437-443.
BODINE, J. H., O. M. RAY, T. H. ALLEN, AND L. D. CARLSON, 1939. Enzymes in
ontogenesis (Orthoptera). VIII. Changes in the properties of the natural
activators of protyrosinase during the course of embryonic development.
Jour. Cell, and Comp. Physiol., 14: 173-181.
BOELL, E. J., 1935. Respiratory quotients during embryonic development (Or-
thoptera). Jour. Cell, and Comp. Physiol., 6: 369-385.
CARLSON, L. D., 1940. Lipolytic enzymes during the development of the grass-
hopper egg. A not. Rcc., 78, Suppl., 160 (Abstr.).
EVANS, T. C., 1936. Qualitative and quantitative changes in radiosensitivity of
grasshopper eggs during early development. Physiol. Zool., 9: 443-454.
FALK, K. G., 1924. Chemistry of Enzyme Actions. Chemical Catalogue Co.
FALK, K. G., AND K. SUGIURA, 1915. Studies on enzyme action. XII. The esterase
and lipase of castor beans. Jour. Am. Chcm. Soc., 37: 217-230.
FIESSINGER, N., AND A. GAjoos, 1936. Le Ferment Lipolytique de Galleria mel-
lonella. Compt. Rend, Soc. Biol., 121: 1152-1154.
NEEDHAM, J., 1931. Chemical Embryology, Vol. Ill, p. 1295. University Press,
Cambridge.
OPPENHEIMER, C., 1936. Die Fermente und Ihre Wirkungen. Supplement I.
The Hague.
ESTERASES IN THE GRASSHOPPER EGG 387
SLIFER, E. H., 1930. Insect development. I. Fatty acids in the grasshopper egg.
Physiol. Zoo}., 3: 503-518.
— , 1931. Insect development. II. Mitotic activity in the grasshopper embryo.
Jour. Morph. and Physiol., 51 : 613-618.
— , 1932. Insect development. V. Qualitative studies on the fatty acids from
grasshopper eggs. Physiol. Zoo/.. 5: 448-456.
STUART, R. R., 1935. The development of the mid-intestine in Melanoplus differ-
entialis (Acrididae: Orthoptera). Jour. Morph., 58: 419-437.
TIMON-DAVID, T., 1927-28. Quoted from Wigglesworth, V. B., 1939. Insect
Physiology. Methuen.
ENZYMES IN ONTOGENESIS (ORTHOPTERA)
XIX. PROTYROSINASE AND MORPHOLOGICAL INTEGRITY OF
GRASSHOPPER EGGS *
JOSEPH HALL BODINE AND THOMAS HUNTER ALLEN
(From the Zoological Laboratory, State University of loiva)
Although protyrosinase has been found in extracts of grasshopper
eggs, no evidence for its existence within the intact egg has been pre-
sented. In view of the possibility that the very process of extraction
might inactivate the enzyme, it seems desirable to examine the relation
of protyrosinase to morphological integrity. It should be possible to
perform such a test by subjecting eggs to one of those treatments which
cause the activation of extracted protyrosinase. An increased rate of
oxygen uptake and the appearance of melanin in the intact egg should
then indicate that protyrosinase had been present before its transition
into tyrosinase. This paper deals with results of experiments showing
the occurrence of protyrosinase within the intact egg of a grasshopper,
Melanoplus differentials (Thomas).
The data which are graphically illustrated in the accompanying figure
were obtained from recordings of a Warburg apparatus operated at
24.9° C. The time course of oxygen uptake was plotted for groups of
100 intact eggs which had just previously been heated for five minutes
in water kept at certain indicated temperatures. The rates of oxygen
uptake of diapause eggs heated between 62° to 85° C. remained constant
through the first 100 cu.nim. but declined as a limiting volume of 225
to 230 cu.mm. was approached. However, the rates of oxygen uptake
of eggs which had been exposed to temperatures below 50° C. were
constant. Relative values for the velocity of oxygen uptake may thus
be given by the reciprocal of the time in minutes for the utilization of
the initial 100 cu.mm. of oxygen. When these values are compared, a
complex temperature effect is found (see figure). It is proposed to
interpret this effect according to the properties and occurrence of
protyrosinase.
If an egg extract containing protyrosinase is heated for five minutes
at temperatures between 60° and 85° C., a tyrosinase is formed (Bodine
1 Aided by a grant from the Rockefeller Foundation for work in cellular
physiology.
388
PROTYROSINASE WITHIN INTACT EGGS
389
and Allen, 1938). Heating seems to affect the stability of both pro-
tyrosinase and tyrosinase. With ascending temperature the former is
activated, while the latter is destroyed. Consequently, the tyrosinase
activity of an extract increases from 60° to 75° but declines from 75°
20.
5-
0.
0_J
30
I
50
70
90
FIG. 1. The effect of heat treatment on the oxygen uptake of grasshopper eggs
in various stages of development. Ordinate, reciprocal of the time in minutes for
the uptake of 100 cu.mm. of oxygen multiplied by 1000 ; abscissa, temperatures in
°C. to which eggs were exposed for five minutes. Curve B, 7-day eggs (predia-
pause) ; curve A, 60-day eggs (diapause) ; curve C, eggs 3 days post-diapause.
to 90°. A similar differential effect of heat is found for the velocity
of oxygen uptake of intact diapause or post diapause eggs (see figure,
curve A and C). Since protyrosinase and a naturally occurring sub-
strate can be extracted from eggs of these stages (Bodine, Allen, and
390 J. H. BODINE AND T. H. ALLEN
Boell, 1937), it appears that the increased velocity of oxygen uptake of
the intact egg must be due to the heat-induced enzymic oxidation of the
native substrate. Curve C is presumably higher than curve A, because
in post diapause there is more native substrate than in diapause (Bodine,
Allen, and Boell, 1937).
The latter interpretation also seems to be supported by the eventual
formation of melanin, by the low value for the " respiratory, quotient,"
and by the sensitivity to cyanide. Diapause eggs, which six hours pre-
viously had been heated between 62° to 84° C., changed from a pale
lemon yellow to a dark olive-green color. Upon dissection it seemed that
the darker color was due to the presence of a brown pigment — melanin-
located in the " liquid-filled space" (Slifer, 1937) between the serosa
and cuticle. Similar eggs heated below 62° and above 84° C. remained
a lemon yellow, because their protyrosinase supposedly had either not
been activated or else had been destroyed. From measurements of the
oxygen uptake and carbon dioxide production performed according to
the indirect method of Warburg (Dixon, 1934), an R.Q. of 0.1 to 0.2
was found for eggs that had been heated at 75°. Such a value is to be
expected during the production of melanin (Raper, 1928). Potassium
cyanide in a concentration of 0.01 M abolished the oxygen uptake pro-
duced by heat activation. These properties are usually considered to
be characteristic of a tyrosinase reaction.
Since protyrosinase has not been found in extracts of eggs younger
than. eight to nine days of age (Bodine, Allen, and Boell, 1937), one
should not expect an increased velocity of oxygen uptake for seven-day
eggs that have been exposed to those various degrees of heat sufficient
for activating protyrosinase. The occurrence of such a phenomenon
would serve essentially as a control experiment for the heat treatment
of those eggs containing protyrosinase (see figure, curve B}. The
respiratory processes of prediapause and diapause eggs are evidently
susceptible to the effects of heat. Perhaps the normally working re-
spiratory enzymes are destroyed at 56° C. (see figure). If such be the
case, it may then be supposed that the portion of the curve for diapause
or post-diapause eggs between 62° and 85° C. pertains entirely to the
activation of protyrosinase and the destruction of tyrosinase.
The addition of an activator followed by the formation of an enzyme
presumably should indicate through cause and effect relations that a
proenzyme had once been present. It therefore seems that heat treat-
ment has demonstrated the occurrence of protyrosinase as a constituent
of diapause and post-diapause grasshopper eggs. This demonstration of
protyrosinase seems to be independent of the trituration and dilution
PROTYROSINASE WITHIN INTACT EGGS 391
inherent to an extraction process. Thus it appears that protyrosinase
exists within the intact grasshopper egg and that this protyrosinase does
not lose characteristic properties as a result of extraction. Moreover,
these deductions on the occurrence of the inactive rather than the active
enzyme would lead to the conclusion that oxidations coupled with a
tyrosinase reaction (Allen and Bodine, 1940) can hardly be expected to
complement the respiratory processes of these eggs. Although extracted
protyrosinase can be activated by an oil native to these eggs (Bodine,
Allen, and Boell, 1937), this lipide is probably bound to various proteins
or isolated in such a way that it is inaccessible to the protyrosinase of
intact eggs (Bodine and Carlson, 1940).
SUMMARY
Protyrosinase occurs in the intact egg of the grasshopper, Melano-
plus differcntialis, and shows properties similar to those for extracts
prepared by trituration of the eggs. Moreover, it seems that protyro-
sinase, as a naturally occurring entity, is not an artefact produced by
extraction procedures.
LITERATURE CITED
ALLEN, T. H., AND J. H. BODINE, 1940. Enzymes in ontogenesis (Orthoptera).
XIII. Activation of protyrosinase and the oxidation of ascorbic acid.
Jour. Gen. Physio!., 24 : 99-103.
BODINE, J. H., AND T. H. ALLEN, 1938. Enzymes in ontogenesis (Orthoptera).
V. Further studies on the activation of the enzyme, tyrosinase. Jour. Cell,
and Comp. Physiol., 12 : 71-84.
BODINE, J. H., T. H. ALLEN, AND E. J. BOELL, 1937. Enzymes in ontogenesis
(Orthoptera). III. Activation of naturally occurring enzymes (tyrosi-
nase). Proc. Soc. Exp. Biol. and Med., 37: 450-453.
BODINE, J. H., AND L. D. CARLSON, 1940. Enzymes in ontogenesis (Orthoptera).
X. The effects of temperature on the activity of the naturally occurring
and other activators of protyrosinase. Jour. Cell, and Comp. Physiol., 16:
71-83.
DIXON, M., 1934. Manometric Methods. Cambridge.
RAPER, H. S., 1928. The aerobic oxidases. Physiol. Ret'., 8 : 245-282.
SLIFER, E. H., 1937. The origin and fate of the membranes surrounding the grass-
hopper egg, etc. Quart. Jour. Micr. Sci, 79 : 493-506.
THE FOUNDING OF ANT COLONIES
LAURENCE J. LAFLEUR
The normal method of founding colonies among the more typical of
formicine species is generally understood to be as follows. The males
and females swarm on a given day and copulate in the air, the males
subsequently dying. Each female then descends to earth, tears off her
wings, and finds a suitable spot for her colony. According to Wheeler x :
" In her cloistered seclusion the queen now passes days, weeks, or even
months, waiting for the eggs to mature in her ovaries. When these eggs
have reached their full volume at the expense of her fat-body and degen-
erating wing-muscles, they are laid, after having been fertilized with a
few of the many thousand spermatozoa stored up in her spermatheca
during the nuptial flight. The queen nurses them in a little packet till
they hatch as minute larvae. These she feeds with a salivary secretion
derived by metabolism from the same source as the eggs, namely, from
her fat-body and wing-muscles. The larvae grow slowly, pupate pre-
maturely and hatch as unusually small but otherwise normal workers.
In some species it takes fully ten months to bring such a brood of minim
workers to maturity, and during all this time the queen takes no nourish-
ment, but merely draws on her reserve tissues. As soon as the workers
mature, they break through the soil and thereby make an entrance to the
nest and establish a communication with the outside world. They en-
large the original chamber and continue the excavation in the form of
galleries. They go forth in search of food and share it with their
exhausted mother, who now exhibits a further and final change in her
behavior. She becomes so exceedingly timid and sensitive to the light
that she hastens to conceal herself on the slightest disturbance to the
nest. She soon becomes utterly indifferent to her progeny, leaving them
entirely to the care of the workers, while she limits her activities to lay-
ing eggs and imbibing liquid food from the tongues of her attendants."
To this general picture I wish to suggest three types of modification ; as
to fasting, hazards to the colony, and cooperative founding.
Experiments have shown that queens can live for ten months or
more without food, and bring up young in the meanwhile. Experiments
have even been made with the precaution of furnishing nothing but
distilled water. But it does not follow that because ants are able to fast
1 William Morton Wheeler, Ants, Columbia University Press, 1910, p. 185.
392
FOUNDING OF ANT COLONIES 393
for long periods that they regularly do so, any more than the fact that
these same ants can withstand immersion implies that they regularly
stay under water for any considerable duration : certainly no one sup-
poses that in nature the queens abide by a diet of distilled water. And
there are a number of reasons to believe that queens regularly leave their
incipient nests to look for food.
For many years I have kept nests of ants, principally small ones
developed from queens taken during swarms or from incipient nests less
than a year old. In searching for incipient nests it has been my experi-
ence that I find as many queens wandering at large as I find queens in
their nests. This does not mean, clearly, that half the queens of in-
cipient nests are away from them at any given time, since the nests may
be very hard to find. One clay this June, for instance, I spent a few
hours in a wooded area looking for young nests under loose bark. Ap-
propriate trees were rare, and most of them were preempted by well-
established colonies, so that I failed to find a single incipient nest. But
there may have been several so well hidden that I did not find them, and
there were undoubtedly hundreds of such nests in the soil, where I was
not concerned to look for them. If any of these queens left her nest,
however, the chances were good that I would spot her, and I did in
fact so find a queen of an earth-nesting species that had probably
swarmed the previous fall. This and many other similar experiences
serve to convince me that it is by no means rare for a queen to leave
her nest.
What is the purpose of these excursions? For several days after a
swarm queens may be observed in decreasing numbers. Some of these
may be late swarmers, but not many, as winged females are not found in
comparable numbers. Here the reason is undoubtedly the search for
more suitable quarters. During the remainder of the year, queens are
less commonly observed, and the motive of their wandering is hunger.
Invariably, when these queens are put in artificial nests, their first act is
to eat heartily. The queen referred to in the previous paragraph spent
more than an hour and a half continuously imbibing honey.
It may well be that many queens, attempting to found a nest on the
starvation basis heretofore described, fail, and after consuming all their
progeny wander forth to be detected by the myrmecologist. This is
directly suggested by the fact that in several recorded experiments where
queens failed to rear colonies, they eventually sought to escape. There
is also the possibility, more significant if true, that queens may leave
their incipient colonies in a perfectly healthy state while they forage for
food. In artificial nests they frequently leave their brood to obtain
honey or other foodstuffs some distance away within the nest. It should
394 LAURENCE J. LAFLEUR
also be mentioned that a few queens in artificial nests show no interest
in food made available to them. In only one case in my experience,
however, has the queen died, and as this occurred with a species with
which I have been uniformly unsuccessful, the single case of the queen
that died without eating is hardly significant in view of the two score
that ate and died too. The situation here is artificial, in that no bar-
riers are placed between the queen and the food, and she does not have
to tunnel out or tear down a wall as would usually be the case in nature.
One observation of mine, however, throws direct light on this situa-
tion. In September, 1940, while waiting for the ferry at Hadlyme,
Connecticut, I removed the sole piece of bark from an old fallen log
and discovered underneath a typical incipient nest of Camponotus penn-
sylvanicus containing three pupae besides some eggs and small larvae.
The queen was absent, but I soon saw her hurrying in a straight line
for her nest. She was about two yards away wrhen I first noticed her.
Here we have a case where the queen, without the intervention of any
artificial circumstances, was absent from her nest, possibly in the search
for food, while that nest was in a perfectly healthy condition. On the
other hand, a few queens in my own nests have failed to eat food pro-
vided within the nest, but these queens have been unsuccessful in every
case.
Likewise, one occasionally finds incipient nests with such a consid-
erable quantity of young that it is extremely unlikely that the queens
have existed on a starvation basis. For example, in a very populous
stump in Arlington, Vermont, I found a number of isolated queens of
Camponotus noveboraccnsis that had undoubtedly swarmed the same
year. In a few cases I was able to examine the cavity carefully and to
take a census that was accurate as to pupae and larvae, although possibly
incomplete in the count of eggs. One queen had four pupae, two larvae,
and three eggs ; another three pupae, three larvae of pupal size, and five
eggs; a third three pupae, two large larvae, and fifteen eggs. Pupae
and larvae about to pupate have consumed all the food that they eat
before they emerge as adults ; consequently these queens had provided
the food for four, six, and five workers respectively, and had consider-
able numbers of other young as well. The queen of Camponotus pciiu-
sylvanicus previously mentioned as having been taken while returning
from a foraging trip had three pupae besides a considerable number
of small larvae and eggs.
If starvation is less of a hazard to ant queens than has been generally
supposed, there is nevertheless a terrific mortality from other causes
even when the queen survives the day of the swarm. Many queens
must be killed while foraging, or have their nests invaded by other ants
FOUNDING OF ANT COLONIES
or hostile insects ; some succumb to parasites or to fungoid growths ; and
many must find themselves in such an unfavorable environment that
their callow offspring, on emerging, find insufficient food or are killed
or captured by other ants.
Besides these obvious perils, experience with a large number of
incipient colonies in my artificial nests has indicated the importance of
a number of other factors, an importance wrhich seems to me not inferior
to that of obtaining food. These factors are the following:
1. There is a very considerable mortality rate among queens during
the first few weeks after swarming. This I estimate at 20 per cent, a
figure which has held true from species to species, year in and year out,
despite the best of laboratory care and even when the queens are
adopted by workers of their own species or are given callow young
immediately.
2. Some queens fail to take necessary sanitary precautions, fouling
their nests and allowing mold to destroy their young and themselves.
This is one of the many faults I have noticed in Prenolcpsis queens.
3. Some queens fail to lay eggs at all; or, having laid some, cease to
do so. Unless ovipositing recommences within a month or two this has
always led, in my experience, to the death of the queens. Six queens
of Formica subscricea were taken by me on August 8, 1940. One of
them died in the first few weeks, leaving five, of which one failed to lay
eggs. She died on January 16, 1941. Another queen of this group
experienced difficulty in laying an egg ; and on February 5 I observed
her bent double for over ten minutes, an egg occasionally visible in her
cloacal orifice. Later, however, she became normal in her egg-laying.
A third queen of this group was badly mauled by the others on January
2, but was successful with her colony until she ceased laying eggs about
February 20. She died on April 18. Of seven Camponotus novebora-
censis queens taken on July 6, 1941, and previously mentioned in this
article, one died on July 11 for no apparent reason, and a second, failing
to lay any eggs after being taken, died on July 24.
4. Sometimes queens neglect to collect their eggs, allowing them to
die and become moldy wherever dropped. Prcnolepsis is particularly
prone to do this. A similar fault is to drop eggs into crevices whence
the queen is later unable to extract them. Prcnolepsis is again a fre-
quent offender in this way, and I have observed it not infrequently with
Cremastogaster. Of course, any ant may occasionally lose an egg in a
crack, if one exists in the vicinity. A curious incident occurred, how-
ever, with one queen of Formica subsericea, who developed a mania for
hiding her eggs. A number were shoved as far as possible under the
rim of a bowl, where she could not extricate them, and others were
396 LAURENCE J. LAFLEUR
hidden individually in wet cotton, where they were forgotten. When
moved to another nest and given a worker, this queen became perfectly
normal and successful.
5. Some queens eat all their progeny regardless of the presence of
other food. In mid-August, 1923, a period when other queens had
many larvae, pupae, and even callow young, I took a queen of Canipo-
iwtus pennsylvanicus without young. On July 26, 1941, I took a
Camponotus novcboraccnsis queen with only one medium-sized larva.
On the twenty-seventh the larva had disappeared and an egg was pres-
ent, although there was honey and meat in her cell. By July 31 the
egg, together with a second laid subsequently, had disappeared ; and for
the next two wreeks eggs were eaten shortly after being laid. A queen
of Formica subscricca laid four eggs on November 26, 1940. One was
eaten that evening. Another was laid on the twenty-seventh, and one
eaten November 28. Two more were laid November 29, but all were
eaten by December 12. While this article was being written, two
affiliated queens of this species devoured the fifteen eggs they had col-
lected, and have not acquired any more in the two weeks since that event.
6. That a few eggs should be eaten to further the development of
the more advanced young is natural enough, but I once saw a Campono-
tus pennsylvanicus eat her most advanced larva, and on several occa-
sions larvae have been consumed in the presence of both pupae and eggs ;
and once or twice pupae have been eaten in the presence of older pupae,
as well as larvae and eggs. A Camponotus ferrugineus queen and one
worker devoured three well-formed naked pupae, but saved a medium-
sized larva.
7. In one nest larvae twice failed to grow, either because of some
constitutional defect or because they were not fed. This instance is
that of the Camponotus pennsylvanicus queen taken in September, 1940
and already mentioned in another connection. Of the three pupae, two
developed normally and one was stillborn : the small larvae remained
small, however, from September until the following June, when all but
one disappeared during some affiliation experiments. In this period
eggs developed normally into larvae, but failed to grow in the larval
stage. The larvae appeared healthy enough, except that their skins were
less shiny than normal. At the conclusion of the affiliation experiment
this nest contained only the queen and one larva of the queen's first year
progeny, two alien workers, and five alien males. A new lot of eggs
began to be laid on June 1, just before the experiment, and these started
hatching July 17. Once more, however, despite the presence of a new
lot of workers, the larvae failed to grow.
FOUNDING OF ANT COLONIES 397
8. When larvae pupate and commence to spin, the queens sometimes
allow eggs and small larvae to become entangled in the spun silk of the
pupating larva, usually with fatal results. This failure might be much
more rare under natural conditions, where the pupating larva can be
buried in earth. The same Camponotus queen previously mentioned
invariably put her whole stock of eggs and small larvae on any cne of
the introduced male larvae which happened to be pupating at the moment.
If these were rescued, she restored the status quo ante as soon as the
human intervention was over. In fact, it is to this behavior that I
ascribe the eventual death of the first-year eggs and larvae some nine
months from their first appearance.
9. A not infrequent fault in incipient nests is the failure to open
cocoons, though this is the fault of workers more commonly than of
queens. One Formica nitidiucntris queen lost ten out of fifteen cocoons
in this way. Four were saved only because I opened them, and the fif-
teenth was tenderly guarded by the queen, long after its demise was
evident (on close application) even to human nostrils. These four were
removed to another nest, after which the sixteenth and seventeenth
cocoons were successfully opened.
10. It is- less easy to understand why cocoons are sometimes partially
opened, and the unborn ant allowed vainly to struggle with one leg or
antenna protruding, or perhaps a whole head. Yet this happens not
infrequently, while the queen and sister workers pay no attention. The
third worker of the same Camponotus pcnnsylvanlcus queen struggled
for forty hours with only its head emerged, before I took pity on it and
released it.
11. When the cocoons are opened, the membranes may not be com-
pletely removed. As these dry, they contract and twist the still supple
exoskeleton into awkward and useless shapes. At best this results in
a curving and weakening of a leg — most commonly one of the hind
ones — at worst it results in the complete incapacitation of one or more
members. Some examples may be given: a Camponotus noveboracensis
queen left her third worker hampered by membranes around both hind
legs, and I removed these membranes thirty-six hours after birth. A
Formica snbsericca colony worked on the tenth callow to be born for six
hours without removing all the membranes. A Crcmastogaster queen
left her first worker swathed in membranes for twenty-four hours, after
which my efforts to save it were unsuccessful. In a colony that con-
tained three queens of Formica subsericca, fighting among the queens
prevented proper attention to the young, and several died ; one had
badly deformed antennae ; and two were saved only by my intervention.
In the nest of Formica nitidiventris, already mentioned, the third worker
398 LAURENCE J. LAFLEUR
born (exclusive of the fifteen cocoons of early vintage) was left with
membranes binding one hind leg to the gaster.
12. The infant mortality rate, among ants as among other animals,
is higher than the rate at any other period. In a healthy and well-
developed nest it may be negligible, with incipient nests I expect to lose
between 5 and 10 per cent of callows through stillbirth and death in the
first two weeks of life. This estimate does not include losses through
failure to open cocoons or remove membranes.
13. The young workers, when they arrive, may be deficient in very
much the same ways as the queens. One additional defect, however, is
failure to forage. During the nine months that her two workers lived
with the Camponotns pennsylvanicus queen before mentioned, they
failed to seek food, but obtained it instead by regurgitation from the
queen.
14. Workers may fail to keep the nest clean. In my experience this
is particularly true of Formica ncoclncrca, and of slave-making ants
where this species is used as slave. The related Formica subscricca is
also addicted to this carelessness, although to a lesser extent.
15. Misdirected activity with regard to eggs is not unknown among
workers. In a nest of Lasius which I collected in 1936, all the pupae
were allowed to soak in the water compartment. As this would kill the
pupae in short order, I turned out the whole nest. A few hours later
one worker began putting the pupae in the water once more, but I
removed them and the act was not repeated.
16. Workers may eat the young. In a nest of Formica subsericea
containing one queen and one worker, the latter ate the eggs laid by the
queen despite the presence of other food. She never molested one egg,
but would eat any excess over this number. Finally, after the death of
the queen, she was left alone with this one, and nursed it to medium-
large size. Then she affiliated with another queen, and did not return
to her infantivorous practices. In another nest of this species the two
workers consumed the only young then present, which consisted of five
eggs.
17. In small nests, inexperienced workers are more prone than queens
to leave cocoons unopened. In larger nests, one at least of the workers
will usually be successful in caring for the young, and the others take
their cue from her. In nests started with nothing more than larvae and
pupae, some of the latter being opened by hand, it is usually necessary
to continue opening operations for some time before the workers take
over. Somewhat the same conditions prevail in an incipient nest, and
if the queen immediately leaves everything to the first few workers born,
which she frequently does, one or two callows are apt to die or be born
crippled before the workers become adept at their jobs. In one nest of
FOUNDING OF ANT COLONIES 399
Formica sitbscricca the eighteenth worker to be born was left helpless
in a half -opened cocoon and was relieved of her cocoon and membranes
by me some ten hours after her birth.
It is interesting to notice, in passing, that when callows are being
relieved of membranes, they almost invariably submit willingly to the
necessary treatment. When picked up, the callow tries to escape, al-
though its efforts are much less violent than those of a full-grown ant.
But as soon as the point of a teasing needle is applied in an effort to
remove the membrane, the callow remains quiet without being held until
the operation is over. I have known adult ants to act in the same way
when I have attempted to remove wax which adhered to them and inter-
fered with their movements.
Another point of interest is that when a queen shows a failure in
one aspect of nest-building, that queen and her progeny are very apt
to show degeneracy in other ways as well. Consider the case of the
Camponotus pennsylvanicus queen. Her first worker was the only one
born successfully. The second was stillborn ; the third required my aid
at birth. Though they were ready enough to defend the nest, these two
workers failed to aid the queen in caring for the young, or in foraging
for food. And in two successive seasons, the larvae failed to grow
beyond minimum size. Such a series of mishaps could not occur in
nature, for any one might well be fatal, and any two would almost
certainly be so.
We now pass to the third and last modification of the usual picture
of colony foundation. While queens are capable of founding nests un-
aided, they may receive aid in doing so, and this method seems to me to
be more important than has been generally realized. It is well known
that certain species are temporarily or permanently parasitic; and that
in other species minims accompany the queen on the marriage flight.
It is also known that queens sometimes collaborate in the founding of
nests and that a large colony may retain many of its own females as
additional queens, but these facts have not been given their full weight.
My own experience is that in the two highest subfamilies — and these
include all the well-known ants of the north temperate zone — polygynous
colonies are just as typical as monogynous. In some cases, indeed, the
number of queens is fantastic. Windsor - reports that he removed
forty-nine dealeated queens of Formica neocinerea from one spadeful
of earth, and in opening a nest of Formica sanguinca subintegra his im-
pression was that the queens were almost as numerous as the workers.
Furthermore, on the day of swarming and for a few days thereafter,
the queens of most species are exceptionally ready to form alliances, and
- Reported to me by letter and to be published in " Anti-Social Behavior among
Ants," Journal of Comparative Psychology, circa April, 1942.
400 LAURENCE J. LAFLEUR
any number can be put together successfully. This even applies to
queens of different species, if they happen to swarm within a day or so
of each other. This was forcefully demonstrated while this paper was
being written, for Lasiiis and AcaiitJwuiyops 3 swarmed on the same day
and two boys who had been asked to collect queens for me put sixty of
the former and eighteen of the latter in one jar. The group was entirely
peaceable, and no casualties whatever resulted from the strange mixture.
The rule is not true of all species, however, nor of all queens of any
species. When Crcmastogastcr swarmed this year, I collected three
groups of six queens each, and one of four. In one group one queen
killed all the others; in a second only one queen died, while no injuries
occurred in the other two.
An instance is on record where a nest divided into two sections,
which gradually separated and became distinct colonies. This process
of colony formation is probably quite important: that it is so is indi-
cated by the fact that many nests in a given area fraternize (sic) with
each other.
It must happen not infrequently that colonies lose their queens and
descend towards extinction. Under such circumstances the workers are
exceptionally ready to adopt queens of their own or even of a related
species. The same is probably true of colonies whose queens have be-
come infertile, and possibly for colonies that have undergone other dis-
couraging misadventures. When large colonies are deprived of their
queen, their morale may be shattered and groups of workers migrate
into suitable holes and shelters in the vicinity. In such cases it is pos-
sible for several groups to adopt queens and for one colony to aid in
the development of several new nests. The evidence for this consists
partly of the fact, easily determined by experiment, that queens and
workers affiliate more readily than do queens alone or workers alone.
It is also easy to show that small, queenless, or demoralized groups of
workers affiliate more readily than do normal colonies, and this principle
has been successfully applied by me in artificially inducing affiliation.
On several occasions, indeed, such groups have actively sought affilia-
tion, as when a group of about two hundred queenless workers of
Tctramorium cacspititm forced their way into an alien colony of fifty
workers and several females and affiliated with them. Third, I have
noticed in both natural and artificial nests that when deprived of a queen
and of young the workers exhibit a tendency to wander, and to congre-
gate in small groups of from five to fifty. And lastly, the well-known
behavior of many of the permanent parasites demonstrates that queens
can get themselves accepted by another colony.
3 Acanthomyops inurphyi. determination by William S. Creighton. The Lasiiis
were principally L. amcricanns, with two -mix t us and one ncarcticiis.
FOUNDING OF ANT COLONIES- 401
We now come to the last method by which queens may receive aid
in founding a colony. In nature, worker ants must occasionally wander
so far that they are unable to find their way home, and others must be
carried away by wind or water, or be transferred to new areas by cling-
ing to birds, animals, or to human transport. Solitary workers will die
in a day or so at most, but I seriously doubt whether the majority of
strays die in this manner. It appears to me, on the contrary, that most
of these strays will enter other formicaries and there be killed or adopted,
largely dependent upon the size of the community entered. If an object
being explored by an ant be removed to some considerable distance from
her nest, the typical behavior may be readily observed. At first the
worker shows the normal exploratory behavior : she examines crevices
for food, and will capture any available. If she meets an alien ant she
avoids her, but without any appearance of panic. Sooner or later she
apparently becomes aware that she is lost : her movements are now more
rapid, she usually ignores food (except for an unusually luscious tidbit
such as a drop of honey), and she exhibits fright in contact with alien
ants. If she is put into another nest during this period she tries to
escape but rarely fights back if attacked. Still later another change may
be noted : the ant apparently abandons hope of finding her own nest,
and there are two conditions frequently met. In one the worker be-
comes quiescent, with only a quivering of an antenna or leg to indicate
life, and dies within a day — sometimes within an hour.. In the second
typical condition the worker becomes interested in other ants and will
actively seek alliances with them. In either case the worker forms al-
liances readily, although only passively in the first type. Even after
making alliances, however, workers of the first type occasionally die or
wander off in a few days. It is quite clear that these stray workers, who
cannot be excessively rare, form an accessible auxiliary to queens who
are founding nests.
I have observed a related type of behavior in — so far — only a single
species. Almost any worker of Camponotus novcboracensis will affili-
ate with any solitary queen if she wanders or is put into the latter's
nest. On several occasions I have seen such workers feed the queen
and care for the young for a day or so, and then seek to leave. This
raises the question whether this temporary affiliation can occur at all
frequently in nature. Somewhat similar behavior has been previously
reported of Acanthomyops, which are said to feed alien workers that
enter the nest. I have been unable to verify this behavior. If either
type occurs, it would almost amount to a confederation of a whole
species for mutual aid, a condition previously unknown in the insect
world, and infrequent elsewhere.
STUDIES ON EXPERIMENTAL HAPLOIDY IN
SALAMANDER LARVAE
II. CYTOLOGICAL STUDIES ON ANDROGENETIC EGGS OF TRITURUS
VIRIDESCENS
CORNELIUS T. KAYLOR
(From the Department of Anatomy, Medical College, Syracuse University and the
Marine Biological Laboratory, Woods Hole, Massachusetts')
INTRODUCTION
One of the outstanding features of the experiments on androgenesis
with eggs of salamanders has been the high rate of mortality during
cleavage and gastrulation (Fankhauser, 1934a; Kaylor, 1937). How-
ever, there have been surprisingly few investigations on the cytology of
failure of development in these early stages of androgenetic develop-
ment. The most extensive observations have been those of Fankhauser
(1934, a and b) on androgenetic egg fragments of Triton paltnatus and
more recently of Fankhauser and Moore (1941) on androgenetic eggs
of Triturus viridesccns. There have been a number of cytological
studies on parthenogenesis in eggs of frogs (review of literature, Par-
menter, 1933), but these have been concerned more with the role of the
nucleus in early development of the egg (Dalcq, 1932) or with the origin
of diploid and higher numbers of chromosomes (Parmenter, 1933, 1940)
in cells of eggs and embryos rather than some of the factors underlying
a failure of development beyond certain stages.
In view of the scarcity of studies on the cytology of early stages of
androgenetic development in salamanders, the present study seemed to
be indicated. It is a survey of the microscopical evidences of the causes
of cessation of development in androgenetic eggs of Triturus viridescens.
A preliminary cytological examination (Kaylor, 1939) showed that an
irregular distribution of chromosomes had taken place in these eggs, as
in the merogonic eggs of Fankhauser, and was probably responsible for
the arrested development, since in this type of experiment no injury to
the existing organization of the egg is possible.
MATERIAL AND METHODS
Material
During the course of experiments on androgenesis in Triturus viri-
descens (Kaylor, 1937, and later experiments not published) consider-
402
HAPLOIDY IN SALAMANDER LARVAE 403
able material was preserved for future cytological studies. Of this ma-
terial, 63 eggs which had ceased development during early cleavage,
blastula or gastrula stages were selected for cytological examination.
Fifty-nine of these eggs had actually completed their developmental pos-
sibilities; they were fixed either after they had remained in the same
stage for 12 hours or more or at the onset of cytolysis as indicated by a
beginning discoloration of some of the cells. Four of the eggs were
preserved because of broken yolk membranes.
Methods
Experimental. — The technique used in obtaining these androgenetic
eggs has already been described in detail (Kaylor, 1937). It consists
essentially of the removal of the second maturation spindle from the
egg by puncturing the polar area containing the spindle with a fine glass
needle and sucking a small amount of material into a capillary pipette.
The egg then develops with only the male, haploid set of chromosomes.
Fixation, Sectioning, Staining. — All eggs wrere fixed in Bouin's fluid,
cleared from 95 per cent alcohol through wintergreen oil, and imbedded
in paraffin containing about 5 per cent bayberry wax. This fixative
hardens the yolk, but satisfactory sections were obtained by soaking the
imbedded eggs in water for 12 to 24 hours, after the first 10 or 12 sec-
tions were cut and mounted : the method used by Fankhauser and
Moore (1941). After this soaking, a complete ribbon of perfect sec-
tions was obtained. The sections were cut at 15 /A, parallel to the animal-
vegetal axis. The sections were stained in Harris' acid-haemalum for
the nuclear stain, eosin as a counterstain for the yolk granules, and
Light green for the spindle fibers. They were then cleared from 95
per cent alcohol through pure aniline oil and mounted in an aniline-
balsam mixture. The use of aniline was necessary since the use of
xylene after the staining and dehydration processes always cracked the
sections.
Figures 1 and 9 were drawn at a magnification of 80 and reduced to
one-half in reproduction.
OBSERVATIONS
Observations on the Living Eggs
To review briefly the former observations on the living androgenetic
eggs of Triturus viridescens (Kaylor, 1937), it was found first of all
that although the majority of the androgenetic eggs underwent irregular
cleavage and died prior to gastrulation, this abnormal cleavage was not
404 CORNELIUS T. KAYLOR
entirely responsible for the early cessation of development, since approxi-
mately one-half of the normally segmenting eggs failed to develop be-
yond the gastrula stage. Secondly, there existed no correlation be-
tween the type of cleavage of an androgenetic egg and the number of
spermatozoa present in the egg at the time of operation. It was ob-
vious, then, that a detailed cytological study of the early development
of androgenetic eggs might determine the causes of the early arrested
development.
FIG. 1. Drawing of a median section of the egg 30.4e, sectioned parallel to the
egg axis. All nuclei projected into this section from neighboring sections. Three
degenerating sperm nuclei : two in prophase, one in telophase. One cytaster. In-
dication of a furrow.
Cytological Observations
The following stages of development of androgenetic eggs were ex-
amined in sections :
Stage of Development Number of Eggs Examined
a. Irregular beginning cleavage
1 . Abortive cleavage 7
2. Early irregular cleavage 5
b. Early blastula 19
c. Late blastula 23
d. Gastrula 9
Total 63
a. Irregular Beginning Cleavage Stages. — 1. Abortive cleavage.
Seven eggs were fixed 25 to 36 hours after operation, during which
time only a few irregular, incomplete furrows had appeared on the egg
surface. These furrows were still visible at the time the eggs were
preserved. Surprisingly enough, in the sections there was no evidence
of furrows in six of the seven eggs (Table I). One egg showed definite
irregular furrowing, not connected with mitotic activity within the egg.
HAPLOIDY IN SALAMANDER LARVAE
405
In each of these eggs there was evidence of early mitotic activity
on the part of the sperm nuclei. The evidence is summarized in Table
I. The cytological condition of each egg showed very little variation.
The majority of sperm nuclei degenerated either before or after early
mitotic activity. Cytasters were present in most of the eggs. Figure 1
illustrates the typical cytological condition encountered. In this par-
ticular egg, three or four sperm entrance marks were present on the
egg surface at the time of operation. Three degenerating nuclei and
TABLE I
Summary of cytological conditions in abortive cleavage stages
Egg
No.
No.
Sperm
Marks
Age
External Appearance
Cytological Condition
hours
26.1c
1
32
Irregular furrows at
animal pole
No furrows, 2 nuclei degenerating in pro-
phase, 1 spindle, no chromosomes
27.5e
3
36
Irregular furrows at
animal pole
No furrows, 1 nucleus degenerating in
prophase
28.6e
3
26
Irregular furrows at
animal pole
No furrows, 3 nuclei degenerating in
prophase
30.4e
3-4
26
One irregular
furrow
Indication of furrows, 2 nuclei degenerating
in prophase, 1 nucleus degenerating in
telophase, 1 cytaster
30.5e
2
36
Irregular furrows at
animal pole
No furrows, 1 cytaster, cytolysis
30.6e
4
25
Irregular furrows at
animal pole
No furrows, 1 degenerating nucleus. 4
cytasters
101. le
6
28
Irregular furrows at
animal pole
No furrows, many degenerating nuclei
one cytaster were actually found in the egg ; two of the nuclei were
degenerating at prophase, and one at telophase.
2. Early Irregular cleavage. Five eggs, fixed 24 to 26 hours after
operation, were examined in this group. The cytological condition of
each of these eggs is summarized in Table II.
From this table it is clear that the sperm nuclei in these eggs began
to divide at the same or nearly the same time. One nucleus divided
sooner than the others, but succeeded in forming only a few small,
sperm nuclei either degenerated dur-
irregular cells.
The " accessory "
406
CORNELIUS T. KAYLOR
ing early mitosis, or continued to divide haphazardly. In any case, the
presence of so many constellations in the egg does not lead to the
formation of complete cleavage furrows.
Figures 2 and 3 illustrate the cytological condition of two of the
most interesting eggs of this group. In the egg shown in Fig. 2, five
TABLE II
Summary of cytological conditions in early irregular cleavage stages
Egg
No.
No.
Sperm
Marks
Age
External Appearance
Cytological Condition
hours
32. le
6
26
Irregular cleavage
4-6 irregular "cells," no nuclei. In un-
segmented region: 6 nuclei degenerating
in prophase (monaster), 1 cytaster
34.3e
7 +
26
Irregular cleavage
6-8 irregular cells, degenerating nuclei.
In unsegmented region: 3 nuclei degen-
erating in prophase (monaster), 3 nuclei
•
degenerating in metaphase (bipolar),
1 degenerating nucleus
61. le
7
25
Irregular cleavage
6-8 irregular cells, degenerating nuclei.
In unsegmented region: 3 nuclei degen-
erating in prophase (monastral), 1 nu-
cleus degenerating in bipolar mitosis, 2
cytasters
63. le
6
24
Irregular cleavage
4 irregular cells, mitosis in each. In unseg-
mented region: 5 nuclei degenerating in
prophase (monastral), 9 cytasters, 1
triaster
64.2e
3
26
Irregular cleavage
many irregular cells, nuclei in majority
degenerating. In unsegmented region:
4 small bipolar spindles, 7 degenerated
nuclei, 3 large triastral mitoses with large
•
number of chromosomes, 2 large tetras-
tral mitoses with large number of chro-
•
mosomes, 10 cytasters
of the six spermatozoa entering the egg are degenerating after a begin-
ning monastral mitosis. It is probable that the sixth sperm nucleus
divided in a normal manner and was responsible for the formation of
the few cells in the upper part of the egg. In four of these cells, a
normal haploid mitosis is in progress. The nine cytasters scattered
through the unsegmented part of the egg apparently have no connection
with any of the sperm nuclei and for this reason probably originated
HAPLOIDY IN SALAMANDER LARVAE
407
de novo in the cytoplasm, as they do in egg fragments of Triton (Fank-
hauser, 1934a). The large triaster is probably a fusion of three cy-
tasters.
The cytological condition of the egg in Fig. 3 is much more complex.
An inventory of the contents of this egg is given in Table II and in the
explanation of Fig. 3. Since there were only three sperm entrance
FIG. 2. Drawing of a median section of the egg 63. le, sectioned parallel to the
egg axis. All nuclei and cytasters projected into this section from neighboring
sections. Irregular cells, mitosis in each. Five degenerating sperm nuclei, nine
cytasters, one triaster in the unsegmented yolk region.
FIG. 3. Drawing of a median section of the egg 64.2e, sectioned parallel to the
egg axis. All nuclei projected into this section from neighboring sections. Ir-
regular cells, degenerating nuclei in most cells. In the unsegmented region : four
small bipolar mitoses, seven degenerated nuclei, three large triastral mitoses and
two large tetrastral mitoses with large numbers of chromosomes, ten cytasters.
408
CORNELIUS T. KAYLOR
marks on the living egg, it seems probable that only two sperm nuclei
could have been responsible for the large number of irregular mitotic
figures present, while the third sperm nucleus initiated the formation of
the few small, irregular cells in the upper part of the egg. Several
chromosome counts were made in the figures present in the yolk region.
FIG. 4. Drawing of a median section of the egg 28.5e, sectioned parallel to the
egg axis. Nuclei projected from neighboring sections into the cells and unseg-
mented region. Many cells non-nucleated, some with single asters, others with
degenerating sperm nuclei in the yolk region.
FIG. 5. Drawing of a median section of the egg 30.7e, sectioned parallel to the
egg axis. Nuclei projected from neighboring sections into the cells. Majority of
cells non-nucleated. Fairly normal blastula.
In one normal anaphase figure, seventeen chromosomes were identified ;
eight at one pole and nine at the other (Fig. 10). In another anaphase,
18 chromosomes were identified, while in a nearby metaphase plate, 13
chromosomes could be counted. Several large, irregular triasters and
tetrasters were in this yolk region. Large numbers of chromosomes
were present in each of these figures.
HAPLOIDY IN SALAMANDER LARVAE
409
b. Early Blastulae. — Nineteen eggs were fixed approximately 28
hours after operation, when they failed to develop beyond the mid-
blastula stage. The most conspicuous features of the sections of these
eggs were, first, that in 13 eggs a large area of the vegetative region was
unsegmented. Only 6 eggs were completely segmented. Secondly,
closer examination revealed a large number of abnormal mitotic figures
TABLE III
Chromosome numbers in early androgenetic blastulae
Total
Number of
Chromosome Numbers
Egg No.
Analyzable
Mitotic
Figures
9
10-11
11
11-12
12-14
14-16
16-18
18 +
22
22 +
26.9e
6
2
2
2
20.21e*
6
1
2
1
2
28.5e
2
1
1
34.2e
25
2
3
5
6
?
2
4
1
37Ae
11
2
1
1
1
6
52Ae
6
1
3
2
53Ae*
6
1
1
2
2
54. 3e
12
1
3
8
68Ae
->
2
70.2e
11
3
3
3
2
208. 16e
8
4
2
2
00. E
18
8
3
3
4
* Preserved while still developing.
in the cells and unsegmented regions; in the 19 eggs a total of 312
mitoses of the following types were observed :
(a) Pluripolar mitoses.
(b) Monastral mitoses.
(c) Bipolar mitoses with degenerating chromosomes.
(d) Bipolar mitoses with no chromosomes.
Figure 4 is an example of an egg of the group of thirteen eggs with
the undivided vegetal region. The cytology of this egg is given in
detail since, although the egg does not possess all of the irregularities
listed above, it is in general illustrative of the cytological condition of
this group of eggs. A large sector of the roof of this blastula is com-
posed of cells without nuclei ; each cell contains a small bipolar spindle
with no chromosomes. Other cells nearby contain only a single aster.
In a few cells, bipolar mitoses are in progress, but in several of these
figures chromosomes are showing signs of degeneration. Figure 11 il-
410
CORNELIUS T. KAYLOR
lustrates an anaphase spindle in one of these cells. Four chromosomes
are lagging on the spindle and show definite abnormal swelling. The
large unsegmented yolk region of this egg contains three nuclei which
are degenerating in early prophase. The undivided yolk region of
similar eggs, however, contained a larger number of abnormal figures
FIG. 6. Drawing of a median section of the egg 25.3e, sectioned parallel to the
egg axis. Abnormal late blastula. Large areas of the yolk region unsegmented.
No blastocoele.
FIG. 7. Drawing of a median section of the egg 23.2e, sectioned parallel to the
egg axis. Abnormal late blastula. Cells with pycnotic nuclei in segmentation
cavity.
than are seen in this egg. Figures 12 and 13 show two of these mitoses.
The egg shown in Fig. 5 illustrates the typical condition in the
group of six completely segmented eggs. The majority of cells contain
bipolar spindles with no chromosomes; spindles similar to the one
shown in Fig. 14. A few cells contain single cytasters. In other cells,
the nuclei are degenerating. The small blastocoele has a few fragments
HAPLOIDY IN SALAMANDER LARVAE
411
of cytoplasm containing no chromatin. The other five eggs did not have
as many cells without nuclei.
Chromosome counts were possible in some of the cells of twelve of
the nineteen eggs (Table III). In all but one of the eggs, the counts
deviated from the haploicl number (11 chromosomes) in the majority
of cells. The one blastula which was haploid happened to have been
fixed while still developing. It is doubtful that this egg could have
reached an advanced stage of development because the cleavage was very
irregular.
TABLE IV
Chromosome numbers in late androgenetic blastulae
Egg
Total
Number of
Chromosome Numbers
*-*&&
No.
Analyzable
Mitotic
Figures
7-8
9-10
10-11
11
11-12
12-14
15-18
22
27 +
30-33
23. 2e
23
12
2
3
6
25.3e
17
1
1
1
2
3
8
1
25.9e
22
2
2
4
2
3
5
4
26.5e
40
5
1
4
14
10
3
1
2
26.6e*
40
14
15
8
2
1
27.1e*
26
3
5
5
9
4
36.1e
19
1
4
6
6
1
1
61.5e
11
1
3
5
2
64. le
11
2
7
2
86.2e
9
1
2
4
1
1
AA.e
11
1
1
2
7
3Ae
15
1
1
4
2
2
4
1
56Ae
13
2
3
3
3
2
60Ae
12
1
1
4
1
1
2
2
* Preserved while still developing.
c. Late Blastulae. — Of the 23 eggs fixed in the late blastula stage,
only two were fixed while still developing. The following description
will cover first of all the 21 eggs which had ceased development.
Although in external appearance each of these eggs resembled a
normal blastula, the sections showed that all eggs were abnormal.
Eighteen eggs were incompletely segmented in certain areas of the
vegetal region. Only three eggs were completely segmented. All of
the irregularities of mitosis observed in the earlier cleavage stages could
be identified in the cells of these blastulae.
Since it would be impossible to describe the cytology of each of these
eggs, the blastula shown in Fig. 6 was selected as representative of the
group of 18 incompletely divided eggs. No blastocoele is present in the
412
CORNELIUS T. KAYLOR
egg. The upper half of the egg is composed of regularly segmented
cells, while in the lower half the boundaries of many of the cells are
incomplete. In sixty or more cells, the nuclear conditions were abnor-
mal. The nuclei in the majority of these were degenerating, and in
FIG. 8. Drawing of a median section of the egg 26.6e which was preserved
while still developing. Sectioned parallel to the egg axis. Fairly normal late
blastula. Irregular mitoses beginning. Tetrastral mitosis in cell of vegetal region
at right of drawing, one triastral mitosis in nearby cell.
FIG. 9. Drawing of a median section of the egg 37.3e, sectioned parallel to the
egg axis. Abortive gastrula. Incomplete invagination of the yolk. Many cells
with pycnotic nuclei in the blastocoele.
other cells, mitoses, still in progress, were frequently of a monastral
type. Chromosome counts in 17 cells varied from 7 to 22 in number,
indicating that irregular distributions of the chromosomes had occurred
earlier in the cleavage history. Several mitotic figures in this egg
showed stages of chromosome elimination. Figure 15 illustrates a
HAPLOIDY IN SALAMANDER LARVAE
413
metaphase figure in which all of the chromosomes have degenerated.
In another cell (Fig. 16), the chromatin is completely removed from the
spindle. Other mitoses were observed in which the elimination of
chromosomes was occurring more gradually ; a few chromosomes at a
time were being lost from the spindle. This is seen in Fig. 17. At
least two and probably six chromosomes are not included in the meta-
phase group and will remain outside the nucleus in one of the two
daughter cells. A telophase in a cell from another egg (Fig. 18) shows
several degenerating chromosomes near the new cell membranes. These
chromosomes will not be included in the daughter nuclei.
Each of the three completely segmented eggs possessed a segmenta-
tion cavity. Figure 7 illustrates one of these blastulae. About one-
TABLE V
Chromosome numbers in androgenetlc gastrulae
Total
Chromosome Numbers
Egg
Mr>
Number of
Analyzable
*N L>.
Mitotic
Figures
7-8
9-10
10
10-11
11
11-12
12
12-14
15-17
20-21
22 +
26.1 le
10
7
3
31. le
14
14
35. le
12
8
4
35. 2e
18
6
10
2
35.9e
6
4
2
37. 2e
25
2
.1
7
3
9
1
2
37.3e
21
1
2
2
7
4
2
3
75. le
10
2
2
2
4
76.3e
9
2
2
1
1
3
half of the roof of this blastula is composed of a double row of cells.
The vegetal region still has abnormally large cells. A number of cells
with pycnotic nuclei have separated from the yolk into the blastocoele.
Although abnormal mitoses were not observed, an irregular distribution
of chromosomes had occurred in earlier stages of development since
chromosome numbers in 23 cells varied from 7 to 16 or 18.
Even though development was at a standstill in most of these eggs,
mitoses were still frequent. The chromosomes of metaphase plates
could be counted accurately in 14 eggs (Table IV). From Table IV it
is clear that none of these blastulae were completely haploid.
Two late blastulae were preserved because of ruptured yolk mem-
branes. One of these, Fig. 8, is most interesting because, unlike the
majority of operated eggs, its cleavage had been undelayed and perfectly
normal. There had been no suspicion, therefore, that the female nucleus
414 CORNELIUS T. KAYLOR
was actually out of the egg. In Fig. 8 it is seen that the egg was a
fairly normal blastula. The first few chromosome counts were all
haploid. Then the following mitoses were observed: (a) two normal
bipolar figures with 22 chromosomes (the diploid number) ; (fr) one
triatral mitosis with 33 chromosomes (Fig. 19) ; and (r) a tetrastral
figure with a large number of chromosomes, presumably the tetraploid
number. One other cell (Fig. 20) contained a telophase figure with
fragments of chromosomes at the center of the spindle. In view of
the small number of cells with slightly irregular cytological conditions,
this egg could probably have developed to a more advanced stage. The
other egg possessed irregular chromosome numbers in the majority of
cells. For this reason it probably would not have developed farther.
d. Gastnilac. — The last group of androgenetic eggs consisted of nine
eggs fixed at the end of their development in the gastrula stage. In
section, all of these eggs were found to be abortive gastrulae. The
process of invagination of cells into the blastocoele was incomplete. In
most of these eggs yolk cells with pycnotic nuclei were accumulating in
the blastocoele (Fig. 9).
Although mitoses were not frequent in these gastrulae, a few chro-
mosome counts were made in each egg (Table V). In all but two eggs,
the majority of cells were not haploid. It is interesting to note that one
gastrula had only 10 chromosomes in every cell clear enough for analy-
sis. Apparently the lack of even one chromosome may be sufficient to
disturb the processes of differentiation occurring for the first time at
gastrulation.
The abnormal gastrulation of the two eggs which were completely
haploid is not surprising since in later stages of development, as for
example the formation of the neural plate, haploid embryos frequently
have serious difficulties. This was observed in an earlier report (Kay-
lor, 1937), and in the experiments on the androgenetic development of
frog embryos (Porter, 1939).
DISCUSSION
The cytological conditions found in these eggs explain fully the high
mortality rate during cleavage and gastrulation. In eggs fixed after
irregular beginning cleavage, it was observed that either none of the
sperm nuclei was sufficiently active to form cleavage furrows, or, quite
the opposite, all of the sperm nuclei divided at the same or nearly the
same time causing incomplete and irregular cleavage of the egg. The
cytological conditions were somewhat the same in eggs which ceased de-
HAPLOIDY IN SALAMANDER LARVAE
415
PLATE I
EXPLANATION OF FIGURES
Figures 10 to 14 were drawn at a magnification of 1200 and reduced to ca. 400
in reproduction.
FIG. 10. Anaphase figure in the yolk region of the egg in Text Fig. 3. Nine
chromosomes at the upper pole and eight at the lower.
FIG. 11. Anaphase figure in a cell of the egg in Text Fig. 4. Four chromo-
somes, lagging on the spindle, show beginning degeneration.
FIG. 12. Pluripolar figure in the yolk region of the egg 37Ae. Apparently
the fusion of several nuclei.
FIG. 13. Triastral figure in the yolk region of the egg 37Ae. Degenerating
nucleus.
FIG. 14. Bipolar figure without chromatin in a cell of the egg 20.21e. The
spindle shows a reduction in the number of spindle fibers.
416
CORNELIUS T. KAYLOR
15
17
PLATE II
EXPLANATION OF FIGURES
Figures IS to 20 drawn at a magnification of 1200 and reduced to ca. 600 in
reproduction.
Figures 15 to 18, different stages in elimination of chromatin.
FIG. 15. Metaphase figure in a cell of the egg in Text Fig. 6. The chromo-
somes have degenerated into a pycnotic mass on the center of the spindle.
FIG. 16. Metaphase figure, polar view, in a cell of the egg in Text Fig. 6.
The chromatin is completely removed from the spindle.
FIG. 17. Metaphase figure in a cell of the vegetal region of the egg in Text
Fig. 6. Six chromosomes lagging on the spindle.
FIG. 18. Telophase mitosis in the egg AA.e. Several chromosomes lagging
near the new cell membranes.
FIG. 19. Triastral figure in a cell of the egg in Text Fig. 8. Thirty-three
chromosomes present.
FIG. 20. Anaphase figure in a cell of the egg in Text Fig. 8. Several chromo-
somes lagging on the spindle.
HAPLOIDY IN SALAMANDER LARVAE 417
velopment during the early blastula stage. The majority of these eggs
were incompletely segmented and contained abnormal nuclei in the cells
and in the undivided areas, indicating the early irregular division of
more than one sperm nucleus. The few completely segmented blastulae,
although fairly normal in their cleavage, were, nevertheless, very irregu-
lar in their nuclear conditions. In these cases, the division of only
one sperm nucleus probably initiated the almost normal cleavage, but
even this early mitosis must have been extremely irregular.
The majority of eggs which had ceased development during the late
blastula stage were incompletely segmented in certain areas of the egg.
Only a few were normally formed blastulae. Chromosome counts in
these eggs showed conclusively that irregular distribution of the male
chromosomes had occurred earlier, and, indeed, was still going on in
many cells at the time the eggs were preserved. It was of interest to
note in the case of the normal androgenetic blastulae preserved while
still developing, that one of these eggs possessed irregular chromosome
numbers in the majority of mitoses analyzed. In the other egg, it was
observed that irregular mitoses were just beginning. Cytological con-
ditions such as these in normally developing androgenetic blastulae would
be of importance in experiments involving the transplantation of haploid
cells.
The nuclear conditions of the gastrulae were abnormal. About 80
per cent of the eggs ceasing development in this stage were not haploid.
All of these gastrulae were abortive. Since it has been shown pre-
viously (Kaylor, 1937) that all androgenetic embryos which develop
beyond the gastrula are haploid, it is apparent that the early gastrula is
as far as an androgenetic egg can develop unless it possesses at least the
haploid number of chromosomes in all of its cells.
These observations are in exact agreement with Fankhauser's (1934,
b) conclusions from his excellent analysis of chromosome numbers and
chromosome individuality in andro-merogonic Triton eggs. A complete
discussion of the indispensability of a balanced set of chromosomes in
early development is found in Fankhauser's papers.
These experiments on androgenesis have recently been extended to
eggs of the Japanese newt, Triturus pyrrhogaster (Kaylor, 1940). In
this species, a smaller percentage of the operated eggs die during blastula
or gastrula stages. The more normal development of these eggs as
compared with that in Triturus viridescens must be connected, then, with
a more normal behavior of the sperm nuclei in early cleavage.
418 CORNELIUS T. KAYLOR
SUMMARY
1. Androgenetic eggs of Triturus viridescens most frequently cease
development in the following stages: a. Irregular beginning cleavage;
b. Early blastula ; c. Late blastula ; d. Gastrula.
2. The causes of arrested development were investigated cytologi-
cally in eggs fixed in each of these stages.
3. Eggs of the first group were of two types, i.e., abortive cleavage,
and early irregular cleavage in which a few cells were formed near the
animal pole. In seven eggs of the first type, it was found that the
sperm nuclei had degenerated either before or during early mitosis and
cleavage furrows had disappeared. In five eggs of the second type,
either all sperm nuclei had degenerated during early mitosis or one sperm
nucleus divided more or less normally while " accessory " sperm nuclei
either degenerated or divided irregularly in the unsegmented part of the
egg-
4. In nineteen early blastulae, thirteen were incompletely segmented
and six, although irregularly segmented, were fairly normal blastulae.
Associated with these abnormalities in the thirteen eggs were the inde-
pendent division of sperm nuclei in the yolk region without segmentation
of the cytoplasm, and the presence of abnormal mitoses in the majority
of cells. In the six almost normal mid-blastulae, the greater number of
cells contained abnormal nuclei. Chromosome counts varied from 9 to
22 -|- in twelve of the nineteen eggs in which analyses could be made.
5. In twenty-three late blastulae sectioned, the same abnormalities as
found in the earlier blastulae were observed. The majority of eggs were
incompletely segmented and all of the eggs contained abnormal mitotic
figures in some of the cells. Chromosome counts were made in fourteen
eggs. None of these blastulae were completely haploid.
6. Nine gastrulae examined were abortive. No abnormal mitotic
figures were found in these eggs, but in seven gastrulae the chromosome
numbers varied above and below the haploid number, indicating that
abnormal mitoses had occurred during earlier cleavage stages. Two
gastrulae were haploid and it is assumed that these are examples of the
abnormalities which many haploid embryos exhibit when differentiation
of parts or of structures first takes place.
7. These observations confirm and extend those of Fankhauser and
of Fankhauser and Moore. In order to develop beyond the gastrula
stage, an anclrogenetic egg must be at least completely haploid.
BIBLIOGRAPHY
DALCQ, A., 1932. Contribution a 1'analyse des fonctions nucleaires dans 1'onto-
genese de la grenouille. IV. Modifications de la formula chromosomiale.
Arch, dc Biol, 43 : 343-366.
HAPLOIDY IN SALAMANDER LARVAE 419
FANKHAUSER, G., 1934a. Cytological studies on egg fragments of the salamander
Triton. IV. The cleavage of egg fragments without the egg nucleus.
Jour. Exper. Zool., 67 : 349-393.
FANKHAUSER, G., 1934&. Cytological studies on egg fragments of the salamander
Triton. V. Chromosome number and chromosome individuality in the
cleavage mitoses of merogonic fragments. J our. E.vpcr. Zool., 68 : 1-57.
FANKHAUSER, G., AND CAROLINE MOORE, 1941. Cytological and experimental
studies of polyspermy in the newt, Triturus viridescens. II. The behavior
of the sperm nuclei in androgenetic eggs (in the absence of the egg
nucleus). Jour. Morph., 68: 387-423.
KAYLOR, C. T., 1937. Experiments on androgenesis in the newt, Triturus viri-
descens. Jour. Expcr. Zool., 76 : 375-394.
KAYLOR, C. T., 1939. Cytological studies on androgenetic embryos of Triturus
viridescens which have ceased development. Biol. Bull., 77 : 334.
KAYLOR, C. T., 1940. Studies on experimental haploidy in salamander larvae. I.
Experiments with eggs of the newt, Triturus pyrrhogaster. Biol. Bull.,
79: 397-408.
PARMENTER, C. L., 1933. Haploid, diploid, triploid, and tetraploid chromosome
numbers and their origin in parthenogenetically developed larvae and frogs
of Rana pipiens and Rana palustris. Jour. Expcr. Zool., 66 : 409-453.
PARMENTER, C. L., 1940. Chromosome numbers in Rana fusca parthenogenetically
developed from eggs with known polar body and cleavage histories. Jour.
Morph., 66 : 241-260.
PORTER, K. R., 1939. Androgenetic development of the egg of Rana pipiens. Biol.
Bull, 77 : 233-257.
REVERSAL OF SEX PRODUCTION IN MICROMALTHUS
ALLAN SCOTT
(From Union College, Schcncctady, N. Y. and the Marine Biological Laboratory,
Woods Hole, Mass.)
INTRODUCTION
There is only one known example of paedogenesis in the Coleoptera
and there are relatively few cases in the whole insect class. Hence any
information which relates to the nature of paedogenesis in the beetle Mi-
crotnalthus debilis has a general biological importance. One important
question relative to paedogenesis in the beetle Microvnalthus is : what
is the mechanism which determines the strict separation of male pro-
duction from female production in two types of larval mothers? This
paper shows that the mechanism has an environmental rather than a
genetic basis.
Many groups of animals produce unisexual broods. Thus some,
aphids, Hymenoptera, Diptera, certain rotifers and Isopoda and some
nematodes etc., produce broods of one sex and in some cases a partial
explanation of the mechanism of this unisexual propagation is known.
In many cases the broods are consistently female and involve a more or
less constant process of diploid parthenogenesis, but in a few cases
unisexual male progenies also occur. In the genus Sciara, Metz (1931)
has disclosed a genetic basis which determines the sex of brood and a
sex-linked gene is responsible. The thelytokous wasp, N enter it is can-
escens, studied cytologically by Speicher (1937) is a perfect example of
constant female production ; no males were found in some fifty gener-
ations ! The mechanism here also appears to have a genetic basis. It
controls sex of progeny by determining a constant type of maturation.
in the paedogenetic fly, Miastor metraloas, unisexual broods are appar-
ently almost inviolably the rule and although Gabritschevsky (1928)
ascribes this to a genetic mechanism, the contrary conclusion is indi-
cated by Ulrich's (1936) work on Oligarccs. Ulrich has shown that
in the paedogenetic fly, Oligarccs parado.vus, many broods show uni-
sexual propagation although some broods contain both sexes. Heredi-
tary differences among the larvae of Oligarccs are not the determiners of
the sex of the brood. It is the environment that is of primary impor-
tance.
420
PAEDOGENESIS IN MICROMALTHUS 421
It is the physiological state of the presumptive paedogenetic mother
and the environment which primarily determine the sex of the brood in
Micromalthus, just as in Oligarccs. It is the purpose of this paper to
describe a reversal of sex of brood which can be made to occur experi-
mentally in the larval male-producer of Micromalthus. All the members
of the first brood are male and all of the second brood are female.
REVIEW OF THE REPRODUCTIVE TYPES
In a previous paper (1938) I have described extensively the life
history of Micromalthus and have outlined the reproductive anatomy
of the various types. It is necessary for the purpose of discussion to
review the reproductive types and more especially to describe the male
producer with considerable care.
There are in the American variety of Micromalthus deb His five sex-
ually mature reproductive types: (1) an adult female, (2) a female-
producing paedogenetic larva, (3) a male-producing paedogenetic larva,
(4) an adult male, and (5) a paedogenetic female larva with a mixed
brood. This last is a modified male-producing larva and is the subject
of research reported here. It is essential to note that the modified
male-producer (amphoterotokous female) is simply a later developmental
stage of the male producer. They rarely occur in nature but can be
produced in large numbers experimentally. It is of incidental interest
to refer here to the apparent absence of male-producers in the South
African variety of Micromalthus which has recently been reported by
Pringle (1938).
NORMAL HISTORY OF THE MALE PRODUCER
The male-producer (arrhenotokous female) is the only source of
the adult male. She arises viviparously from a female-producing paedo-
genetic mother (thelytokous paedogenetic female) as one member of a
large brood, sometimes twenty or more. In the first instar all these
viviparous larvae possess legs which are lost at an early moult. They
are all identical in appearance, indeed, it is impossible to distinguish the
male-producer from other types until shortly before the last moult when
inspection by dissection shows an ovary of a very special character in
the male-producer. This early ovary is often recognizably distinct when
only about 80 microns in length when a few egg cells (from one to five
in each ovary) first begin to grow (Speicher, 1937). These continue
to grow until they are of relatively large size and have become the
shape of a hen's egg. The eggs of the thelytokous paedogenetic female
are elongate so that the sex of the embryo resulting from either type
422 ALLAN SCOTT
egg is predictable long before maturation. This adds another animal to
the list showing sexual dimegaly of the ova (Wilson, 1925). When
they are mature, the eggs of the male-producer begin development by
haploid parthenogenesis in contrast to the diploid parthenogenetic devel-
opment of the viviparous young (Scott, 1936). The one male that is
successful in emerging from the mother is shed as a very young embryo
in late June or early July. It is most peculiar, however, that although
several embryos may be present in the ovary, only one is born. This
new-born male remains for some four or five days adherent to the out-
side of the mother as is shown in Fig. 2 of Plate I. By that time he
has developed sufficiently to insert his head into her genital aperture,
which is shown at the arrow in Plate I, Fig. 1. Within a few days
more the male has devoured his mother completely. These canabalistic
males pupate and soon emerge as male adults.
This astonishing form of reproduction raises several perplexing
questions. (1) Why is but one embryo shed by the male-producer when
others equally advanced in development are present? (2) Is any one
of the embryos more likely to be born than any other; viz., (a) does the
position of the male in the mother have any bearing on successful emer-
gence? Or (&) does the age of the embryo affect his ability to emerge?
(3) Can any one of the other embryos be shed if the one that has been
born is not allowed to feed upon the mother? (4) What becomes of
the male-producer if her son is prevented from eating her?
THE BIRTH PROCESS
Why is only one male embryo shed by the male-producer? I can
give no answer to the question but can only indicate some additional
facts. Only seven male-producers in a group of three hundred and
fifty-seven have given birth to two embryos. Fifty-eight male-producers
have shed their male embryos in isolation. The females had previously
been placed each in a shallow depression made in black wax and kept in
a moist chamber. It is apparent from this that removal from their
gallery in the wood does not affect their ability to give birth to the male
embryo. Four of the fifty-eight individuals which shed in isolation,
PLATE I
FIG. 1. Feulgen preparation of male producer with one egg visible. The
genital aperture is shown at the arrow.
FIG. 2. Male producer and successful male offspring.
FIG. 3. Ovary of the male producer with three embryos, all in the same
developmental stage.
FIG. 4. Ovary of the reversing male producer with a newly-developed female
embryo, and an exceptionally well-developed male embryo still within its follicle.
PAEDOGENESIS IN MICROMALTHUS
423
I
:-
'•*
•
.. ••
PLATE I
424 ALLAN SCOTT
shed two eggs, thus it is possible that removal from the wood favors
the birth of a second embryo.
Two other factors might conceivably affect the birth process, that
is, — (1) position in the mother and (2) stage of development of
embryo within the mother. I have previously shown that there is no
favored position in the ovary from which an embryo is shed. (Scott,
1938, Fig. 13.) The successful male embryo may have occupied any
position within the ovary. Indeed, the successful embryo may some-
times occupy such a position within the mother that it must experience
some mechanical difficulty at birth, since other embryos appear to block
its exit.
I do not think that the most mature male embryo is necessarily the
most likely to emerge, for frequently the difference in age of the embryos
is negligible, as is shown in Plate I, Fig. 3, and moreover, an embryo
may occasionally develop into a rather well-developed larva while still
within the follicle of the ovary, as illustrated in Plate I, Fig. 4.
No factual explanation of the mechanism governing this uniparity
is available. However, a very plausible hypothesis can be formulated
from the point of view of natural selection. The male has no other
source of food during his larval life than his mother's body and since a
second male would compete for this food supply, a process may have
been developed which prevents this competition. This process very
probably involves the active cooperation of the mother in that some in-
ternal physiological mechanism prevents a second male from being born.
This mechanism is quite conceivably a failure of the muscular contrac-
tions which normally expel the egg. The continued presence of the
born male on the mother is not necessary to prevent birth of the re-
maining embryos. The birth of one in some way sets the mother against
further activity of the ovary and ducts. Does a hormone govern the
contractions involved in the ovulation-birth process?
A NEW BROOD IN MALE PRODUCERS
The fate of the mother after the emerged male has been removed
is quite unexpected and is, I believe, a quite unprecedented observation.
In practically every surviving case after approximately four weeks time
a new, small brood is born. The members of the new broods have not
yet been reared, nor have chromosome counts been possible, but the off-
spring are judged to be females with considerable certainty for the
following reasons: (1) the shape of the egg is in every way similar to
that of the female-producing female, (2) the development is in every
way identical with that of the ordinarily produced females, and is vastly
PAEDOGENESIS IN MICROMALTHUS 425
different from the development of the male embryo, (3) the appearance
of the newly-hatched embryos is identical with that of the more normally
produced female larvae. That is, these second brood embryos possess
well-developed legs and well-differentiated jaws, whereas new-born male
TABLE I
Dead
Dissected
7/24
Alive
Dissected
7/25
Total Percentage
Females showing female embryos on
dissection
1
43
44
21.8
Females showing no female embryos
Females in which ovaries were not located
47
13
2
4
49
17
24.3
8.4
Died before examination
91
45.2
Total number involved in experiment — 201.
Mortality— 75.7%
embryos are rarely beyond the germ band stage. The second brood
larvae will, therefore, subsequently be referred to as females.
It should be stressed that this process is not an occasional one but is
quite normal for those larval mothers that survive long enough. Thus
in the summer of 1938, 93 females from which the male had been re-
moved gave rise to a new female brood. Of this number, 21 mothers
shed their brood and the rest showed female embryos on dissection.
Since mortality records were not kept in 1938, the experiment was
repeated in 1939.
On July 1, 1939, 201 females, each with the shed male removed,
were isolated in black wax depressions in Syracuse dishes, 20 to each
TABLE II
New brood born before July 24 4
Found with female brood on dissection 7/24 10
Dead when dissected, ovaries disintegrated
Dead, no new brood developing
Lost - - 1
Total . 25
dish and kept in a moist chamber. The mortality was severe, therefore
the larvae were dissected before they could have given birth to their new
brood. Table I summarizes the results.
In another experiment 25 male-producers were removed from the
wood and the adherent male was removed from each one. They were
likewise placed in a moist chamber at 35° C. ± 1°. The results are
shown in Table II.
426 ALLAN SCOTT
In this group 56 per cent developed a new brood of female embryos.
It is impossible from these data to decide whether or not every male-
producer can, under favorable conditions, give rise to a new female brood
but it is certainly indicated by the fact that out of the 63 animals that
were still alive at the end of their respective experiments, only 6 did
not show indications of a new brood. It is reasonable to expect that
the larvae that died during the course of the experiment would also have
given rise to a female brood had they survived.
The mortality, high in both experiments, is less severe at higher
temperatures. The difficulty is largely due to the susceptibility of the
larvae to mold. Perhaps a sterile technique' would obviate the trouble.
Apparently no structural feature of the male producer prevents
viviparity of the new female brood since a considerable number have
been kept long enough to allow normal birth. The birth process is in
every respect similar to that which takes place in the normal female-
producing, paedogenetic female.
The size of the second brood of the reversing male-producer is
intermediate between the size of normal female broods and the size of
male broods. Normal female broods are frequently more than ten
while male broods are rarely as many as four. An examination of the
ovaries of forty reversing male-producers showed that the average
number of new eggs formed was 4.2.
Study of the ovaries of this same group of forty reversing male-
producers showed that a few females failed to shed even one male, yet
they developed a new brood of female larvae notwithstanding.
The production of a new brood is not, therefore, absolutely de-
pendent upon the experimental removal of the emerged male from his
mother. Male-producers whose emerged male embryo dies will evi-
dently give birth to a new all- female brood in the natural course of
events. Indeed, in August, 1937, I found eighteen individuals with a
new brood developing, obviously the result of this natural event. Dis-
section of these eighteen larval mothers showed an empty follicle from
which a male had emerged and apparently died.
It seems altogether possible that a third brood might be produced by
the original male-producer if it lived long enough. However, a single
individual that lived thirty days after the production of the second
brood showed no sign of new eggs when dissected.
It will be of some interest to test similarly the further reproductive
capacity of the thelytokous paedogenetic female after the birth of her
first brood.
PAEDOGENESIS IN MICROMALTHUS
427
TEXT FIG. 1. A diagrammatic representation of the developmental possibili-
ties of the basic ovary as it occurs in the several reproductive forms : (a) basic
ovary (schematic) showing several undeveloped ovarioles in each ovary; (fr) the
ovary of the adult female with three ovarioles developing; (c) the ovary of the
male producer (three ovarioles and eggs greatly enlarged, four others have re-
mained small, see asterisk) ; (d) the ovary of the reversing male producer experi-
mentally produced (the rudimentary ovarioles have enlarged and are developing
female-producing eggs; (c) the ovary of the female producer with numerous
ovarioles developing.
428 ALLAN SCOTT
HISTOLOGY OF THE REVERSING OVARY
Studies of the ovaries of the male-producer indicate that the cells
which give rise to the new crop of female-producing eggs are already
present on the oviducts of the male-producer before the male embryo is
born ; indeed, they were probably present at the time of the first differ-
entiation of the male-producing ovary. In the mature ovary of the
male-producer these cells are located in little clusters around the ventral
and lateral surfaces of the oviducts at the point of junction of oviduct
and follicle. (Text Fig. lr at the asterisk and Plate II, Fig. 5&).
Structurally these groups of cells are undeveloped ovarioles. They
doubtless represent ovarioles which did not enlarge during the first
period of development of the male-producing eggs. The detailed struc-
ture of these ovarioles is shown in Plate II, Figs. 5 and 6. In both
figures some differentiation can be seen within the ovariole and although
no single egg cell can be identified with certainty, still, terminal cells of
the germarium, nurse cells, and duct cells can be seen in Plate II, Figs.
6a, 6b, and 6c respectively.
In Micromalthus the ovaries of the four reproductive types are fun-
damentally similar. The general plan of the ovary in each of the female
reproductive types is meroistic and acrotrophic, since the nutritive cells
are all located at the apex of the ovariole. The variations in structure
which the ovaries of the several reproductive types present may all be
considered as modifications of a basic, undifferentiated ovary illustrated
in Text Fig. la. This basic ovary possesses multiple ovarioles at the
ends of a forked oviduct ; it is the development or non-development of
these potential ovarioles that determines the nature of the mature ovary.
If the ovary develops within an adult female, then three or 'four of the
ovarioles will enlarge with their contained eggs, as indicated in Text
Fig. \b. When, however, the basic ovary develops within a female-
producing paedogenetic mother, a number of eggs, each in a different
PLATE II
FIG. 5. Frontal section of ovary of male producer: (a) Follicle; (&) unde-
veloped ovarioles (the follicle on the right side appears in another section) ; (c)
oviduct; (d) last ganglion ; (c) vagina.
FIG. 6. Oil immersion photograph of undeveloped ovarioles of the male-
producing ovary. No enlargement has as yet taken place: (a) germarium; (fr)
potential nurse cells; (c) potential duct segment; (d) oviduct.
FIG. 7. Total Feulgen preparation of a reversing male-producing ovary : (a)
unshed male egg; (b) female-producing egg; (c) oviduct; (d) new segment of
oviduct; (e) empty follicle (out of focus) ; (/) vagina or terminal duct.
FIG. 8. Total Feulgen preparation of the ovary of a male producer with a
new brood of female embryos: (a) female embryo; (b) empty follicle; (c) re-
tained degenerating male embryo.
PAEDOGENESIS IN MICROMALTHUS
420
~
PLATE II
430 ALLAN SCOTT
ovariole, enlarge to determine the characteristic ovary of that type of
larva. This is illustrated in Text Fig. Ic. When the basic ovarioles
develop within a male-producer, again only a few of the ovarioles en-
large. In this case, too, only a few eggs develop, one in each ovariole.
so that the fully developed ovary of the male-producer, shown in Text
Fig. If, still possesses a number of undeveloped ovarioles at the base of
the enlarged follicles. It is these undeveloped ovarioles that enlarge to
give rise to the second all-female brood under the conditions noted above
and illustrated in Text Fig. Id.
The development of the new eggs involves the production of other
new parts of the reproductive system, for although the old oviducts and
vagina are utilized by the larvae of the second brood on emergence, it
will be apparent from Plate II, Figs. 6r and 7d, that a new segment of
oviduct is added during the development of the new crop of eggs. The
potentialities of the ovariole tissue are such, therefore, that it gives
rise to the following reproductive structures: (a) oviduct, (/>) follicle
cells, (Y) eggs, one per ovariole, ( d ) nurse cells and (Y ) germarium.
The new oviduct segment is at first relatively long, but it is inconspicuous
in late development, as Plate II, Fig. 8 shows. Perhaps it is incor-
porated into the follicle as the egg grows.
It should be added that the development of the new eggs is not
particularly related to the stage of development of the retained males,
for the latter may be in any stage of development from a post-matura-
tional stage to a well-developed larva. Frequently, indeed, the retained
males undergo an abnormal type of development which also has no
apparent effect on the new brood.
SUMMARY
1. The paedogenetic, arrhenotkous female in the beetle, Micromal-
thits dcbilis (Leconte), gives birth to but one male embryo, although
unshed males also develop.
2. Factors which determine this uniparity are still uncertain, but
neither greater age nor more favorable position in the mother are
determining factors.
3. When the single successful male offspring is not allowed to
devour his mother, a new crop of eggs develops in the ovary.
4. These new eggs are all of the elongate female type. They de-
velop into a larva identical in appearance with the first stage larva of
the thelytokous paedogenetic female.
5. Histologically the new eggs originate from undeveloped ovarioles
which failed to develop during the first period of growth of the male-
producing eggs.
PAEDOGENESIS IN MICROMALTHUS 431
6. Sex of brood in Micromalthus is obviously determined by environ-
ment, intrinsic or extrinsic, and not by the hereditary constitution of
the mother.
BIBLIOGRAPHY
GABRITSCHEVSKY, E., 1928. Bull. Biol. dc la Prance ct Bclg., vol. 62.
METZ, C. W., 1931. Genetics, vol. 16.
PRINGLE, J. A., 1938. Trans. Roy. Ent. Soc., vol. 87.
SCOTT, A. C., 1936. Jour. Morph., vol. 59.
SCOTT, A. C., 1938. Zeitschr. fur Morph. und Okol., Bd. 33.
SPEICHER, B. R., 1937. Jour. Morph., vol. 61.
ULRICH, HANS, 1936. Zeitschr. fiir ind. Abst. und Vcrcrb., Bd. 71.
WILSON, E. B., 1925. The Cell in Development and Heredity. Third Edition.
The Macmillan Co., New York.
THE TIME-TEMPERATURE RELATION OF DIFFERENT
STAGES OF DEVELOPMENT
FRANCIS JOSEPH RYAN
(From the Department of Zoology, Columbia I'mvcrsity, Nciv York City)
The phenomenon of development appears as a series of processes
which are visibly unlike. Many investigators have demonstrated that
some of these events can also be separated on the grounds that their
rates possess different temperature coefficients. For example, the rate
of growth of the gill filaments in the frog is more depressed by a low
temperature than is the rate of body growth (Atlas, 1935 ; Doms, 1915).
Again, the rate of embryo formation in Salino has a higher temperature
coefficient than the rate of growth in wet weight (Gray. 1928). In
view of this and other evidence it 1ms always seemed curious that
development could yield approximately the same differentiated product
over a wide range of temperatures. As a possible solution to the prob-
lem, Tyler (1936«) has shown that in some marine invertebrates the
temperature coefficients of various cleavages are not only the same but
are also identical with those for later stages of differentiation. Yet his
results are not comparable with those on the frog obtained by Hertwig
(1898) and Krogh (1914) whose data show, although the authors do
not point it out, that the temperature relation of cleavage is different
from that of later development. The experiments on the egg of the
frog to be reported here were designed to discover the temperature rela-
tions of some of the more clear-cut events of development which could
be accurately measured.
Rana pipicns from Vermont were caused to ovulate at 15° by pitui-
tary injection. Batches of about 25 eggs were shed into finger bowls
and fertilized artificially. The sperm suspension was replaced after 5
minutes by 200 cc. of 10 per cent Ringer's solution at the temperature
at which the eggs were to be kept and the bowls were then distributed
to constant temperature environments. When the jelly swelled, the egg
mass in each container was cut into bunches containing about 5 eggs
apiece. The 10 per cent Ringer's was replaced daily by solution at the
same temperature. Cold rooms, water baths and incubators were used
to maintain constant temperatures. Generally the temperatures were
constant within 0.1° C. (except at 10° and 8.5° where ice-boxes were
used; the maximum observed variations here were 1.0° and 0.5° re-
432
TIME-TEMPERATURE RELATION 433
spectively). The temperature in the finger bowls seldom changed more
than 0.5° during observation on the stage of a binocular microscope and
observation never lasted as much as 5 minutes. Mortality during early
development was less than 5 per cent and development was normal at
temperatures between 25° and 8°. Above and below these tempera-
tures mortality increased and abnormalities became frequent, so that at
29.6° usually less than 50 per cent of the eggs hatched and abnormalities
were very common.
The times to various cleavages, gastrulation and gill circulation were
measured from fertilization for embryos remaining constantly at one of
the several temperatures. Cleavage was considered begun when the first
slight furrowing was seen on the surface of the egg ; gastrulation when
the dark line of pigment associated with the initial dorsal lip invagination
appeared ; and gill circulation when the initial blood corpuscles could be
seen circulating in the anterior gill. When the critical time approached,
repeated observations were made until about 50 per cent of a batch of
eggs had reached the initiation point, at which time it was considered
that the stage was entered. All of the embryos in a group of 25 entered
a stage well within 10 per cent of the total time necessary to reach that
stage. The maximum deviation of any batch of eggs from the average
time was about 10 per cent.
In order to portray the relation between the times to different stages
at different temperatures a semi-logarithmic plot was chosen (see fig-
ures). The logarithm of time was placed along the ordinates and the
abscissae represent either temperature, in which case the curves are for
different developmental intervals, or stages, in which case the curves are
for different temperatures. The choice of one of these abscissae was
made so as to employ the largest number of points per curve. In either
event it is possible to compare what types of function of time the stages
are at different temperatures. Spacing of temperatures along the ab-
scissa was obtained by plotting the data for one stage as a straight line.
This arbitrary abscissa was then used as a base for the times to other
stages. Stages wrere spaced along the abscissa in a similar fashion.
This method is preferred to the comparison of temperature coefficients
inasmuch as: (1) it does not entail a selection of points but involves all
of the data; (2) it avoids attributing one of the several controversial
numerical constants to the temperature relation; and (3) the linear ar-
rangement of poinfs obtained by a distortion of one axis permits imme-
diate visual comparison of the time-temperature relation.
Figure 1 compares the temperature relation of the different stages of
development in Rana pipicns. The curve for time between gastrulation
and gill circulation (Stages 10-20) has the greatest slope. The curve
434
FRANCIS J. RYAN
for time between fourth cleavage and gastrulation has a lesser slope
which is, however, greater than the slope of the curves for all the cleav-
ages. These differences are real. If the curves actually were parallel
to that for gastrulation to gill circulation, a time error of 25 per cent
would have to be assumed at both ends of the gastrulation curve, an
error of about 45 per cent at both ends of the curve for first cleavage and
an error of about 45 per cent at both ends of the curve for second, third
and fourth cleavage. Such errors are highly improbable because the
Ul
O
o
_J
4TH CL.- ST. 10
PERT.- IST CL
1ST, 2ND 8. 3RD CL.
23.0 20.2 180
10.0
24.5 21.0
19.0 157
TEMPERATURE
11.2
8.5
FIG. 1. The relation, at different temperatures, of developmental intervals to
time in Rana pipicns. Ordinate, logarithm of time in minutes between the specified
stages; abscissa, temperature in °C. The data for development between stages 10
and 20 are plotted as a straight line by the arbitrary distortion of the temperature
axis. The latter is used as a base for the data for other intervals. The upper
complete curve describes development between gastrulation (stage 10 of Pollister
and Moore, 1937) and gill circulation (stage 20) ; the next, between fourth cleavage
and gastrulation ; the next, between fertilization and first cleavage. In the lowest
curve, the circles describe development between first and second cleavage, the
squares, between second and third, and the triangles, between third and fourth.
Twenty points are single determinations ; the remaining thirty-four points are the
averages of from two to ten determinations. One hundred and seventy-two deter-
minations were made in all. The broken lines were drawn through the points at
24.5° parallel to the curve for stages 10 to 20. These broken lines emphasize the
real nature of the slope differences among the curves for different intervals.
TIME-TEMPERATURE RELATION 435
maximum deviation of a point from any one of the curves is only 10 per
cent. The deviations of points for the cleavages from the parallel
curves drawn through them are at random and are about the size of the
expected experimental error (10 per cent). Hence, in Rana pipiens the
temperature relations of cleavages are alike, but they are different from
those of later development.1 Differences in temperature relation are
apparent even in rate-temperature plots (Ryan, 1941) where, in addition
to a difference in /*. values, the curve for later development shows a
" break " at about 18° C., while the curves for cleavage " break " around
14° C.
The data of Krogh (1914) for Rana butyrhina when placed on the
semi-logarithmic plot (Fig. 2) completely confirm this difference between
cleavage and later development. Even though the times to later stages
are from fertilization and must include some time during cleavage when
the temperature relation is like that of the upper curve, a significant
difference in slope is visible. If all the curves actually were parallel,
an error of about 25 per cent must be postulated at both ends of the
cleavage curve. Such errors are extremely improbable with Krogh's
method. His precision in measuring cleavages should be better than
that for later stages and yet no such errors are visible in his later stage
data (for example, the maximum deviation of a point from the straight
lines in Fig. 2 is equivalent to an error in time of only 5 per cent).
Despite this difference between cleavage and later development, the
parallelism of curves among different stages of later development in
Fig. 2 shows that the temperature relations of the latter are alike.- Ap-
parently in contradiction to this, Belehradek (1926) has calculated for
Krogh's data a series of b values increasing from 1.76 to 2.52 between
medullary groove closure and 7.8 mm. tadpole formation. However,
from the same data and for the same stages b values of 1.6 and 1.7 (and
Q10's decreasing from 4.2 to 3.5) can be calculated according to the
points selected for comparison.
1 Atlas' (1935) Fig. 8 indicates the same temperature characteristic over the
low temperature range for the rates of different stages of development in Rana
pipiens, but the column he uses to include all the points obscures the difference be-
tween the temperature relations of the different stages. At higher temperatures
the temperature relations of cleavages, of gastrulation, and of later development
show the same sort of differences as are visible in Fig. 1 of this paper. Neither
the data for Rana pipiens described in this paper nor Atlas' data (Fig. 2) for the
same animal show the adaptation in the rate of later cleavages found by Hoadley
and Brill (1937) in Arbacia and Chactopterus, although this may be because the
temperatures used were not close enough to the maximum.
- Times from fertilization or first cleavage, instead of the length of develop-
mental intervals, are used in this and all subsequent figures in order not to exag-
gerate errors in timing the events of later development which are difficult to
measure.
tffc
436
FRANCIS J. RYAN
Confirmation of the similarity in the temperature relations of events
in later development can be found in semi-logarithmic plots of the data
of Moore (1939) for Rana pipiens, R. sylvatica, R. damitans and R.
palustris, and of Knight (1938) for Triton alpestris. The curves for
24
o
O
20
2.2
1.8
O
O
1.4
15.3
'26.0°
168 1295 MO 85
TEMPERATURE
77
MI2 14 16 18
STAGE
20
Fir,. 2 (left). Krogh's (1914) data for Rana Inityrhina showing" the relation
at different temperatures of stage to time from fertilization.3 Ordinate represents
the logarithm of time from fertilization ( for cleavages in minutes, for later stages
in hours) ; abscissa, temperature in °C. The symbols over the curves represent
first cleavage, 7.8 and 7 mm. length, branched gills, external gills, and medullary
groove closure. For the sake of ready comparison, the data for the formation of
external gills are plotted as a straight line by the arbitrary distortion of the tem-
perature axis. The latter are used as a base for the data for other stages.
FIG. 3 (right). Moore's (1939) data for Rana pipiens showing the relation,
at different temperatures, of stages between gastrulation and gill circulation to
time from first cleavage. Ordinate represents time from first cleavage in hours ;
abscissa, stage of development (Pollister and Moore, 1937). For the sake of ready
comparison the data for 18.6° are plotted as a straight line by the arbitrary distor-
tion of the stage axis. The latter are used as a base for the data from other
temperatures.
later development are all parallel. For example, in Fig. 3 Moore's
data for Rana pipiens are presented. These supplement the data in
3 It should be pointed out that the similarity in temperature relation among
these stages does not necessarily imply that each step in the formation of a given
stage has the same temperature relation. There may be differences of short dura-
tion which might be in opposite directions and cancel one another. At any rate, if
there are such differences, they are not additive, for the overall sort of examination
made does not reveal them. The stages in between gastrulation and stage 20 are
not clear-cut enough to obtain easily sufficiently accurate determinations to solve
this problem.
TIME-TEMPERATURE RELATION 437
Fig. 1 inasmuch as they show that many different events between yolk
plug and gill circulation have the same temperature relation in Ran a
pipiens. Indeed, the significance of the difference between the tempera-
ture relation of the period from fourth cleavage to gastrulation and that
of the interval between gastrulation and gill circulation (Fig. 1) is
dubious. The process of gastrulation might have the same temperature
relation as other stages of later development, but when the measured
time also includes cleavages (between fourth cleavage and gastrulation),
the observed overall temperature relation would be intermediate. As-
suming that the curve for fourth cleavage to gastrulation is just such a
composite, a calculation shows that the cleavage temperature relation
would prevail into early blastula stages. However, it is very difficult
to measure blastula stages accurately enough to break this period into its
components and settle the problem.
Peter (1905) computed the Q10's for Hertwig's (1898) data on Raua
fiisca .and claimed that the temperature coefficients gradually increased
with the age of the animal. However, when the O10's between 15° and
24° are compared for different cleavages, there is no significant difference
from the average of 1.37. The same holds for later stages where there
is no significant difference from the average of 2.36. Peter's difficulty
resides in the fact that he included O10's computed for low lethal tem-
peratures where development began but was never completed. In this
range the difference between high and low temperatures becomes pro-
gressively greater with age. When Hertwig's data are put on a semi-
logarithmic plot (Fig. 4), it can be seen from the parallelism of curves
that the times to stages of later development are the same type of func-
tion of temperature but are a different type from that for times to the
first three cleavages. Hertwig could have a 25 per cent time error at
both ends of each of his three cleavage curves. But since the maximum
deviation of a point from the straight lines in Fig. 4 A is only 10 per
cent, the difference between cleavage and later development is probably
real. The displacement of the points for gastrulation from the curves in
Fig. 4B may be real (at 10° there is a time discrepancy of 20 per cent).
This is in accordance with the fact shown in Fig. 1 that the rate of
gastrulation increases to a degree intermediate between cleavage and
later development with a temperature rise. Since gastrulation is com-
pared with cleavage in Fig. 4A, the difference between the temperature
relations of cleavage and later development is all the more convincing.
In summary, it is definite that in Amphibia not only is the temperature
relation different for cleavages and later stages, but there are extremely
long periods during cleavage and during embryo formation over which
the temperature relation remains constant.
438
FRANCIS J. RYAN
It is not surprising that a difference should exist between the tem-
perature relations of cleavages and morphogenesis because visibly these
phenomena are unlike and probably have different causes. The amazing
thing is the similarity in response of so many different stages to tem-
perature. Second, third, and fourth cleavages are enough alike so that
it is not hard to believe that they are the result of the same process.
Between fertilization and first cleavage, however, there occur the com-
pletion of the second maturation division, release of the second polar
0.6
o
o
0-3
1.2
0.8
O
-I 0.4
.0
B
24° 20° 17.5 14.5
TEMPERATURE
12°
P T B E
STAGE
75 9
FIG. 4. Hertwig's (1898) data for Rana fnsca showing the relation, at differ-
ent temperatures, of stage to time from fertilization. A. Ordinate represents log-
arithm of time from fertilization (for cleavages in hours, for later stages in days) ;
abscissa, temperature in °C. B. Ordinate, logarithm of time from fertilization in
days ; abscissa, stage of development from gastrulation to limb bud formation. At
temperatures of 6° and below the parallel relation does not hold, but also develop-
ment does not go to completion at these temperatures. The abscissae in A and B,
using the data for second cleavage and for 20° respectively as the base curves, have
been distorted as in Figs. 1 and 3.
body, and the fusion of pronuclei, before the process begins to resemble
later cleavages. Since the total process of first cleavage is, then, differ-
ent from that of later cleavages, it would be expected a priori that the
temperature relations should differ. But they do not (Fig. 1). The
coincidence may be chance or due to the independence of the cytoplasmic
cleavage (which is being measured) from the nuclear phenomena
(wherein lies the difference between first and later cleavages) or due
to a fundamental process which controls all of the phenomena and im-
poses its temperature relation upon them. The latter seems more likely
TIME-TEMPERATURE RELATION 439
inasmuch as it affords an explanation of the even .more astonishing
similarity in the temperature relations of stages between gastrulation
and stage 20 (Fig. 3). Here such strikingly different processes as
neurulation, tail bud and gill formation, onset of circulation etc. have
the same relation. Either each operation independently has achieved
this or all are controlled by an underlying process which imposes its
temperature relation upon its various expressions. Atlas (1938) has
shown that in Rana pipicns the temperature coefficient of the rate of
oxygen consumption during development is approximately the same as
that of the rate of development. In many marine eggs temperature
affects the rate of early development and the rate of respiration in the
same way (Tyler, 1936&). These correlations suggest that the " pri-
mary gear shaft " (Needham, 1933) integrating developmental processes
is some part of the respiratory metabolism.
If this were so, then there should be a difference in the type of
metabolism during cleavage in the frog's egg from that prevalent during
morphogenesis. Brachet (1934) has, indeed, shown that in the frog
the respiratory quotient changes abruptly at gastrulation from about 0.7
to 1.0. Again, in Tyler's (1936a) studies of marine invertebrates, there
should be the same type of metabolism during cleavage as during later
development because the temperature relations of both processes are the
same. Accordingly, in Urechis where the temperature coefficients of
the first four cleavages are the same (Tyler), the respiratory quotient
remains unchanged over an equivalent period of time (2% hours at
20° C.) (Horowitz, 1940). Thus there is real evidence for the belief
that the coordinator of the various processes of differentiation, the
factor which permits development to be reproducible over a wide range
of temperatures, is the respiratory metabolism.
SUMMARY
1. Stages in embryo formation among Amphibia between yolk plug
and gill circulation have similar time-temperature relations.
2. The time-temperature relation of cleavage, although constant from
first to fourth cleavages, differs from that of embryo formation.
3. It is suggested that the different time-temperature relations of
cleavage and of morphogenesis represent different controlling processes ;
while the similarity of the time-temperature relations among cleavages
and among stages of later development is the expression of a common
controlling process in each case. These controlling processes are prob-
ably parts of the respiratory metabolism and they prevent temperature
from disorganizing development.
I wish to thank Dr. H. B. Steinbach for criticisms of the manuscript.
440 FRANCIS J. RYAN
LITERATURE CITED
ATLAS, M., 1935. The effect of temperature on the development of Rana pipiens.
Physiol. Zool, 8: 290-310.
ATLAS, M., 1938. The rate of oxygen consumption of frogs during embryonic
development and growth. Physiol. Zool., 11: 278-291.
BELEHRADEK, J., 1926. Protoplasmic viscosity as determined by a temperature co-
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BRACKET, J., 1934. fitude du metabolisme de 1'oeuf de Grenouille (Rana fusca)
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DOMS, H., 1915. Uber den Einfluss der Temperatur auf Wachstum und Differ-
enzierung der Organe wahrend der Entwicklung von Rana esculenta.
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GRAY, J., 1928. The growth of fish. III. The effect of temperature on the devel-
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HERTWIG, O., 1898. Ueber den Einfluss der Temperatur auf die Entwicklung von
Rana fusca und Rana esculenta. Arch. mikr. Anat., 51 : 319-381.
HOADLEY, L., AND E. R. BRILL, 1937. Temperature and the cleavage rate of
Arbacia and Chaetopterus. Growth, 1 : 234-244.
HOROWITZ, N. H., 1940. The respiratory metabolism of the developing eggs of
Urechis caupo. Jour. Cell. Comp. Physiol, 15: 299-308.
KNIGHT, F. C. E., 1938. Die Entwicklung von Triton alpestris bei verschiedenen
Temperaturen mit Normaltafel. Arch. Entiv.-incch., 137: 461-473.
KROGH, A., 1914. On the influence of temperature on the rate of embryonic de-
velopment. Zcitschr. allgcin. Physiol., 16 : 163-193.
MOORE, J. A., 1939. Temperature tolerance and rates of development in the eggs
of Amphibia. Ecology, 20 : 459-478.
NEEDHAM, J., 1933. On the dissociability of the fundamental processes in onto-
genesis. Biol. Rev., 8: 180-223.
PETER, K., 1905. Der Grad der Beschleunigung tierischer Entwicklung durch
erhohte Temperatur. Arch. Entiv.-incch., 20: 130.
POLLISTER, A. W., AND J. A. MOORE, 1937. Tables for the normal development of
Rana sylvatica. Anat. Rcc., 68: 489-496.
POWSNER, L., 1935. The effects of temperature on the duration of the develop-
mental stages of Drosophila melanogaster. Physiol. Zool., 8 : 474—520.
RYAN, F. J., 1941. Temperature change and the subsequent rate of development.
Jour. E.i-pcr. Zool, 88 : 25-54.
TYLER, A., 1936a. On the energetics of differentiation. III. Comparison of the
temperature coefficients for cleavage and later stages in the development
of the eggs of some marine animals. Biol Bull, 71 : 59-81.
TYLER, A., 1936&. On the energetics of differentiation. IV. Comparison of the
rates of oxygen consumption and of development at different temperatures
of eggs of some marine animals. Biol Bull, 71 : 82-100.
INDEX
, R. G., AND I. H. PAGE. Be-
havior of the arterioles in hyperten-
sive rabbits, and in normal rabbits
following injections of angiotonin
(abstract), 293.
Acetylcholine, action on intestine of
Daphnia magna, 105.
Action potential, related to protoplasmic
streaming in Nitella and Chara (ab-
stract), 296.
Activation of Cumingia and Arbacia
eggs by bivalent cations, 261.
ADDISON, W. H. F. The distribution of
elastic tissue in the arterial path-
way to the carotid bodies in the
adult dog (abstract), 293.
Adrenaline, hypersensitization of catfish
melanophores to, by denervation
(abstract), 302.
ALLEN, T. H. See Bodine and Allen,
388.
Allometry in normal and regenerating
antennal segments, Daphnia, 119.
Alopias vulpinus, interrenal body (ab-
stract), 299.
ALSUP, F. W. Photodynamic studies on
Arbacia eggs (abstract), 297.
Amaroucium constellatum, regeneration
in early zooid (abstract), 287.
Ameiurus melas, role of hypophysis in
melanogenesis, 352.
Ammonia, utilization of, by Chilomonas
(abstract), 285.
Amoeba verrucosa, cytology of (ab-
stract), 299.
ANDERSON, B. G., AND H. L. BUSCH.
Allometry in normal and regenerat-
ing antennal segments in Daphnia,
119.
Androgenetic eggs of Triturus virides-
cens, 402.
Angiotonin, effect on arterioles, hyper-
tensive and normal rabbits (ab-
stract), 293.
Annual report of the Marine Biological
Laboratory, 1.
Ant colonies, founding of, 392.
441
Antifertilizin and fertilization, sea-
urchin eggs, 364.
Arbacia egg, cleavage, acceleration of,
in hypotonic sea water (abstract),
288.
-, intracellular pH (abstract),
305.
— , lipo-protein complexes in
(abstract), 296.
— , lipo-protein complexes in
(abstract), 296.
— , permeability to potassium
(abstract), 295.
- eggs, effect of centrifugation on
oxygen consumption (abstract),
303.
-, photodynamic studies on (ab-
stract), 297.
larva, ectodermization of (ab-
stract), 304.
• punctulata egg, centrifuged, vital
staining of, 114.
eggs and sperm, intermediary
carbohydrate metabolism before and
after fertilization (abstract), 289.
- sperm, effect of sea water on radio-
sensitivity of (abstract), 282.
Arterioles, behavior following injections
of angiotonin, in hypertensive and
normal rabbits (abstract), 293.
Ascidian larvae, metamorphosis (ab-
stract), 286.
Atropine, action on intestine of Daphnia,
105.
Auditory vesicle, induction, effect of dif-
ferences between stages of donor
and host upon, from foreign ecto-
derm in salamander embryo (ab-
stract), 306.
Axon, giant, of squid, rectifying prop-
erty of (abstract), 277.
Azide, effect on Cypridina luciferin (ab-
stract), 283.
T) ALL, E. G. The source of pancre-
atic juice bicarbonate (program title
only), 277.
442
INDEX
BARRON, E. S. G., AND J. M. GOLDINGER.
Intermediary carbohydrate metabo-
lism of eggs and sperm of Arbacia
punctulata before and after fertiliza-
tion (abstract), 289.
EARTH, L. G. Sec Goldin and Earth,
177.
BENEDICT, D. Sec Navez, Crawford,
Benedict and DuBois (abstract),
289.
Bicarbonates, catalysis of ionic ex-
changes by (abstract), 294.
— , role of carbonic anhydrase in
catalysis of ionic exchanges (ab-
stract), 294.
BIRMINGHAM, L. Regeneration in the
early zooid of Amaroucium con-
stellatum (abstract), 287.
Blastoderm, chick, respiratory rates of
different regions during early stages
(abstract), 283.
Blood, effects of desoxycortico-sterone
and related compounds on mam-
malian red cell (abstract), 295.
— , equilibrium between hemoglobin
and oxygen of whole and hemo-
lyzed, tautog, 307.
— , heat produced by, of Tautoga and
Mustelus (abstract), 305.
BODINE, J. H., AND T. H. ALLEN. En-
zymes in ontogenesis (Orthoptera).
XIX. Protyrosinase and morpho-
logical integrity of grasshopper
eggs, 388.
Bresslaua, feeding mechanisms and nu-
trition in, 221.
BROOKS, M. M. Further interpretations
of the effects of CO and CN on ox-
idations in living cells (abstract),
284.
BROWN, F. A., JR., AND O. CUNNING-
HAM. Upon the presence and dis-
tribution of a chromatophorotropic
principle in the central nervous sys-
tem of Limulus, 80.
BUSCH, H. L. See Anderson and
Busch, 119.
GABLE, R. M., AND A. V. HUNNI-
NEN. Studies on the life history of
Siphodera vinaledwardsii, a trema-
tode parasite of the toadfish (ab-
stract), 279.
Carbonic anhydrase, role in catalysis of
ionic exchanges by bicarbonates
(abstract), 294.
Carbohydrate metabolism, intermediary,
of Arbacia eggs and sperm before
and after fertilization (abstract),
289.
CARLSON, L. D. Enzymes in ontogene-
sis (Orthoptera). XVIII. Ester-
ases in the grasshopper egg, 375.
Catalysis of ionic exchanges by bicar-
bonates (abstract), 294.
- of ionic exchanges by bicarbonates,
role of carbonic anhydrase in (ab-
stract), 294.
Catfish melanophores, responses to er-
gotamine, 163.
— , role of hypophysis in melanogene-
sis, 352.
Cations, bivalent, activation of Cumingia
and Arbacia eggs by, 261.
Cell oxidation, effects of CO and CN
(abstract), 284.
Cellular respiration, fractionation by
narcotics (abstract), 282.
Central nervous system of Limulus, lo-
calization of neurosecretory cells,
96.
Centrifugation, effect on oxygen con-
sumption of Arbacia eggs (ab-
stract), 303.
Chaos nobilis Penard in permanent cul-
ture (abstract), 303.
Chara, protoplasmic streaming and ac-
tion potential (abstract), 296.
CHASE, A. M. Effect of azide on Cy-
pridina luciferin (abstract), 283.
— , — . — . Observations on lumines-
cence in Mnemiopsis (abstract),
296.
Chilomonas paramecium, ammonia uti-
lization (abstract), 285.
Chromatin bridges and irregularity of
mitotic coordination in Peromatus
notatus, 149.
Chromatophorotropic principle in cen-
tral nervous system of Limulus, 80.
Ciliary movement, coordination of, in
Modiolus gill (abstract), 290.
Ciliates, thiamin synthesis by (abstract),
285.
CLAFF, C. L., V. C. DEWEY AND G. W.
KIDDER. Feeding mechanisms and
nutrition in three species of Bress-
laua, 221.
CLARK, L. B. Factors in the lunar cy-
cle which may control reproduction
in the Atlantic palolo (abstract),
278.
INDEX
443
CLAUDE, A. Chemical composition of
mitochondria and secretory granules
(program title only), 286.
Cleavage, acceleration of, in Arbacia
egg, in hypotonic sea water (ab-
stract), 288.
Clymenella, implants of young buds
formed in anterior regeneration and
nerve cord of adjacent old part (ab-
stract), 302.
CN, effects on oxidations in living cells
(abstract), 284.
CO, effects on oxidations in living cells
(abstract), 284.
COE, W. R. Sexual phases in wood-
boring mollusks, 168.
Colchicine, disruption of mitosis by, in
Colchicum (abstract), 297.
Colchicum, mitosis disrupted by colchi-
cine in (abstract), 297.
COLE, K. S. Sec Guttman and Cole
(abstract), 277.
CORNMAN, I. Characteristics of the ac-
celeration of Arbacia egg cleavage
in hypotonic sea water (abstract),
288.
— , — . Disruption of mitosis in Col-
chicum by means of colchicine (ab-
stract), 297.
Crangon armillatus, moulting, factors
influencing, 215.
CRAWFORD, J. D. Sec Navez, Craw-
ford, Benedict and DuBois (ab-
stract), 289.
Crayfish kidney, urine-formation by, as
shown by secretion of inulin, xylose
and dyes, 235.
— , micturition in, 134.
— , nephric tubule, outward secretion
of water by, 127.
Culture, permanent, of Chaos nobilis
Penard (abstract), 303.
Cunner, melanophore control (abstract),
300.
CUNNINGHAM, O. Sec Brown and
Cunningham, 80.
Cynthia partita, larva of, " eye-spot "
and light responses (abstract), 287.
Cypridina luciferin, effect of azide on
(abstract), 283.
"T^APHNIA, allometry in normal and
regenerating antennal segments, 119.
- magna, effect of acetylcholine, atro-
pine and physostigmine on intestine,
105.
DAVSON, H. See Shapiro and Davson
(abstract), 295.
Decomposition and regeneration of ni-
trogenous organic matter in sea wa-
ter, 63.
Desoxycortico-sterone, effects on mam-
malian red cell (abstract), 295.
Development, time-temperature relation
of different stages of, 431.
DEWEY, V. C. Sec Claff, Dewey and
Kidder, 221.
— , — . — ., AND G. W. KIDDER. The
possibility of thiamin synthesis by
ciliates (abstract), 285.
Dog, distribution of elastic tissue in ar-
terial pathway to carotid bodies
(abstract), 293.
DuBois, A. B. See Navez, Crawford,
Benedict and DuBois (abstract),
289.
Dyes, effect on response to light in Pe-
ranema trichophorum (abstract),
285.
— , secretion of, and urine-formation
by crayfish kidney, 235.
DYTCHE, M. See Wolf, Dytche, O'Neal
and Schaffel (abstract), 305.
DZIEMIAN, A. J. The permeability and
the lipid content of the erythrocytes
in experimental anemia (abstract),
277.
gCHINODERM hybrids, maternal in-
heritance in (abstract), 288
Ectodermization of Arbacia larva (ab-
stract), 304.
Elasmobranch interrenal (abstract), 299.
Entamoeba muris, food habits, 324.
Entamoeba ranarum, food habits, 324.
Ergotamine, responses of catfish mela-
nophores to, 163.
Erythrocytes, permeability and lipid con-
tent in experimental anemia (ab-
stract), 277.
EVANS, T. C. The effect of roentgen
radiation on the jelly of the Nereis
zygote (abstract), 298.
— , — . — ., AND J. C. SLAUGHTER. Ef-
fect of sea water on the radiosensi-
tivity of Arbacia sperm (abstract),
282.
— , — . — ., J. C. SLAUGHTER, E. P.
LITTLE AND G. FAILLA. The in-
fluence of the medium on the radio-
sensitivity of sperm (abstract), 291.
Esterases in the grasshopper egg, 375.
444
INDEX
" Eye-Spot " and light responses of
larva of Cynthia partita (abstract),
287.
"pAILLA, G. See Evans, Slaughter,
Little and Failla (abstract), 291.
Feather germs, pigment deposition, chick
embryos (abstract), 280.
Feeding mechanisms and nutrition in
three species of Bresslaua, 221.
Fertilization, intermediary carbohydrate
metabolism of Arbacia eggs and
sperm before and after (abstract),
289.
Fertilization, metabolism and, in star-
fish egg (abstract), 278.
Fertilization, role of antifertilizin in,
sea-urchin eggs, 364.
Fertilization, sea-urchin eggs, role of
fertilizin, 190.
Fertilizin, role of, in fertilization of sea-
urchin eggs, 190.
FISHER, K. C. The fractionation of cel-
lular respiration by the use of nar-
cotics (abstract), 282.
Flounder, factors influencing pigmenta-
tion of regenerating scales on ven-
tral surface (abstract), 301.
— , summer, origin of artificially de-
veloped melanophores, 341.
Food habits of Entamoeba muris, 324.
QALTSOFF, P. S. Accumulation of
manganese and the sexual cycle in
Ostrea virginica (abstract), 278.
GATES, R. R. Tests of nucleoli and
cytoplasmic granules in marine eggs
(abstract), 298.
GOLDIN, A., AND L. G. BARTH. Regen-
eration of coenosarc fragments re-
moved from the stem of Tubularia
crocea, 177.
GOLDINGER, J. M. See Barren and Gol-
dinger (abstract), 289.
Grasshopper egg, esterases in, 375.
— , protyrosinase, effect on mor-
phological integrity, 388.
GUTTMAN AND K. S. COLE. The recti-
fying property of the giant axon of
the squid (abstract), 277.
GRAVE, C. Further studies of metamor-
phosis of ascidian larvae (abstract),
286.
— , — . The " eye-spot " and light-
responses of the larva of Cynthia
partita (abstract), 287.
^[ABROBRACON, sex-linkage of
stubby (sb) in (abstract), 298.
HAGER, R. P. Sex-linkage of stubby
(sb) in Habrobracon (abstract),
298.
HAMILTON, H. L. The influence of
hormones on the differentiation of
melanophores in birds (abstract),
281.
Haploidy, experimental, in salamander
larvae, 402.
HARVEY, E. B. Maternal inheritance in
echinoderm hybrids (abstract), 288.
— , — . — . Vital staining of the cen-
trifuged Arbacia punctulata egg,
114.
— , E. N. Stimulation by intense
flashes of ultra-violet light (ab-.
stract), 291.
HASSETT, C. C. The effect of dyes on
the response to light in Peranema
trichophorum (abstract), 285.
HAYES, E. R. The elasmobranch inter-
renal ; a preliminary note. The in-
terrenal body of Alopias vulpinus
(Bonnaterre) (abstract), 299.
Heart, metabolism of, clam (abstract),
289.
Hearts, myogenic and neurogenic, com-
parative pharmacology of (ab-
stract), 292.
Heat produced by respiring whole blood
of Tautoga and Mustelus (ab-
stract), 305.
Hemoglobin-oxygen equilibrium in whole
and hemolyzed blood of tautog, 307.
HESS, W. N. Factors influencing
moulting in the crustacean, Crangon
armillatus, 215.
HILL, S. E. The relation between pro-
toplasmic streaming and the action
potential in Nitella and Chara (ab-
stract), 296.
HOLLINGSWORTH, J. Activation of Cu-
mingia and Arbacia eggs by biva-
lent cations, 261.
HOPKINS, D. L. The cytology of
Amoeba verrucosa (abstract), 299.
Hormones, influence of, on differentia-
tion of melanophores in birds (ab-
stract), 281.
Host-relations, specificity and, in Zoo-
gonus, 205.
HUNNINEN, A. V. Sec Cable and Hun-
ninen (abstract), 279.
INDEX
445
HUNTER, G. W., Ill, AND E. WASSER-
MAN. Observations on the melano-
phore control of the cunner Tau-
togolabrus adspersus (Walbaum)
(abstract), 300.
HUTCHENS, J. O. The utilization of
ammonia by Chilomonas parame-
cium (abstract), 285.
Hypersensitization of catfish melano-
phores to adrenaline by denervation
(abstract), 302.
Hypophysis, role in melanogenesis in
catfish, 352.
JMMUNITY to infection, pathology
and, by heterophyid trematodes (ab-
stract), 279.
Implants of young buds, formed in an-
terior regeneration, plus nerve cord
of adjacent old part, Clymenella
(abstract), 302.
Inheritance, maternal, in echinoderm hy-
brids (abstract), 288.
Interrenal body of Alopias vulpinus
(abstract), 299.
Intestine, action of acetylcholine, atro-
pine and physostigmine on, Daphnia
magna, 105.
Inulin, secretion of, and bearing on
urine-formation by kidney of cray-
fish, 235.
IRVING, L. See Root and Irving, 307.
JACOBS, M. H. See Netsky and Ja-
cobs (abstract), 295.
— , — . — . Sec Stewart and Jacobs
(abstract), 294.
— , — . — ., AND D. R. STEWART. Ca-
talysis of ionic exchanges by bicar-
bonates (abstract), 294.
IT AYLOR, C. T. Studies on experi-
mental haploidy in salamander lar-
vae. II. Cytological studies on
androgenetic eggs of Triturus viri-
descens, 402.
KIDDER, G. W. See Claff, Dewey and
Kidder, 221.
— , — . — . See Dewey and Kidder
(abstract), 285.
, L. J. The founding of
ant colonies, 392.
Light, response to, effect of dyes on, in
Peranema trichophorum (abstract),
285.
responses of larva of Cynthia par-
tita (abstract), 287.
Limulus, chromatophorotropic principle
in central nervous system, 80.
— , localization of neurosecretory cells
in central nervous system, 96.
Lipid content of erythrocytes in experi-
mental anemia (abstract), 277.
Lipo-protein complexes in Arbacia egg
(abstract), 296.
LITTLE, E. P. Sec Evans, Slaughter,
Little and Failla (abstract), 291.
LUCAS, A. M., AND J. SNEDECOR. Co-
ordination of ciliary movement in
the Modiolus gill (abstract), 290.
Luminescence in Mnemiopsis (abstract),
296.
Lunar cycle, and reproduction in At-
lantic palolo (abstract), 278.
]\/f ACTRA egg cells, studies on (ab-
stract), 303.
MALUF, N. S. R. Experimental cyto-
logical evidence for an outward se-
cretion of water by the nephric tu-
bule of the crayfish, 127.
, — . — . — . Micturition in the
crayfish and further observations on
the anatomy of the nephron of this
animal, 134.
, — . — . — . Secretion of inulin,
xylose and dyes and its bearing on
the manner of urine-formation by
the kidney of the crayfish, 235.
Manganese, accumulation of, and sexual
cycle in Ostrea virginica (abstract),
278.
Marine Biological Laboratory, annual
report, 1.
Melanogenesis, role of hypophysis in,
in catfish, 352.
Melanophore control of cunner (ab-
stract), 300.
differentiation, influence of hor-
mones on, in birds (abstract), 281.
hormone, distribution and develop-
ment of, in pituitary of chick (ab-
stract), 281.
system, organization of, in bony
fishes (abstract), 280.
Melanophores, artificially developed, ori-
gin of, in summer flounder, 341.
— , catfish, hypersensitization of, to
adrenaline by denervation (ab-
stract), 302.
446
INDEX
MENDOZA, G. The reproductive cycle
of the viviparous teleost, Neotoca
bilineata, a member of the family
Goodeidae, 70.
Metabolism and fertilization in starfish
egg (abstract), 278.
— , carbohydrate, intermediary, of
eggs and sperm of Arbacia before
and after fertilization (abstract),
289.
- of heart of Venus mercenaria (ab-
stract), 289.
Metamorphosis of ascidian larvae (ab-
stract), 286.
Micromalthus, reversal of sex produc-
tion, 420.
Micturition in the crayfish, 134.
MILNE, L. J. Preparing an animated
diagram of somatic mitosis (ab-
stract), 290.
Mitosis, disruption of, in Colchicum by
colchicine (abstract), 297.
— , somatic, preparing an animated
diagram of (abstract), 290.
Mnemiopsis, luminescence in (abstract),
296.
Modiolus gill, coordination of ciliary
movement (abstract), 290.
Mollusks, wood-boring, sexual phases,
168.
MOOG, F. The influence of temperature
on reconstitution in Tubular ia (ab-
stract), 300.
Moulting, factors influencing, in Cran-
gon, 215.
Mustelus, heat produced by respiring
whole blood (abstract), 305.
JS^ACHMANSOHN, D. Electrical
potential and activity of choline
esterase in nerves (program title
only), 286.
Narcotics, fractionation of cellular respi-
ration by (abstract), 282.
NAVEZ, A. E., J. D. CRAWFORD, D. BEN-
EDICT AND A. B. DuBois. On me-
tabolism of the heart of Venus mer-
cenaria (abstract), 289.
Neotoca bilineata, reproductive cycle, 70.
Nephron, anatomy of. crayfish, 134.
Nereis zygote jelly, effect of Roentgen
radiation (abstract), 298.
NETSKY, M. G., AND M. H. JACOBS.
Some effects of desoxycortico-ster-
one and related compounds on the
mammalian red cell (abstract), 295.
Neurosecretory cells, localization of, in
central nervous system, Limulus, 96.
Nitella, protoplasmic streaming and ac-
tion potential (abstract), 296.
Nitrogenous organic matter, in sea wa-
ter, decomposition and regeneration,
63.
Nucleoli, tests of, and cytoplasmic gran-
ules in marine eggs (abstract), 298.
Nutrition, feeding mechanisms and, in
three species of Bresslaua, 221.
QBRESHKOVE, V. The action of
acetylcholine, atropine and physo-
stigmine on the intestine of Daphnia
magna, 105.
O'MELVENY, K. See Tyler and O'Mel-
veny, 364.
O'NEAL, J. D. See Wolf, Dytche,
O'Neal and Schaffel (abstract),
305.
OSBORN, C. M. Factors influencing the
pigmentation of regenerating scales
on the ventral surface of the sum-
mer flounder (abstract), 301.
, — . — . Studies on the growth of
integumentary pigment in the lower
vertebrates. I. The origin of arti-
ficially developed melanophores on
the normally unpigmented ventral
surface of the summer flounder
(Paralichthys dentatus), 341.
— , — . — . Studies on the growth of
integumentary pigment in the lower
vertebrates. II. The role of the
hypophysis in melanogenesis in the
common catfish (Ameiurus melas),
352.
Ostrea virginica, accumulation of man-
ganese and sexual cycle (abstract),
278.
Oxidations, living cells, effects of CO
and CN (abstract), 284.
Oxygen consumption, effect of centrifu-
gation on, Arbacia eggs (abstract),
303.
—hemoglobin equilibrium in whole
and hemolyzed blood of tautog, 307.
pAGE, I. H. See Abell and Page
(abstract), 293.
Palolo, Atlantic, lunar cycle and repro-
duction in (abstract), 278.
Paralichthys dentatus, origin of artifi-
cially developed melanophores, 341.
INDEX
447
Paramecium bursaria, zoochlorellae-free
(abstract), 304.
Parasite, malarial, conditions affecting
survival in vitro (abstract), 284.
PARKER, G. H. Hypersensitization of
catfish melanophores to adrenaline
by denervation (abstract), 302.
— , — . — . The organization of the
melanophore system in bony fishes
(abstract), 280.
— , — . — . The responses of catfish
melanophores to ergotamine, 163.
PARPART, A. K. Lipo-protein complexes
in the egg of Arbacia (abstract) ,
296.
Pathology and immunity to infection
with heterophyid trematodes (ab-
stract), 279.
Peranema trichophorum, effect of dyes
on response to light (abstract), 285.
Permeability of Arbacia egg to potas-
sium (abstract), 295.
of erythrocytes in experimental
anemia (abstract), 277.
Peromatus notatus, chromatin bridges
and irregularity of mitotic coordina-
tion, 149.
pH, intracellular, in Arbacia egg (ab-
stract), 305.
Pharmacology of myogenic and neuro-
genic hearts (abstract), 292.
PHILIPS, F. S. Comparison of the re-
spiratory rates of different regions
of the chick blastoderm during early
stages of development (abstract),
283.
Photodynatnic studies on Arbacia eggs
(abstract), 297.
Physostigmine, action on intestine of
Daphnia magna, 105.
Pigmentation, factors influencing, of re-
generating scales on ventral sur-
face of summer flounder (abstract) ,
301.
Pigment disposition, some aspects of, in
feather germs of chick embryos
(abstract), 280.
Pituitary of chick, distribution and de-
velopment of melanophore hormone
in (abstract), 281.
Plasmodium lophurae, conditions affect-
ing survival in vitro (abstract) , 284.
Potassium, permeability of Arbacia egg
to (abstract), 295.
PROSSER, C. L., AND G. L. ZIMMERMAN.
Comparative pharmacology of myo-
genic and neurogenic hearts (ab-
stract), 292.
Protoplasmic streaming, related to ac-
tion potential, in Nitella and Chara
(abstract), 296.
Protyrosfnase, effect on morphological
integrity of grasshopper eggs, 388.
{^ADIOSENSITIVITY of Arbacia
sperm, effect of sea water (ab-
stract), 282.
RAHN, H. The distribution and devel-
opment of the melanophore hormone
in the pituitary of the chick (ab-
stract), 281.
RAKESTRAW, N. W. Sec von Brand and
Rakestraw, 63.
Reconstitution in Tubularia, influence of
temperature (abstract), 300.
Regeneration in early zooid of Amarou-
cium constellatum (abstract), 287.
• of coenosarc fragments removed
from Tubularia stem, 177.
Reproduction, lunar cycle and, in At-
lantic palolo (abstract), 278.
Reproductive cycle, of Neotoca biline-
ata, 70.
Respiration, cellular, fractionation of, by
narcotics (abstract), 282.
Respiratory rates of chick blastoderm,
comparison of, during early stages
of development (abstract), 283.
Roentgen radiation, effect on jelly of
Nereis zygote (abstract), 298.
ROOT, R. W., AND L. IRVING. The equi-
librium between hemoglobin and
oxygen in whole and hemolyzed
blood of the tautog, with a theory
of the Haldane effect, 307.
Ryan, F. J. The time-temperature rela-
tion of different stages of develop-
ment, 431.
gALAMANDER embryo, effect of dif-
ferences between stages of donor
and host on induction of auditory
vesicle from foreign ectoderm (ab-
stract), 306.
SAYLES, L. P. Implants consisting of
young buds, formed in anterior re-
generation in Clymenella, plus the
nerve cord of the adjacent old part
(abstract), 302.
SCHAEFFER, A. A. Chaos nobilis Pe-
nard in permanent culture (ab-
stract), 303.
448
INDEX
SCHAFFEL, M. Sec Wolf, Dytche,
O'Neal and Schaffel (abstract),
305.
SCHARRER, BERTA. Neurosecretion. IV.
Localization of neurosecretory cells
in the central nervous system of
Limulus, 96.
SCHECHTER, V. Aging phenomena, and
factors influencing the longevity of
Mactra eggs (program title only),
283.
— , — . Further studies on Mactra
egg cells (abstract), 303.
SCHRADER, F. Chromatin bridges and
irregularity of mitotic coordination
in the pentatomid Peromatus nota-
tus Am. and Serv., 149.
SCOTT, A. Reversal of sex production
in Micromalthus, 420.
Sea-urchin eggs, antifertilizin and fer-
tilization, 364.
— , fertilization in fertilization of,
190.
Sea water, decomposition and regenera-
tion of nitrogenous organic matter
in, 63.
— , effect on radiosensitivity of
Arbacia sperm (abstract), 282.
Sex-linkage of stubby (sb) in Habro-
bracon (abstract), 298.
Sex phases in wood-boring mollusks,
168.
- production, reversal of, in Micro-
malthus, 420.
Sexual cycle, and manganese accumula-
tion in Ostrea (abstract), 278.
SHAPIRO, H. Metabolism and fertiliza-
tion in the starfish egg (abstract),
278.
— , — ., AND H. DAVSON. Permeabil-
ity of the Arbacia egg to potassium
(abstract), 295.
Siphodera vinaledwardsii, life history of
(abstract), 279.
SLAUGHTER, J. C. See Evans and
Slaughter (abstract), 282.
— , — . — . See Evans, Slaughter, Lit-
tle and Failla (abstract), 291.
SNEDECOR, J. Sec Lucas and Snedecor
(abstract), 290.
Specificity and host-relations in Zoogo-
nus, 205.
Squalus acanthias, yolk absorption,
structures concerned with (ab-
stract), 292.
Squid, giant axon, rectifying property
of (abstract), 277.
Starfish egg, metabolism and fertiliza-
tion in (abstract), 278.
STEWART, D. R. See Jacobs and Stew-
art (abstract), 294.
— , — . — ., AND M. H. JACOBS. The
role of carbonic anhydrase in the
catalysis of ionic exchanges by bi-
carbonates (abstract), 294.
Stimulation by intense flashes of ultra-
violet light (abstract), 291.
STUNKARD, H. W. Spectificity and
host-relations in the trematode ge-
nus Zoogonus, 205.
— , — . ., AND C. H. WlLLEY. Pa-
thology and immunity to infection
with heterophyid trematodes (ab-
stract), 279.
HPAUTOGA, heat produced by respir-
ing whole blood (abstract) , 305.
Tautog, equilibrium between hemoglobin
and oxygen of whole and hemolyzed
blood, 307.
Tautogolabrus adspersus, melanophore
control (abstract), 300.
Temperature, influence on reconstitution
in Tubularia (abstract), 300.
TEWINKEL, L. E. Structures concerned
with yolk absorption in Squalus
acanthias (abstract), 292.
Thiamin synthesis, possibility of, by cili-
ates (abstract), 285.
Time-temperature relation of different
stages of development, 431.
Tissue, elastic, distribution of, in ar-
terial pathway to carotid bodies in
adult dog (abstract), 293.
TRACER, W. Studies on conditions af-
fecting the survival in vitro of a
malarial parasite (Plasmodium lo-
phurae), 284.
Trematodes, heterophyid, pathology and
immunity to infection with (ab-
stract), 279.
Triturus viridescens, androgenetic eggs
of, 402.
Tubularia crocea, regeneration of coeno-
sarc fragments removed from stem,
177.
— , temperature and reconstitution
(abstract), 300.
TYLER, A. The role of fertilizin in the
fertilization of eggs of the sea
urchin and other animals, 190.
INDEX
449
-, — ., AND K. O'MELVENY. The role
of antifertilizin in the fertilization of
sea-urchin eggs, 364.
[JLTRA-VIOLET light, stimulation
by intense flashes of (abstract), 291.
Urine-formation, as shown by secretion
of inulin, xylose and dyes, crayfish
kidney, 235.
yELICK, S. F. The effect of centrif-
ugation upon the oxygen consump-
tion of Arbacia eggs (abstract),
303.
Venus mercenaria, metabolism of heart
of (abstract), 289.
Vital staining of centrifuged Arbacia
egg, 114.
VON BRAND, T., AND N. W. RAKESTRAW.
Decomposition and regeneration of
nitrogenous organic matter in sea
water. IV, 63.
\yASSERMAN, E. Sec Hunter and
Wasserman (abstract), 300.
WATERMAN, A. Ectodermization of the
larva of Arbacia (abstract), 304.
Water secretion by nephric tubule of
crayfish, 127.
WATTERSON, R. L. Some aspects of
pigment deposition in feather germs
of chick embryos (abstract), 280.
WENRICH, D. H. Observations on the
food habits of Entamoeba muris and
Entamoeba ranarum, 324.
\VICHTERMAN, R. Studies on zoochlo-
rellae-free Paramecium bursaria
(abstract), 304.
WIERCINSKI, F. J. An experimental
study of intracellular pH in the
Arbacia egg (abstract), 305.
WILLEY, C. H. Sec Stunkard and Wil-
ley (abstract), 279.
WOLF, E. A., M. DYTCHE, J. D. O'NEAL,
AND M. SCHAFFEL. Heat produced
by respiring whole blood of Tautoga
onitis and Mustelus canis (ab-
stract), 305.
WKIXCII, D. Native proteins and the
structure of cytoplasm (program
title only), 286.
"VYLOSE secretion and urine-forma-
tion by crayfish kidney, 235.
VOLK absorption in Squalus acanthias
(abstract), 292.
YNTEMA, C. L. Effect of differences
between stages of donor and host
upon induction of auditory vesicle
from foreign ectoderm in the sala-
mander embryo (abstract), 306.
£IMMERMAN, G. L. See Prosser
and Zimmerman (abstract), 292.
Zoochlorellae-free Paramecium bursaria
(abstract), 304.
Zo»e;onus, specificity and host-relations,
205.
Volume LXXXI Number 1
\\
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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.
BIOLOGY SUPPLIES
The Supply Department of the Marine Biological Labora-
tory has a complete stock of excellent plain preserved and
latex injected materials, and would be pleased to quote prices
on your summer school needs.
PRESERVED SPECIMENS
for
Zoology, Botany, Embryology,
and Comparative Anatomy
LIVING SPECIMENS
for
Zoology and Botany
including Protozoan and
Drosophila Cultures, and
Animals for Experimental and
Laboratory Use.
MICROSCOPE SLIDES
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Histology, Bacteriology, and
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Catalogues promptly sent on request.
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MARINE
BIOLOGICAL LABORATORY
Woods Hole, Mass.
CONTENTS
Page
ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY. ... i
VON BRAND, THEODOR AND NORRIS W. RAKESTRAW
Decomposition and Regeneration of Nitrogeneous Organic
Matter in Sea Water. IV 63
MENDOZA, GUILLERMO
The Reproductive Cycle of the Viviparous Teleost, Neotoca
bilineata, a Member of the Family Goodeidae 70
BROWN, FRANK A., JR., AND ONA CUNNINGHAM
Upon the Presence and Distribution of a Chromatophorotropic
Principle in the Central Nervous System of Limulus 80
SCHARRER, BERTA
Neurosecretion. IV. Localization of neurosecretory cells in
the central nervous system of Limulus 96
OBRESHKOVE, VASIL
The Action of Acetylcholine, Atropine and Physostigmine on
the Intestine of Daphnia magna 105
HARVEY, ETHEL BROWNE
Vital Staining of the Centrifuged Arbacia punctulata Egg. . . 114
ANDERSON, B. G., AND H. L. BUSCH
Allometry in Normal and Regenerating Antennal Segments in
Daphnia 119
MALUF, N. S. RUSTUM
Experimental Cytological Evidence for an Outward Secretion
of Water by the Nephric Tubule of the Crayfish 127
MALUF, N. S. RUSTUM
Micturition in the Crayfish and Further Observations on the
Anatomy of the Nephron of this Animal 134
SCHRADER, FRANZ
Chromatin Bridges and Irregularity of Mitotic Coordinationfin
the Pentatomid Peromatus notatus Am. and Serv. . 149
Volume LXXXI Number 2
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
E. N. HARVEY, Princeton University CARL R> MOORE, University of Chicago
SELIG HECHT Columbia University GEQRGE T M MissQuri
LEIGH HOADLEY, Harvard University „, TT ,,
L. IRVING, Swarthmore College aGAN' Callforma 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
OCTOBER, 1941
Printed and Issued by
LANCASTER PRESS, Inc.
PRINCE &. LEMON STS.
LANCASTER, PA.
ANNOUNCEMENT
x/N INDEX of the Biological Bulletin, Volumes 61 to 80,
^— ^-^ will be published about October 25, 1941. This Index
contains an alphabetical list of authors, showing the titles of their
papers, and a classified index of subjects. The published abstracts
of papers presented at the Marine Biological Laboratory are also
indexed in this volume.
The edition of the Index is limited. Orders should be sent
to the Marine Biological Laboratory, Woods Hole, Mass.
The price of the Index is $2.50 postpaid. The Index to Vols.
1-60 and the Index to Vols. 61-80 are now offered at a combi-
nation price of $5.00.
ORDER FORM
MARINE BIOLOGICAL LABORATORY,
WOODS HOLE, MASSACHUSETTS.
Please enter my order for one copy of the Index of the
Biological Bulletin, Volumes 61 to 80, at the price of $2.50.
(check here)
Please enter my order for the Index to Vols. 1-60 and
the Index to Vols. 61-80 at a total price of $5.00.
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NAME
ADDRESS
Message to Biologists
Some biologists take Biological Abstracts for granted. It's just one
of those things they couldn't very well get along without — but they don't
stop to think that they have any responsibility in the matter.
If Biological Abstracts were discontinued what would it mean to you?
—how would you keep abreast of all the important literature in your field?
Many leading scientists have said that Biological Abstracts is one journal
they just could not afford to dispense with.
Biological Abstracts is the biologists' journal. It is a co-operative,
non-profit enterprise published by biologists themselves. As a result of
the war many foreign subscriptions were lost and if this invaluable ab-
stracting service is to be continued without interruption, the loss must be
made up in the Western Hemisphere.
Biological Abstracts needs the active support of every biologist. Send
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low priced sections in addition to the complete issue.
BIOLOGICAL ABSTRACTS
University of Pennsylvania
Philadelphia, Pa.
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.
THIS .MARK
ON YOUR SCIENTIFIC
ILLUSTRATIONS
MEANS IT WAS
PRODUCED BY EXPERIENCED
CRAFTSMEN IN THE OLDEST
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PLANT IN AMERICA.
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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 (414x7
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.
MARINE AQUARIA
SETS
During the past fifteen years, the Supply Depart-
ment has shipped out several thousand balanced
Marine Aquaria Sets, and has had extraordinary
success in delivering these animals alive and in per-
fect condition. Sets such as these give inland stu-
dents the same advantages in the study of living
forms as students near the ocean where marine ma-
terial may be had abundantly.
From November 1st to March 1st we guarantee
live delivery on these sets to points as far west as
the Mississippi and as far south as Georgia. We
have successfully shipped sets to other parts of the
country and during other months, but we do not
guarantee live delivery on such shipments.
Our new 1941 catalogue which will be issued
within a few weeks lists these Marine Aquaria sets,
as well as an excellent stock of other living material
and preserved specimens for school courses. We
shall be glad to furnish quotations on any of this
material if desired.
Catalogues sent on request
Supply Department
MARINE
BIOLOGICAL LABORATORY
Woods Hole Est. isoo Massachusetts
CONTENTS
Page
PARKER, G. H.
The Responses of Catfish Melanophores to Ergotamine 163
COE, WESLEY R.
Sexual Phases in Wood-boring Mollusks 168
GOLDIN, A., AND L. G. EARTH
Regeneration of Coenosarc Fragments Removed from the
Stem of Tubularia crocea 177
TYLER, ALBERT
The Role of Fertilizin in the Fertilization of Eggs of the Sea
Urchin and Other Animals 190
STUNKARD, HORACE W.
Specificity and Host-relations in the Trematode Genus
Zoogonus 205
HESS, WALTER N.
Factors Influencing Moulting in the Crustacean, Crangon
armillatus 215
CLAFF, C. L., V. C. DEWEY AND G. W. KIDDER
Feeding Mechanisms and Nutrition in Three Species of
Bresslaua . . 221
MALUF, N. S. R.
Secretion of Inulin, Xylose and Dyes and its Bearing on the
Manner of Urine-formation by the Kidney of the Crayfish . . . 235
HOLLINGSWORTH, JOSEPHINE
Activation of Cumingia and Arbacia Eggs by Bivalent Cations 261
PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS PRESENTED
AT THE MARINE BIOLOGICAL LABORATORY, SUMMER OF
1941 276
Volume LXXXI Number 3
THE
BIOLOGICAL BULLETIN
PUBLISHED BY
THE MARINE BIOLOGICAL LABORATORY
Editorial Board
GARY N. CALKINS, Columbia University H. S. JENNINGS, Johns Hopkins University
E. G. CONKLIN, Princeton University FRANK R. LlLLDE, University of Chicago
E. N. HARVEY, Princeton University CARL R. MOORE, University of Chicago
SELIG HECHT, Columbia University GEORGE T. MOORE, Missouri Botanical Garden
LEIGH HOADLEY, Harvard University T. H. MORGAN, California Institute of Technology
L. IRVING, Swarthmore College G. H. PARKER, Harvard University
M. H. JACOBS, University of Pennsylvania F. SCHRADER, Columbia University
ALFRED C. REDFIELD, Harvard University
Managing Editor
DECEMBER, 1941
Printed and Issued by
LANCASTER PRESS, Inc.
PRINCE 8C LEMON STS.
LANCASTER, PA.
ANNOUNCEMENT
,^N INDEX of the Biological Bulletin, Volumes 61 to 80,
^ — -^ was published in October, 1941. This Index contains
an alphabetical list of authors, showing the titles of their papers,
and a classified index of subjects. The published abstracts of
papers presented at the Marine Biological Laboratory are also
indexed in this volume.
The edition of the Index is limited. Orders should be sent
to the Marine Biological Laboratory, Woods Hole, Mass.
The price of the Index is $2.50 postpaid. The Index to Vols.
1-60 and the Index to Vols. 61-80 are now offered at a combi-
nation price of $5.00.
ORDER FORM
MARINE BIOLOGICAL LABORATORY,
WOODS HOLE, MASSACHUSETTS.
Please enter my order for one copy of the Index of the
Biological Bulletin, Volumes 61 to 80, at the price of $2.50.
(check here)
Please enter my order for the Index to Vols. 1-60 and
the Index to Vols. 61-80 at a total price of $5.00.
(check here)
NAME
ADDRESS
Message to Biologists
Some biologists take Biological Abstracts for granted. It's just one
of those things they couldn't very well get along without — but they don't
stop to think that they have any responsibility in the matter.
If Biological Abstracts were discontinued what would it mean to you?
—how would you keep abreast of all the important literature in your field ?
Many leading scientists have said that Biological Abstracts is one journal
they just could not afford to dispense with.
Biological Abstracts is the biologists' journal. It is a co-operative,
non-profit enterprise published by biologists themselves. As a result of
the war many foreign subscriptions were lost and if this invaluable ab-
stracting service is to be continued without interruption, the loss must be
made up in the Western Hemisphere.
Biological Abstracts needs the active support of every biologist. Send
your subscription or ask for a sample copy now. It is published in five
low priced sections in addition to the complete issue.
BIOLOGICAL ABSTRACTS
University of Pennsylvania
Philadelphia, Pa.
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.
THIS MARK
ON YOUR SCIENTIFIC
ILLUSTRATIONS
MEANS IT WAS
PRODUCED BY EXPERIENCED
CRAFTSMEN IN THE OLDEST
COLLOTYPE PRINTING
PLANT IN AMERICA.
WRITE US TODAY FOR
SAMPLES AND ESTIMATES
HELIOTYPE CORPORATION
172 GREEN ST. JAMAICA PLAIN
TELEPHONE ARNOLD 1312
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 July 1 and October 1 and to the Department of
Zoology, Columbia University, New York City, 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 (4^4 x 7
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.
BIOLOGY MATERIALS
MARINE AQUARIA SETS
During the past eight years we have
sent out several thousand living marine
aquaria sets to schools all over the coun-
try, and have had extraordinary success in
delivering the animals alive and in splen-
did condition.
From November 1st to March 1st we
guarantee live delivery on these speci-
mens to points indicated in the living ma-
terial section of our catalogue. The sets
listed have proved most successful, but
should any customer wish substitutions on
the sets or materials not listed, we would
be pleased to quote prices on same.
NEW CATALOGUE
A new issue of our catalogue has just
been printed, and we shall be glad to send
a copy free of charge on request.
Supply Department
MARINE
BIOLOGICAL LABORATORY
Woods Hole Est. isoo Massachusetts
CONTENTS
Page
ROOT, R. W., AND L. IRVING
The Equilibrium between Hemoglobin and Oxygen in Whole
and Hemolyzed Blood of the Tautog, with a Theory of the
Haldane Effect 307
WENRICH, D. H.
Observations on the Food Habits of Entamoeba muris and
Entamoeba ranarum 324
OSBORN, C. M.
Studies on the Growth of Integumentary Pigment in the Lower
Vertebrates. I. The origin of artificially developed melano-
phores on the normally unpigmented ventral surface of the
summer flounder (Paralichthys dentatus) 341
OSBORN, C. M.
Studies on the Growth of Integumentary Pigment in the
Lower Vertebrates. II. The role of the hypophysis in
melanogenesis in the common catfish (Ameiurus melas) .... 352
TYLER, A., AND K. O'MELVENY
The Role of Antifertilizin in the Fertilization of Sea-urchin
Eggs ... 364
CARLSON, L. D.
Enzymes in Ontogenesis (Orthoptera). XVIII. Esterases in
the grasshopper egg 375
BODINE, J. H., AND T. H. ALLEN
Enzymes in Ontogenesis (Orthoptera). XIX. Protyrosinase
and morphological integrity of grasshopper eggs 388
LAFLEUR, L. J.
The Founding of Ant Colonies 392
KAYLOR, C. T.
Studies on Experimental Haploidy in Salamander Larvae.
II. Cytological studies on androgenetic eggs of Triturus
viridescens 402
SCOTT, A.
Reversal of Sex Production in Micromalthus 420
RYAN, F. J.
The Time-Temperature Relation of Different Stages of
Development 431
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