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THE 


BIOLOGICAL  BULLETIN 


PUBLISHED  BY 

THE  MARINE  BIOLOGICAL  LABORATORY 


Editorial  Board 


HAROLD  C.  BOLD,  University  of  Texas 
FRANK  A.  BROWN,  JR.,  Northwestern  University 
JOHN  B.  BUCK,  National  Institutes  of  Health 
T.  H.  BULLOCK,  University  of  California, 

Los  Angeles 

E.  G.  BUTLER,  Princeton  University 
J.  H.  LOCHHEAD,  University  of  Vermont 


ARTHUR  W.  POLLISTER,  Columbia  University 
C.  L.  PROSSER,  University  of  Illinois 
MARY  E.  RAWLES,  Carnegie  Institution  of 

Washington 

WM.  RANDOLPH  TAYLOR,  University  of  Michigan 
A.  R.  WHITING,  University  of  Pennsylvania 
CARROLL  M.  WILLIAMS,  Harvard  University 


DONALD  P.  COSTELLO,  University  of  North  Carolina 
Managing  Editor 


VOLUME  115 

AUGUST  TO  DECEMBER,  1958 


Printed  and  Issued  by 

LANCASTER  PRESS,  Inc. 

PRINCE  8C  LEMON  STS. 

LANCASTER,  PA. 


11 


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

Subscriptions  and  similar  matter  should  be  addressed  to  The 
Biological  Bulletin,  Marine  Biological  Laboratory,  Woods  Hole, 
Massachusetts.  Agent  for  Great  Britain:  Wheldon  and  Wesley, 
Limited,  2,  3  and  4  Arthur  Street,  New  Oxford  Street,  London, 
W.  C.  2.  Single  numbers  $2.50.  Subscription  per  volume  (three 
issues),  $6.00. 

Communications  relative  to  manuscripts  should  be  sent  to  the 
Managing  Editor,  Marine  Biological  Laboratory,  Woods  Hole, 
Massachusetts,  between  June  1  and  September  1,  and  to  Dr. 
Donald  P.  Costello,  P.O.  Box  429,  Chapel  Hill,  North  Carolina, 
during  the  remainder  of  the  year. 


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


LANCASTER    PRESS,    INC.,    LANCASTER,    PA. 


CONTENTS 


No.  1.  AUGUST,  1958  PAGE 

Annual  Report  of  the  Marine  Biological  Laboratory 1 

BOOLOOTIAN,  RICHARD  A.,  AND  ARTHUR  C.  GIESE 

Coelomic  corpuscles  of  echinoderms 53 

BOROUGHS,  HOWARD,  AND  DELLA  F.  REID 

The  role  of  the  blood  in  the  transportation  of  strontium90-yttrium90  in 
teleost  fish 64 

BROOKBANK,  JOHN  W. 

Dispersal  of  the  gelatinous  coat  material  of  Mellita  quinquiesperforata 
eggs  by  homologous  sperm  and  sperm  extracts 74 

BROWN,  FRANK  A.,  JR. 

An  exogenous  reference-clock  for  persistent,  temperature-independent, 
labile,  biological  rhythms 81 

EPPLEY,  RICHARD  W.,  AND  CARLTON  R.  BOVELL 

Sulfuric  acid  in  Desmarestia 101 

GRIFFIN,  D.  R.,  A.  NOVICK  AND  M.  KORNFIELD 

The  sensitivity  of  echolocation  in  the  fruit  bat,  Rousettus 107 

HODGSON,  EDWARD  S. 

Electrophysiological  studies  of  arthropod  chemoreception.   111.   Chemo- 
receptors  of  terrestrial  and  fresh-water  arthropods 114 

MATHEWSON,    ROBERT,    ALEXANDER    MAURO,    ERNEST    AMATNIEK    AND 

HARRY  GRUNDFEST 

Morphology  of  main  and  accessory  electric  organs  of  Narcine  brasiliensis 
(Olfers)  and  some  correlations  with  their  electrophysiological  properties . .    126 

ROTHSCHILD,  LORD,  AND  ALBERT  TYLER 

The  oxidative  metabolism  of  eggs  of  Urechis  caupo 136 

\YATKINS,  MARGARET  J. 

Regeneration  of  buds  in  Botryllus 147 

No.  2.  OCTOBER,  1958 

BLACK,  ROBERT  E.,  SAMUEL  EPSTEIN  AND  ALBERT  TYLER 

The  oxidation  of  carbon  monoxide  by  fertilized  eggs  of  Urechis  caupo 
shown  by  use  of  a  C13  label 153 

FANGE,  R.,  K.  SCHMIDT-NIELSEN  AND  H.  OSAKI 

The  salt  gland  of  the  herring  gull 162 

FANGE,  RAGNAR,  AND  JONATHAN  B.  WITTENBERG 

The  swimbladder  of  the  toadfish  (Opsanus  tau  L.) 172 


in 


iv  CONTENTS 

FLEMISTER,  LAUNCE  J. 

Salt  and  water  anatomy,  constancy  and  regulation  in  related  crabs  from 
marine  and  terrestrial  habitats.  . .  180 

FLICKINGER,  REED  A. 

Regional  localization  of  neural  and  lens  antigens  in  the  frog  embryo  in 
relation  to  induction 201 

HOYLE,  GRAHAM 

Studies  on  neuromuscular  transmission  in  Limulus .  .    209 

LANE,  CHARLES  E.,  AND  ELEANOR  DODGE 

The  toxicity  of  Physalia  nematocysts 219 

MANWELL,  CLYDE 

On  the  evolution  of  hemoglobin.      Respiratory  properties  of  the  hemo- 
globin of  the  California  hagfish,  Polistotrema  stouti 227 

MUN,  ALTON  M. 

Toxic  effects  of   normal   sera   and   homologous  antisera   on   the   chick 
embryo 239 

RYTHER,  J.  H.,  C.  S.  YENTSCH,  E.   M.  HULBURT  AND  R.  F.  VACCARO 

The  dynamics  of  a  diatom  bloom 257 

SCHERBAUM,  OTTO  H.,  ALLAN  L.  LOUDERBACK  AND  THEODORE  L.  JAHN 

The  formation  of  subnuclear  aggregates  in   normal  and  synchronized 
protozoan  cells 269 

STUNKARD,  HORACE  W.,  AND  JOSEPH  R.  UZMANN 

Studies    on    digenetic    trematodes    of    the    genera    Gymnophallus    and 
Parvatrema 276 

\YKBB,  H.  MARGUERITE,  AND  FRANK  A.  BROWN,  JR. 

The  repetition  of  pattern  in  the  respiration  ol  Uca  pugnax 303 

Abstracts  of  papers  presented  at  the  Marine  Biological  Laboratory  : 

Tuesday  Evening  Seminars 319 

Electrobiology  Seminars 329 

General  Meetings 332 

Lalor  Fellowship  Reports 371 

No.  3.  DECEMBER,  1958 

ANDERSON,  JOHN  MAXWELL,  AND  JEANNE  CAROL  JOHANN 

Some  aspects  of  reproductive  biology  in  the  tresh-water  triclad  turbel- 
larian,  Cura  foremanii 375 

AUCLAIR,  WALTER,  AND  DOUGLAS  MARSLAND 

Form-stability  of  ciliates  in  relation  to  pressure  and  temperature 384 

DAVENPORT,  DEMOREST,  AND  KENNETH  S.  NORRIS 

Observations  on  the  symbiosis  of  the  sea  anemone  Stoichactis  and  the 
pomacentrid  fish,  Amphiprion  percula 397 

DENT,  JAMES  NORMAN,  AND  W.  GARDNER  LYNN 

A  comparison  of  the  effects  of  goitrogens  on  thyroid  activity  in  Triturus 
viridescens  and  Desmognathus  fuscus 411 

Fox,  WADE,  AND  HERBERT  C.  DESSAUER 

Responses  of  the  male  reproductive  system  of  lizards  (Anolis  carolinen- 
sis)  to  unnatural  day-lengths  in  different  seasons 421 


CONTENTS  V 

HASTINGS,  J.  WOODLAND,  AND  BEATRICE  M.  SWEENEY 

A  persistent  diurnal  rhythm  of  luminescence  in  Gonyaulax  polyedra  ....    440 

HEILBRUNN,  L.  V.,  FRANCIS  T.  ASHTON,  CARL  FELDHERR  AND  WALTER  L. 

WILSON 
The  action  of  insulin  on  cells  and  protoplasm 459 

HILL,  ROBERT  B. 

The  effects  of  certain  neurohumors  and  of  other  drugs  on  the  ventricle 
and  radula  protractor  of  Busycon  canaliculatum  and  on  the  ventricle  of 
Strombus  gigas 471 

HUTCHISON,  VICTOR  H.,  AND  CARL  S.  HAMMEN 

Oxygen  utilization  in  the  symbiosis  of  embryos  of  the  salamander, 
Ambystoma  maculatum  and  the  alga,  Oophila  amblystomatis 483 

ROYS,  CHESTER  C. 

A  comparison  between  taste  receptors  and  other  nerve  tissues  of  the 
cockroach  in  their  responses  to  gustatory  stimuli 490 

SCHARRER,  BERTA,  AND  MARIANNE  VON  HARNACK 

Histophysiological  studies  on  the  corpus  allatum  of  Leucophaea  maderae. 

I.  Normal  life  cycle  in  male  and  female  adults 508 

VON  HARNACK,  MARIANNE 

Histophysiological  studies  on  the  corpus  allatum  of  Leucophaea  maderae. 

II.  The  effect  of  starvation 521 

SCHNEIDERMAN,    HOWARD    A.,    AND    LAWRENCE    I.    GILBERT 

Substances  with  juvenile  hormone  activity  in  Crustacea  and  other 
invertebrates 5.30 

TERZIAN,  LEVON  A.,  AND  NATHAN  STABLER 

A  study  of  some  effects  of  gamma  radiation  on  the  adults  and  eggs  of 
Aedes  aegypti -. 536 

WELSH,  JOHN  H.,  AND  PEGGY  B.   PROCK 

Quaternary  ammonium  bases  in  the  coelenterates 551 


Vol.  115,  No.  1  August,  1958 

THE 

BIOLOGICAL  BULLETIN 

PUBLISHED  BY  THE  MARINE  BIOLOGICAL  LABORATORY 


THE  MARINE  BIOLOGICAL  LABORATORY 
SIXTIETH  REPORT,  FOR  THE  YEAR  1957 — SEVENTIETH  YEAR 

I.     TRUSTEES  AND  EXECUTIVE  COMMITTEE  (AS  OF  AUGUST  10,  1957)    ....  1 

STANDING  COMMITTEES 

II.     ACT  OF  INCORPORATION  3 

III.  BY-LAWS  OF  THE  CORPORATION  4 

IV.  REPORT  OF  THE  DIRECTOR   6 

Statement    7 

Memorials     8 

Addenda : 

1.  The  Staff 12 

2.  Investigators,  Lalor  and  Lillie  Fellows,  and  Students 15 

3.  Fellowships  and  Scholarships 24 

4.  Tabular  View  of  Attendance,  1953-1957 24 

5.  Institutions  Represented    25 

6.  Evening  Lectures 26 

7.  Shorter  Scientific  Papers  (Seminars)    26 

8.  Members  of  the  Corporation    28 

V.     Report  of  the  LIBRARIAN 46 

VI.     REPORT  OF  THE  TREASURER  47 


I.     TRUSTEES 

EX  OFFICIO 

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

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

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

Syracuse 

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

EMERITI 

EUGENE  DuBois,  Cornell  University  Medical  College 
G.  H.  A.  CLOWES,  Lilly  Research  Laboratory 
W.  C.  CURTIS,  University  of  Missouri 

1 


2  MARINE  BIOLOGICAL  LABORATORY 

PAUL  S.  GALTSOFF,  Woods  Hole,  Mass. 
Ross  G.  HARRISON,  Yale  University 

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

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

F.  P.  KNOWLTON,  Syracuse  University 
W.  J.  V.  OSTERHOUT,  Rockefeller  Institute 
CHARLES  PACKARD,  Woods  Hole,  Mass. 

LAWRASON  RIGGS,  74  Trinity  Place,  New  York  6,  N.  Y. 

TO   SERVE  UNTIL    1961 

D.  W.  BRONK,  Rockefeller  Institute 

G.  FAILLA,  Columbia  University,  College  of  Physicians  &  Surgeons 

E.  NEWTON  HARVEY,  Princeton  University 
R.  T.  KEMPTON,  Vassar  College 

L.  H.  KLEINHOLZ,  Reed  College 

IRVING  M.  KLOTZ,  Northwestern  University 

ALBERT  SZENT-GYORGYI,  Marine  Biological  Laboratory 

WM.  RANDOLPH  TAYLOR,  University  of  Michigan 

TO  SERVE  UNTIL   1960 

H.  F.  BLUM,  Princeton  University 

K.  S.  COLE,  National  Institutes  of  Health 

L.  V.  HEILBRUNN,  University  of  Pennsylvania 

S.  W.  KUFFLER,  Johns  Hopkins  Hospital 

C.  B.  METZ,  Florida  State  University 

G.  T.  SCOTT,  Oberlin  College 

A.  H.  STURTEVANT,  California  Institute  of  Technology 

E.  ZWILLING,  University  of  Connecticut 

TO  SERVE  UNTIL   1959 

E.  G.  BUTLER,  Princeton  University 

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

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

M.  KRAHL,  University  of  Chicago 

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

TO  SERVE  UNTIL   1958 

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

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

AURIN  CHASE,  Princeton  University 

ALBERT  I.  LANSING,  Emory  University 

DANIEL  MAZIA.  University  of  California 

S.  MERYL  ROSE,  University  of  Illinois 

MARY  SEARS,  Woods  Hole  Oceanographic  Institution 

ALBERT  TYLER,  California  Institute  of  Technology 


TRUSTEES 

EXECUTIVE  COMMITTEE  OF  THE  BOARD  OF  TRUSTEES 

GERARD  SVVOPE,  JR.,  Chairman  E.  G.  BUTLER 

A.  K.  PARPART  RUDOLPH  KEMPTON 

J.   H.  WlCKERSHAM  D.   P.   COSTELLO 

P.  B.  ARMSTRONG  H.  B.  STEINBACH 

K.  S.  COLE  EDGAR  ZWILLING 

THE  LIBRARY  COMMITTEE 

MARY  SEARS,  Chairman  E.  G.  BUTLER 

HAROLD  F.  BLUM  J.  P.  TRINKAUS 

E.  T.  MOUL  RALPH  CHENEY 

THE  APPARATUS  COMMITTEE 

C.  LLOYD  CLAFF,  Chairman  ALBERT  I.  LANSING 

M.  V.  EDDS 

THE  SUPPLY  DEPARTMENT  COMMITTEE 

RUDOLPH  KEMPTON,  Chairman  ROBERT  DAY  ALLEN 

C.  B.  METZ  L.  V.  HEILBRUXN 

THE  EVENING  LECTURE  COMMITTEE 

P.  B.  ARMSTRONG,  Chairman  L.  V.  HEILBRUNN 

E.  G.  BALL  W.  D.  MCELROY 

THE  INSTRUCTION  COMMITTEE 

S.  MERYL  ROSE,  Chairman  C.  L.  PROSSER 

L.  H.  KLEINHOLZ  I.  M.  KLOTZ 

THE  BUILDINGS  AND  GROUNDS  COMMITTEE 

EDGAR  ZWILLING,  Chairman  C.  B.  METZ 

RALPH  WICHTERMAN  SEARS  CROWELL 

THE  RADIATION  COMMITTEE 

G.  FAILLA,  Chairman  ROBERTS  RUGH 

CLAUDE  VILLEE  MONES  BERMAN 

WALTER  L.  WILSON  ROGER  L.  GREIF 


II.     ACT  OF  INCORPORATION 

No.  3170 

COMMONWEALTH  OF  MASSACHUSETTS 

Be  It  Known,  That  whereas  Alpheus  Hyatt,  William  Sanford  Stevens,  William  T. 
Sedgwick,  Edward  G.  Gardiner,  Susan  Minns,  Charles  Sedgwick  Minot,  Samuel  Wells, 
William  G.  Farlow,  Anna  D.  Phillips,  and  B.  H.  Van  Vleck  have  associated  themselves 


4  MARINE  BIOLOGICAL  LABORATORY 

with  the  intention  of  forming  a  Corporation  under  the  name  of  the  Marine  Biological 
Laboratory,  for  the  purpose  of  establishing  and  maintaining  a  laboratory  or  station  for 
scientific  study  and  investigation,  and  a  school  for  instruction  in  biology  and  natural  his- 
tory, and  have  complied  with  the  provisions  of  the  statutes  of  this  Commonwealth  in  such 
case  made  and  provided,  as  appears  from  the  certificate  of  the  President,  Treasurer,  and 
Trustees  of  said  Corporation,  duly  approved  by  the  Commissioner  of  Corporations,  and 
recorded  in  this  office; 

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

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

[SEAL]  HENRY  B.  PIERCE, 

Secretary  of  the  Commonwealth. 


III.     BY-LAWS   OF  THE  CORPORATION   OF  THE   MARINE 

BIOLOGICAL  LABORATORY 

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

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

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

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

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

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

VII.  The  Annual  Meeting  of  the  Trustees  shall  be  held  promptly  after  the  Annual 
Meeting  of  the  Corporation  at  the  Laboratory  in  Woods  Hole,  Mass.     Special  meetings 
of  the  Trustees  shall  be  called  by  the  President,  or  bv  anv  seven  Trustees,  to  be  held  at 


BY-LAWS  OF  THE  CORPORATION 

such  time  and  place  as  may  be  designated,  and  the  Secretary  shall  give  notice  thereof  by 
written  or  printed  notice,  mailed  to  each  Trustee  at  his  address  as  shown  on  the  records 
of  the  Corporation,  at  least  one  ( 1 )  week  before  the  meeting.  At  such  special  meeting 
only  matters  stated  in  the  notice  shall  be  considered.  Seven  Trustees  of  those  eligible  to 
vote  shall  constitute  a  quorum  for  the  transaction  of  business  at  any  meeting. 

VIII.  There  shall  be  three  groups  of  Trustees: 

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

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

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

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

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

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

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

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


0  MARINE  BIOLOGICAL  LABORATORY 

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


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

Gentlemen : 

I  submit  herewith  the  report  of  the  seventieth  session  of  the  Marine  Biological 
Laboratory. 

During  the  past  year  the  Laboratory  made  significant  progress  in  rehabilitating 
some  of  its  research  space  and  facilities  and  also  funds  were  obtained  for  a  new 
research  building  and  additional  housing. 

1.  Crane  Building 

The  Officers  of  the  Laboratory  held  several  conferences  during  the  winter 
(1957)  with  representatives  of  our  architectural  firm,  Coolidge,  Shepley,  Richardson 
and  Abbott,  to  develop  plans  for  the  rehabilitation  of  the  Crane  Building  under 
the  National  Science  Foundation  Grant  of  $415,000.  Planning  was  completed 
early  in  the  summer ;  the  contracts  and  subcontracts  were  let  in  August.  A  de- 
tailed schedule  of  the  operation  was  developed  by  the  general  contractor,  the 
building  was  evacuated  by  the  Laboratory  immediately  after  Labor  Day  and  the 
remodeling  started.  The  schedule  called  for  the  completion  of  the  job  by  May  1, 
1958.  Present  indications  are  that  the  work  will  be  completed  on  time,  and  that 
the  equipment  can  be  moved  in  for  occupancy  by  the  investigators  not  later  than 
June  1. 

Out  of  this  remodeling  the  Laboratory  will  have  an  essentially  new  building 
with  facilities  for  any  type  of  research  in  biology  and  the  cognate  sciences.  The 
rearrangement  of  the  standard  facilities  within  the  individual  laboratories  will 
result  in  a  much  more  efficient  use  of  available  space. 

2.  New  Research  Laboratory 

In  1938  it  was  recommended  by  an  ad  hoc  committee  set  up  to  formulate  policy 
on  the  future  development  of  the  Marine  Biological  Laboratory  that  the  wooden 
buildings  should  be  replaced  by  a  modern  brick  laboratory  building.  At  the  Annual 
Meeting  of  the  Board  of  Trustees,  August  16,  1957,  the  Officers  of  the  Laboratory 
were  authorized  to  seek  funds  to  implement  this  recommendation.  Applications 
were  made  to  the  Rockefeller  Foundation,  the  National  Science  Foundation  and  the 
Public  Health  Service  for  the  necessary  funds.  Early  in  December  the  Laboratory 
was  notified  of  a  grant  from  the  Rockefeller  Foundation  of  $738,500,  providing 
one-half  the  necessary  funds.  Subsequently,  grants  were  obtained  from  the  Na- 
tional Science  Foundation  and  the  Public  Health  Service  which  shared  equally  in 
providing  the  other  half  of  the  cost  of  the  new  building  and  its  equipment.  Plan- 
ning for  the  new  building  is  already  under  way,  construction  to  start  in  the  fall  of 
1959  with  occupancy  planned  for  the  spring  of  1961.  It  will  be  a  three-story  and 
basement  building,  almost  entirely  devoted  to  research  and  research  service  labora- 
tories. 


REPORT  OF  THE  DIRECTOR  7 

3.  Housing 

The  1938  ad  hoc  Committee  on  the  Development  of  the  Laboratory  also  ex- 
pressed its  concern  with  the  problems  of  housing  and  adequate  care  of  the  large 
number  of  persons  attracted  to  the  community  by  reason  of  their  Laboratory  activi- 
ties. It  was  pointed  out  that  the  housing  needs  have,  from  the  beginning,  been 
recognized  as  one  of  the  primary  responsibilities  of  the  Laboratory  and  that  the 
arrangements  then  existing  were  not  adequate.  Since  that  report,  three  frame 
houses  in  the  immediate  vicinity  of  the  Laboratory  have  been  acquired  and  con- 
verted to  dormitory  use.  Since  World  War  II  there  has  developed  an  acute  short- 
age of  housing  for  younger  married  investigators  with  children.  Toward  the  end 
of  the  year  (1957)  the  Laboratory  made  application  to  the  National  Science 
Foundation  for  a  grant  of  $175,000  to  erect  25  housekeeping  cottages  on  the 
Laboratory's  Devils  Lane  Property.  Favorable  action  was  taken  by  the  National 
Science  Foundation  on  this  grant  request.  Plans  have  been  developed  for  these 
cottages  which  will  be  erected  for  1959  occupancy. 

Also,  the  Board  of  Trustees  voted  to  discontinue  any  further  sale  of  lots  from 
the  Devils  Lane  Property  so  that  the  Laboratory  will  retain  title  to  the  remaining 
75  acres  for  future  Laboratory  use,  either  housing  or  scientific. 

4.  Grants,  Contracts  and  Contributions 

The  total  income  to  the  Laboratory  from  these  sources  of  support  amounted  to 
$210,000  in  1957.  This  represents  32%  of  the  total  income  and  consists  of  the 
following  accounts : 

American  Cancer  Soc. — R-7G — Fundamental  Studies  in  Radiobiology  $  6,600.00 
A.E.C. — 1343 — Program  of  Research  on  the  Physiology  of  Marine 

Organisms  Using  Radioisotopes  9,545.00 

N.I.H. — 4359 — Biological  Research  on  the  Morphology,  Ecology, 

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

N.I.H'— 5143— Training  Program  in  Nerve-Muscle  Physiology 40,342.00 

National  Science  Found. — G2142 — Funds  for  Biological  Research  .  .  .  25,000.00 

National  Science  Found. — G3608 — Optical  Equipment 11,500.00 

National  Science  Found. — G3987 — Centrifuge  Equipment 10,000.00 

O.N.R.— 1497— Studies  in  Marine  Biology  15,000.00 

O.N.R.— 09701— Studies  on  Isolated  Nerve  Fibers 7,670.00 

O.N.R.— 09702— Studies  in  Ecology 5,268.00 

M.B.L.  Associates 3,481.00 

Abbott  Laboratories  1,000.00 

American  Philosophical  Society  2,500.00 

Ciba  Pharmaceutical  Products,  Inc 1,000.00 

Eli  Lilly  Company  5,000.00 

Merck  and  Company,  Inc 1,000.00 

Rockefeller  Foundation  20,000.00 

Sobering  Corporation  1,000.00 

Smith,  Kline,  and  French  Foundation 3,000.00 

The  Upjohn  Company  1,000.00 

$209,906.00 


8  MARINE  BIOLOGICAL  LABORATORY 

5.  Boats 

Late  in  the  year  the  Laboratory  contracted  with  the  Riverside  Boat  Company 
of  Newcastle,  Maine,  for  two  24-foot  boats  for  trap  work  and  inshore  collecting. 
These  boats  will  replace  the  old  Sogitta  and  Tern,  both  of  which,  after  years  of 
service,  outlived  their  usefulness.  The  new  boats  are  to  be  delivered  in  May 
(1958). 

Respectfully  submitted, 

PHILIP  B.  ARMSTRONG, 

Director 

MEMORIAL 

BENJAMIN  M.  DUGGAR 

by 
Win.  Randolph  Taylor 

Benjamin  Minge  Duggar,  late  Emeritus  Trustee  of  this  Laboratory,  died  10  Sept. 
1956  in  New  Haven,  Conn.  Dr.  Duggar  was  born  in  Gallion,  Alabama  in  1872.  His 
early  education  in  private  schools  was  followed  by  studies  in  civil  engineering  at  the 
University  of  Alabama  and  the  Mississippi  Agricultural  and  Mechanical  College,  where 
his  interest  shifted  to  botany,  and  be  received  his  B.S.  in  1891.  He  then  completed  his 
work  for  the  M.S.  at  Alabama  Polytechnic,  but,  going  to  Harvard  University,  qualified 
there  for  the  A.B.  and  A.M.  degrees,  transferring  to  Cornell  University  where  he  received 
the  doctorate  in  1898,  completing  his  formal  studies.  He  subsequently  worked  in  several 
laboratories  in  Europe,  further  widening  his  experience.  His  government  and  academic 
appointments  were  numerous,  but  four  institutions  claimed  his  services  as  full  Professor 
before  retirement:  first  the  University  of  Missouri,  then  Cornell,  and  then  for  much  longer 
periods  Washington  University  and  the  University  of  Wisconsin. 

His  distinguished  academic  career  was  marked  by  a  happy  combination  of  physiology 
and  pathology,  in  both  of  which  fields  he  contributed  notably  in  research  and  produced 
textbooks  of  exceptional  merit,  that  in  plant  pathology  remaining  useful  to  this  day.  He 
contributed  his  share,  also,  to  one  of  the  most  successful  American  elementary  botanical 
text-books  ever  produced,  that  prepared  by  the  Wisconsin  group  and  still  in  use.  During 
his  period  at  Wisconsin  the  Department  of  Botany  strengthened  its  position  as  one  of  the 
most  notable  in  the  country.  His  researches  covered  a  considerable  range  of  endeavor, 
but  those  on  virus  diseases,  particularly  the  mosaic  disease  of  tobacco,  are  most  often 
remembered. 

Dr.  Duggar  does  not  seem  first  to  have  appeared  at  the  Marine  Biological  Laboratory 
as  a  student  or  as  an  investigator,  as  is  commonly  the  case.  In  1909  he  was  appointed 
to  what  was  termed  the  "Research  Staff" "in  botany,  while  Professor  of  Physiology  at 
Cornell.  In  1911  the  course  in  botany  was  divided,  the  first  three  weeks  dealing  with  the 
algae,  the  second  with  "The  Physiology  and  Ecology  of  Marine,  Strand  and  Bog  Vege- 
tation" with  Lewis  Knudsen,  also  from  Cornell,  as  his  associate.  Knudsen  was  replaced 
in  1912  by  W.  ].  Robbins,  best  known  as  the  Director  of  the  New  York  Botanical  Gar- 
den, but  the  course  was  dropped  in  1915.  "Investigation  Staff"  replaced  the  old  term 
for  the  advisory  group,  and  Dr.  Duggar  served  Botany  on  this  board  from  1926  to  1941. 
He  was  elected  to  the  Corporation  in  1911  and  to  the  Trustees  of  the  Laboratory  in  1928, 
retiring  Emeritus  in  1944.  During  all  these  years  he  was  frequently  in  residence  through 
the  summer,  and  always  helpful  to  those  at  the  Laboratory  whose  enquiries  fell  within  his 
field  of  interest. 


REPORT  OF  THE  DIRECTOR 

The  discernment  shown  by  Dr.  Duggar  respecting  the  affairs  of  the  Marine  Biological 
Laboratory  was  appreciated  by  other  institutions,  and  he  served  as  Trustee  of  the 
Bermuda  Biological  Station  1933-1937,  and  of  the  Woods  Hole  Oceanographic  Institute 
from  its  inception  in  1931  until  1938.  Honorary  degrees  were  bestowed  on  him  by 
Washington  University,  the  University  of  Missouri  and  the  University  of  Wisconsin; 
he  was  elected  to  the  most  distinguished  of  our  learned  and  professional  societies. 

On  retirement  from  Wisconsin  Dr.  Duggar  promptly  joined  the  research  staff  of  the 
Lederle  Laboratories  of  the  American  Cyanamid  Company,  and  settled  down  to  research 
on  the  discovery,  production  and  evaluation  of  antibiotics  from  various  Actinomycete 
bacteria.  All  reports  from  the  company  describe  with  admiration  his  quiet  industry  and 
the  keen  mind  he  placed  most  generously  at  the  disposal  of  his  fellow  workers.  His  most 
spectacular  success  was  in  the  discovery  in  1945  of  Aureomycin,  a  very  effective  anti- 
biotic, which  has  gone  into  extensive  commercial  production.  He  continued  active  in 
research  until  his  final  illness. 

Dr.  Duggar  lost  his  first  wife  in  1922;  his  second  wife,  several  children  and  grand- 
children survive  him.  To  them  we  wish  to  express  our  appreciation  of  his  many  con- 
tributions to  science  and  our  sympathy  in  the  loss  they  have  suffered. 

Mr.  President,  I  move  that  a  copy  of  this  memorial  be  placed  in  the  minutes  of  this 
meeting,  and  that  a  copy  be  sent  to  Mrs.  Duggar. 

MEMORIAL 

E.  S.  G.  BARRON 

by 
H.  B.  Steinbach 

E.  S.  G.  Barren,  "Achito"  to  many,  died  this  summer  and  is  buried  in  the  cemetery 
at  his  beloved  Woods  Hole.  His  scientific  studies  achieved  world-wide  recognition  as 
did  the  charming  personality  of  the  man  responsible  for  them. 

While  Barron  was  truly  a  scientist  of  the  world,  his  ties  to  the  Marine  Biological 
Laboratory  were  strong  and  his  affection  for  the  area  was  great.  He  was  elected  a 
member  of  the  Corporation  in  1933,  a  trustee  in  1949  and  again  in  1952.  He  served  as 
instructor  in  the  Physiology  Course  from  1945  until  1948  when  he  assumed  the  headship 
for  a  five-year  period.  Under  his  guidance  the  course  continued  its  strong  development 
and  became  especially  well  known  on  the  international  scene.  He  was  largely  instru- 
mental in  obtaining  much  of  the  special  equipment  that  is  now  in  use.  He  conducted  the 
special  session  of  the  course  in  honor  of  his  revered  teacher,  Leonor  Michaelis,  and  edited 
the  volume  "Modern  Trends  in  Physiology  and  Biochemistry"  which  carried  the  fame 
of  the  MBL  even  farther  than  before. 

For  the  past  several  years  Barron  found  it  necessary  to  give  up  his  attendance  here 
to  carry  out  a  labor  of  love  dear  to  his  heart,  spending  his  summers  in  teaching  and  con- 
sulting in  South  America  as  his  contribution  to  the  advancement  of  science  in  those  areas, 
especially  the  country  of  his  birth,  Peru. 

While  he  was  perhaps  best  known  for  his  studies  on  oxidative  mechanisms,  Achito's 
interests  and  activities  were  very  broad  indeed,  ranging  from  a  classical  work  on 
bilirubinemia  to  the  effects  of  ionizing  radiations  on  crystalline  proteins.  However  he 
was  preeminently  a  biologist  and,  in  his  mind,  all  his  studies  were  fundamentally  directed 
at  understanding  cellular  oxidations  and  their  regulation.  Shortly  before  his  death,  his 
plans  for  future  work  were  keyed  largely  to  a  comprehensive  comparative  study  of  cellu- 
lar oxidations  with  the  hope  of  finding  critical  keys  to  physiological  regulations. 

Barron  was  born  in  Huari,  Peru,  in  1898.     Following  two  years  in  France  he  came 


10  MARINE  BIOLOGICAL  LABORATORY 

to  this  country  in  1927,  first  as  a  Rockefeller  Fellow  and  then  as  instructor  in  Johns 
Hopkins  University.  In  1930  he  moved  to  the  University  of  Chicago,  his  University 
until  his  death.  During  World  War  II,  he  did  scientific  work  for  both  the  AEC  and 
the  Medical  Division  of  the  Chemical  Warfare  Service.  He  was  especially  well  fitted 
to  carry  out  the  important  studies  on  effects  of  ionizing  radiations  and  the  biological 
actions  of  nitrogen  mustards  and  related  compounds. 

Achito  was  a  remarkable  teacher  even  though  his  position  at  Chicago  did  not  involve 
conducting  formal  classes.  He  had  a  keen  and  incisive  sense  of  humor  and  a  fine  critical 
attitude  towards  science.  Many  have  benefitted  from  his  wisdom  and  have  been  delighted 
with  his  conversation.  He  had  a  strong  sense  of  the  necessity  for  training  minds  in 
intellectual  pursuits.  This  led  him  to  his  fruitful  efforts  in  the  Woods  Hole  course  and 
in  the  training  programs  in  South  America.  When  he  purchased  his  new  home  in  Chi- 
cago some  years  ago,  his  greatest  delight  was  that  he  had  a  large  pleasant  room  with  a 
big  blackboard.  Here  he  could  invite  his  students  and  colleagues  for  seminars  and  dis- 
cussions and  here  many  of  the  ideas  for  experiments  by  himself  and  collaborators  were 
born. 

Achito,  his  wife  Cora,  and  his  son  Richard  constituted  a  family  it  was  a  pleasure  and 
privilege  to  know.  And  while  we  are  expressing  our  gratitude  to  Achito  for  his  many 
contributions  to  us,  we  must  include  his  wife  and  son  for  providing  the  setting  for  such 
a  fruitful  career. 

As  an  experimentalist,  as  a  teacher,  as  one  who  travelled  widely  and  spread  the  tradi- 
tions of  science  and  inspired  the  young.  Barren  was  at  the  height  of  his  activity  when 
he  became  ill  and  died.  At  such  a  time  it  is  not  trite  to  say  that  a  man's  death  is  untimely. 

MEMORIAL 

ROBERT  CHAMBERS 

by 
B.  W.  Zweifach  and  G.  H.  A.  Clowes 

With  the  death  of  Robert  Chambers  at  the  age  of  75  on  July  22nd,  the  Marine  Bio- 
logical Laboratory  lost  one  of  the  most  illustrious  members  of  its  old  guard — marking 
as  it  were  the  passing  of  an  era  in  which  microscopy  as  a  fine  art  was  utilized  to  its  fullest 
extent  for  the  study  of  cellular  behavior  and  protoplasmic  structure.  Chambers'  associa- 
tions here  in  Woods  Hole  were  long  and  deep-rooted.  He  first  came  to  the  M.  B.  L. 
in  the  summer  of  1911  as  a  graduate  investigator  and  in  1912  was  on  the  teaching  staff 
in  Zoology  and  Embryology — a  course  in  those  days  associated  with  such  names  as 
Calkins,  Lillie,  Conklin,  Morgan  and  Wilson.  By  1914  it  became  apparent  that  Chambers' 
interests  were  not  along  the  lines  of  conventional  zoology  and  he  was  thereafter  listed  in 
the  annual  reports  of  the  M.  B.  L.  as  an  investigator  in  Physiology — an  indication  that 
the  science  of  cellular  physiology  had  come  of  age. 

By  training  Robert  Chambers  was  a  histologist  and  embryologist.  He  was  born  and 
raised  in  Turkey,  where  his  parents  resided  as  missionaries.  The  rough,  harsh  life  dur- 
ing his  formative  years  left  an  indelible  imprint  on  his  makeup  and  was  to  a  considerable 
extent  responsible  for  his  great  compassion  for  the  underdog  and  his  willingness  to 
champion  humanitarian  causes.  It  was  at  Roberts  College  that  his  interest  in  nature  was 
crystallized  and  his  future  course  indicated.  Later,  under  the  aegis  of  Hertwig  and  Gold- 
schmidt  in  Munich,  where  he  received  his  Ph.D  in  1908,  Chambers  was  indoctrinated 
into  the  field  of  histophysiology  and  developed  a  keen  interest  in  basic  embryology.  He 
returned  to  Canada,  the  early  home  of  the  Chambers  family,  and  eventually  joined  Cornell 
Medical  College  in  1915.  These  were  his  most  fruitful  years — his  outstanding  contribu- 
tions in  large  part  derived  from  his  ingenious  researches  at  Woods  Hole.  His  laboratorv 


REPORT  OF  THE  DIRECTOR  H 

here  in  Room  328,  in  association  with  the  Eli  Lilly  group,  was  a  beehive  of  activity  where 
Chambers'  dynamic  personality  infused  all  who  worked  with  him.  Few  could  keep  pace 
with  his  amazing  physical  stamina  and  drive.  At  an  early  age  Robert  Chambers  became 
virtually  a  legendary  figure,  not  only  because  of  his  scientific  stature  but  because  of  the 
anecdotes  which  grew  up  around  his  prodigious  unconcern  for  practical  matters.  There 
are  many  here  who  knew  him  during  these  inspiring  years  as  a  most  attractive  and  con- 
genial personality.  Woods  Hole  was  the  center  of  the  social  and  scientific  life  of  Robert 
and  Bertha  Chambers.  They  practically  raised  their  four  sons  at  Bobtuckett  Cottage  and 
many  of  the  delightful  and  entertaining  experiences  of  the  Chambers  family  have  attained 
the  stature  of  local  folklore.  Robert  might  be  found  at  almost  any  time,  day  or  night, 
in  his  M.  B.  L.  laboratory  and  the  Chambers  family  regarded  the  remainder  of  the  year 
as  an  unavoidable  intrusion  into  the  Woods  Hole  continuum. 

In  1928  he  transferred  from  the  Anatomy  Department  at  Cornell  to  the  Department 
of  Biology  at  New  York  University,  where  he  maintained  until  his  retirement  in  1949 
a  research  center  which  attracted  students  from  every  country  of  Europe,  from  Asia,  and 
from  South  America,  many  of  whom  are  today  outstanding  figures  in  scientific  research. 

The  magnitude  of  Robert  Chambers'  contribution  becomes  all  the  more  impressive 
when  it  is  considered  that  he  published  over  230  scientific  articles,  bearing  in  mind  the 
fact  that  writing  was  extremely  burdensome  for  Chambers.  A  great  deal  of  what  he  did, 
he  left  for  others  to  put  into  words.  He  unflaggingly,  to  the  point  of  self-denial,  gave  his 
time  and  counsel  to  a  never-ending  stream  of  students,  associates,  cronies  and  visitors. 
Time  was  a  meaningless  entity  to  him. 

In  1912,  at  the  M.  B.  L.  seminar  sessions,  Chambers  was  greatly  stimulated  by  a 
lecture  in  which  G.  L.  Kite  showed  that  it  was  possible  to  interfere  with  the  develop- 
ment of  marine  ova  with  glass  rnicrotools.  In  retrospect,  we  can  see  that  this  event 
proved  to  be  the  turning  point  in  his  scientific  career.  The  potentialities  of  this  approach 
appealed  so  much  to  Chambers  that  he  developed  and  applied  the  microsurgical  technique 
extensively,  his  name  becoming  synonymous  with  the  micromanipulative  method.  In  his 
early  work,  principally  at  Woods  Hole,  he  clearly  showed  the  importance  of  sol-gel 
transformations  in  relation  to  aster  formation  and  cell  division.  There  followed  the 
beautiful  demonstrations,  accompanied  by  motion  pictures,  of  the  capacity  of  the  cyto- 
plasm and  cell  surface  to  recover  from  various  forms  of  microinjury  in  the  presence  of 
the  proper  ionic  environment.  He  made  the  earliest  measurements  of  the  pH  of  the 
cytoplasm  in  intact  cells,  using  indicator  dyes.  His  enthusiasm  was  such  that  every 
aspect  of  cellular  behavior  intrigued  him,  the  cohesion  of  blastomeres  in  developing 
embryos,  the  action  of  salts  on  protoplasm,  the  nature  of  vital  staining,  the  interfacial 
tension  at  protoplasmic  surfaces,  the  acid  of  injury,  etc. 

Later,  Chambers  combined  tissue  culture  with  microtechniques.  Especially  note- 
worthy were  his  studies  on  malignant  cells,  the  secretory  activity  of  kidney  tubules  and 
chemotactic  phenomena.  During  World  War  II,  he  devoted  a  goodly  part  of  his  energies 
to  studies  on  capillary  permeability  and  to  the  vascular  sequelae  of  experimental  shock. 
New  and  important  concepts  of  circulatory  homeostasis  were  originated. 

Numerous  honors  were  bestowed  upon  Chambers.  As  early  as  1926  he  gave  his  first 
Harvey  lecture  on  the  living  cell.  During  this  period  he  received  the  Traill  medal  from 
the  Linnean  Society  of  London,  the  John  Scott  medal  from  the  City  of  Philadelphia,  the 
medal  of  L' Academic  Nationale  de  Medecine  of  Paris,  was  made  a  Fellow  of  the  Royal 
Microscopical  Society  of  England,  and  was  given  an  honorary  LL.D.  from  Queens  Uni- 
versity. He  was  active  in  the  affairs  of  many  societies,  having  been  a  Trustee  of  the 
Marine  Biological  Laboratory,  a  member  of  the  Board  of  Directors  of  the  Long  Island 
Biological  Association,  President  of  the  American  Society  of  Zoologists,  the  Harvey 
Society,  the  Union  of  American  Biological  Sciences,  and  vice-president  of  the  American 
Association  of  Anatomists. 


12  MARINE  BIOLOGICAL  LABORATORY 

When  one  attempts  to  give  an  account  of  a  man's  life  in  a  few  hundred  words,  the 
impossibility  of  the  task  becomes  increasingly  apparent.  In  the  case  of  Robert  Chambers, 
his  human  qualities  transcended  even  his  outstanding  scientific  achievements.  His  later 
years  were  saddened  by  the  loss  of  his  oldest  son,  Robert,  in  World  War  II,  and  by  the 
protracted  illness  and  death  of  his  wife  Bertha.  It  would  be  a  mere  platitude  to  say 
that  we  shall  miss  him,  but  we  hope  that  the  imprint  of  his  indomitable  spirit  will  live 
on  in  those  of  us  who  were  fortunate  enough  to  know  him  and  to  contribute  some  small 
part  to  the  fruits  of  his  labor. 

1.  THE  STAFF,  1957 

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

ZOOLOGY 

I.  CONSULTANTS 

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

II.  INSTRUCTORS 

THEODORE  H.  BULLOCK,  Professor  of  Zoology.  University  of  California,  Los  Angeles; 
in  charge  of  course 

JOHN  M.  ANDERSON,  Associate  Professor  of  Zoology,  Cornell  University 

JOHN  B.  BUCK,  Senior  Biologist,  National  Institutes  of  Health 

CLARK  P.  READ,  Associate  Professor,  School  of  Hygiene  and  Public  Health,  Johns  Hop- 
kins University 

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

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

CADET  HAND,  Assistant  Professor  of  Zoology.  University  of  California,  Berkeley 

HOWARD  A.  SCHNEIDERMAN,  Assistant  Professor  of  Zoology,  Cornell  University 

III.     LABORATORY  ASSISTANTS 

ROBERT  V.  KIRCHEN,  Columbia  University 
PETER  PICKENS,  University  of  California 

EMBRYOLOGY 

I.     INSTRUCTORS 

M.  V.  EDDS,  JR.,  Professor  of  Biology,  Brown  University ;  in  charge  of  course 
N.  T.  SPRATT,  JR.,  Professor  of  Zoology,  University  of  Minnesota 
M.  SUSSMAN,  Associate  Professor  of  Biological  Sciences,  Northwestern  University 
J.  P.  TRINKAUS,  Associate  Professor  of  Zoology,  Yale  University 
P.  B.  WEISZ,  Associate  Professor  of  Zoology,  Brown  University 

E.  ZWILLING,  Program  Director,  National  Science  Foundation  (on  leave  from  University 
of  Connecticut) 

II.     LABORATORY  ASSISTANTS 

R.  G.  BEARD,  Carnegie  Institution  of  Washington,  Department  of  Embryology 
C.  M.  FULTON,  Rockefeller  Institute  for  Medical  Research 


REPORT  OF  THE  DIRECTOR  13 

PHYSIOLOGY 

I.  CONSULTANTS 

MERKEL  H.  JACOBS,  Professor  of  Physiology,  University  of  Pennsylvania 

ARTHUR  K.  PARPART,  Professor  of  Biology,  Princeton  University 

ALBERT  SZENT-GYORGYI,  Director,  Institute  for  Muscle  Research,  Woods  Hole 

II.  INSTRUCTORS 

W.  D.  MCELROY,  Professor  of  Biology,  Johns  Hopkins  University;  in  charge  of  course 
FRANCIS  D.  CARLSON,  Assistant  Professor  of  Biophysics,  Johns  Hopkins  University 
BERNARD  D.  DAVIS,  Professor  of  Pharmacology,  New  York  University,  College  of  Medi- 
cine 

DONALD  GRIFFIN,  Professor  of  Zoology,  Harvard  University 
HOWARD  SCHACHMAN,  Virus  Laboratory,  University  of  California,  Berkeley 
ANDREW  SZENT-GYORGYI,  Institute  for  Muscle  Research,  Marine  Biological  Laboratory 

III.     LABORATORY  ASSISTANT 
ROGER  THEIS,  Rockefeller  Institute 

BOTANY 
I.     CONSULTANT 
WM.  RANDOLPH  TAYLOR,  Professor  of  Botany,  University  of  Michigan 

II.     INSTRUCTORS 

HAROLD  C.  BOLD,  Professor  of  Biology,  Vanderbilt  University ;  in  charge  of  course 
ROBERT  \Y.  KRAUSS,  Associate  Professor  of  Botany,  University  of  Maryland 
RICHARD  C.  STARR,  Associate  Professor  of  Botany,  Indiana  University 

III.  LECTURER 

RUTH  PATRICK,  Curator  of  Limnology,  Academy  of  Natural  Sciences  of  Philadelphia 

IV.  COLLECTOR 
GINA  ARCE,  Vanderbilt  University 

V.     LABORATORY  ASSISTANTS 

EUGENE  Fox,  Indiana  University 

RAYMOND  A.  GALLOWAY,  University  of  Maryland 

MARINE  ECOLOGY 
I.     CONSULTANTS 

PAUL  GALTSOFF,  U.  S.  Fish  and  Wildlife  Service,  Woods  Hole 
ALFRED  C.  REDFIELD,  Woods  Hole  Oceanographic  Institution 
JOHN  S.  RANKIN,  University  of  Connecticut 


14 


MARINE  BIOLOGICAL  LABORATORY 


II.     INSTRUCTORS 

EUGENE  P.  ODUM,  Professor  of  Zoology,  University  of  Georgia ;  in  charge  of  course 

EDWIN  T.  MOUL,  Associate  Professor  of  Botany,  Rutgers  University 

JOHN  H.  RYTHER,  Marine  Biologist,  Woods  Hole  Oceanographic  Institution 

III.     LABORATORY  ASSISTANT 
JOANNE  VAN  DYK,  University  of  New  Hampshire 


THE  LABORATORY  STAFF,  1957 
HOMER  P.  SMITH,  General  Manager 


MRS.   DEBORAH   LAWRENCE  HARLOW, 

Librarian 
CARL  O.  SCHWEIDENBACH,  Manager, 

Supply  Department 


ROBERT  KAHLER,  Superintendent, 
Buildings  and  Grounds 

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


GENERAL  OFFICE 
IRVINE  L.  BROADBENT,  Office  Manager 

MRS.  LILA  S.  MYERS  NANCY  WIGLEY 

GEORGIANA  MARKS 


MARY  A.  ROHAN 


LIBRARY 


ALBERT  K.  NEAL 


NAOMI  BOTELHO 


MAINTENANCE  OF  BUILDINGS  AND  GROUNDS 


ROBERT  ADAMS 
EDMOND  A.  BOTELHO 
ARTHUR  D.  CALLAHAN 
ROBERT  GUNNING 
JOHN  H.  HEAD 


DONALD  B.  LEHY 
RALPH  H.  LEWIS 
RUSSELL  F.  LEWIS 
ALTON  J.  PIERCE 
TAMES  S.  THAYER 


DEPARTMENT  OF  RESEARCH  SERVICE 


GAIL  M.  CAVANAUGH 
JOHN  P.  HARLOW 


SEAVER  R.  HARLOW 
LUDIE  A.  JOHNSON 


SUPPLY  DEPARTMENT 


DONALD  P.  BURN  HAM 
MILTON  B.  GRAY 
GERALDINE  E.  KEELER 
ROBERT  M.  PERRY 


GEOFFREY  J.  LEHY 
ROBERT  O.  LEHY 
BRUNO  TRAPASSO 
H.  S.  WAGSTAFF 


REPORT  OF  THE  DIRECTOR  15 

2.  INVESTIGATORS,  LALOR  AND  LILLIE  FELLOWS,  AND  STUDENTS 

Independent  Investigators,  1957 

AIELLO,  EDWARD,  Assistant  in  Zoology,  Columbia  University 

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

ANDERSON,  JOHN  MAXWELL,  Associate  Professor  of  Zoology,  Cornell  University 

ARMSTRONG,   PHILIP   B.,   Professor  of  Anatomy,   State  University  of   New   York,   College  of 

Medicine,  at  Syracuse 

ARNOLD,  WILLIAM  A.,  Scientific  Investigator,  Oak  Ridge  National  Laboratory 
BACON,  DONALD  F.,  Assistant  in  Department  of  Microbiology,  Yale  Medical  School 
BANG,  FREDERIK,  Professor  of  Pathology,  Johns  Hopkins  University  School  of  Hygiene 
BARTON,  JAY,  II,  Associate  Professor  of  Biology,  Collegeville,  Indiana 
BENESCH,  REINHOLD,  Investigator,  Marine  Biological  Laboratory 

BENNETT,  MICHAEL,  Research  Worker,  Columbia  University,  College  of  Physicians  and  Surgeons 
BENNETT,  MIRIAM  F.,  Instructor  in  Biology,  Sweet  Briar  College 
BERG,  WILLIAM  E.,  Associate  Professor  of  Zoology,  University  of  California 
BERGER,  CHARLES  A.,  Chairman,  Department  of  Biology,  Fordham  University 
BISHOP,  NORMAN  I.,  Research  Associate,  University  of  Chicago 
BRADY,  ROSCOE,  Section  Chief,  National  Institutes  of  Health 
BRAAMS,  RENIER,  Research  Associate,  Yale  University 
BRIDGMAN,  JOSEPHINE,  Professor  of  Biology,  Agnes  Scott  College 
BROWN,  FRANK  A.,  JR.,  Professor  of  Biology,  Northwestern  University 
BRYANT,  S.  H.,  Professor  of  Pharmacology,  University  of  Cincinnati,  College  of  Medicine 
BUCKMANN,  DETLEF,  Zoologisches  Institut,  Saarstrabe  21,  Mainz,  Germany 
BULLOCK,  THEODORE  H.,  Professor  of  Zoology,  University  of  California,  Los  Angeles 
BURGEN,  ARNOLD,  Professor  of  Physiology,  McGill  University 
CAMPBELL,  MILDRED  A.,  Instructor  in  Zoology,  Smith  College 

CARLSON,  FRANCIS  D.,  Associate  Professor  of  Biophysics,  Johns  Hopkins  University 
CASE,  JAMES  F.,  Assistant  Professor  of  Zoology,  Iowa  State  University 
CHAET,  ALFRED  B.,  Instructor  in  Physiology,  Boston  University  School  of  Medicine 
CHANG,  JOSEPH  J.,  Member  of  Laboratory  of  Biophysics,  National  Institutes  of  Health 
CHASE,  AURIN  M.,  Associate  Professor  of  Biology,  Princeton  University 
CHENEY,  RALPH  H.,  Professor  of  Biology;  Director  Physiology  Division,  Brooklyn  College 
CLAFF,  C.  LLOYD,  Research  Associate  in  Surgery,  Harvard  Medical  School 
CLARK,  GORDON  M.,  Research  Associate,  University  of  Michigan 
CLEMENT,  A.  C.,  Professor  of  Biology,  Emory  University 
CLOWES,  G.  H.  A.,  Research  Director  Emeritus,  Lilly  Research  Laboratories 
COLE,  KENNETH  S.,  Chief,  Laboratory  of  Biophysics,  National  Institutes  of  Health 
COHEN,  MELVIN  J.,  Instructor  in  Biology,  Harvard  University 
COLLIER,  JACK  R.,  Instructor  in  Zoology,  University  of  Vermont 
COLWIN,  ARTHUR  L.,  Associate  Professor,  Queens  College 
COLWIN,  LAURA  HUNTER,  Lecturer,  Queens  College 
CONNELLY,  CLARENCE  M.,  Associate,  Rockefeller  Institute 
COOPERSTEIN,  SHERWIN  J.,  Associate  Professor  of  Anatomy,  Western  Reserve  University  School 

of  Medicine 

COSTELLO,  DONALD  P.,  Kenan  Professor  of  Zoology,  University  of  North  Carolina 
CRANE,  ROBERT  K.,  Associate  Professor  of  Biological  Chemistry,  Washington  University  Medi- 
cal School 

CROWELL,  SEARS,  Associate  Professor  of  Zoology,  Indiana  University 
CSAPO,  ARPAD  L,  Associate  Professor,  Rockefeller  Institute  for  Medical  Research 
GUSHING,  JOHN  ELDRIDGE,  Associate  Professor  of  Biological  Sciences,  University  of  California, 

Santa  Barbara  College 

DEVOE,  ROBERT,  Graduate  Fellow,  Rockefeller  Institute  for  Medical  Research 
DIETER,  CLARENCE  D.,  Head  of  Department  of  Biology,  Washington-Jefferson  College 
EDDS,  MAC  V.,  JR.,  Professor  of  Biology,  Brown  University 

EDWARDS,  CHARLES,  Professor  of  Physiological  Optics,  Johns  Hopkins  University 
ELLIOTT,  ALFRED  M.,  Professor  of  Zoology,  University  of  Michigan 


16  MARINE  BIOLOGICAL  LABORATORY 

ENGLE,  RALPH  L.,  JR.,  Assistant  Professor  of  Medicine,  Cornell  University  Medical  College 

FAILLA,  G.,  Professor,  Columbia  University 

FRYE,  B.  E.,  Postdoctoral  Fellow,  Princeton  University 

GALL,  JOSEPH  G.,  Assistant  Professor  of  Zoology,  University  of  Minnesota 

GREEN,  HOWARD,  Assistant   Professor  of  Chemical   Pathology,   New  York  University   College 

of  Medicine 

GREEN,  MAURICE,  Assistant  Professor  of  Microbiology,  St.  Louis  University  School  of  Medicine 
GREENBERG,  SYLVIA  S.,  Damon  Runyon  Cancer  Research  Fellow,  New  York  University 
GREGG,  JAMES  H.,  Associate  Professor  of  Biology,  University  of  Florida 
GREIF,  ROGER  L.,  Associate  Professor  of  Physiology,  Cornell  University  Medical  College 
GROSCH,  DANIEL  S.,  Associate  Professor  of  Genetics,  N.  C.  State  College 
GROSS,  PAUL,  Assistant  Professor  of  Biology,  New  York  University 

GRUNDFEST,  HARRY,  Associate  Professor  of  Neurology,  College  of  Physicians  and  Surgeons 
GUDERNATSCH,  FREDERICK,  1300  York  Avenue,  New  York  21 
GUTTMAN,  RITA,  Assistant  Professor  of  Biology,  Brooklyn  College 
HAND,  CADET,  Assistant  Professor  of  Zoology,  University  of  California,  Berkeley 
HARVEY,  ETHEL  BROWNE,  Research  in  Biology,  Princeton  University 
HARVEY,  E.  NEWTON,  Professor  Emeritus  of  Biology,  Princeton  University 
HAYASHI,  TERU,  Associate  Professor  of  Zoology,  Columbia  University 
HEILBRUNN,  L.  V.,  Professor  of  Zoology,  University  of  Pennsylvania 
HENLEY,  CATHERINE,  Research  Associate,  University  of  North  Carolina 
HERVEY,  JOHN  P.,  Electronic  Engineer,  Rockefeller  Institute  for  Medical  Research 
HILL,  ROBERT  B.,  Instructor  in  Zoology,  University  of  Maine 

HOBERMAN,  HENRY  D.,  Associate  Professor  of  Biochemistry,  Albert  Einstein  College  of  Medicine 
HOROWITZ,  SAMUEL  B.,  Research  Fellow,  Eastern  Pennsylvania  Psychiatric  Institute 
HOYLE,  GRAHAM,  Glasgow  University,  Scotland 

HYDE,  BEAL  B.,  Assistant  Professor  of  Plant  Sciences,  University  of  Oklahoma 
ISENBERG,  IRVIN,  Research  Associate,  Institute  for  Muscle  Research 
JENNER,  CHARLES  E.,  Associate  Professor  of  Zoology,  University  of  North  Carolina 
KALCKAR,  BARBARA  W.,  Biochemist,  National  Institutes  of  Health 
KEMP,  NORMAN  E.,  Assistant  Professor  of  Zoology,  University  of  Michigan 
KEMPTON,  RUDOLF  T.,  Professor  of  Zoology,  Vassar  College 

KLOTZ,  IRVING  M.,  Professor  of  Chemistry  and  Biology,  Northwestern  University 
KUFFLER,  STEPHEN  W.,  Professor  of  Ophthalmic   Physiology  and   Biophysics,  Johns   Hopkins 

University 

LANSING,  ALBERT  I.,  Professor  of  Anatomy,  University  of  Pittsburgh 

LAZAROW,  ARNOLD,  Professor  and  Head  of  Department  of  Anatomy,  University  of  Minnesota 
LAWLER,  H.  CLAIR,  Associate  in  Biochemistry,  College  of  Physicians  and  Surgeons 
LAWRENCE,  H.  SHERWOOD,  Associate  Professor  of  Medicine,  New  York  University  College  of 

Medicine 

LEIGHTON,  JOSEPH,  Assistant  Professor  of  Pathology,  University  of  Pittsburgh 
LEVY,  MILTON,  Professor  and  Chairman,  Department  of  Biochemistry,   New  York  University 

College  of  Dentistry 

LEWIN,  RALPH  A.,  National  Institutes  of  Health 

LINDBERG,  OLOV,  Professor  and  Head  of  Wenner-Grens  Institute,  Sweden 
LITT,  MORTIMER,  Research  Fellow  in  Bacteriology,  Harvard  Medical  School 
LOCH  HEAD,  JOHN  H.,  Professor  of  Zoology,  University  of  Vermont 
LORAND,  L.,  Assistant  Professor  of  Chemistry,  Northwestern  University 
LOWENSTEIN,  O.  E.,  Professor  of  Zoology,  University  of  Birmingham,  England 
LUBIN,  MARTIN,  Associate  in  Pharmacology,  Harvard  Medical  School 
MCELROY,  W.  D.,  Chairman,  Biology  Department,  Johns  Hopkins  University 
MAAS,  WERNER  K.,  Assistant  Professor  of  Pharmacology,  New  York  University  Medical  College 
MARSHAK,  ALFRED,  Marine  Biological  Laboratory 

MARSLAND,  DOUGLAS,  Professor  of  Biology,  New  York  University,  Washington  Square  College 
MENKIN,  VALY,  Head  of  Experimental  Pathology,  Temple  University  School  of  Medicine 
METZ,  CHARLES  B.,  Associate  Professor  of  Zoology,  Florida  State  University 
METZ,  CHARLES  W.,  Professor  of  Zoology,  University  of  Pennsylvania 
MIDDLEBROOK,  W.  ROBERT,  Institute  for  Muscle  Research 


REPORT  OF  THE  DIRECTOR  17 

MILLS,  KENNETH  S.,  Instructor  of  Biophysics,  University  of  California  Medical  Center 

MOORE,  JOHN  W.,  Associate  Chief,  National  Institutes  of  Health 

MULNARD,  JACQUES  G.,  Chef  De  Travau,  University  of  Brussels,  Belgium 

MULLINS,  L.  J.,  Associate  Professor  of  Biophysics,  Purdue  University 

NACE,    PAUL  F.,   Associate    Professor   of    Biology,    Hamilton    College,    McMaster    University, 

Ontario 

Niu,  MAN-CHIANG,  Associate,  Rockefeller  Institute  for  Medical  Research 
ODUM,  EUGENE  P.,  Professor  of  Zoology,  University  of  Georgia 

OSTERHOUT,  W.  J.  V.,  Member  Emeritus,  Rockefeller  Institute  for  Medical  Research 
PADAWER,  JACQUES,  Assistant  Professor  of  Biochemistry,  Albert  Einstein  College  of  Medicine 
PARPART,  ARTHUR  K.,  Professor  and  Chairman,  Department  of  Biology,  Princeton  University 
PERSON,  PHILIP,  Chief,  Dental  Research,  V.  A.  Hospital,  Brooklyn 
PERT,  JAMES  H.,  Professor  in  Medicine,  Cornell  University  Medical  College 
PLOUGH,  HAROLD  H.,  Professor  of  Biology,  Amherst  College 
PROSSER,  C.  LADD,  Professor  of  Physiology,  University  of  Illinois 
READ,  CLARK  P.,  Associate  Professor  of  Pathobiology,  Johns  Hopkins  University 
REBHUN,  LIONEL  L,  Instructor  in  Anatomy,  University  of  Illinois  College  of  Medicine 
RIESER,  PETER,  Research  Associate,  University  of  Pennsylvania 
ROCKSTEIN,    MORRIS,   Associate    Professor   of   Physiology,    New    York   University    College   of 

Medicine 

ROGERS,  K.  T.,  Assistant  Professor  of  Zoology,  Oberlin  College 
ROSENBERG,    EVELYN    K.,   Assistant    Professor    of    Pathology,    New    York    University-Bellevue 

Medical  Center 

ROTH,  JAY  S.,  Associate  Professor  of  Biochemistry,  Hahnemann  Medical  College 
RUGH,  ROBERTS,  Associate  Professor  of  Radiology,  Columbia  University 
SCHECHTER,  VICTOR,  Associate  Professor  of  Biology,  City  College  of  New  York 
SCHNEIDERMAN,  HOWARD  A.,  Associate  Professor  of  Zoology,  Cornell  University 
SCHOFFENIELS,  ERNEST,  Research  Associate,  College  of  Physicians  and  Surgeons 
SCHUH,  JOSEPH  E.,  Professor  and  Chairman,  Department  of  Biology,  St.  Peter's  College 
SCHULMAN,  MARTIN  P.,  Assistant  Professor  of  Biochemistry,  State  University  of  New  York, 

College  of  Medicine  at  Syracuse 
SCOTT,  DWIGHT  B.   McNAiR,   Assistant   Professor  of  Physiology,  University  of  Pennsylvania 

Medical  School 
SCOTT,  SISTER  FLORENCE  MARIE,  Professor  and  Chairman,  Department  of  Biology,  Seton  Hill 

College 

SCOTT,  GEORGE  T.,  Professor  of  Zoology,  Oberlin  College 
SHANES,  A.  M.,  Physiologist,  National  Institutes  of  Health 
SHAW,  EVELYN,  Research  Fellow,  American  Museum  of  Natural  History 
SLIFER,  ELEANOR  H.,  Associate  Professor  of  Zoology,  State  University  of  Iowa 
SMELSER,  GEORGE  K.,  Professor  of  Anatomy,  College  of  Physicians  and  Surgeons 
SPEIDEL,  CARL  C.,  Professor  and  Chairman,  Department  of  Anatomy,  University  of  Virginia 
SPERELAKIS,  NICK,  Teaching  Assistant,  University  of  Illinois 
SPIEGEL,  MELVIN,  Assistant  Professor  of  Biology,  Colby  College 
SPRATT,  NELSON  T.,  Professor  of  Zoology,  University  of  Minnesota 
SPYROPOULOS,  CONSTANTINE,  National  Institutes  of  Health 
STARR,  RICHARD  C.,  Assistant  Professor  of  Botany,  Indiana  University 
STEELE,  RICHARD  H.,  Institute  for  Muscle  Research 
STEINBACH,  H.  B.,  Professor  of  Zoology,  University  of  Chicago 
STEPHENS,  GROVER  C.,  Assistant  Professor  of  Zoology,  University  of  Minnesota 
STEPHENSON,  WILLIAM  K.,  Assistant  Professor  of  Biology,  Earlham  College 
STETTEN,  DEWnr,  Associate  Director  in  Charge  of  Research,  National  Institutes  of  Health 
STOREY,  ALMA  G.,  Professor  Emeritus,  Mount  Holyoke  College  ; 
STONE,  WILLIAM,  JR.,  Massachusetts  Eye  and  Ear  Infirmary 
STUNKARD,  HORACE  W.,  Research  Biologist,  U.  S.  Fish  and  Wildlife  Service 
SUSSMAN,  MAURICE,  Associate  Professor  of  Biological  Sciences,  Northwestern  University 
SZENT-GYORGYI,  ALBERT,  Chief  Investigator,  Institute  for  Muscle  Research 
SZENT-GYORGYI,  ANDREW  G.,  Investigator,  Institute  for  Muscle  Research 
TASAKI,  TCHIJI,  Chief,  Special  Senses  Section,  National  Institutes  of  Health 


18  MARINE  BIOLOGICAL  LABORATORY 

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

TE\VINKEL,  Lois  E.,  Professor  of  Zoology,  Smith  College 

TRINKAUS,  JOHN  PHILIP,  Associate  Professor  of  Zoology,  Yale  University 

TROLL,  WALTER,  Assistant  Professor,  New  York  University  College  of  Medicine 

TWAROG,  BETTY  MACK,  Research  Fellow,  Harvard  University 

TWEEDELL,  KENYON  S.,  Arsistant  Professor  of  Zoology,  University  of  Maine 

ULLBERG,  SVEN  G.  F.,  Royal  Veterinary  College,  Stockholm,  Sweden 

DEViLLLAFRANCA,  GEORGE  W.,  Assistant  Professor  of  Zoology,  Smith  College 

VILLEE,  CLAUDE  A.,  Associate  Professor  of  Biological  Chemistry,  Harvard  Medical  School 

VINCENT,  WALTER  S.,  Instructor  in  Anatomy,  State  University  of  New  York,  Medical  Center 

at  Syracuse 

WAINIO,  WALTER  W.,  Associate  Professor  of  Biochemistry,  Rutgers  University 
WEBB,  H.  MARGUERITE,  Assistant  Professor  of  Physiology,  Goucher  College 
WEIGLE,  WILLIAM  O.,  Research  Associate,  University  of  Pittsburgh  School  of  Medicine 
WESTHEIMER,  GERALD,  Assistant  Professor  of  Physiological  Optics,  Ohio  State  University 
WHITING,  ANNA  R.,  Lecturer  in  Zoology,  University  of  Pennsylvania 
WHITING,  P.  W.,  Professor  of  Zoology  Emeritus,  University  of  Pennsylvania 
WICHTERMAN,  RALPH,  Professor  of  Biology,  Temple  University 

WILBER,  CHARLES  G.,  Chief,  Comparative  Physiology  Branch,  Army  Chemical  Center 
WILLEY,  C.  H.,  Chairman,  Department  of  Biology,  New  York  University 
WILSON,  DONALD  M.,  Teaching  Assistant,  University  of  California,  Los  Angeles 
WILSON,  T.  HASTINGS,  Assistant   Professor  of  Biological   Chemistry,  Washington  University 

School  of  Medicine 
WILSON,  WALTER  L.,  Assistant   Professor  of  Physiology,  University  of  Vermont   College  of 

Medicine 
WITTENBERG,  JONATHAN    B.,   Assistant   Professor   of   Physiology,   Albert   Einstein   College  of 

Medicine 

WOODS,  KENNETH  R.,  Research  Associate,  Cornell  University  Medical  College 
WRIGHT,  PAUL  A.,  Associate  Professor  of  Zoology,  University  of  Michigan 
ZWEIFACH,  BENJAMIN  W.,  Associate  Professor  of  Pathology,  New  York  University-Bellevue 

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

Beginning  Investigators,  1957 

ALSUP,  PEGGY,  University  of  Pennsylvania 

BENSUSAN,  HOWARD  B.,  Western  Reserve  University 

BURKE,  JOSEPH,  S.J.,  Fordham  University 

CAGLE,  JULIEN,  Princeton  University 

CASCARANO,  JOSEPH,  University  of  Minnesota  Medical  School 

CERT,  JEAN  A.,  University  of  California 

CHANCE,  ELEANOR  K.,  University  of  Pennsylvania 

DINGLE,  A.  D.,  McMaster  University 

GANGI,  DOMINICK  P.,  Upstate  Medical  Center,  State  University  of  New  York 

HARDIMAN,  CLARENCE  W.,  Florida  State  University 

KANE,  ROBERT  E.,  Johns  Hopkins  University 

MASHIMA,  HIDENOBER,  Rockefeller  Institute  for  Medical  Research 

MASON,  DAVID  T.,  Reed  College 

Moos,  CARL,  Northwestern  University 

NAGLER,  ARNOLD  L.,  Bellevue  Medical  Center 

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

RUGGIERI,  GEORGE,  St.  Louis  University 

SCHWARTZ,  JAMES  H.,  New  York  University  College  of  Medicine 

SMITH,  ROBERT  G.,  Washington  University  Medical  School 

STROHMAN,  RICHARD  C.,  Columbia  University 

THEIS,  ROGER  E.,  Rockefeller  Institute  for  Medical  Research 

TURNER,  BARBARA,  Johns  Hopkins  University  School  of  Medicine 

WESSELLS,  NORMAN  KEITH,  Yale  University 


REPORT  OF  THE  DIRECTOR  19 

Research  Assistants,  1957 


ALBERT,  MORRIS,  Boston  University 

AMATNIEK,  ERNEST,  Columbia  University 

AUCLAIR,  WALTER,  New  York  University,  Washington  Square  College 

BARN  HART,  B.  J.,  Indiana  University 

BARNWELL,  FRANKLIN  H.,  Northwestern  University 

BARROW,  PATIENCE  C,  University  of  Toronto 

BENOIT,  RICHARD,  Massachusetts  Eye  and  Ear  Infirmary 

BLANCHARD,  ROBERTA,  Woods  Hole,  Mass. 

BRUCK,  STEPHEN  D.,  duPont  de  Nemours  &  Company 

GATHER,  JAMES  N.,  Emory  University 

CLARK,  WILLIAM  R.,  JR.,  Boston  University 

CORNER,  M.,  Rockefeller  Institute 

CROWLEY,  ELIZABETH  M.,  University  of  Pittsburgh 

DIBBELL,  DAVID  G.,  University  of  Pennsylvania 

DOUGLAS,  STEVEN,  Cornell  University 

ERDMAN,  HOWARD  E.,  North  Carolina  State  College 

FEINBERG,  HARRIET  ADELE,  University  of  Pennsylvania 

FELDMAN,  RICHARD,  Rockefeller  Institute  for  Medical  Research 

Fox,  J.  EUGENE,  Indiana  University 

FRIEDMAN,  LEONARD,  Rutgers  University 

GEBHART,  JOHN  H.,  National  Institutes  of  Health 

GIFFORD,  CAMERON  E.,  Harvard  University 

GIFFORD,  CHARLES  A.,  University  of  Minnesota 

GORDON,  ROBERT,  Massachusetts  Institute  of  Technology 

GORKENANT,  INGEBURG,  Woods  Hole,   MaSS. 

GOUDSMIT,  ESTHER  M.,  University  of  Michigan 

GRINNELL,  ALAN,  Harvard  University 

HIATT,  HOWARD,  Harvard  Medical  School 

INGLIS,  LAURA  H.,  Hahnemann  Medical  College 

JONES,  HELEN,  Massachusetts  Eye  and  Ear  Infirmary 

JOSEPHSON,  ROBERT  K.,  University  of  California 

KARAKASHIAN,  STEPHEN  J.,  Drew  University 

KAUFMAN,  SHARON  L.,  Smith  College 

KEREVYI,  THOMAS,  Harvard  Medical  School 

KERNAN,  RODERICK  P.,  Rockefeller  Institute  for  Medical  Research 

KIRCHEN,  ROBERT  V.,  Columbia  University 

KOPMAN,  AARON,  Queens  College 

KRASSNER,  STUART,  Johns  Hopkins  University 

LEVI,  COLETTE  P.,  Northwestern  University 

LIEBERMAN,  HARRY,  New  York  Univcrsity-Bellevue  Medical  Center 

LORING,  JANET,  Harvard  Medical  School 

LUHRS,  CARO,  Harvard  Medical  School 

MATHESON,  GAIL  E.,  Yale  University 

McCANN,  FRANCIS,  University  of  Connecticut 

METCALF,  CARROLL,  Colby  College 

MORRISON,  ELAINE,  Massachusetts  Eye  and  Ear  Infirmary 

NASS,  SYLVAN,  New  York  University 

OLSON,  JOANNE  M.,  University  of  Minnesota 

PAULSEN,  ELIZABETH,  Rutgers  University 

PLUMB,  MARY  ELLEN,  Vassar  College 

POLLOCK,  BRIAN,  Brooklyn  V.  A.  Hospital 

REICH,  MELVIN,  Rutgers  University 

RICHARDS,  ELMER  G.,  University  of  California 

ROBERTSON,  MRS.  C.  W.,  United  States  Fish  and  Wildlife  Service 

ROOT,  RICHARD,  University  of  Michigan 

ROOT,  ELIZABETH,  University  of  Michigan 


20  MARINE  BIOLOGICAL  LABORATORY 

ROSENBLUTH,  RAJA,  Columbia  University 

Ross,  SHIRLEY  EILEEN,  Washington  University 

ROSSILLO,  LUDWIG  A.,  St.  Peter's  College 

RUBINOFF,  IRA,  American  Museum  of  Natural  History 

SCHINSKE,  ROBERT,  University  of  Minnesota 

SCHELTEMA,  AMELIE  H.,  University  of  North  Carolina 

SHAY,  JONATHAN,  Temple  Medical  School 

SHEPARD,  DAVID,  University  of  Chicago 

SIMMONS,  JOHN  E.,  Johns  Hopkins  University 

SMILEY,  SHELDON,  New  York  State  University  at  Syracuse 

STADLER,  JOAN,  Swarthmore  College 

STAUB,  HERBERT  W.,  Rutgers  University 

TITUS,  CHARLES  C.,  Western  Reserve  University 

TREMOR,  JOHN,  University  of  Michigan 

WAITE,  RICHARD  E.,  University  of  Pennsylvania 

WARWICK,  ANNE  C.,  Johns  Hopkins  University 

WEISBLUM,  BERNARD,  State  University  of  New  York 

WELLINGTON,  FREDERICA,  Harvard  Medical  School 

WHITBECK,  ELAINE,  Smith  College 

WYTTENBACH,  CHARLES  R.,  Carnegie  Institute 

Library  Readers,  1957 

ALLFREY,  VINCENT  G.,  Associate,  Rockefeller  Institute  for  Medical  Research 
AMBERSON,  WILLIAM  R.,  Professor  of  Physiology,  University  of  Maryland  School  of  Medicine 
BALL,  ERIC  G.,  Chairman,  Division  of  Medical  Sciences,  Harvard  Medical  School 
BERNHEIMER,  ALAN  W.,  Associate  Professor  of  Microbiology,  New  York  University  College 

of  Medicine 

BLOCK,  ROBERT,  Associate  Editor,  Biological  Abstracts,  University  of  Pennsylvania 
BODANSKY,  OSCAR,  Sloan-Kettering  Institute 

BROOKS,  MATILDA  M.,  Research  Associate  in  Physiology,  University  of  California 
CHANUTIN,  ALFRED,  Professor  of  Biochemistry,  Medical  School,  University  of  Virginia 
CLARK,  ELIOT  R.,  Professor  Emeritus  of  Anatomy,  University  of  Pennsylvania  School  of  Medi- 
cine 

COHEN,  SEYMOUR  S.,  Professor  of  Biochemistry,  Children's  Hospital 
DEANE,  HELEN  WENDLER,  Harvard  Biological  Laboratories 
DIXON,  FRANK  J.,  JR.,  Chairman,  Department  of  Pathology,  University  of  Pittsburgh  School 

of  Medicine 
DuBois,  ARTHUR  D.,  Associate  Professor  of  Physiology,  University  of  Pennsylvania  School  of 

Medicine 

EICHEL,  HERBERT  J.,  Hahnemann  Medical  College 
EISEN,  HERMAN  N.,  Professor  of  Medicine,  Washington  University 
GABRIEL,  MORDECAI  L.,  Associate  Professor  of  Biology,  Brooklyn  College 
GAFFRON,  HANS,  Professor  of  Biochemistry,  University  of  Chicago 
GOLDTHWAIT,  DAVID  A.,  Western  Reserve  University 

GREEN,  JAMES  W.,  Associate  Professor  of  Physiology,  Rutgers  University 
JACOBS,  M.  H.,  Emeritus  Professor  of  General  Physiology,  University  of  Pennsylvania  School 

of  Medicine 

KAAN,  HELEN  W.,  Indexer,  National  Research  Council 

KARUSH,  FRED,  Associate  Professor  of  Immunology,  University  of  Pennsylvania 
LIONETTI,  FABIAN  J.,  Associate  Professor  of  Biochemistry,  Boston  University  School  of  Medicine 
LONDON,  IRVING  M.,  Professor  and  Chairman,  Department  of  Medicine,  Albert  Einstein  College 

of  Medicine 

LOVE,  Lois  H.,  Research  Associate,  National  Research  Council 

MCDONALD,  SISTER  ELIZABETH,  Chairman,  Department  of  Biology,  College  of  Mt.  St.  Joseph 
MOORE,  GEORGE  M.,  Professor  and  Chairman  of  Zoology,  University  of  New  Hampshire 
NOVIKOFF,  ALEX  B.,  Research  Associate  Professor  of  Pathology,  Albert  Einstein   College  of 

Medicine 


REPORT  OF  THE  DIRECTOR  21 

PICK,  JOSEPH,  Professor  of  Anatomy,  New  York  University-Bellevue  Medical  Center 

ROOT,  WALTER  S.,  Professor  of  Physiology,  College  of  Physicians  and  Surgeons 

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

SCHLESINGER,  R.  WALTER,  Director,  Department  of  Microbiology,  St.  Louis  University  School 
of  Medicine 

SCOTT,  ALLAN,  Professor  of  Biology  and  Chairman  of  Department,  Colby  College 

SHERMAN,  FRANK  E.,  Assistant  Professor  of  Pathology,  University  of  Pittsburgh 

STEINHARDT,  JACINTO,  Director,  Operations  Evaluation  Group,  Massachusetts  Institute  of  Tech- 
nology 

SULKIN,  S.  EDWARD,  Professor  and  Chairman,  Department  of  Microbiology,  University  of 
Texas,  Southwestern  Medical  School 

WAGNER,  ROBERT  R.,  Assistant  Professor  of  Medicine,  Johns  Hopkins  University  School  of 
Medicine 

WARNER,  ROBERT  C,  Associate  Professor  of  Biochemistry,  New  York  University  College  of 
Medicine 

WHEELER,  GEORGE  E.,  Instructor  of  Biology,  Brooklyn  College 

WHITEHOUSE,  MICHAEL  W.,  Instructor  of  Biochemistry,  University  of  Pennsylvania  School  of 
Medicine 

YNTEMA,  CHESTER  L.,  Professor  of  Anatomy,  State  University  of  New  York  College  of  Medi- 
cine 

ZORZOLI,  ANITA,  Associate  Professor  of  Physiology,  Vassar  College 

LALOR  FELLOWS,  1957 

BACON,  DONALD,  Yale  Medical  School 

BISHOP,  NORMAN,  University  of  Chicago 

BRYANT,  S.  H.,  University  of  Cincinnati 

BUCKMANN,  DETLEF,  Zoologisches  Institut,  Mainz,  Germany 

BURGEN,  A.  S.  V.,  McGill  University 

EDWARDS,  CHARLES,  Johns  Hopkins  University 

ENGLE,  RALPH,  Cornell  University  Medical  College 

LORAND,  L.,  Northwestern  University 

LINDBERG,  OLOV,  Wcnner-Grens  Institute,  Sweden 

LUBIN,  MARTIN,  Harvard  Medical  School 

SCHULMAN,  MARTIN,  State  University  of  New  York,  College  of  Medicine  at  Syracuse 

STEPHENSON,  W.  K.,  Earlham  College 

WHITEHOUSE,  MICHAEL,  University  of  Pennsylvania  School  of  Medicine 

WILSON,  T.  HASTINGS,  Washington  University  School  of  Medicine 

WOODS,  KENNETH,  Cornell  University  Medical  School 

Lillie  Fellow,  1957 
Niu,  MAN-CHIANG,  Rockefeller  Institute  for  Medical  Research 

Students,  1957 
BOTANY 

ABELES,  FRED  B.,  Cornell  University 
ARNOLD,  ELIZABETH  L,  University  of  Rochester 
ARONSON,  FLORA  P.,  Brooklyn  College 
BOUCK,  GEORGE  B.,  Columbia  University 
COOK,  PHILIP  W.,  University  of  Vermont 
CZELUSNIAK,  MARILYN  M.,  Smith  College 
FRANKEL,  JOSEPH,  Yale  University 
HERSKOWITZ,  JULIA,  Antioch  College 
KEELER,  CARL  R.,  JR.,  Northwestern  University 
KLEPPER,  ELIZABETH,  Vanderbilt  University 


MARINE  BIOLOGICAL  LABORATORY 

KUENZLER,  EDWARD  J.,  University  of  Georgia 
MORELAND,  RALPH  E.,  JR.,  Indiana  University 
MUSCHIO,  HENRY  M.,  Fordham  University 
PAIR,  HYANGJU,  Wellesley  College 
PAOLI,  GISELA,  Chatham  College 
PARKER,  BRUCE  C.,  Yale  University 
PROTA,  CARL  D.,  Fordham  University 
RICE,  ELEANOR,  Wheaton  College 
TEWS,  LEONARD  C.,  Indiana  University 
WALSER,  STEPHANIE  L.,  Radcliffe  College 

EMBRYOLOGY 

ARKLESS,  RICHARD,  University  of  Pennsylvania  Medical  School 

CASTON,  J.  DOUGLAS,  University  of  North  Carolina 

GOERINGER,  GERALD  C.,  Johns  Hopkins  University 

GRIFFIN,  JOE  LEE,  Princeton  University 

HANKS,  JAMES  E.,  University  of  New  Hampshire 

HERSH,  GEORGE  L.,  University  of  California 

KARAKASHIAN,  STEPHEN  J.,  Drew  University 

KERR,  NORMAN  S.,  Northwestern  University 

KESSEL,  RICHARD  G.,  State  University  of  Iowa 
i  KIRCHEN,  ROBERT  V.,  Columbia  University 

KRAM,  FLEURETTE  L.,  Northwestern  University 

LOVE,  DAVID  S.,  University  of  Colorado 

LOWE,  JANET  M.,  University  of  Minnesota 

MATHIESEN,  GEORGE  C.,  Harvard  University 
t  MELLON,  DEFOREST,  JR.,  Yale  University 

NELSON,  SHIRLEY,  Northwestern  University 

ROSEWATER,  JOSEPH,  Harvard  University 

SPARANO,  BENJAMIN  M.,  Fordham  University 

TALBOT,  WILLIAM  H.,  Rockefeller  Institute 
9  TYSON,  GRETA  E.,  University  of  New  Hampshire 

VAN  DYK,  N.  JOANNE,  University  of  New  Hampshire 

WALCOTT,  CHARLES,  Cornell  University 

WATKINS,  MARGARET  J.,  University  of  Minnesota 

WHITE,  JEAN  ANN,  Mount  Holyoke  College 

WYLIE,  RICHARD  M.,  Harvard  University 

PHYSIOLOGY 

CLARK,  ALVIN  JOHN,  Harvard  University 
Cox,  RODY  P.,  University  of  Pennsylvania 
DAVIDSON,  MORTON,  Bellevue  Medical  College 
DUBNAU,  DAVID  A.,  Columbia  University 
ERWIN,  JOSEPH  A.,  Syracuse  University 

FAHN,  STANLEY,  University  of  California  School  of  Medicine 
FELIX,  MARIE  D.,  Cornell  University  Medical  School 
HAFT,  DAVID  E.,  University  of  Rochester  School  of  Medicine 
HALPEREN,  SIDNEY,  University  of  Texas 
KAHLBROCK,  MARGIT,  Columbia  University 
*KIRSCH,  JACK  F.,  Rockefeller  Institute 

MCCLUSKEY,  ROBERT  T.,  New  York  University-Bellevue  Medical  Center 
MAZUR,  PETER,  Princeton  University 

MEDINA,  HEITOR  S.,  Inst.  de  Biolojia — Curitiba,  Paroni,  Brazil 
MINDICH,  LEONARD  E.,  Rockefeller  Institute 
NAGLER,  ARNOLD  L.,  Bellevue  Medical  School 
OTERO,  Luis  R.,  University  of  Puerto  Rico 
RABINOWITZ,  LAWRENCE,  University  of  California 
RAWITSCHER,  ERIKA,  American  Museum  of  Natural  History 


REPORT  OF  THE  DIRECTOR  23 


ROBERTS,  PATRICIA  R.,  Duke  University 

SCHNEIDER,  JOHN  H.,  University  of  Wisconsin 

SIGER,  ALVIN,  Johns  Hopkins  University 

STERN,  DANIEL  N.,  Albert  Einstein  College  of  Medicine 

STONE,  NANCY  J.,  Columbia  University 

TAKEUCHI,  IKUO,  Princeton  University 

WEEKS,  BOYD  M.,  University  of  California 

WILLIAMS,  FRANK  ROBERT,  Oberlin  College 

WILLIAMS,  FREDERICK  M.,  Yale  University 

WILLIS,  JOHN  S.,  Harvard  University 

INVERTEBRATE  ZOOLOGY 
ASHER,  DAVID  M.,  Harvard  University 
AUGENFELD,  JOHN  M.,  University  of  Wisconsin 
BECKER,  JOYCE  E.,  Evansville  College 
BRANNING,  ARLEEN,  City  College  of  New  York 
BRAVERMAN,  MAXWELL  H.,  University  of  Illinois 
CAMP,  DONALD  B.  M.,  Acadia  University 
CLARKE,  ARTHUR  H.,  JR.,  Cornell  University 
COLEMAN,  CHASE,  Vassar  College 
CONCANNON,  BRO.  JOSEPH,  St.  John's  University 
COOPER,  MADELINE,  American  Museum  of  Natural  History 
COOPER,  KENNETH  K.,  American  Museum  of  Natural  History 
CROWELL,  JANE,  Oberlin  College 
DIAMOND,  JARED  M.,  Harvard  University 
DOBBEN,  PHYLLIS  A.,  Rocky  River  16,  Ohio 
DOBBS,  HARRY  D.,  Wofford  College 
EGLOFF,  DAVID  A.,  Amherst  College 
GFELLER,  SISTER  MARION  D.,  Marquette  University 
GUZE,  CAROL  D.,  Washington  University 
HAFENER,  PAUL  A.,  JR.,  Franklin  and  Marshall  College 
HECHTEL,  GEORGE  J.,  Yale  University 
HILD,  DAVID  H.,  Wesleyan  University 

HORVATH,  NANCY,  10121  S.  Parnell  Avenue,  Chicago  38,  Illinois 
HORWITZ,  JUDITH,  Radcliffe  College 
ISAAC,  DONALD  E.,  University  of  California 
JENSEN,  DONALD  DALE,  Yale  University 
JOHNSON,  B.  THOMAS,  University  of  California 
JORDAN,  ELKE,  Goucher  College 
KAUFMAN,  JOHN  H.,  University  of  California 
KRASSNER,  STUART,  Johns  Hopkins  University 
LANE,  ROSEMARY  M.,  Dalhousie  University 
LEISY,  ELSA,  University  of  California 
LONGACRE,  HARRIETTS,  Mount  Holyoke  College 
LORENZO,  MICHAEL  A.,  St.  Louis  University 
LOWE,  MILDRED  E.,  Tulane  University 
MCMANUS,  LAWRENCE  ROBERT,  Cornell  University 
MENAKER,  MICHAEL,  Princeton  University 
NEWBERRY,  ANDREW  TODD,  Stanford  University 
POULSON,  THOMAS  L.,  University  of  Michigan 
PRAGER,  JAN  C,  University  of  Cincinnati 
REESE,  ERNST  S.,  University  of  California 
ROOT,  RICHARD  B.,  University  of  Michigan 
Ross,  SHIRLEY  E.,  Washington  University 
SHERMAN,  IRWIN  W.,  City  College  of  New  York 
SMITH,  S.  CLARKE,  Wabash  College 
SMITH,  SUSAN,  Earlham  College 
THOMPSON,  JANE  F.,  University  of  Massachusetts 
THOMPSON,  MARTHA  JANE,  Oberlin  College 


24 


MARINE  BIOLOGICAL  LABORATORY 


TROTTER,  NANCY  L.,  Brown  University 

VITOLS,  ANDRIS  T.,  University  of  Minnesota 

WILHELM,  ROBERT  C,  Cornell  University 

WILLIS,  JOHN  S.,  Harvard  University 

WITTRY,  SISTER  ESPERANCE,  College  of  St.  Catherine 

WOOD,  LANGLEY  H.,  Cornell  University 

Yow,  FRANCIS  W.,  Emory  University 

ECOLOGY 

ABELES,  FRED,  Cornell  University 

BARBER,  RICHARD  I.,  Brown  University 

BARTH,  ROBERT  H.,  JR.,  Harvard  University 

BLUNT,  SISTER  MARION  XAVIER,  Marquette  University 

BOTHNER,  RICHARD  C.,  Fordham  University 

ELLSWORTH,  JOANNE,  Elmira  College 

GIFFORD,  CAMERON  E.,  Harvard  University 

RANDALL,  DONALD,  Oberlin  College 

STORY,  LAWRENCE  P.,  Drew  University 

3.  FELLOWSHIPS  AND  SCHOLARSHIPS,  1957 

Lucretia  Crocker  Scholarship : 

GEORGE  B.  BOUCK,  Botany  Course 
Conklin  Scholarship : 

ROBERT  KIRCHEN,  Embryology  Course 
Merkel  Jacobs  Scholarship : 

MARGIT  KAHLBROCK,  Physiology  Course 
Calkins  Scholarship : 

THOMAS  L.  POULSON,  Invertebrate  Zoology  Course 
Bio  Club  Scholarships : 

ARLEEN  BRANNING,  Invertebrate  Zoology  Course 

IRWIN  W.  SHERMAN,  Invertebrate  Zoology  Course 
Linton  Memorial  Fund : 

C.  D.  DIETER,  Washington-Jefferson  College 


4.  TABULAR  VIEW  OF  ATTENDANCE,  1953-1957 

1953  1954  1955 

INVESTIGATORS — TOTAL 310  298  250 

Independent  176  180  162 

Under  Instruction  37  20  9 

Library  Readers  46  52  54 

Research  Assistants  51  46  25 

STUDENTS — TOTAL  136  134  148 

Zoology  55  56  56 

Embryology 30  29  30 

Physiology 31  28  30 

Botany 11  12  19 

Ecology 9  9  13 

TOTAL  ATTENDANCE 446  432  398 

Less  persons  represented  as   both   investigators  and 

students  5 


446 


427 


398 


7956 

304 

184 

20 

50 

50 

140 

55 

28 

30 

18 

9 

444 
2 

442 


1957 

326 

186 
23 
42 
75 

139 
55 
27 
30 
18 
9 

465 

3 

462 


REPORT  OF  THE  DIRECTOR 


25 


INSTITUTIONS  REPRESENTED — TOTAL    155  136  129  130  129 

By   investigators    90  104  95  97  94 

By  students  65  32  34  33  35 

SCHOOLS  AND  ACADEMIES  REPRESENTED 

By   investigators    2  3  3  5 

By  students   1  1  2  1  1 

FOREIGN  INSTITUTIONS  REPRESENTED 

By  investigators    15  11  8  9  11 

By  students   6  13  6  6  5 


5.  INSTITUTIONS  REPRESENTED,  1957 


Amherst  College 

American  Museum  of  Natural  History 

Boston  University  School  of  Medicine 

Brooklyn  College 

Brown  University 

Bryn  Mawr  College 

Chatham  College 

Children's  Hospital  of  Philadelphia 

City  College  of  New  York 

Colby  College 

College  of  Mt.  St.  Joseph  on  the  Ohio 

Columbia    University,    College    of    Physicians 

and  Surgeons 

Columbia  University,  Zoology  Dept. 
Cornell  University 
Cornell  University  Medical  School 
Corporation  of  Roman  Catholic  Clergymen 
Duke  University 

Albert  Einstein  College  of  Medicine 
Elmira  College 
Emory  University 
Florida  State  University 
Fordham  University 
Hahnemann  Medical  College 
Harvard  University 
Harvard  University  Medical  School 
Indiana  University 
Institute  for  Muscle  Research 
Johns  Hopkins  University 
Johns  Hopkins  University  Medical  School 
Eli  Lilly  and  Company 
Marquette  University 
National  Institutes  of  Health 
New  York  University — Heights 
New  York  University  College  of  Medicine 
New    York    University,    Washington    Square 

College 

North  Carolina  State  College 
Northwestern  University 
Oberlin  College 
Princeton  University 
Purdue  University 


Radcliffe  College 

Rockefeller  Institute  for  Medical  Research 

Rutgers  University 

Saint  Joseph's  College 

St.  Louis  University 

St.  Louis  University,  School  of  Medicine 

Single  Cell  Foundation 

Sloan-Kettering  Institute 

Southwestern  Medical  College 

State  University  of  Iowa 

State    University    of    New    York,    College    of 

Medicine  at  Syracuse 
Syracuse  University 
Temple  University 
University  of  Chicago 
University  of  Florida 
University  of  Illinois 

University  of  Illinois,  College  of  Medicine 
University  of  Maine 
University  of  Michigan 
University  of  Minnesota 
University  of  New  Hampshire 
University  of  North  Carolina 
University  of  Oklahoma 
University  of  Pennsylvania 
University  of  Pennsylvania  Medical  School 
University  of  Pittsburgh 
University  of  Rochester 
University  of  Vermont 
University  of  Virginia,  School  of  Medicine 
University  of  Wisconsin 
U.  S.  Fish  and  Wildlife  Service 
Vassar  College 

Veterans  Administration  Hospital  of  Brooklyn 
Wabash  College 

Washington  and  Jefferson  College 
Washington  University  School  of  Medicine 
Wellesley  College 
Wesleyan   University 
Wheaton  College 
Wilson  College 
Yale  University 


26  MARINE  BIOLOGICAL  LABORATORY 

SUPPORTING  INSTITUTIONS  AND  AGENCIES,  1957 

Abbott  Laboratories  Eli  Lilly  and  Company 

American  Cancer  Society  Merck  and  Company,  Inc. 

American  Philosophical  Society  National  Institutes  of  Health 
Associates  of  the   Marine   Biological  Labora-       National  Science  Foundation 

tory  Office  of  Naval  Research 

Atomic  Energy  Commission  The  Rockefeller  Foundation 

Ciba  Pharmaceutical  Products,  Inc.  Schering  Corporation 

The  Grass  Foundation  Smith,  Kline  and  French  Foundation 

Kellogg  Foundation  The  Upjohn  Company 
The  Lalor  Foundation 

FOREIGN  INSTITUTIONS  REPRESENTED,  1957 

Zoologisches  Institut,  Mainz,  Germany  University  of  Oslo,  Sweden 

McGill  University,  Montreal,  Canada  University  of  Brussels,  Belgium 

Glasgow  University,  Scotland  Royal  Veterinary  College,  Sweden 

University  College,  London,  England  Utrecht  University,  The  Netherlands 

Wenner-Grens  Institute,  Sweden  McMaster  University,  Hamilton  College,  Can- 
University  of  Birmingham,  England  ada 

6.  EVENING  LECTURES,  1957 

July  5 

BENTLEY   GLASS    "In  pursuit  of  a  gene" 

July   12 

K.  LINDERSTROM-LANG "Deuterium  exchange  of  proteins  in  aqueous 

solution" 
July  19 

OLOV  LIXDBERG "Functional-structural   correlations   in  mito- 
chondria" 
July  26 

ALBERT  1.  LANSING   "Chemical   morphology  of  the  elastic   fiber" 

August  2 

JAMES  D.  EBERT "The  acquisition  of  biological  specificity" 

August  9 

J.  C.  ECCLES   "The  behavior  of  nerve  cells" 

August  16 

FRANCIS  J.  RYAN  "Mutation  as  an  error  in  gene  duplication" 

August  23 

SEYMOUR  S.  COHEN   "The   chemical   pathology   of   the   virus    in- 
fected cell" 

7.  TUESDAY  EVENING  SEMINARS,  1957 

July  2 

CHARLES  B.  METZ  "The  enhancement  of  starfish  sperm  motility 

and  respiration  by  metals  and  metal  bind- 
ing agents" 

NORMAN  E.  KEMP  "Differentiation  of  cortical  cytoplasm  and 

extra-cellular  membranes  of  oocytes.  in- 
cluding changes  at  fertilization" 


REPORT  OF  THE  DIRECTOR  27 

LAURA  HUNTER  COLWIN  and  ARTHUR 

L.  COLWIN   "Lytic  and  other  activities  of  the  individual 

spermatozoon  during  the  early  events  of 
sperm  entry  (Hydroides,  Saccoglossus, 
and  several  other  invertebrates)" 

July  9 

A.  M.  SHANES "Ion  movement  in  vertebrate  nerve" 

WILLIAM  STEPHENSON   "Relationships  between  ion  movements  and 

membrane  potential  changes  in  muscle" 
G.  HOYLE  "Nervous  control  of  muscular  contraction  in 

arthropods" 
W.  H.  FREYGANG,  JR "Evidence    for    electrical     inexcitability    of 

neuron  soma" 

July  16 

T.  R.  TOSTESON,  S.  A.  FERGUSON  and 

L.  V.  HEILBRUNN "Further  studies  of  the  antimitotic  and  car- 

cinostatic  action  of  ovarian  extracts" 
L.  V.  HEILBRUNN,  FRANCIS  ASHTON, 

CARL  FELDHERR  and  W.  L.  WILSON  . .  "The  action  of  insulin  on  living  cells" 

FRANCIS  ASHTON   "Magnetic  studies  on  cells  and  protoplasm" 

CARL  FELDHERR    "The  metachromatic  reaction  in  various  types 

of  protoplasm" 

PETER  RIESER   "Effect  of  x-rays  on  fibrinogen" 

PAUL   R.   GROSS,    SYLVAN   NASS   and 

WILLIAM  PEARL "Mechanisms   of   sol-gel   transformations    in 

the  cytoplasm" 

July  23 

R.  E.  BENESCII  and  R.  BENESCH  "Sulfur  linkages  in  hemoglobins" 

A.  CHASE "Uricase  inactivation  by  urea" 

L.  LORAND  "Clotting  of  blood :  a  study  of  the  polymeri- 
zation of  proteins" 

H.  K.  SCHACHMAN   "Structural    considerations    on    bushy    stunt 

virus" 

July  30 

LUIGI   PROVASOLI    .' "Effect  of  plant  hormones  on  sea  weed" 

DU-KIHT  McNAiR  SCOTT '\  hanges  in  RNA  during  synchronous  di- 
vision of  E.  coli" 

TAY  S.  ROTH  "Observations  on  the  RNase  system  of  rat 

liver" 

BERNARD  DAVIS "Bacterial  permease  systems" 

August  6 

JOSEPH  GALL "Thymidine  incorporation  into  the  macro- 
nucleus  of  Euplotes  (Protozoa)" 

BEAL  B.  HYDE "The  effect  of  Versene  on  the  sulfhydryls  of 

chromatin" 

C.  W.  METZ "Interactions  between  chromosomes  and  cy- 
toplasm during  early  embryonic  develop- 
ment in  Sciara  (Diptera)" 


MARINE  BIOLOGICAL  LABORATORY 

August  13 

BOSTWICK  H.  KETCHUM   "Marine  ecology  and  its  place  in  biological 

research" 

EUGENE  P.  ODUM "Studies  on  simple  natural  ecosystems" 

JOHN  H.  RYTHER "On  the  efficiency  of  photosynthesis   in  the 

sea" 

THOMAS  S.  AUSTIN   "The  ecology  of  the  biota  of  the  equatorial 

Pacific" 
August  20 

L.  LORAND,  J.  MOLNAR  and  C.  Moos  ....  "Biochemical   studies   of   relaxation   in   gly- 

cerinated  muscle" 

F.  D.  CARLSON  and  A.  SIGER "Creatine    phosphate    and    adenosintriphos- 

phate  breakdown   in   iodoacetate  poisoned 
muscle" 
A.  G.  SZENT-GYORGYI  and  CAROLYN 

COHEN "Structural  aspects  of  muscle  proteins" 

T.   HAYASHI,  R.   STROHMAN  and  R. 

ROSENBLUTH    "Myosin  and  actin  interaction,  and  construc- 
tion" 


8.  MEMBERS  OF  THE  CORPORATION,  1957 
1.     LIFE  MEMBERS 

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

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

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

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

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

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

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

DUNGAY,  DR.  NEIL  S.,  Carleton  College,  Northfield,  Minnesota 

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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


REPORT  OF  THE  DIRECTOR  29 

2.     REGULAR  MEMBERS 

ABELL,  DR.  RICHARD  G.,  7  Cooper  Road,  New  York  City,  New  York 

ADAMS,  DR.  A.  ELIZABETH,  Mount  Holyoke  College,  South  Hadley,  Massachusetts 

ADDISON,  DR.  W.  H.  F.,  286  East  Sidney  Avenue,  Mount  Vernon,  New  York 

ADOLPH,  DR.  EDWARD  F.,  University  of  Rochester,  School  of  Medicine  and  Dentis- 
try, Rochester,  New  York 

ALBERT,  DR.  ALEXANDER,  Mayo  Clinic,  Rochester,  Minnesota 

ALLEN,  DR.  M.  JEAN,  Department  of  Biology,  Wilson  College,  Chambersburg, 
Pennsylvania 

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

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

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

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

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

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

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

ARNOLD,  DR.  WILLIAM  A.,  Oak  Ridge  National  Laboratory,  Oak  Ridge,  Tennessee 

ATWOOD,  DR.  KIMBALL  C,  68.]  Outer  Drive,  Oak  Ridge,  Tennessee 

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

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

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

BAKER,  DR.  H.  B.,  Zoological  Laboratory,  University  of  Pennsylvania,  Philadel- 
phia, Pennsylvania 

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

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

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

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

BARTH,  DR.  L.  G.,  Department  of  Zoology,  Columbia  University,  New  York  City, 
New  York 

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

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

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

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


30  MARINE  BIOLOGICAL  LABORATORY 

BEHRE,  DR.  ELINOR  H.,  Louisiana  State  University,  Baton  Rouge,  Louisiana 
BENESCH,  DR.  REINHOLD,  Marine  Biological  Laboratory,  Woods  Hole,  Massachu- 
setts 

BENESCH,  DR.  RUTH,  Marine  Biological  Laboratory,  Woods  Hole,  Massachusetts 
BENNETT,  DR.  MIRIAM,  Department  of  Biology,  Sweet  Briar  College,  Sweet  Briar, 

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

California 
BERMAN,  MR.  MONES,  Sloan-Kettering  Institute,  410  E.  68th  Street,  New  York  21, 

New  York 
BERNSTEIN,  DR.  MAURICE,  Virus  Laboratory,  University  of  California,  Berkeley  4, 

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

York  16,  New  York 

BERTHOLF,  DR.  FLOYD  M.,  College  of  the  Pacific,  Stockton,  California 
BEVELANDER,  DR.  GERRIT,  New  York  University  School  of  Medicine,  New  York 

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

Cambridge,  Massachusetts 

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

BLANCHARD,  DR.  K.  C.,  Johns  Hopkins  Medical  School,  Baltimore,  Maryland 
BLOCK,  DR.  ROBERT,  c/o  Biological  Abstracts,  3815  Walnut  Street,  Philadelphia  4, 

Pennsylvania 

BLUM,  DR.  HAROLD  F.,  24  Rue  de  Babylone,  Paris  VII,  France 
BODANSKY,  DR.  OSCAR,  Department  of  Biochemistry,  Memorial  Cancer  Center,  444 

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

Baltimore  5,  Maryland 

BOELL,  DR.  EDGAR  J.,  Yale  University,  New  Haven,  Connecticut 
BOETTIGER,  DR.  EDWARD  G.,  Department  of  Zoology,  University  of  Connecticut, 

Storrs,  Connecticut 

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

Massachusetts 

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

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

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

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

Arbor,  Michigan 
BROWN,  DR.  FRANK  A.,  JR.,  Department  of  Biological   Sciences,   Northwestern 

University.  Evanston,  Illinois 


REPORT  OF  THE  DIRECTOR  31 

BROWNELL,  DR.  KATHERINE  A.,  Ohio  State  University,  Columbus,  Ohio 

BUCK,  DR.  JOHN  B.,  Laboratory  of  Physical  Biology,  National  Institutes  of  Health, 

Bethesda,  Maryland  (10405  Muir  Place,  Kensington,  Maryland) 
BULLINGTON,  DR.  W.  E.,  Randolph-Macon  College,  Ashland,  Virginia 
BULLOCK,  DR.  T.  H.,  Department  of  Zoology,  University  of  California,  Los  An- 
•    geles  24,  California 

BURBANCK,  DR.  WILLIAM  D.,  Box  834,  Emory  University,  Georgia 
BURDICK,  DR.  C.  LALOR,  The  Lalor  Foundation,  4400  Lancaster  Pike,  Wilmington, 

Delaware 
BURKENROAD,  DR.  M.  D.,  c/o  Lab.  Nal.  de  Pesca,  Apartado  3318,  Estofeta  #1, 

Olindania,  Republic  of  Panama 
BUTLER,  DR.  E.  G.,  Department  of  Biology,  Princeton  University,  Princeton,  New 

Jersey 

CAMERON,  DR.  J.  A.,  Baylor  College  of  Dentistry,  Dallas,  Texas 
CANTONI,  DR.  GIULIO,  National  Institutes  of  Health,  Mental  Health,  Bethesda  14, 

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

Baltimore,  Md. 

CARPENTER,  DR.  RUSSELL  L.,  Tufts  College,  Medford  55,  Massachusetts 
CARSON,  Miss  RACHEL,  204  Williamsburg  Drive,  Silver  Spring,  Maryland 
CATTELL,  DR.  McKEEN,  Cornell  University  Medical  College,  1300  York  Avenue, 

New  York  City,  New  York 

CATTELL,  MR.  WARE,  Cosmos  Club,  Washington  5,  D.  C. 
CHAET,  DR.  ALFRED  B.,  Boston  University  School  of  Medicine,  80  E.  Concord 

Street,  Boston  18,  Massachusetts 
CHAMBERS,  DR.  EDWARD,  Department  of  Physiology,  University  of  Miami  Medical 

School,  Coral  Gables,  Florida 
CHANG,  DR.  JOSEPH  J.,  National  Institute  of  Neurological  Diseases  and  Blindness, 

National  Institutes  of  Health,  Bethesda,  Maryland 
CHASE,  DR.  AURIN  M.,  Department  of  Biology,  Princeton  University,  Princeton, 

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

New  York 

CLAFF,  MR.  C.  LLOYD,  5  Van  Beal  Road,  Randolph,  Massachusetts 
CLARK,  DR.  A.  M.,  Department  of  Biological  Sciences,  University  of  Delaware, 

Newark,  Delaware 

CLARK,  DR.  E.  R.,  The  Wistar  Institute,  Woodland  Avenue  and  36th  Street,  Phila- 
delphia 4,  Pennsylvania 
CLARK,  DR.  LEONARD  B.,  Department  of  Biology,  Union  College,   Schenectady, 

New  York 
CLARKE,  DR.  GEORGE  L.,  Harvard  University,  Biological  Laboratory,  Cambridge 

38,  Massachusetts 

CLELAND,  DR.  RALPH  E.,  Indiana  University,  Bloomington,  Indiana 
CLEMENT,  DR.  A.  C.,  Department  of  Biology,  Emory  University,  Emory,  Georgia 
CLOWES,  DR.  G.  H.  A.,  Eli  Lilly  and  Company,  Indianapolis,  Indiana 
COE,  DR.  W.  R.,  183  Third  Avenue,  Chula  Vista,  California 
COHEN,  DR.  SEYMOUR  S.,  Department  of  Physiological  Chemistry,  University  of 

Pennsylvania,  Philadelphia,  Pennsylvania 


32  MARINE  BIOLOGICAL  LABORATORY 

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

Maryland 

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

Rouge,  Louisiana 

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

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

York 

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

COPELAND,  DR.  D.  E.,  1027  N.  Manchester  Street,  Arlington  5,  Virginia 
COPELAND,  DR.  MANTON,  Bowdoin  College,  Brunswick,  Maine 
COPLEY,  DR.  ALFRED  L.,  Centre  National  cle  Transfusion  Sanguine,  6,  Rue  Alex- 

andra-Cobonel,  Paris  XVe,  France 

CORNMAN,  DR.  IVOR,  Hazleton  Laboratories,  Box  333,  Falls  Church,  Virginia 
COSTELLO,  DR.  DONALD  P.,  Department  of  Zoology,  University  of  North  Carolina, 

Chapel  Hill,  North  Carolina 

COSTELLO,  DR.  HELEN  MILLER,  Department  of  Zoology,  University  of  North  Caro- 
lina, Chapel  Hill,  North  Carolina 
CRANE,  MR.  JOHN  O.,  Woods  Hole,  Massachusetts 

CROASDALE,  DR.  HANNAH  T.,  Dartmouth  College,  Hanover,  New  Hampshire 
GROUSE,  DR.  HELEN  V.,  Goucher  College,  Baltimore,  Maryland 
CROWELL,  DR.  P.  S.,  IR.,  Department  of  Zoology,  Indiana  University,  Bloomington, 

Indiana 
CSAPO,  DR.  ARPAD  I.,  Rockefeller  Institute  for  Medical  Research,  New  York  21, 

New  York 

CURTIS,  DR.  MAYNIE  R.,  University  of  Miami,  Box  1015,  South  Miami,  Florida 
CURTIS,  DR.  W.  C,  University  of  Missouri,  Columbia,  Missouri 
DAN,  DR.  JEAN  CLARK,  Misaki  Biological  Station,  Misaki,  Japan 
DAN,  DR.  KATSUMA,  Misaki  Biological  Station,  Misaki,  Japan 

DANIELLI,  DR.  JAMES  F.,  Department  of  Zoology,  King's  College,  London,  England 
DAVIS,  DR.  BERNARD  D.,  Department  of  Pharmacology,  New  York  University  Col- 
lege of  Medicine,  New  York  16,  New  York 

DAWSON,  DR.  A.  B.,  Harvard  University,  Cambridge  38,  Massachusetts 
DAWSON,  DR.  T.  A.,  College  of  the  City  of  New  York,  New  York  City,  New  York 
DEANE,  DR.  HELEN  W.,  Albert  Einstein  College  of  Medicine,  New  York  61,  New 

York 

DILLER,  DR.  IRENE  C.,  Institute  for  Cancer  Research,  Philadelphia,  Pennsylvania 
DILLER,  DR.  WILLIAM  F.,  2417  Fairhill  Avenue,  Glenside,  Pennsylvania 
DIXON,  DR.  FRANK  J.,  Department  of  Pathology,  University  of  Pittsburgh  School 

of  Medicine,  Pittsburgh,  Pennsylvania 
DOODS,  DR.  G.  S.,  West  Virginia  University  School  of  Medicine,   Morgantown, 

West  Virginia 

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


REPORT  OF  THE  DIRECTOR  33 

DONALDSON,  DR.  JOHN  C,  University  of  Pittsburgh  School  of  Medicine,  Pitts- 
burgh, Pennsylvania 

DOTY,  DR.  MAXWELL  S.,  Department  of  Biology,  University  of  Hawaii,  Honolulu, 
T.  H. 

DuBois,  DR.  EUGENE  F.,  200  East  End  Avenue,  New  York  28,  New  York 

DURYEE,  DR.  WILLIAM  R.,  George  Washington  University  School  of  Medicine, 
Department  of  Physiology,  Washington  5,  D.  C. 

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

EDWARDS,  DR.  CHARLES,  Wilmer  25,  Johns  Hopkins  Hospital,  Baltimore  5,  Mary- 
land 

EICHEL,  DR.  BERTRAM,  Bureau  of  Biological  Research,  Box  515,  Rutgers  Univer- 
sity, New  Brunswick,  New  Jersey 

EICHEL,  DR.  HERBERT  J.,  Hahnemann  Medical  College,  Philadelphia,  Pennsylvania 

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

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

FAILLA,  DR.  G.,  College  of  Physicians  and  Surgeons,  Columbia  University,  New 
York  City,  New  York 

FAURE-FREMIET,  DR.  EMMANUEL,  College  de  France,  Paris,  France 

FERGUSON,  DR.  F.  P.,  Department  of  Physiology,  University  of  Maryland  Medical 
School,  Baltimore  1,  Maryland 

FERGUSON,  DR.  JAMES  K.  W.,  Connought  Laboratories,  University  of  Toronto, 
Ontario,  Canada 

FIGGE,  DR.  F.  H.  J.,  University  of  Maryland  Medical  School,  Lombard  and  Green 
Streets,  Baltimore  1,  Maryland 

FINGERMAN,  DR.  MILTON,  Department  of  Zoology,  Newcomb  College,  Tulane  Uni- 
versity, New  Orleans  18,  Louisiana 

FISCHER,  DR.  ERNST,  Department  of  Physiology,  Medical  College  of  Virginia, 
Richmond  19,  Virginia 

FISHER,  DR.  JEANNE  M.,  Department  of  Biochemistry,  University  of  Toronto, 
Toronto,  Canada 

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

FORBES,  DR.  ALEXANDER,  Biological  Laboratories,  Harvard  University,  Cambridge 
38,  Massachusetts 

FRAENKEL,  DR.  GOTTFRIED  S.,  Department  of  Entomology,  University  of  Illinois, 
Urbana,  Illinois 

FREYGANG,  DR.  WALTER  H.,  JR.,  Essex  Fells,  New  Jersey 

FRIES,  DR.  ERIK  F.  B.,  Box  605,  Woods  Hole,  Massachusetts 

FRISCH,  DR.  JOHN  A.,  Canisius  College,  Buffalo,  New  York 

FURTH,  DR.  JACOB,  18  Springdale  Road,  Wellesley  Farms,  Massachusetts 

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

GAFFRON,  DR.  HANS,  Research  Institutes,  University  of  Chicago,  5650  Ellis  Ave- 
nue, Chicago  37,  Illinois 

GALL,  DR.  JOSEPH  G.,  Department  of  Zoology,  University  of  Minnesota,  Minne- 
apolis 14,  Minnesota 


34  MARINE  BIOLOGICAL  LABORATORY 

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

GASSER,  DR.  HERBERT  S.,  Rockefeller  Institute,  New  York  21,  New  York 

GEISER,  DR.  S.  W.,  Southern  Methodist  University,  Dallas,  Texas 

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

Gables,  Florida 

GINSBERG,  DR.  HAROLD  S.,  Western  Reserve  University  School  of  Medicine,  Cleve- 
land, Ohio 
GOODCHILD,  DR.  CHAUNCEY  G.,  Department  of  Biology,  Emory  University,  Emory 

University,  Georgia 

GOODRICH,  DR.  H.  B.,  Wesleyan  University,  Middletown,  Connecticut 
GOTTSCHALL,  DR.  GERTRUDE  Y.,  315  E.  68th  Street,  New  York  21,  New  York 
GOULD,  DR.  H.  N.,  Biological  Sciences  Information  Exchange,  1113  Dupont  Circle 

Building,  Washington,  D.  C. 

GRAHAM,  DR.  HERBERT,  U.  S.  Fish  and  Wildlife  Service,  Woods  Hole,  Massa- 
chusetts 
GRAND,  MR.  C.  G.,  Dade  County  Cancer  Institute,  1155  N.  W.  15th  Street,  Miami, 

Florida 

GRANT,  DR.  M.  P.,  Sarah  Lawrence  College,  Bronxville,  New  York 
GRAY,  DR.  IRVING  E.,  Duke  University,  Durham,  North  Carolina 
GREEN,    DR.   JAMES   W.,    Department   of    Physiology,    Rutgers   University,    New 

Brunswick,  New  Jersey 
GREEN,  DR.  MAURICE,  Department  of  Biochemistry,  University  of  Pennsylvania, 

Philadelphia,  Pennsylvania 

GREGG,  DR.  JAMES  H.,  University  of  Florida,  Gainesville,  Florida 
GREGG,  DR.  J.  R.,  Department  of  Zoology,  Columbia  University,  New  York  27, 

New  York 

GREIF,  DR.  ROGER  L.,  Department  of  Physiology,  Cornell  University  Medical  Col- 
lege, New  York  21,  New  York 
GROSCH,  DR.  DANIEL  S.,  Department  of  Zoology,  North  Carolina  State  College, 

Raleigh,  North  Carolina 
GROSS,    DR.    PAUL,    Department   of    Biology,    New   York   University,    University 

Heights,  New  York  53,  New  York 

GRUNDFEST,  DR.   HARRY,   Columbia  University,   College  of   Physicians  and   Sur- 
geons, New  York  City,  New  York 

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

Street,  Detroit  1,  Michigan 
GUTTMAN,   DR.   RITA,   Department   of   Physiology,   Brooklyn    College,    Brooklyn, 

New  York 

GUYER,  DR.  MICHAEL  F.,  University  of  Wisconsin,  Madison,  Wisconsin 
HAJDU,  DR.  STEPHEN,  U.  S.  Public  Health  Institute,  Bethesda  14,  Maryland 
HALL,  DR.  FRANK  G.,  Duke  University,  Durham,  North  Carolina 
HAMBURGER,  DR.  VIKTOR,   Department  of  Zoology,  Washington  University,   St. 

Louis,  Missouri 

HAMILTON,  DR.  HOWARD  L.,  Iowa  State  College,  Ames,  Iowa 
HANCE,  DR.  ROBERT  T.,  Box  108,  R.  R.  #3,  Loveland,  Ohio 

HARDING,  DR.  CLIFFORD  V.,  JR.,  705  N.  Wayne  Street,  Apt.  305,  Arlington  1, 
Virginia 


REPORT  OF  THE  DIRECTOR  35 

HARMAN,  DR.  MARY  T.,  Box  68,  Camden,  North  Carolina 

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

York  City,  New  York 

HARRISON,  DR.  Ross  G.,  Yale  University,  New  Haven,  Connecticut 
HARTLINE,  DR.  H.  KEFFER,  Rockefeller  Institute  for  Medical  Research,  New  York 

21,  New  York 

HARTMAN,  DR.  FRANK  A.,  Hamilton  Hall,  Ohio  State  University,  Columbus,  Ohio 
HARVEY,  DR.  ETHEL  BROWNE,  48  Cleveland  Lane,  Princeton,  New  Jersey 
HARVEY,  DR.  E.  NEWTON,  Guyot  Hall,  Princeton  University,  Princeton,  New  Jersey 
HAUSCHKA,  DR.  T.  S.,  Roswell  Park  Memorial  Institute,  663  North  Oak  Street, 

Buffalo  3,  New  York 

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

City,  New  York 

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

HENDLEY,  DR.  CHARLES  D.,  615  South  Second  Avenue,  Highland  Park,  New  Jersey 
HENLEY,  DR.  CATHERINE,  Department  of  Zoology,  University  of  North  Carolina, 

Chapel  Hill,  North  Carolina 
HENSHAW,  DR.  PAUL  S.,  17th  Floor,  501  Madison  Avenue,  New  York  22,  New 

York 

HERVEY,  DR.  JOHN  P.,  Box  735,  Woods  Hole,  Massachusetts 
HESS,  DR.  WALTER  N.,  Hamilton  College,  Clinton,  New  York 
HIBBARD,  DR.  HOPE,  Department  of  Zoology,  Oberlin  College,  Oberlin,  Ohio 
HILL,  DR.  SAMUEL  E.,  135  Brunswick  Road,  Troy,  New  York 
HINRICHS,  DR.  MARIE,  Board  of  Education,  Bureau  of  Health  Service,  228  North 

LaSalle  Street,  Chicago,  Illinois 

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

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

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

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

Pennsylvania 
HOLLAENDER,  DR.  ALEXANDER,  P.  O.  Box  W,  Clinton  Laboratories,  Oak  Ridge, 

Tennessee 
HOPKINS,  DR.  HOYT  S.,  New  York  University  College  of  Dentistry,  New  York 

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

Colombia,  South  America 

HUTCHENS,  DR.  JOHN  O.,  Department  of  Physiology,  University  of  Chicago,  Chi- 
cago 37,  Illinois 


36  MARINE  BIOLOGICAL  LABORATORY 

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

New  York 

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

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

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

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

New  Jersey 

JONES,  DR.  E.  RUFFIN,  JR.,  Department  of  Biology,  University  of  Florida,  Gaines- 
ville, Florida 

KAAN,  DR.  HELEN  W.,  Marine  Biological  Laboratory,  Woods  Hole,  Massachusetts 
RABAT,  DR.  E.  A.,  Neurological  Institute,  College  of  Physicians  and   Surgeons, 

New  York  City,  New  York 

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

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

Arbor,  Michigan 

KEMPTON,  DR.  RUDOLF  T.,  Vassar  College,  Poughkeepsie,  New  York 
KEOSIAN,  DR.  JOHN,  Department  of  Biology,  Rutgers  University,  Newark  2,  New 

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

Massachusetts 

KILLE,  DR.  FRANK  R.,  Carleton  College,  Northneld,  Minnesota 
KIND,  DR.  C.  ALBERT,  Department  of  Chemistry,  University  of  Connecticut,  Storrs, 

Connecticut 

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

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

Angeles  24,  California 
KOPAC,  DR.  M.  J.,  New  York  University,  Washington  Square  College,  New  York 

City,  New  York 
KORR,  DR.   I.  M.,  Department  of  Physiology,   Kirksville   College  of  Osteopathy, 

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

Illinois 


REPORT  OF  THE  DIRECTOR  37 

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

KREIG,  DR.  WENDELL  J.  S.,  303  East  Chicago  Avenue,  Chicago,  Illinois 

KUFFLER,  DR.  STEPHEN,  Department  of  Ophthalmology,  Johns  Hopkins  Hospital, 
Baltimore  5,  Maryland 

KUNITZ,  DR.  MOSES,  Rockefeller  Institute,  66th  Street  and  York  Avenue,  New 
York  21,  New  York 

LACKEY,  DR.  JAMES  B.,  University  of  Florida,  College  of  Engineering,  Gainesville, 
Florida 

LANCEFIELD,  DR.  D.  E.,  Queens  College,  Flushing,  New  York 

LANCEFIELD,  DR.  REBECCA  C,  Rockefeller  Institute,  66th  Street  and  York  Avenue, 
New  York  21,  New  York 

LANDIS,  DR.  E.  M.,  Harvard  Medical  School,  Boston  15,  Massachusetts 

LANGE,  DR.  MATHILDA  M.,  Box  307,  Central  Valley,  New  York 

LANSING,  DR.  ALBERT  I.,  Department  of  Anatomy,  University  of  Pittsburgh  Medi- 
cal School,  Pittsburgh  13,  Pennsylvania 

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

LAVIN,  DR.  GEORGE  I.,  3714  Springdale  Avenue,  Baltimore,  Maryland 

LAZAROW,  DR.  ARNOLD,  Department  of  Anatomy,  University  of  Minnesota,  Medi- 
cal School,  Minneapolis  14,  Minnesota 

LEDERBERG,  DR.  JOSHUA,  Department  of  Genetics,  University  of  Wisconsin,  Madi- 
son 6,  Wisconsin 

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

LEFEVRE,  DR.  PAUL  G.,  Brookhaven  Apartments,  Upton,  Long  Island,  New  York 

LEHMANN,  DR.  FRITZ,  Zool.  Inst,  University  of  Berne,  Berne,  Switzerland 

LESSLER,  DR.  MILTON  A.,  Department  of  Physiology,  Ohio  State  University,  Co- 
lumbus, Ohio 

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

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

LEWIN,  DR.  RALPH  A.,  Marine  Biological  Laboratory,  Woods  Hole,  Massachusetts 

LEWIS,  DR.  I.  F.,  1110  Rugby  Road,  Charlottesville,  Virginia 

LING,  DR.  GILBERT,  Eastern  Pennsylvania  Psychiatric  Inst.,  Henry  Avenue  and 
Abbottsford  Road,  Philadelphia  29,  Pennsylvania 

LITTLE,  DR.  E.  P.,  150  Causeway  Street,  Anderson  Nichols  &  Company,  Boston  24, 
Massachusetts 

LLOYD,  DR.  DAVID  P.  C.,  Rockefeller  Institute,  66th  Street  &  York  Avenue,  New 
York  21,  New  York 

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

LOEB,  DR.  LEO,  40  Crestwood  Drive,  St.  Louis  5,  Missouri 

LOEB,  DR.  R.  F.,  Presbyterian  Hospital,  620  West  168th  Street,  New  York  32, 
New  York 

LOEWI,  DR.  OTTO,  155  East  93rd  Street,  New  York  City,  New  York 

LORAND,  DR.  LASZLO,  Department  of  Chemistry,  College  of  Liberal  Arts,  North- 
western University,  Evanston,  Illinois 


38  MARINE  BIOLOGICAL  LABORATORY 

LOVE,  DR.  Lois  H.,  4233  Regent  Street,  Philadelphia  4,  Pennsylvania 
LOVE,  DR.  WARNER  E.,  1043  Marian  Drive,  Baltimore,  Maryland 
LUBIN,  DR.  MARTIN,  Department  of  Pharmacology,  Harvard  Medical  School,  Bos- 
ton 15,  Massachusetts 
LYNCH,  DR.  CLARA  J..  Rockefeller  Institute,  66th  Street  and  York  Avenue,  New 

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

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

Iowa 
LYNN,  DR.  WILLIAM  G.,  Department  of  Biology,  Catholic  University  of  America, 

Washington,  D.  C. 
MACDOUGALL,  DR.  MARY  S.,  Mt.  Vernon  Apartments,  423   Clairmont  Avenue, 

Decatur,  Georgia 
McCoucH,  DR.  MARGARET  SUMWALT,  University  of  Pennsylvania  Medical  School, 

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

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

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

Baltimore  18,  Maryland 
MAAS,  DR.  WERNER  K.,  New  York  University  College  of  Medicine,  New  York 

City,  New  York 

MACKLIN,  DR.  CHARLES  C.,  37  Gerard  Street,  London,  Ontario,  Canada 
MAGRUDER,  DR.  SAMUEL  R.,  Department  of  Anatomy,  Tufts  Medical  School,  136 

Harrison  Avenue,  Boston,  Massachusetts 

MANWELL,  DR.  REGINALD  D.,  Syracuse  University,  Syracuse,  New  York 
MARSHAK,  DR.  ALFRED,  Marine  Biological  Laboratory,  Woods  Hole,  Massachu- 
setts 
MARSLAND,  DR.  DOUGLAS  A.,  New  York  University,  Washington  Square  College, 

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

York 

MATHEWS,  DR.  A.  P.,  Glenwood  Boulevard,  Schenectady,  New  York 
MATHEWS,  DR.  SAMUEL  A.,  Thompson  Biological  Laboratory,  Williams  College, 

Williamstown,  Massachusetts 

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

California 

MEDES,  DR.  GRACE,  Lankenau  Research  Institute,  Philadelphia,  Pennsylvania 
MEIGS,  MRS.  E.  B.,  1736  M  Street,  N.  W.,  Washington,  D.  C. 
MEINKOTH,  DR.  NORMAN  A.,  Department  of  Biology,  Swarthmore  College,  Swarth- 

more,  Pennsylvania 

MEMHARD,  MR.  A.  R.,  Riverside,  Connecticut 
MENKIN,  DR.  VALY,  Agnes  Barr  Chase  Foundation  for  Cancer  Research,  Temple 

University  Medical  School,  Philadelphia,  Pennsylvania 


REPORT  OF  THE  DIRECTOR  39 

METZ,  DR.  C.  B.,  Oceanographic  Institute,  Florida  State  University,  Tallahassee, 

Florida 

METZ,  DR.  CHARLES  W.,  University  of  Pennsylvania,  Philadelphia,  Pennsylvania 
MIDDLEBROOK,  DR.  ROBERT,  Institute  for  Muscle  Research,  Marine  Biological  Lab- 
oratory, Woods  Hole,  Massachusetts 

MILLER,  DR.  J.  A.,  Basic  Science  Building,  Emory  University,  Georgia 
MILNE,  DR.  LORUS  J.,  Department  of  Zoology,  University  of  New  Hampshire, 

Durham,  New  Hampshire 
MOE,  MR.  HENRY  A.,  Secretary  General,  Guggenheim  Memorial  Foundation,  551 

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

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

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

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

New  Jersey 

MOUNTAIN,  MRS.  J.  D.,  9  Coolidge  Avenue,  White  Plains,  New  York 
MULLER,   DR.   H.  J.,  Department  of  Zoology,   Indiana  University,   Bloomington, 

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

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

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

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

University,  Hamilton,  Ontario 

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

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

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

5,  Washington 

NEWMAN,  DR.  H.  H.,  173  Devon  Drive,  Clearwater,  Florida 
NICOLL,  DR.  PAUL  A.,  Indiana  Contract,  Box  K,  A.  P.  O.  474,  San  Francisco, 

California 
Niu,  DR.  MAN-CHIANG,  Rockefeller  Institute  for  Medical  Research,  New  York 

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

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

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

Mawr,  Pennsvlvania 


40  MARINE  BIOLOGICAL  LABORATORY 

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

Maryland 
OSTERHOUT,  DR.  W.  J.  V.,  Rockefeller  Institute,  66th  Street  and  York  Avenue, 

New  York  21,  New  York 
OSTERHOUT,  MRS.   MARION   IRWIN,  Rockefeller   Institute,  66th   Street  and  York 

Avenue,  New  York  21,  New  York 
PACKARD,  DR.  CHARLES,  Woods  Hole,  Massachusetts 
PAGE,  DR.  IRVINE  H.,  Cleveland  Clinic,  Cleveland,  Ohio 
PARMENTER,  DR.  CHARLES  L.,  Department  of  Zoology,  University  of  Pennsylvania, 

Philadelphia,  Pennsylvania 

PARPART,  DR.  ARTHUR  K.,  Department  of  Biology,  Princeton  University,  Prince- 
ton, New  Jersey 
PASSANO,  DR.  LEONARD  M.,  Osborn  Zoological  Laboratories,  Yale  University,  New 

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

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

Chicago  37,  Illinois 

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

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

PIERCE,  DR.  MADELENE  E.,  Vassar  College,  Poughkeepsie,  New  York 
PLOUGH,  DR.  HAROLD  H.,  Amherst  College,  Amherst,  Massachusetts 
POLLISTER,  DR.  A.  W.,  Columbia  University,  New  York  City,  New  York 
POND,  DR.  SAMUEL  E.,  53  Alexander  Street,  Manchester,  Connecticut 
PRATT,  DR.  FREDERICK  H.,  105  Hundreds  Road,  Wellesley  Hills  82,  Massachusetts 
PROCTOR,  DR.  NATHANIEL,  Department  of  Biology,  Morgan  State  College,  Balti- 
more 12,  Maryland 
PROSSER,  DR.  C.  LADD,  401  Natural  History  Building,  University  of  Illinois,  Ur- 

bana,  Illinois 
PROVASOLI,  DR.  LUIGI,  Department  of  Biology,  Haskins  Laboratories,  305  E.  43rd 

Street,  New  York  17,  New  York 
QUASTEL,  DR.  JUDA  H.,  Department  of  Biochemistry,  McGill  University,  Montreal, 

Canada 

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

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

Foot  East  15th  Street,  New  York  9,  New  York 

RAY,  DR.  CHARLES,  JR.,  Department  of  Biology,  Emory  University,  Emory,  Georgia 
READ,  DR.  CLARK  P.,  Johns  Hopkins  University,  Baltimore,  Maryland 
REBHUN,  DR.  LIONEL  I.,  Department  of  Anatomy,  University  of  Illinois,  College 

of  Medicine,  Chicago,  Illinois 

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


REPORT  OF  THE  DIRECTOR  41 

REDFIELD,  DR.  ALFRED  C,  Woods  Hole,  Massachusetts 

REINER,  DR.  J.  M.,  Columbia-Presbyterian  Medical  Center,  622  W.  168th  Street, 

New  York  32,  New  York. 
RENN,  DR.  CHARLES  E.,  509  Ames  Hall,  Johns  Hopkins  University,  Baltimore  18, 

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

New  York  City,  New  York 

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

of  Minnesota,  St.  Paul,  Minnesota 

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

RIESER,  DR.  PETER,  Marine  Biological  Laboratory,  Woods  Hole,  Massachusetts 
ROCKSTEIN,  DR.  MORRIS,  Department  of  Physiology,  New  York  University,  College 

of  Medicine,  New  York  16,  New  York 

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

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

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

York  City,  New  York 

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

Illinois 
ROSENTHAL,  DR.  THEODORE  B.,  Department  of  Anatomy,  University  of  Pittsburgh 

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

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

Philadelphia  2,  Pennsylvania 
ROTHENBERG,  DR.  M.  A.,  Chief,  Chemical  Laboratories,  Dugway  Proving  Ground, 

Dugway,  Utah 
RUGH,  DR.  ROBERTS,  Radiological  Research  Laboratory,  College  of  Physicians  and 

Surgeons,  New  York  City,  New  York 

RUNNSTROM,  DR.  JOHN,  Wenner-Grens  Institute,  Stockholm,  Sweden 
RUTMAN,   DR.   ROBERT  J.,   Department  of  Zoology,   University  of   Pennsylvania, 

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

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

North  Carolina 

SAUNDERS,  MR.  LAWRENCE,  R.  D.  7,  Bryn  Mawr,  Pennsylvania 
SCHAEFFER,  DR.  ASA  A.,  Department  of  Biology,  Temple  University,  Philadelphia, 

Pennsvlvania 


42  MARINE  BIOLOGICAL  LABORATORY 

SCHARRER,  DR.  ERNST  A.,  Albert  Einstein  College  of  Medicine,   1710  Newport 

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

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

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

Technology,  Cambridge,  Massachusetts 

SCHMITT,  DR.  O.  H.,  Department  of  Physics,  University  of  Minnesota,  Minne- 
apolis 14,  Minnesota 
SCHNEIDERMAN,   DR.   HOWARD  A.,   Department  of  Zoology,   Cornell  University, 

Ithaca,  New  York 
SCHOLANDER,  DR.   P.   F.,   Institute  of  Zoophysiology,  University   of   Oslo,   Oslo, 

Norway 
SCHOTTE,    DR.    OSCAR   E.,    Department   of   Biology,   Amherst    College,    Amherst, 

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

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

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

Indiana 

SCOTT,  DR.  ALLAN  C.,  Colby  College,  Waterville,  Maine 

SCOTT,  DR.  D.  B,  McNAiR,  Department  of  Biochemistry,  University  of  Pennsyl- 
vania Hospital,  Philadelphia,  Pennsylvania 

SCOTT,  SISTER  FLORENCE  MARIE,  Seton  Hill  College,  Greensburg,  Pennsylvania 
SCOTT,  DR.  GEORGE  T.,  Oberlin  College,  Oberlin,  Ohio 

SEARS,  DR.  MARY,  Woods  Hole  Oceanographic  Institution,  Woods  Hole,  Massa- 
chusetts 
SEVERINGHAUS,  DR.  AURA  E.,  Department  of  Anatomy,  College  of  Physicians  and 

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

Institutes  of  Health,  Bethesda  14,  Maryland 

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

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

New  York  21,  New  York 

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

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

School  of  Medicine,  Baltimore,  Maryland 
SMITH,  DR.  EDWARD  H.,  Woods  Hole  Oceanographic  Institution,  Woods  Hole, 

Massachusetts 


REPORT  OF  THE  DIRECTOR  43 

SMITH,  MR.  HOMER  P.,  General  Manager,  Marine  Biological  Laboratory,  Woods 
Hole,  Massachusetts 

SMITH,  MR.  PAUL  FERRIS,  Marine  Biological  Laboratory,  Woods  Hole,  Massa- 
chusetts 

SMITH,  DR.  RALPH  I.,  Department  of  Zoology,  University  of  California,  Berkeley 
4,  California 

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

SONNENBLICK,  DR.  B.  P.,  40  Rector  Street,  Newark  3,  New  Jersey 

SPEIDEL,  DR.  CARL  C.,  University  of  Virginia,  University,  Virginia 

SPIEGEL,  DR.  MELVIN,  Department  of  Biology,  Colby  College,  Waterville,  Maine 

SPRATT,  DR.  NELSON  T.,  Department  of  Zoology,  University  of  Minnesota,  Minne- 
apolis 14,  Minnesota 

STARR,  DR.  RICHARD  C.,  Department  of  Botany,  Indiana  University,  Bloomington, 
Indiana 

STEINBACH,  DR.  HENRY  BURR,  Department  of  Zoology,  University  of  Chicago, 
Chicago  15,  Illinois 

STEINBERG,  DR.  MALCOLM  S.,  Department  of  Embryology,  Carnegie  Institution  of 
Washington,  Baltimore  5,  Maryland 

STEPHENS,  DR.  GROVER  C.,  Department  of  Zoology,  University  of  Minnesota,  Min- 
neapolis 14,  Minnesota 

STEWART,  DR.  DOROTHY,  Rockford  College,  Rockford,  Illinois 

STOREY,  DR.  ALMA  G.,  Department  of  Botany,  Mount  Holyoke  College,  South 
Hadley,  Massachusetts 

STRAUS,  DR.  W.  L.,  JR.,  Johns  Hopkins  University,  Baltimore  18,  Maryland 

STUNKARD,  DR.  HORACE  W.,  American  Museum  of  Natural  History,  New  York  24, 
New  York 

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

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

SWOPE,  MR.  GERARD,  JR.,  570  Lexington  Avenue,  New  York  22,  New  York 

SZENT-GYORGYI,  DR.  ALBERT,  Marine  Biological  Laboratory,  Woods  Hole,  Massa- 
chusetts 

SZENT-GYORGYI,  DR.  ANDREW  G.,  Marine  Biological  Laboratory,  Woods  Hole, 
Massachusetts 

TASAKI,  DR.  ICHIJI,  Laboratory  of  Neurophysiology,  National  Institute  of  Neuro- 
logical Diseases  and  Blindness,  Bethesda  14,  Maryland 

TASHIRO,  DR.  SHIRO,  University  of  Cincinnati  Medical  College,  Cincinnati,  Ohio 

TAYLOR,  DR.  ROBERT  E.,  Laboratory  of  Neurophysiology,  National  Institute  of 
Neurological  Diseases  and  Blindness,  Bethesda  14,  Maryland 

TAYLOR,  DR.  WM.  RANDOLPH,  Department  of  Botany,  University  of  Michigan, 
Ann  Arbor,  Michigan 

TEWINKEL,  DR.  Lois  E.,  Department  of  Zoology,  Smith  College,  Northampton, 
Massachusetts 

TRACY,  DR.  HENRY  C.,  P.  O.  Box  54,  Oxford,  Mississippi 

TRACER,  DR.  WILLIAM,  Rockefeller  Institute,  66th  Street  and  York  Avenue,  New 
York  21,  New  York 


44  MARINE  BIOLOGICAL  LABORATORY 

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

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

College  of  Medicine,  New  York  City,  New  York 
TWEEDELL,  DR.  KENYON  S.,  Department  of  Zoology,  University  of  Maine,  Orono, 

Maine 

TYLER,  DR.  ALBERT,  California  Institute  of  Technology,  Pasadena  4,  California 
UHLENHUTH,  DR.  EDWARD,  University  of  Maryland  School  of  Medicine,  Baltimore, 

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

Illinois 

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

VILLEE,  DR.  CLAUDE  A.,  Harvard  Medical  School,  Boston  15,  Massachusetts 
VINCENT,  DR.  WALTER  S.,  Department  of  Anatomy,  State  University  of  New  York 

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

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

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

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

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

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

New  York  21,  New  York 

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

York  21,  New  York 

WHITE,  DR.  E.  GRACE,  Wilson  College,  Chambersburg,  Pennsylvania 
WHITING,  DR.  ANNA  R.,  University  of  Pennsylvania,  Philadelphia,  Pennsylvania 
WHITING,  DR.  PHINEAS  W.,  Zoological  Laboratory,  University  of  Pennsylvania, 

Philadelphia,  Pennsylvania 

WICKERSHAM,  MR.  JAMES  H.,  530  Fifth  Avenue,  New  York  36,  New  York 
WICHTERMAN,  DR.  RALPH,  Biology  Department,  Temple  University,  Philadelphia, 

Pennsylvania 

WIEMAN,  DR.  H.  L.,  Box  485,  Falmouth,  Massachusetts 

WIERCINSKI,  DR.  FLOYD  J.,  Department  of  Physiology,  Hahnemann  Medical  Col- 
lege, Philadelphia,  Pennsylvania 

WILBER,  DR.  C.  G.,  Medical  Laboratories,  Applied  Physiology  Branch,  Army  Chem- 
ical Center,  Maryland 
WILLIER,  DR.  B.  H.,  Department  of  Biology,  Johns  Hopkins  University,  Baltimore, 

Maryland 
WILSON,  DR.  J.  W.,  Brown  University,  Providence  12,  Rhode  Island 


REPORT  OF  THE  DIRECTOR 


45 


WILSON,  DR.  WALTER  L.,  Department  of  Physiology,  University  of  Vermont  Col- 
lege of  Medicine,  Burlington,  Vermont 

WITSCHI,  DR.  EMIL,  Department  of  Zoology,  State  University  of  Iowa,  Iowa  City, 
Iowa 

WOLF,  DR.  ERNST,  Pendleton  Hall,  Wellesley  College,  Wellesley,  Massachusetts 

WOODWARD,  DR.  ARTHUR  A.,  Army  Chemical  Center,  Maryland  (Applied  Physiol- 
ogy Branch,  Army  Chemical  Corps,  Medical  Laboratory) 

WRIGHT,  DR.  PAUL  A.,  Department  of  Zoology,  University  of  Michigan,  Ann 
Arbor,  Michigan 

WRINCH,  DR.  DOROTHY,  Department  of  Physics,  Smith  College,  Northampton, 
Massachusetts 

YNTEMA,  DR.  C.  L.,  Department  of  Anatomy,  State  University  of  New  York  Col- 
lege of  Medicine,  Syracuse  10,  New  York 

YOUNG,  DR.  D.  B.,  Main  Street,  North  Hanover,  Massachusetts 

ZINN,  DR.  DONALD  J.,  Department  of  Zoology,  University  of  Rhode  Island,  Kings- 
ton, Rhode  Island 

ZIRKLE,  DR.  RAYMOND  E.,  Department  of  Radiobiology,  University  of  Chicago, 
Chicago  37,  Illinois 

ZORZOLI,  DR.  ANITA,  Department  of  Physiology,  Vassar  College,  Poughkeepsie, 
New  York 

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

ZWILLING,  DR.  E.,  Department  of  Genetics,  University  of  Connecticut,  Storrs, 
Connecticut 


3.  ASSOCIATE  MEMBERS 


ALDRICH,  Miss  AMY  OWEN 
ALTON,  DR.  AND  MRS.  BENJAMIN  H. 
ARMSTRONG,  DR.  AND  MRS.  P.  B. 
BACON,  MRS.  ROBERT 
BARBOUR,  MR.  Lucius 
BARB,  MR.  ROBERT  P. 
BARTOW,  MR.  AND  MRS.  CLARENCE 
BARTOW,  MRS.  FRANCIS  D. 
BARTOW,  MR.  AND  MRS.  PHILIP 
BELL,  MRS.  ARTHUR 
BRADLEY,  MR.  ALBERT  L. 
BRADLEY,  MRS.  CHARLES  CRANE 
BROWN,  MRS.  THORNTON 
BURLINGAME,  MRS.  F.  A. 
CAHOON,  MRS.  SAMUEL 
CALKINS,  MR.  G.  NATHAN,  JR. 
CALKINS,  MRS.  GARY  N. 
CARLETON,  MRS.  WINSLOW 
CLAFF,  MR.  AND  MRS.  C.  LLOYD 
CLARK,  DR.  AND  MRS.  ALFRED  HULL 
CLARK,  MRS.  LEROY 


CLARK,  MR.  W.  VAN  ALAN 

CLOWES,  MR.  ALLEN  W. 

CLOWES,  MRS.  G.  H.  A. 

CLOWES,  DR.  AND  MRS.  GEORGE,  JR. 

COLTON,  MR.  H.  SEYMOUR 

CRANE,  Miss  LOUISE 

CRANE,  MRS.  W.  CAREY 

CRANE,  MRS.  W.  MURRAY 

CROWELL,  MR.  PRINCE  S. 

CURTIS,  DR.  W.  D. 

DANIELS,  MR.  AND  MRS.  F.  HAROLD 

DAY,  MR.  AND  MRS.  POMEROY 

DRAPER,  MRS.  MARY  C. 

DREYER,  MRS.  FRANK 

ELSMITH,  MRS.  DOROTHY 

ENDERS,  MR.  FREDERICK 

EWING,  MR.  FREDERICK 

FASEY,  MRS.  PAULINE  M. 

FAY,  MRS.  BRUCE  CRANE 

FRANCIS,  MR.  LEWIS,  JR. 

FROST,  MRS.  EUGENIA 


46 


MARINE  BIOLOGICAL  LABORATORY 


GALTSOFF,  MRS.  EUGENIA 
GlFFORD,  MR.  AND  MRS.  JOHN  A. 

GlLDEA,  DR.  AND  MRS.   E.  F. 

GREEN,  Miss  GLADYS  W. 
HAMLEN,  MR.  J.  MONROE 
HARRELL,  MR.  AND  MRS.  JOEL  E. 
HARRINGTON,  MR.  AND  MRS.  A.  W. 
HARRINGTON,  MR.  ROBERT  D. 

HlRSCHFELD,    MRS.    NATHAN 

HOUSTON,  MR.  AND  MRS.  HOWARD  E. 

JEWETT,  MRS.  GEORGE  F. 

KEITH,  MR.  AND  MRS.  HAROLD  C. 

KING,  MR.  FRANKLIN 

KOLLER,  MRS.  LEWIS 

LEMANN,  MRS.  SOLEN  B. 

LOBB,  MRS.  JOHN 

LURDON,  MR.  W.  R. 

McKELOY,  MR.  JOHN 

MARVIN,  MRS.  WALTER  T. 

MAST,  MRS.  S.  O. 

MEIGS,  MRS.  EDWARD  B. 

MEIGS,  DR.  AND  MRS.  J.  WISTER 

MITCHELL,  MRS.  JAMES  McC. 

MIXTER,  MRS.  JASON 

MOSSER,  MRS.  FLORENCE  M. 

MOTLEY,  MRS.  THOMAS 

NEWTON,  Miss  HELEN  K. 


NICHOLS,  MRS.  GEORGE 
NIMS,  MRS.  E.  D. 
PACKARD,  DR.  AND  MRS.  CHARLES 
PACKARD,  MRS.  LAURENCE  B. 
PARK,  MR.  MALCOLM  S. 
PENNINGTON,  Miss  ANNE  H. 
REDFIELD,  MRS.  ALFRED 
REZNIKOFF,  DR.  PAUL 
RIGGS,  MRS.  LAWRASON 
RIVINUS,  MR.  AND  MRS.  F.  MARKOE 
ROOT,  MRS.  WALTER 
ROZENDOAL,  MR.  H.  M. 
RUDD,  MRS.  H.  W.  DWIGHT 
SANDS,  Miss  ADELAIDE  G. 
SAUNDERS,  MRS.  LAWRENCE 
SHIVERICK,  MRS.  MARY 
STONE,  MR.  AND  MRS.  S.  M. 
SWIFT,  MR.  AND  MRS.  E.  KENT 
SWOPE,  MR.  AND  MRS.  GERARD,  JR. 
SWOPE,  Miss  HENRIETTA  H. 
TILNEY,  MRS.  ALBERT  A. 
TOMPKINS,  MR.  AND  MRS.  B.  A. 
WEBSTER,  MRS.  EDWIN  S. 
WHITELY,  Miss  MABEL  W. 

WlCKERSHAM,  MR.  AND  MRS.  JAMES  H. 

WILLISTON,  Miss  EMILY 

WOLFINSOHN,   MRS.  WOLFE 


V.     REPORT  OF  THE  LIBRARIAN 

In  1957,  seventy-six  new  journals  were  acquired,  bringing  the  total  number  of 
currently  received  titles  to  1635.  Of  these  titles,  there  were  490  (15  new)  Marine 
Biological  Laboratory  subscriptions;  617  (14  new)  exchanges  and  192  (21  new) 
gifts;  90  (9  new)  were  Woods  Hole  Oceanographic  Institution  subscriptions;  191 
(7  new)  were  exchanges  and  55  (10  new)  were  gifts.  During  the  past  ten  years, 
we  averaged  60  new  journals  per  year.  The  ever  growing  number  of  new  journals 
being  issued  far  exceeds  the  number  which  cease  publication. 

The  Laboratory  purchased  151  books,  received  61  complimentary  copies  (4 
from  authors  and  57  from  publishers),  and  accepted  13  miscellaneous  gifts.  The 
Institution  purchased  39  titles  and  received  10  gifts.  The  total  number  of  books 
accessioned  amounted  to  274. 

By  purchase  and  by  gift  the  Laboratory  completed  13  journal  sets  and  partially 
completed  19.  The  Institution  completed  4  sets  and  partially  completed  3.  There 
were  3920  reprints  added  to  the  collection,  of  which  2055  were  of  current  issue. 

At  the  close  of  the  year,  the  Library  contained  67,961  bound  volumes  and 
206,125  reprints. 


REPORT  OF  THE  TREASURER  47 

The  Library  sent  out  on  inter-library  loan  243  volumes  and  borrowed  115  for 
the  convenience  of  the  scientists.  It  is  hoped  that  a  copying  machine  may  be  pur- 
chased in  the  near  future  so  that  short  papers  may  be  reproduced  for  out-of-town 
loans,  thus  eliminating  some  of  the  depreciation  on  our  volumes.  A  process  such 
as  this  could  also  be  utilized  for  summer  service. 

Reprint  collections  were  received  from  the  estate  of  Dr.  Arthur  Weysse  and 
from  the  University  of  Pittsburgh ;  many  books,  journal  numbers  and  papers  were 
received  from  Drs.  Ethel  B.  Harvey,  C.  Ladd  Prosser,  Rufus  R.  Humphrey,  Phineas 
W.  Whiting,  Paul  S.  Galtsoff,  Ralph  Wichterman,  and  the  Tompkins-McCaw 
Library,  Medical  College  of  Virginia.  Dr.  Alfred  W.  Senft  kindly  donated  back 
volumes  and  a  current  subscription  to  the  "New  England  Journal  of  Medicine." 
Grateful  acknowledgment  is  herewith  extended  to  the  donors  of  these  very  accept- 
able presentations. 

With  a  larger  sum  available  for  the  purchase  of  books,  and  with  the  many  sug- 
gestions so  willingly  submitted  by  the  Library  Advisory  Committee,  we  were  in 
a  position  in  1957  to  add  many  new  titles  to  the  shelves.  An  increase  in  the  binding 
budget  also  enabled  us  to  have  bound  275  back  volumes,  bringing  the  total  to  1110 
for  the  year.  This  same  degree  of  progress  is  anticipated  in  1958. 

Respectfully  submitted, 

DEBORAH  L.  HARLOW, 

Librarian 


VI.     REPORT  OF  THE  TREASURER 

The  market  value  of  both  the  General  Fund  and  the  Library  at  December  31, 
1957,  amounted  to  $1,461,278  as  compared  with  the  total  of  $1,472,265  as  of 
December  31,  1956.  The  average  yield  on  the  securities  was  3.84%  of  market 
value  and  5.60%  of  book  value.  The  total  uninvested  principal  cash  in  the  above 
accounts  as  of  December  31,  1957,  was  $2,248.  Classification  of  the  securities 
held  in  the  Endowment  Funds  appears  in  the  auditor's  report. 

The  market  value  of  the  pooled  securities  as  of  December  31,  1957,  was 
$247,629  with  uninvested  principal  cash  of  $102.  The  book  value  of  the  securities 
in  this  account  was  $236,735.  The  average  yield  on  market  value  was  3.88%  and 
4.06%  of  book  value. 

The  proportionate  interest  in  the  Pooled  Fund  account  of  the  various  Funds 
as  of  December  31,  1957,  is  as  follows: 

Pension  Fund  17.608% 

General  Laboratory  Investment 57.866 

Other : 

Bio  Club  Scholarship  Fund 1.687 

Rev.  Arsenious  Boyer  Scholarship  Fund 2.064 

Gary  N.  Calkins  Fund 1.933 

Allen  R.  Memhard  Fund 374 

F.  R.  Lillie  Memorial  Fund  6.515 


48  MARINE  BIOLOGICAL  LABORATORY 

Lucretia  Crocker  Fund   7.054 

E.  G.  Conklin  Fund 1.194 

M.  H.  Jacobs  Scholarship  Fund 850 

Jewett  Memorial  Fund 626 

Anonymous  Gift 2.229 

The  Pooled  Fund  includes  the  Jewett  Memorial  Fund  and  an  anonymous  Gift 
Fund  which  were  additions  during  1957.  The  Jewett  Memorial  Fund  was  created 
by  gifts  in  memory  of  the  late  George  Frederick  Jewett.  Mr.  Jewett  as  well  as  his 
father  and  mother  and  the  other  members  of  his  family  have  been  keenly  interested 
in  the  Laboratory  since  its  inception.  It  has  not  yet  been  determined  how  the 
Jewett  Fund  and  the  fund  created  by  the  anonymous  gift  will  be  used,  but  the  views 
of  the  Jewett  family  and  the  donor  of  the  latter  fund  will  be  given  first  consideration. 

Considerable  activity  was  recorded  in  the  special  custodian  account  owing  to 
the  purchase  of  short-term  Government  bonds  to  activate  available  cash  which 
would  otherwise  remain  idle  in  our  regular  cash  accounts  pending  payment  of 
construction  expenses.  Income  earned  was  $646.40. 

Inasmuch  as  the  MBL  Club  loan  was  reduced  to  $2,052,  the  securities  pledge 
to  cover  this  loan  was  reduced  to  $3,000. 

Donations  from  MBL  Associates  for  1957  were  $3,481  as  compared  with  $5,255 
in  1956.  Unrestricted  gifts  from  foundations,  societies  and  companies  amounted 
to  $33,000. 

For  the  rehabilitation  of  the  Crane  Building,  the  National  Science  Foundation 
advanced  $415,000  in  1957.  Construction  began  in  September  and  is  scheduled 
for  completion  in  May  of  1958. 

In  April  of  1957  we  paid  off  the  David  House  Mortgage  in  the  amount  of  $5,000. 

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

Following  is  a  statement  of  the  auditors. 

To  the  Trustees  of  the  Marine  Biological  Laboratory,  Woods  Hole,  Massachusetts: 

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

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

LYBRAND,  Ross  BROS.  &  MONTGOMERY 

Boston,  Massachusetts 
May  22,  1958 

JAMES  H.  WICKERSHAM, 

Treasurer 


REPORT  OF  THE  TREASURER  49 

MARINE  BIOLOGICAL  LABORATORY 

BALANCE  SHEET 
December  31,  1957 

Investments 

Investments  held  by  Trustee : 

Securities,  at  cost  (approximate  market  quotation  $1,461,278)    $1,002,682 

Cash   2,248 


1,004,930 


Investments  of  other  endowment  and  unrestricted  funds : 


Pooled  Investments,  at  cost  (approximate  market  quotation  $247,629)    236,735 

Less  temporary  investment  of  current  fund  cash  5,728 


231,007 

Other  investments  (Note  A)    67,323 

Cash   11,263 

Accounts  receivable   5,038 


314,631 


Plant  Assets 


Land,  buildings,  library  and  equipment  (Note  B)   2,517,845 

Less  allowance  for  depreciation  (Note  B)    1,026,681 


1,491,164 

Construction  in  progress    103,856 

Cash  34,560 

U.  S.  Treasury  bills,  due  1/30/58,  at  cost  (face  value  $350,000)    346,815 


1,976,395 

Current  Assets 

Cash  142,160 

U.  S.  Treasury  bills,  at  cost : 

$40,000  face  value  due  2/13/58  39,649 

Temporary  investment  in  pooled  securities  5,728 

Accounts  receivable  (U.  S.  Government  $19,605)    36,274 

Inventories  of  specimens  and  Bulletins  57,282 

Prepaid  insurance  and  other  13,531 


$3,590,580 
Notes : 

A — The  Laboratory  has  guaranteed  a  note  of  approximately  $2,400  of  the  M.B.L.  Club 
and  has  pledged  as  security  therefor  bonds  with  an  original  cost  of  $3,000  included 
in  other  investments. 

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


50  MARINE  BIOLOGICAL  LABORATORY 

MARINE  BIOLOGICAL  LABORATORY 

BALANCE  SHEET 
December  31,  1957 

Endowment  Funds 

Endowment  funds  given  in  trust  for  benefit  of  the  Marine  Biological  Laboratory   ..   $1,004,930 
Endowment  funds  for  awards  and  scholarships  : 

Principal  $     64,415 

Unexpended   income    2,428         66,843 


Unrestricted  funds  functioning  as  endowment   206,378 

Retirement  fund   46,233 

Pooled  investments — accumulated  gain  or    (loss)    ' (4,823) 


314,631 


Plant  Liability  and  Funds 

Funds  expended  for  plant,  less  retirements  $2,551,469 

Less  allowance  for  depreciation  charged  thereto   1,026,681     1,524,788 


Unexpended  plant  funds  381,375 


1,906,163 
Accounts  payable   70,232 


1,976,395 


Current  Liabilities  and  Funds 

Accounts  payable   43,409 

Unexpended  balances  of  gifts  for  designated  purposes  8,744 

Advance  payments  on  research  contracts   94,217 

Current  fund   148,254 


$3,590,580 


REPORT  OF  THE  TREASURER  51 

MARINE  BIOLOGICAL  LABORATORY 

STATEMENT  OF  OPERATING  EXPENDITURES  AND  INCOME 
Year  Ended  December  31,  1957 

Operating  Expenditures 

Direct  expenditures  of  departments : 

Research  and  accessory  services   $146,859 

Instruction  35,237 

Library,  including  book  purchases   32,712 

Biological   Bulletin    16,995 


231,803 

Direct  costs  on  research  contracts  129,983 

Administration  and  general    54,526 

Plant  operation  and  maintenance   81,156 

Dormitories  and  dining  services   143,322 

Plant  additions  from  current  funds  59,581 


700,371 
Less  depreciation  included  in  plant  operation  and  dormitories  and  dining  services 

above  but  charged  to  plant  funds   36,351 


664,020 

Income 
Direct  income  of  departments  : 

Research  fees   43,418 

Accessory  services  (including  sales  of  biological  specimens  $67,562)    103,718 

Instruction  fees    16,980 

Library  fees  and  income 8,239 

Biological  Bulletin,  subscriptions  and  sales   19,846 


192,201 

Reimbursement  and  allowance  for  direct  and  indirect  costs  on  research  contracts 151,444 

Dormitories  and  dining  services  income   108,349 


451,994 
Investment  income  used  for  current  expenses  : 

Endowment  funds    83,984 

Current  fund  investments    1,645 

Gifts  used  for  current  expenses   127,301 

Sundry  income   175 


Total  current  income   6o5,099 


Excess  of  income  1 ,079 


MARINE  BIOLOGICAL  LABORATORY 
STATEMENT  OF  CURRENT  FUND 

Year  Ended  December  31,  1957 

Balance  January  1,  1957   $147,175 

Excess  of  income  over  operating  expenditures  1957  1,079 


Balance  December  31,  1957   $148,254 


52 


MARINE  BIOLOGICAL  LABORATORY 

MARINE  BIOLOGICAL  LABORATORY 

SUMMARY  OF  INVESTMENTS 

December  31,  1957 


Cost 


Approximate 
%  of        Market 
Total     Quotations 


Investment 
%  of      Income 
Total        1957 


Securities  held  by  Trustee: 
General  endowment  fund  : 
U   S    Government  bonds  . 

$     81,000 

9.7 

$     81,000 

6.8 

$  2,359 

Other  bonds     

420,980 

50.2 

403,589 

33.7 

12,246 

Preferred  stocks               •  . 

501,980 
85,788 

59.9 
10.2 

484,589 
71,713 

40.5 
6.0 

14,605 
3,370 

Common  stocks   

251,097 

29.9 

641,355 

53.5 

28,312 

838,865 

100.0 

1,197,657 

100.0 

46,287 

General     Educational     Board 
ment  fund  : 
U    S    Government  bonds 

endow  - 
25,000 

15.3 

25,000 

9.5 

749 

Other  bonds 

70,530 

43.0 

68,813 

26.1 

2,327 

Preferred  stocks         

95,530 
27,281 

58.3 
16.7 

93,813 
24,337 

35.6 
9.2 

3,076 
1,130 

Common  stocks 

41,006 

25.0 

145,471 

55.2 

5,608 

163,817 

100.0 

263,621 

100.0 

9,814 

Total  securities  held  by  Trustee  $1,002,682 

Investments  of  other  endowment  and  un- 
restricted funds : 
Pooled  investments : 


$1,461,278 


67,323 

Total  investments  of  other  en- 
dowment and  unrestricted 
funds  $  304,058 

Total  investment  income  , 

Custodian's  fee  charged  thereto   

Income  of  current  funds  temporarily  invested  in  pooled  securities 


$56,101 


U   S    Government  bonds  





= 

,  B 

833 

Other  bonds      

138,302 

58.4 

141,416 

57.1 

3,105 

Common  stocks    

138,302 
98,433 

58.4 
41.6 

141,416 
106,213 

57.1 
42.9 

3,938 
5,676 

236,735 

100.0 

$   247,629 

100.0 

9,614 

Other  investments  : 
U.  S    Government  bonds     

2,970 

131 

Common  stocks    

43,600 

23,444 

Real  estate  and  mortgage    

20,753 

23,575 


$33,189 

89,290 

(574) 
(204) 


Investment  income  distributed  to  funds 


$88,512 


COELOMIC  CORPUSCLES  OF  ECHINODERMS  1 

RICHARD  A.  BOOLOOTIAN  -  AND  ARTHUR  C.  GIESE 
Hopkins  Marine  Station  of  Stanford  University,  Pacific  Grove,  California 

Although  a  variety  of  corpuscles  have  been  described  during  the  last  century 
by  investigators  of  echinoderm  perivisceral  fluid,  disagreement  exists  among  the 
descriptions  of  different  authors  and  a  re-investigation  of  the  problem  with  newer 
methods  is  desirable  before  the  corpuscles  of  echinoderm  perivisceral  fluid  can  be 
properly  characterized.  These  newer  methods  are  primarily  observation  through 
the  phase  contrast  microscope,  so  effective  in  Gregoire's  studies  (1953)  on  insect 
blood,  and  observation  of  cells  unaltered  by  contact  with  air,  glass  or  chemicals 
which  Hensill  (1949)  found  so  useful  in  his  study  of  crab  blood.  In  addition,  the 
study  of  all  the  transformations  of  a  cell  of  a  given  type  under  gradually  altered  con- 
ditions discloses  changes  from  one  cell  type  to  another  in  some  instances.  Further- 
more, a  comparative  study  made  possible  a  useful  tentative  classification  of  the  cells 
found  in  fifteen  species  of  echinoderms  representing  all  the  living  classes  of 
Echinodermata. 

MATERIALS  AND  METHODS 

The  animals  were  collected  in  the  vicinity  of  the  Monterey  Peninsula  at  low  tide 
in  some  cases  and  by  dredging  in  others.  The  animals  were  used  as  soon  after  col- 
lection as  possible  since  starvation  is  known  to  alter  clotting  (Glavind,  1948). 
Cell  types  of  each  species  were  determined  by  the  examination  of  fluid  drawn  from 
the  perivisceral  cavity  with  the  aid  of  a  siliconized  syringe.  A  drop  of  the  fluid  was 
placed  on  a  siliconized  cover  slip  which  was  inverted  over  a  depression  slide,  and 
examined  immediately  at  magnifications  of  43  X  and  97  X  and  photographed 
periodically. 

The  optical  equipment  consisted  of  a  Spencer  18  ML  phase  microscope  equipped 
with  a  Spencer  phase  turret  condenser,  bright  contrast  objectives  and  wide  field 
oculars.  The  source  of  illumination  was  an  Ortho-Illuminator-B  (American  Op- 
tical Co.),  using  100-300  watt  bulbs. 

The  photomicrographic  equipment  used  was  a  Kine-Exacta  model  VX  camera 
coupled  to  a  Leitz  Micro-Ibso  attachment.  Exposures  were  made  on  Microfile  film 
which  was  developed  in  D-ll  developer  and  printed  on  single  weight  glossy  surface 
DuPont  Varigram  paper. 

Since  contact  with  air  is  known  to  alter  the  morphology  of  cells,  the  perivisceral 
fluid  was  taken  up  into  evacuated  capillaries.  The  capillaries  were  prepared  by 

1  Supported  in  part  by  National  Science  Foundation  Grant  GS-482  and  Public  Health 
Grant  RG-4578  (C). 

We  are  also  indebted  to  Dr.  A.  R.  Moore  for  his  sustained  interest  and  suggestions,  to  Dr. 
L.  Blinks  for  accommodations  and  suggestions,  to  Dr.  R.  L.  Bolin  for  extending  use  of  facilities 
and  for  helpful  criticism,  and  to  Mr.  A.  Farmanfarmaian  for  counsel  and  advice. 

-  Now  at  the  Department  of  Zoology,  University  of  California  at  Los  Angeles. 

53 


54  RICHARD  A.  BOOLOOTIAN  AND  ARTHUR  C.  GIESE 

pulling  5-mm.  Pyrex  tubing  in  such  a  manner  that  the  capillary  diameters  never  ex- 
ceeded 1  mm. 

The  inner  walls  of  the  capillaries  were  coated  with  silicon  (G.E.  Dri-Film)  by 
aspirating  the  reagent  and  subsequent  drying.  They  were  then  flame-sealed  at  one 
end,  evacuated,  and  flame-sealed  at  the  other  end  in  7.5-cm.  segments.  Silicon  was 
used  because  it  coats  the  glass  and  prevents  cytolysis  of  cells  coming  in  contact  with 
clean  glass  (Jacques  ct  at.,  1946).  Each  capillary  was  scratched  with  a  carborun- 
dum point  half  a  centimeter  from  one  end.  The  scratched  end  inserted  through  the 
peristome  (echinoids)  or  a  dermal  branchia  (asteroid)  can  be  broken  at  the  scratch 
by  a  slight  pressure,  and  the  body  fluid  is  aspirated  into  the  capillary.  In  the  case 
of  holothuroids  a  longer  capillary,  scratched  in  the  center,  was  inserted  into  the 
interambulacral  margin  of  the  animal  and  broken  in  the  middle  in  the  same  manner. 
The  open  tip  of  the  capillary  was  covered  with  silicon  grease  upon  removal.  Then 
the  capillary  was  placed  on  a  slide  in  a  channel  filled  with  glycerine  and  covered  with 
a  cover  slip.  With  this  method  it  is  possible  to  study  types  for  at  least  five  minutes 
before  clotting  appears,  and  to  observe  any  changes  which  occur  during  this  time. 
Furthermore,  the  capillary  tubes  can  be  rotated  and  the  nature  of  the  corpuscles  as- 
certained in  three  dimensions.  Clots  also  can  be  studied  effectively  in  such  prepara- 
tions. This  method  readily  lends  itself  to  photography. 

In  order  to  determine  which  coelomic  cells,  if  any,  were  phagocytic,  one  ml.  of 
finely  ground  carmine  suspension  in  sea  water  was  injected  by  a  syringe  through 
the  peristomial  membrane  in  echinoids,  through  a  dermal  branchia  in  asteroids  and 
through  the  body  wall  in  holothuroids.  At  various  time  intervals,  ranging  from 
ten  minutes  to  five  days,  hanging  drop  and  capillary-tube  preparations  of  the  peri- 
visceral  fluid  were  examined  and  photographed. 

CLASSIFICATION  OF  CORPUSCLES  OF  ECHINODERM  BODY  FLUIDS 

The  results  of  the  present  study,  documented  in  succeeding  sections,  revealed 
thirteen  types  of  fairly  distinct  cells  (see  Tables  I  and  II).  Some  of  these  cor- 
puscles appear  to  be  phyletic  in  distribution,  e.g.,  the  bladder  amebocytes  (Fig.  1) 
and  the  filiform  amebocytes  (Fig.  2),  the  first  of  which  occur  in  thirteen  of  the  spe- 
cies examined  and  the  latter  in  twelve  of  the  species  examined.  As  will  be  dis- 
cussed later,  these  two  cell  types  are  different  phases  of  the  same  cell,  e.g.,  in  Pis- 
aster  ochraceus.  The  small  spherical  amebocytes  (Fig.  3)  are  found  in  three  of  the 
asteroids  investigated  and  in  the  ophiuroid  and  the  crinoid.  The  fusiform  cor- 
puscle (Fig.  4),  the  vibratile  corpuscle  (Fig.  6),  the  eleocyte  (Fig.  7),  and  hyaline 
hemocyte  (Fig.  8)  are  found  in  the  sea  urchins  only.  The  colorless  spherical  ame- 
bocyte  (Fig.  5)  is  common  both  to  the  sea  urchins  and  sand  dollars.  The  other 
types  of  cells  have  a  rather  limited  distribution.  The  large  spherical  corpuscle 
(Fig.  9)  and  the  red  corpuscle  (Fig.  10)  are  found  in  the  sand  dollar  and  the  cri- 
noid. The  lobular  corpuscle  (Fig.  11),  on  the  other  hand,  is  limited  to  the  crinoid 
only.  The  hyaline  plasma  amebocyte  (Fig.  12)  is  found  in  the  starfish  Poraniopsis. 
Cells  "staining"  with  osmic  acid  (Fig.  13)  are  observed  only  in  the  sand  dollar. 

CORPUSCLES  OF  ASTEROIDS 

The  fluid  within  the  spacious  coelomic  cavity  of  the  asteroids  contains  coelomo- 
cytes  of  fewer  types  than  occur  in  other  classes.  Two  main  types  of  cells  have  been 


COELOMIC  CORPUSCLKS  OF  ECHINODERMS 


55 


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56 


RICHARD  A.  BOOLOOTIAN  AND  ARTHUR  C.  GIESE 


TABLE  II 

properties  of  coelomic  corpuscles 


Cell  type 

Range  or  size 
in  M* 

Color 

Granules 

Vacuoles 

Function 

Citation 

Bladder  amebocyte 

9-51 

colorless, 
gray 

numerous 
black 

several 

phagocytic 

Kindred,  1921 

Filiform  amrlx  11 

8-55 

gray 

several 
black 

two-many 

clot, 
phagocytic 

Kindred,  1921 

Small  spherical 
amebocyte 

4-8,  7-35 

green, 
yellow,  red 

black  and 
red 

occasional 

clot 

Cuenot,  1888 

Fusiform  corpuscle 

2-12X6-30 

gray 

0 

0 

? 

Cuenot,  1891 

Colorless  splierical 
amebocyte 

8-1  2  X 

13.6-28 

pale  yellow 

lobular 

ii 

li|ii<l 
transport? 

Kindred,  1921 

Vibratile  corpu^  1' 

3-11.7  X 
9-44 

gray 

numerous 
black 

numernii- 
small 

circulation? 

Kindred,  1921 

Eleocyte 

11.2-29 
X6.8-8 

red 

small  red 

0 

O2  transport? 

MacMunn,  1885 

Hyaline  hemocyte 

9.2-13 

pale  yellow 

0 

numerous 

clot 

This  paper 

Large  spherical 
corpuscle 

19-25.6 

slightly 
brownish 

brown 

several 
large 

) 

Bookhout  el  a!., 
1940 

Red  corpus  !• 

5.5-11.  2  X 
8-16 

red 

0 

0 

? 

This  paper 

Lobular  corpuscle 

16-27  X 
19-28.8 

gray 

0 

0 

•) 

Cuenot,  1891 

Hyaline  plasma 
amebocyte 

17.04-31.2 

gray- 

black 

several 

? 

This  paper 

Osmophilic  cells 

10-12.3 

pale  yellow 

0 

(i 

? 

This  paper 

*  Length  or  length  and  width.     A  wide  range  is  observed  in  some  cases  because  the  same  type 
of  corpuscle  is  of  a  different  size  in  different  species  in  which  it  is  found. 

described  by  Theel  (1919),  Kindred  (1924),  Lison  (1930),  Goodrich  (1919), 
Durham  (1888),  Geddes  (1879.  1880),  Cuenot  (1891)  :  amebocytes  with  ordinary 
slender  pseudopodia  (filiform  amebocytes)  and  amebocytes  with  petaloid  or  bladder 
extrusions  called  pseudopodia  (bladder  amebocytes). 

In  the  present  study  both  bladder  amebocytes  (Fig.  1)  and  filiform  amebocytes 
(Fig.  2)  were  found  to  be  abundant  in  all  species  of  asteroids  investigated.  An- 
other cell  type,  a  small  spherical  corpuscle  with  pigmented  granules,  however,  was 
also  found  in  Pycnopodia  helianthoides,  Mediaster  acqualis,  and  Poraniopsis  inflata. 
The  cells  of  the  first  two  species  contain  a  light  red  or  orange  intracellular  pigment 
which  is  particularly  obvious  when  the  cells  are  concentrated  by  centrifugation.  In 
Poraniopsis  inflata  the  pigmented  corpuscles  contain  black  granules,  but  not  in  con- 
spicuous quantity. 


The  body 
studied  most 
dred  (1921), 
burg  (1940), 
described  by 
with  petaloid 


CORPUSCLES  OF  ECHINQIDS 

fluid  of  the  echinoids  contains  several  types  of  cells  which  have  been 
extensively  by  Geddes  (1879),  Cuenot  (1891),  Theel  (1896),  Kin- 
Behre  (1932),  Boliek  (1935),  Kuhl  (1937),  Bookhout  and  Green- 
Liebman  (1950),  and  Schinke  (1950).  Six  types  of  cells  have  been 
various  workers :  amebocytes  with  spiked  pseudopodia,  amebocytes 
pseudopodia,  colorless  spherule  amebocytes.  greenish  and  yellowish 


COELOMIC  CORPUSCLES  OF  ECHINODERMS 


57 


spherule  amebocytes,  red  spherule  amebocytes,  and  vibratile  corpuscles  which  are 
small  spherical  cells  provided  with  a  flagellum. 

Seven  types  of  cells  were  identified  in  the  body  fluid  of  the  echinoids  investigated 
here:    bladder    amebocytes,    filiform    amebocytes,    fusiform    corpuscles,    colorless 


PLATE  I 


FIGURE  1,  bladder  amebocytes  of  Dcndrastcr  excentricus,  FIGURE  2,  filiform  amebocyte  of 
Pisaster  ochraceus.  FIGURE  3,  small  spherical  corpuscle  of  Hclioinetra  i/lacialis.  FIGURE  4, 
fusiform  corpuscle  of  Strongylocentrotus  fraiiciscanus.  FIGURE  5,  colorless  spherule  amebocyte 
of  Strongylocentrotus  pitrpnratus.  FIGURE  6,  vibratile  corpuscle  of  Strongylocentrotus  pitrpitra- 
tits.  FIGURE  7,  eleocyte  of  Strongylocentrotus  franciscanus. 

Photomicrographs  were  taken  at  a  magnification  of  344  X  and  u  t-re  enlarged  subsequently. 


58 


RICHARD  A.  BOOLOOTIAN  AND  ARTHUR  C.  GIESE 


PLATE  II 


FIGURE  8,  hyaline  hemocyte  of  Strongylocentrotus  purpuratus.  FIGURE  9,  large  spherical 
corpuscle  of  Dendraster  c.vccntricns.  FIGURE  10,  red  corpuscle  of  Heliometra  glacialis.  FIGURE 
11,  lobular  corpuscle  of  Heliometra  f/lacialis.  FIGURE  12,  hyaline  plasma  amebocyte  of  Poroniop- 
sis  inflata.  FIGURE  13,  osmophilic  cells  of  Dendraster  exccntriciis  (small  dark  oval  units). 

Photomicrographs  were  taken  at  a  magnification  of  344  X  and  were  enlarged  subsequently. 

spherule  amebocytes,  vibratile  corpuscles,  eleocytes,  and  hyaline  hemocytes  (Figs. 
1,2,4,5,6,7,8). 

The  bladder  amebocytes  with  large  ectoplasmic  extrusions  and  the  filiform  ame- 
bocytes are  phagocytic.  The  eleocytes  contain  red  spherules  of  echinochrome  (Mac- 
Munn,  1885;  Kuhn  and  Wallenfels,  1939).  a  common  echinoid  pigment  which  has 
been  demonstrated  in  the  three  species  of  Strongylocentrotus.  The  colorless 
spherule  amebocytes  with  spherical  inclusions  lack  phagocytic  power  and  move  by 
extending  broad  round  eruptive  (guttata)  pseudopodia.  The  fusiform  corpuscles, 
which  are  few  in  number,  have  no  known  function.  The  hyaline  hemocyte  gives  rise 
to  an  extracellular  clot  after  disintegrating  at  the  site  of  an  injury.  These  disinte- 
grating cells  resemble  the  colorless  spherule  amebocytes  to  a  considerable  degree 
and,  as  a  result,  may  have  escaped  the  observations  of  past  investigators  because 
most  of  their  observations  were  made  on  fixed  and  stained  material. 

The  eleocytes  have  no  known  function,  although  several  have  been  suggested  by 
various  investigators.  Griffiths  (1892)  concluded  that  they  are  associated  with 
oxygen  transport  as  did  Awerinzew  (1911).  Cuenot  (1891)  opposed  this  assump- 


COELOMIC  CORPUSCLES  OF  ECHINODERMS  59 

tion  on  the  basis  that  no  color  change  was  observed  when  pigment  was  allowed  to 
stand  in  air,  and  proposed  that  instead  of  being  an  oxygen-carrying  agent,  the  eleo- 
cyte  was  a  store  of  food  material  which  the  cells  had  taken  from  the  intestine.  The 
concentration  of  eleocytes  in  the  body  fluids  differs  in  the  three  species  of  Strongylo- 
centrotus  studied.  Strongylocentrotus  purpuratus  contains  1100  to  1700,  5\  fran- 
ciscanus,  500  to  700,  and  S.  jragilis,  100  to  150  cells/mm3.  The  red  cells  are  found 
in  the  epithelial  lining  of  the  sea  urchin  test  and  contribute  to  its  color. 

The  vibratile  corpuscles  (Fig.  6)  found  in  the  sea  urchins  are  of  uncertain  origin 
and  function.  They  do  not  participate  in  clotting,  except  as  they  are  incidentally 
caught  in  the  mesh  of  fibers  in  the  extensive  clot  formed  in  sea  urchin  body  fluid. 
It  is  possible  that  the  vibratile  corpuscles  aid  in  the  mixing  of  the  body  fluid  since 
the  current  created  peripherally  by  the  ciliated  epithelium  cannot  extend  far  into  the 
body  mass.  These  cells  were  concentrated  by  fractional  centrifugation  (as  were 
other  types  of  corpuscles)  and  were  kept  alive  in  the  body  fluid  of  the  urchin,  in 
vitro,  for  many  hours,  but  they  were  not  observed  to  divide. 

CORPUSCLES  OF  OPHIUROIDS 

The  coelomic  corpuscles  of  the  ophiuroids  have  been  least  studied.  Cuenot 
(1888)  observed  granular  ameboid  corpuscles  with  short  spiked  pseudopodia  in  an 
ophiuroid,  and  Kindred  (1924)  observed  four  types  of  cells:  active  leucocytes,  pas- 
sive leucocytes,  colorless  spherule  amebocytes,  and  vibratile  corpuscles  in  Ophio- 
pholis  aculeata.  In  the  present  study  two  cell  types  were  observed  in  Gorgono- 
cephalus — small  spherical  corpuscles  (Fig.  3)  and  fusiform  corpuscles  (Fig.  4). 
The  latter  type  of  cell  was  not  observed  by  Cuenot,  while  the  small  spherical  cor- 
puscle resembles  that  found  by  Cuenot.  The  function  of  these  cells  is  unknown, 
although  Cuenot  has  remarked  that  the  ameboid  granular  cells  (small  spherical 
corpuscles)  unite  by  their  short  pseudopodial  tips  and  anastomose  into  a  network, 
but  he  did  not  consider  this  to  be  clot  formation. 

CORPUSCLES  OF  CRINOIDS 

The  coelomocytes  of  the  crinoids  have  been  described  by  Cuenot  (1891),  Ha- 
mann  (1889),  and  Reichensperger  (1912).  Cuenot  (1891)  observed  three  kinds 
of  cells,  a  small  finely  granular  type  with  short  spiked  pseudopods,  a  larger  pyriform 
or  fusiform  slow-moving  cell  filled  with  coarse  spherules,  and  a  cell  filled  with 
safranophil  rods,  which,  according  to  Cuenot,  was  rounded  when  free  in  the  coelomic 
fluid,  pyriform  when  migrating  through  the  tissues.  Hamann  observed  numerous 
wandering  ameboid  cells  in  various  crinoids  and  described  two  types,  neither  of 
which  resembles  Cuenot's  forms.  Reichensperger  also  noticed  two  kinds  of  coelom- 
ocytes in  Antedon:  a  phagocytic  ameboid  form  with  short  spiked  pseudopods  ap- 
parently identical  with  Cuenot's  finely  granular  type  and  an  oblong  cell  filled  with 
numerous  rods  and  granules. 

In  the  species  studied  here.  HeUometra  glacialis,  five  types  of  cells  were  found 
(Table  I),  two  of  which  are  probably  identical  with  the  cells  described  by  Cuenot, 
Hamann,  and  Reichensperger.  The  perivisceral  fluid  contained  an  abundance  of 
ameboid  cells  with  short  pseudopodia  (small  spherical  corpuscles,  Figure  3).  The 
second  cell  type  (fusiform  corpuscle,  Figure  4),  termed  "pyriform"  by  Cuenot,  was 


60  RICHARD  A.  BOOLOOTIAN  AND  ARTHUR  C.  GIESE 

not  pear-shaped  in  H.  glacialis,  but  more  spindle-like.  Occasional  oblong  bodies 
which  approached  pear-form  were  observed.  The  large  spherical  corpuscles,  which 
lacked  pseudopodial  extensions,  were  abundant  in  the  body  fluid.  The  red  cor- 
puscles were  found  in  small  numbers  and  have  not  been  reported  by  other  investi- 
gators. The  lobular  corpuscles  (Fig.  11),  few  in  number,  resemble  an  embryonic 
morula  stage.  The  functions  of  these  cells  were  not  determined  since  the  volume  of 
fluid  from  the  few  specimens  available  was  too  small  for  extensive  studies. 

CORPUSCLES  OF  HOLOTHUROIDS 

The  coelomic  cells  of  holothuroids  have  been  described  by  numerous  workers, 
particularly  by  Herouard  (1889),  Becher  (1907),  Theel  (1921),  Kindred  (1924), 
Ohuye  (1934,  1936a,  1936b)  and  Endean  (1958).  They  have  been  collectively 
called  coelomocytes  (Hyman,  1955).  The  cell  types  present  vary  in  form,  number, 
and  size  in  different  holothuroids,  but  the  following  kinds  are  common  throughout 
the  class :  hemocytes,  phagocytes,  colorless  spherule  amebocytes  and  filiform  amebo- 
cytes. 

Hemocytes  which  have  been  reported  for  many  species  contain  the  pigment  he- 
moglobin, as  shown  by  Howell  (1885,  1886),  Van  der  Hyde  (1922),  Hogben  and 
Van  der  Lingen  (1928),  and  Kobayashi  (1932),  on  Thyone,  Cucuniaria,  Paracau- 
dina  chilensis,  and  Molpadia  roretzii,  respectively.  They  are  not  found  in  Sticlio- 
f>us  calif ornicus,  the  species  studied  here. 

Phagocytes  are  found  in  all  species  so  far  studied.  Various  names  such  as  cells 
with  elongated  pseudopodia  (Herouard),  hyaline  ameboid  corpuscles  (Ohuye),  and 
bladder  amebocytes  (Kindred,  1924)  have  been  applied  to  them.  The  term,  bladder 
amebocyte  (Fig.  1),  is  preferable  since  the  large  bladder-like  projections  are  readily 
observable  when  viewed  three-dimensionally. 

The  colorless  spherule  amebocytes  (Fig.  5)  were  abundant  in  Stichopus  cali- 
f ornicus.  Hamann  (1883)  designated  these  as  plasma  wandering  cells.  Cuenot 
(1891),  who  identified  them  as  muriform  cells,  considered  the  proteinaceous  spher- 
ules to  be  food  reserves. 

The  homogeneous  amebocytes,  which  lack  inclusions,  have  been  reported  by 
Hamann  (1883)  and  Becher  (1907).  This  type  of  cell  is  rare  and  Hyman  (1955) 
considers  it  a  developmental  stage  of  other  cell  types.  It  was  not  found  in  Stichopus. 

Theel,  Kawamoto,  and  Ohuye  observed  crystal-containing  cells  in  several  spe- 
cies of  holothuroids.  The  crystals  are  in  the  cytoplasm  and  are  mostly  rhomboidal 
in  shape.  No  crystal-containing  cells  were  observed  in  Stichopus. 

A  cell  type  which  has  not  been  previously  reported  by  investigators  in  holo- 
thuroids is  the  filiform  amebocyte  (Fig.  2).  In  Stichopus  californicus  these  cells 
are  actively  involved  in  clot  formation  and  also  exhibit  phagocytosis. 

DISCUSSION 

Many  types  of  coelomic  corpuscles  have  been  described  by  various  investigators 
of  echinoderm  body  fluids,  most  of  whom  fixed  and  stained  the  cells  or  used  live 
cells  without  preventing  degenerative  changes  following  contact  with  glass  or  air. 
As  a  consequence  their  results  were  not  entirely  convincing.  In  the  present  study 
in  which  pains  were  taken  to  avoid  the  above  pitfalls,  many  of  the  same  cell  types 


COELOMIC  CORPUSCLES  OF  ECHINODERMS  61 

were  seen.  However,  more  confidence  may  now  be  attached  to  the  cell  types  de- 
scribed by  the  earlier  workers,  since  their  appearance  has  been  checked  with  live  cells 
under  conditions  which  at  least  delay  changes  in  cells  occurring  with  clotting  or 
agglutination. 

Such  coelomic  cells  as  were  not  seen  in  the  preparation  made  here,  but  which 
have  been  described  by  previous  workers,  may  constitute  additional  cell  types  since 
the  species  used  in  the  present  study  were  not  the  same  as  theirs.  Only  future 
work  using  the  same  species  of  organism,  can  resolve  this  uncertainty.  In  the 
special  case  of  the  hemocytes — hemoglobin-containing  cells  of  certain  holothuroids— 
no  question  exists  of  their  reality,  even  though  they  were  not  observed  in  the  species 
of  holothuroid  used  here  (Stichopus  calif ornicus) ,  since  hemocytes  have  been  ob- 
served in  live  specimens  and  recorded  many  times  by  various  authors. 

Some  types  of  coelomocytes  were  observed  in  the  species  examined  here  which 
had  not  been  previously  described,  e.g.  the  red  corpuscles  of  the  sand  dollar  and  the 
crinoid,  and  the  lobular  corpuscles  of  the  crinoid. 

The  existence  of  bladder  amebocytes  need  no  longer  be  questioned,  even  though 
the  bladders  appear  to  be  petaloid  rather  than  vesicular  in  fixed  preparations  (Good- 
rich, 1919).  Examined  in  three  dimensions,  the  bladder-like  nature  of  the  ecto- 
plasmic  extrusions  is  readily  observable. 

It  was  possible  to  resolve  one  controversy  which  occurs  in  the  literature  con- 
cerning the  possible  identity  of  the  bladder  amebocytes  and  the  filiform  amebocytes 
in  asteroids.  Theel  (1919)  and  Kindred  (1924)  state  these  are  merely  phases 
of  one  another  but  cite  no  convincing  evidence,  and  others  question  this  conclusion. 
In  observations  on  body  fluids  of  several  asteroids,  the  fresh  sample  showed  a 
predominance  of  bladder  amebocytes,  but  upon  standing,  the  same  preparation 
shows  a  predominance  of  filiform  amebocytes.  If  the  filiform  amebocytes  represent 
a  pre-coagulation  change,  it  should  be  possible  to  prevent  this  with  an  anti- 
coagulant such  as  cysteine.  Cysteine-treated  coelomic  fluid  was  found  to  contain 
only  bladder  amebocytes  when  examined  at  various  time  intervals  in  Pisaster 
ochraceous  body  fluid.  This  experiment  was  repeated  eight  times  with  the  same 
results.  In  the  control,  samples  of  coelomic  fluid  were  treated  with  sea  water 
equal  in  volume  to  the  sample  of  anticoagulant  and  upon  standing,  both  phases 
were  seen.  Whether  such  transformation  occurs  in  all  echinoderm  coelomic 
fluids  in  which  such  cells  are  found  remains  to  be  seen. 

Some  problems  are  presented  by  the  present  study  of  echinoderm  coelomic  fluid 
which  may  be  of  special  interest  to  comparative  and  cellular  physiologists.  The 
function  of  the  echinochrome-containing  eleocytes  and  the  various  types  of  amebo- 
cytes still  remains  a  challenge.  The  function  of  the  vibratile  corpuscles  of  the  sea 
urchins,  with  the  possibility  that  they  represent  parasites,  is  another  example  of  an 
intriguing  problem.  The  bladder  amebocytes  and  the  explosive  amebocytes  should 
serve  as  interesting  material  for  a  further  study  of  ameboid  movement.  The 
mechanism  of  the  transformation  of  bladder  amebocytes  to  filiform  amebocytes 
offers  still  another  perplexing  problem. 

The  data  so  far  gathered  do  not  permit  evolutionary  speculations  concerning 
the  origin  and  diversification  of  the  different  types  of  coelomocytes.  However,  it 
cannot  escape  mention  that  a  greater  diversity  of  cell  types  appears  in  the  body 
fluid  of  the  more  highly  specialized  forms,  such  as  the  echinoids,  than  in  the 


62  RICHARD  A.  BOOLOOTIAN  AND  ARTHUR  C.  GIESE 

asteroids.  A  more  complete  survey  of  the  coelomic  corpuscles  of  other  species  of 
each  class,  especially  of  the  classes  studied  sparingly  at  present,  may  yield  informa- 
tion making  possible  more  generalizations  than  can  be  made  now. 

SUMMARY 

1.  The  cellular  elements  from  the  body  fluid  of  15  different  species  of  echino- 
derms  were  studied  by  phase  contrast  microscopy.     Thirteen  types  of  corpuscular 
elements  were  identified  and  the  distribution,  properties,  characteristics  and,  where 
possible,  functions,  were  determined. 

2.  Some  types  of  coelomocytes  were  observed  in  the   species  examined  here 
which  had  not  been  previously  described,  e.g.  the  red  corpuscles  of  the  sand  dollar 
and  crinoid,  and  the  lobular  corpuscles  of  the  crinoid.     Some   of  the  coelomo- 
cytes formerly  described  were  also  found  in  the  species  described.     Among  these 
are  the  controversial  bladder  amebocytes  in  which  the  presence  of  bladder  has  been 
questioned.     Present  studies  verify  the  bladders  as  real  structures  easily  seen  in 
three  dimensions.     The  bladder  amebocyte  undergoes  a  transformation  into  the 
filiform  amebocyte  which  represents  a  pre-coagulation  change. 

3.  A  greater  diversity  of  cell  types  was  observed  in  the  body  fluid  of  the  more 
highly  specialized  forms  such  as  the  echinoids  than  in  the  less  specialized  asteroids. 

LITERATURE  CITED 

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Gen.,  ser.  2,  6 :  3-82. 
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GOODRICH,    E.    S.,    1919.     Pseudopodia    of    the    leukocytes    of    invertebrates.     Quart.    J.    Micr. 

Sci.,  64:  19-27. 
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to  coagulation   inhibitors   of   vertebrate  blood.     Biol.   Bull.,   104:    372-393. 
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115:  419-420. 
HAMANN,  O.,  1883.     Beitrage  zur  Histologie  der  Echinodermen.     I.  Die  Holothurien,  Pcdata, 

und  das  Nervensystem  der  Asteriden.     Zeitschr.  zviss.  Zool.,  39:   145. 


COELOMIC  CORPUSCLES  OF  ECHINODERMS  63 

HAMANN,   O.,   1889.     Anatomic  der  Ophiuren  und  Crinoiden.     Jen.  Zeitschr.   Naturwiss.,  23: 

233-388. 
HENSILL,    J.,    1949.     Studies    on    blood    coagulation    in    decapod    Crustacea.     Thesis,    Stanford 

University. 
HEROUARD,  E.,  1889.     Recherches  sur  les  holothuries  des  cotes  de  France.     Arch.  Zool.  Exp. 

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circulation.     Sci.  Rep.   Tohoku  Imp.   Univ.  Biol.,  2:   239-264. 
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41:  144-152. 
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KUHL,  W.,  1937.     Die  Zellelemente  in  der  Liebeshohlenfliissigheit  des  Seeigels  Psammechinus 

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Rep.  Tohoku  Imp.  Univ.  Biol,  9:  47-52. 
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THE  ROLE  OF  THE  BLOOD  IN  THE  TRANSPORTATION  OF 
STRONTIUM90-YTTRIUM90  IN  TELEOST  FISH1'2 

HOWARD  BOROUGHS  3  AND  DELLA  F.  REID 
Hawaii  Marine  Laboratory,  University  of  Ha^vaii,  Honolulu,  Hawaii 

As  the  result  of  global  fallout  and  the  introduction  of  radioactive  wastes  from 
nuclear  reactor  plants  into  the  oceans,  marine  organisms  are  being  subjected  to 
an  environment  which  is  potentially  hazardous  to  themselves  and  to  other  members 
of  the  ecosystems  involved.  During  the  last  few  years,  a  study  has  been  made  in 
this  laboratory  of  various  aspects  of  the  metabolism  of  radiostrontium  by  marine 
fish.  These  fish  may  pick  up  strontium  directly  from  sea  \vater,  by  way  of  the 
skin,  gills,  or  by  swallowing  the  water  (Boroughs,  Townsley  and  Hiatt,  1956). 
They  may  also  take  up  this  element  from  their  food.  In  any  event,  the  transporta- 
tion of  strontium  within  the  fish,  including  its  excretion,  depends  upon  its  trans- 
portation by  the  blood,  except  for  the  strontium  which  is  unabsorbed  from  the 
digestive  tract. 

It  is  the  purpose  of  this  paper  to  report  on  certain  aspects  of  the  transportation 
of  strontium90-yttriumt)0  in  teleost  blood. 

MATERIALS  AND  METHODS 

The  species  used  in  this  experiment  was  Tilapia  mossambica,  a  teleost  fish.  In- 
dividuals weighed  between  50  and  110  grams  each.  They  were  kept  in  tanks 
supplied  with  running  sea  water. 

Two  concentrations  of  Oak  Ridge  Sr90-Y90  were  prepared  by  dilution  with 
saline  solution  approximately  isotonic  with  Tilapia  blood.  Those  fish  which  were 
to  be  bled  a  day  or  more  after  injection  were  given  100/>ic  of  Sr90,  while  the 
fish  killed  at  shorter  time  intervals  were  given  only  10  ju,c.  In  both  instances  the 
dose  injected  was  0.2  ml. 

The  injections  were  made,  and  blood  was  withdrawn  with  the  fishes'  opercula 
in  water.  Separate  fish  were  used  for  each  time  interval  studied  instead  of  using 
a  single  fish  for  repetitive  bleedings.  All  the  fish  were  handled  as  gently  and 
uniformly  as  possible,  and  their  eyes  were  covered  with  the  hand.  We  believe 
this  procedure  results  in  a  minimum  of  trauma. 

The  Sr90-Y90  dose  was  injected  directly  into  the  ventricle  of  the  heart.  At 
predetermined  time  intervals  of  5,  15,  30,  and  45  minutes  and  1,  4,  and  8  days, 
as  much  as  possible  of  each  fish's  blood  was  withdrawn  through  the  kidney  sinus. 
A  red  blood  cell  count  was  made  each  time  a  fish  was  injected  and  again  \vhen 
blood  was  removed. 

1  Contribution  No.  108  Hawaii  Marine  Laboratory,  University  of  Hawaii. 

2  This  work  was   supported  in  part  by  contract   No.   AT(04-3)-56  between   the   U.    S. 
Atomic  Energy  Commission  and  the  University  of  Hawaii. 

3  Present  address :   Institute  Interamericano  de  Ciencias  Agricolas,  Turrialba,   Costa  Rica. 

64 


Sr"0-Yso  IN  TELEOST  FISH  BLOOD  65 

Immediately  after  removing  the  blood  from  the  fish,  triplicate  0.1-ml.  samples 
were  pipetted  onto  circles  of  one  thickness  of  absorbent  tissue  on  aluminum 
planchettes.  Three-tenths-ml.  aliquots  of  the  remaining  blood  were  centrifuged 
for  10  minutes  at  2100  rpm  in  calibrated  small  bore  hematocrit  tubes  in  an  Inter- 
national clinical  centrifuge.  The  separated  blood  in  one  tube  was  used  for  measur- 
ing the  radioactivity  in  the  plasma  and  also  that  associated  with  the  cells.  From 
a  second  tube  the  plasma  was  removed  without  disturbing  the  packed  cells.  Five- 
hundredths  ml.  of  these  cells  were  washed  by  re-suspending  them  twice  in  fresh  saline 
solutions.  All  the  saline  washings  were  pooled.  In  a  third  tube,  the  same  volume 
of  saline-washed  cells  was  lysed  with  distilled  water.  The  ghosts  were  washed 
with  distilled  water  until  no  further  radioactivity  could  be  removed  from  them. 
The  lysing  solution  containing  the  cell  contents  was  added  to  the  distilled  water 
wash  for  measurement  of  the  radioactivity  of  the  cells  exclusive  of  that  bound 
to  the  stroma. 

Separated  organs  and  tissues  were  ashed  and  prepared  for  counting  as  pre- 
viously described  (Boroughs,  Townsley  and  Hiatt,  1956).  Radioactivity  was 
measured  with  a  thin  window  G-M  tube  using  a  commercial  sealer.  Counts  were 
corrected  for  coincidence  whenever  necessary. 

In  order  to  get  an  approximation  of  mixing  time,  SrR5  was  injected  in  the 
heart.  Ten,  20,  and  30  minutes  later,  blood  was  removed  from  the  ventral  aorta 
and  from  the  kidney  sinus,  and  0.1-ml.  samples  were  counted  in  a  well  scintillation 
counter  with  the  aid  of  a  single  channel  pulse  height  analyzer. 

RESULTS  AND  DISCUSSION 
Preliminary  experiments 

Since  very  little  is  known  about  fish  blood,  we  were  at  the  outset  faced  with 
problems  which  were  not  pertinent  to  the  main  idea  of  this  research.  The  first 
problem  to  be  overcome  was  the  bleeding,  because  apparently  very  few  biologists 
have  successfully  removed  blood  directly  from  teleost  fish  (Prosser,  personal  com- 
munication). In  general,  fish  have  been  bled  by  cutting  the  tail  and  allowing  the 
blood  to  drip.  Even  more  refined  methods  have  involved  the  use  of  heparin, 
citrate,  or  other  anticoagulants.  We  have  found  it  difficult  to  withdraw  unclotted 
blood  from  Tilapia  if  the  fish  had  been  kept  out  of  water  for  even  a  short  time. 
There  is  probably  a  dehydration  of  the  blood  in  some  species  of  fish  as  a  result 
of  asphyxiation  (Hall,  Gray  and  Lepkovsky,  1926).  If  Tilapia  were  stressed  by 
prolonged  chasing  with  a  net,  by  rough  handling  or  by  repeated  bleeding,  removal 
of  blood  was  difficult  even  though  they  were  not  taken  from  the  water.  The  cell/ 
plasma  ratio  increased  as  it  did  with  asphyxiation. 

We  had  previously  observed  red  blood  cell  counts  which  varied  between  1  and 
4  X  106/mm3  in  this  species  of  fish,  and  other  workers  (Young,  1949)  have  ob- 
served similar  large  variations  with  other  teleost  fishes.  Table  I  is  a  summary  of 
the  rbc  counts  of  the  fish  used  in  this  experiment  and  shows  that  these  variations 
are  not  intrinsic  and  that  it  is  possible  to  remove  fish  blood  that  has  a  reasonably 
small  fluctuation  in  the  rbc  count.  This  blood  does  not  clot  even  on  prolonged 
standing  at  room  temperature. 

The  tremendous  shift  in  the  number  of  red  blood  cells  observed  in  fish  blood 


66 


HOWARD  BOROUGHS  AND  DELLA  F.  REID 


Time  interval 

between  injection 

and  killing 

5  min. 

5  min. 

5  min. 
15  min. 
15  min. 
30  min. 
30  min. 
45  min. 

1  hr. 

1  hr. 

1  hr. 

2  hr. 
2  hr. 
2  hr. 
4hr. 
4  hr. 
8  hr. 
8  hr. 
1  day 

1  daj' 

2  days 
2  days 
4  days 
4  days 
8  days 
8  days 


TABLE  I 
Red  blood  cell  count  in  Tilapia  mossambica 

RBC/mm.3  of  blood 

Counted  before  Counted  before 

dose  injected  blood  withdrawn 


1.444 
1.150 
1.375 
1.350 
1.209 
1.548 
1.175 
1.125 
1.200 
1.162 
1.050 
1.223 
1.148 
1.150 
1.151 
1.209 
1.199 
1.011 
1.312 
1.649 
1.100 
1.298 
1.103 
1.271 
1.150 
1.018 


X  106 
X  106 
X  106 
X  106 
X  10« 
X  106 
X  106 
X  106 
X  106 
X  106 
X  106 
X  106 
X  106 
X  10" 
X  106 
X  106 
X  106 
X  106 
X  106 
X  106 
X  106 
X  106 
X  106 
X  106 
X  106 
X  106 


1.627 
1.423 
1.400 
2.050 
1.374 
1.525 
1.400 
1.460 
1.600 
1.384 
1.025 
1.347 
1.326 
1.220 
1.169 
1.137 


X  106 
X  106 
X  106 
X  106 
X  10" 
X  106 
X  106 
X  10« 
106 
106 
X  10« 
X  10« 
X  106 
X  106 
X  106 
X  10" 


X 
X 


1.102  X  106 

1.396  X  106 

1.598  X  106 

.199  X  106 

.298  X  106 

.362  X  106 
.273  X  106 
.175  X  106 


could  mean  that  the  plasma,  or  some  portion  of  it,  either  leaves  the  circulatory 
system  or  is  in  effect  removed  by  some  pocketing  device.  The  increase  in  red  blood 
cells  may  also  result  from  the  introduction  into  the  blood  stream  of  cells  previously 
sequestered  in  an  organ  or  tissue.  Studies  on  fish  blood  volume  and  mixing  time 
using  either  classical  techniques  or  radioisotopes  would  be  of  little  value  if  the  fish 
were  stressed. 

The  circulation  of  fish  blood  is  distinguished  from  that  of  higher  animals  in 
that  oxygenated  blood  does  not  necessarily  return  to  the  heart.  All  the  blood 
from  the  heart  goes  to  the  gills,  but  from  the  gills  the  blood  may  go  to  the  head. 


TABLE  II 

The  mixing  time  of  Tilapia  blood 


Blood  source 

Ventral  aorta 
Ventral  aorta 
Ventral  aorta 
Kidney 
Kidney 
Kidnev 


Minutes 
elapsed 

10 
20 
30 
10 
20 
30 


Counts/min. 

249 
79 
51 
40 
45 
50 


Dose:  8477  cpm  in  0.2  nil.  injected  into  ventricle  of  heart. 


r^-Y110  IN  TELEOST  FISH  BLOOD 


67 


Q. 
O 


140 

120 
100 

80 

60 
40 

20 
0 


O     120 
o 

100 


80 


60 


40 


20 


0 


Whole 
Blood 


Plasma 


a. 

o 

o 
o 
o 


120 

100 

80 

60 


20 


0 


15        45 

min. 


2 
HOURS 


III       1 

o  • 

i 

i 

i 

•  

i 

—  — 

—  o 

i 

4     12     | 

MRS. 

2 

3 

4 

5 

6 

7 

8 

DAYS       AFTER     DOSE 

FIGURE  1.     The  disappearance  of  Sr^-Y90  from  the  whole  blood  and 
plasma  of  Tilapia  inossainbica. 


68  HOWARD  BOROUGHS  AND  DELLA  F.  REID 

back  to  the  heart,  or  to  the  remainder  of  the  body.  This  means  that  mixing  is  a 
more  complicated  process  in  fish  than  it  is  in  the  higher  animals. 

The  results  of  studying  mixing  time  in  a  single  fish  are  shown  in  Table  II. 
It  can  be  seen  that  the  bulk  of  the  Srs3  injected  into  the  heart  remained  in  the 
anterior  portion  of  the  fish,  and  that  it  required  about  30  minutes  for  the  blood 
from  the  ventral  aorta  and  that  from  the  kidney  to  reach  the  same  level. 

Since  we  lack  precise  information  about  blood  volume,  we  have  assumed  that 
it  is  roughly  proportional  to  body  weight.  We  have  done  this  not  only  on  the 
basis  of  our  own  work,  but  because  Martin  (1950)  has  suggested  a  similar  relation- 
ship for  other  teleost  fishes. 

Rate  of  disappearance  of  Sr90-Y90  from  the  blood 

Figure  1  shows  the  rate  of  disappearance  of  Sr90-Y9u  from  whole  blood  and 
plasma.  The  numbers  have  been  corrected  for  body  weight.  The  activity  is 
given  in  counts/min./ml.  whole  blood  and  cpm  in  the  plasma  present  in  1  ml.  of 
whole  blood.  Each  point  on  the  curve  represents  the  average  activity  from  at 
least  two  fish.  It  can  be  seen  that  practically  all  the  radioactivity  in  the  whole 
blood  is  carried  in  the  plasma,  and  that  the  formed  elements  can  be  responsible 
for  only  a  very  small  amount.  The  two  curves  are  practically  superimposable. 
The  small  inserts  on  this  graph  show  the  appearance  of  radioactivity  during  the 
first  few  hours,  and  the  larger  graph  extends  the  curves  to  8  days.  Since  all  the 
radioactivity  was  injected  into  the  heart  at  zero  time,  at  first  glance  it  may  seem 
odd  that  the  amount  of  radioactivity  recoverable  from  the  blood  increases  up  to 
30  minutes.  However,  Table  II  indicates  that  this  apparent  increase  is  a  reflection 
of  the  mixing  time.  At  least  two  processes  are  occurring  during  this  time  which 
make  it  extremely  difficult  to  find  out  exactly  how  much  radioactivity  is  in  the 
blood  system.  First,  the  isotopes  are  being  excreted  as  soon  as  they  appear  in 
the  blood,  at  first  principally  by  way  of  the  gills.  Second,  radioactivity  is  rapidly 
accreted  by  the  various  organs  and  tissues,  and  thus  the  concentration  is  decreasing 
continuously.  We  would  like  to  emphasize  that  it  is  the  resultant  of  these  proc- 
esses that  is  being  measured. 

The  radioactivity  was  very  rapidly  lost  from  the  blood  during  the  next  30 
minutes,  and  after  24  hours,  only  between  0.8  and  1.6  per  cent  of  the  injected  dose 
remained  in  the  blood,  assuming  a  blood  volume  of  2-4  per  cent  of  the  body  weight. 
The  shape  of  the  curves  shows  that  more  than  one  rate  process  is  involved  in  the 
disappearance  of  the  radioactivity  from  the  blood.  It  must  be  emphasized  at  this 
point  that  the  above  samples  were  counted  at  least  three  weeks  after  the  fish  was 
killed,  so  that  we  were  observing  the  radioactivity  in  an  equilibrium  mixture  of 
Sr90-Y90.  Strontium110  has  a  half-life  of  about  28  years  and  a  maximum  beta 
energy  of  0.61  Mev.  It  decays  to  form  radioactive  Y90  which  has  a  half-life  of 
2.54  days  and  a  maximum  beta  energy  of  2.18  Mev.  Secular  equilibrium  exists 
when  the  Y90  decays  as  fast  as  it  is  formed,  and  the  radioactivity  of  such  a  mixture 
is  the  sum  of  the  radioactivity  of  the  separate  isotopes. 

In  an  equilibrium  mixture,  therefore,  no  decay  of  radioactivity  would  be  ob- 
servable during  this  experiment  unless  the  two  isotopes  were  separated  by  either 
biological  or  physico-chemical  processes.  Such  a  fractionation  can  be  detected  by 
following  the  counting  rate  of  a  sample  daily.  No  changes  in  this  rate  will  be 


Sr^-Y90  IN  TELEOST  FISH  BLOOD 


69 


observed  if  no  fractionation  has  occurred.  If  the  rate  increases,  Y90  has  been 
removed  and  is  building  up  to  its  equilibrium  value  at  which  point  it  will  level  off. 
If  the  rate  decreases,  the  bulk  of  the  radioactivity  must  be  due  to  the  Y90  which  is 
decaying,  and  the  counts  will  decrease  until  a  level  is  reached  which  is  a  function 
of  the  amount  of  Sr90  present. 

The  role  of  the  blood  fractions  in  the  transport  of  Sr90-Y90 

The  increase  in  the  counts/minute  of  the  whole  blood  and  plasma  in  Figure 
2  is  due  to  the  build  up  of  Y90.  There  are  two  simple  explanations  for  the  loss  of 
yttrium  from  the  blood.  One  is  that  the  yttrium  was  lost  prior  to  its  appearance 
in  the  blood  initially,  that  is,  adsorbed  to  the  glassware  used  in  making  the  dilutions 


o    plasma 

v    whole    blood 


8         9       10        II        12        13        14       15        16 


FIGURE  2.     The  increase  with  time  of  radioactivity  in  samples  of  whole  blood,  plasma, 
and  the  dose,  indicating  the  build-up  of  Y90. 

and  injections.  The  second  explanation  is  that  the  yttrium  was  lost  to  various 
organs  and  tissues  through  which  the  blood  passed.  These  explanations  are  not 
mutually  exclusive  and  we  believe  that  both  processes  occur. 

In  Figure  3,  the  curve  labelled  "dose"  was  obtained  by  counting  planchettes 
prepared  from  the  Sr90-Y90  present  in  the  syringe  used  for  injections.  It  can  be 
seen  that  over  a  period  of  time,  the  cpm  increased,  indicating  that  some  Y90  was 
lost  from  the  equilibrium  mixture.  This  Y90  was  lost  to  the  glassware.  The  curve 
for  whole  blood  and  plasma,  however,  increased  to  a  much  higher  value,  indicating 
that  additional  Y90  had  been  removed  after  the  dose  was  injected. 

Figure  3  shows  the  rate  of  radioactive  decay  of  the  washed  and  unwashed  cells, 
the  saline  washings,  the  washed  ghosts,  and  the  distilled  water  washings  which 
include  the  cell  contents.  The  decay  of  the  unwashed  cells  suggests  that  both  Sr90 
and  Y90  were  associated  with  the  cells.  The  decay  of  the  washed  cells,  saline 


70 


HOWARD  BOROUGHS  AND  DELLA  F.  REID 


wash,  and  ghosts,  however,  suggests  that  the  Sr90  is  readily  removable  either  from 
or  through  the  cell  wall.  The  activity  remaining  in  the  washed  cells  and  ghosts 
indicates  it  to  be  Y90,  because  the  decay  rates  are  very  similar  to  the  rate  for  pure 
Y90.  All  these  conclusions  are  in  harmony  with  the  findings  of  Thomas  et  al. 
(Thomas,  Litovitz,  Rubin  and  Geschickter,  1950).  who  showed  that  radiocalcium. 
metabolically  similar  to  strontium,  was  carried  in  the  plasma  of  rabbit  blood. 


800 
600 
400 

~~i  1  1  1  1  1  r— 

1  O  n  O  -LI  O  ^  O 

l         I          1 

O                     O 

i 

L_u 

*%. 

n  w  a  s  h  e  d 

cells 

130  - 


LL) 

I- 
ID 
Z 


a: 

UJ 

a. 


tn 

i- 


O 
O 


100- 


di  st.  H  0    was  h  — , 

XX  * 


FIGURE  3.  The  radioactive  decay  of  washed  and  unwashed  cells,  the  saline  and  distilled 
water  wash,  and  the  cell  ghosts.  The  decay  of  washed  cells  and  ghosts  indicates  that  they  pick 
up  Y90  rather  than  Sr80. 


90 


Retention  and  distribution  of  Srgo-Y 

Figure  4  shows  the  retention  by  the  fish  of  the  injected  Sr90-Y90  as  a  function 
of  time.  The  upper  curve  represents  the  entire  fish,  and  the  other  curves  represent, 
respectively,  the  bone,  integument,  gills,  muscle,  and  visceral  organs.  Each  point 
is  the  average  of  at  least  two  fish,  and  the  samples  were  counted  at  secular 
equilibrium.  These  results  may  be  compared  with  those  obtained  previously  by 


Sr^-Y90  IN  TELEOST  FISH  BLOOD 


71 


LJ 
</) 

o 

Q 

O 
LJ 

H 
O 
LJ 


entire    fish 


bones- 


z 

LJ 
O 

<r 

UJ 
0- 


viscerol     organs 


I00{ 


80 


60 


40- 


20- 


0 


FIGURE  4.     The  internal  distribution  of  Sr^-Y90  injected  in  the  heart 

of  Tilapia  mossambica. 

Boroughs  et  al.  on  the  retention  of  Sr89  by  Tilapia  following  ingestion  or  intra- 
muscular injection.  In  all  instances,  the  rank  order  of  the  percentage  of  dose 
retained  is  the  same  as  that  in  Figure  4,  indicating  that  once  the  strontium  is  ab- 
sorbed by  the  blood  system,  its  internal  distribution  is  the  same. 

Table  III  emphasizes  the  rapidity  with  which  the  radioactivity  appears  in  the 
various  tissues,  including  the  bones.  A  small  amount  of  vascular  tissue  and  blood 
is  dissected  with  the  bones,  but  this  additional  radioactivity  is  obviously  a  very 
small  percentage  of  the  total  in  the  bones.  This  appearance  and  retention  in  the 
bones  cannot  be  due  to  accretion  by  growth,  but  must  represent  simply  an  exchange 
reaction. 

TABLE  III 

Retention  and  internal  distribution  of  Sr90-  F90  after  intracardial  injection 


s   ,/'            —  —  —  __ 

^\  n  te  g  ument 

2> 

k"^^ 

.gills 
pmuscle 

^rJ 

.2                    -T             -r-          -T- 

HRS.           1                                              3                     4 
DAYS 

1                     1                     I 
567 

t 

8 

Time  interval 


%  injected  dose  remaining  (samples  at  secular  equilibrium) 


Total  of  fish* 

Bones 

Integument 

Gills 

Muscle 

Visceral  organs 

5  min. 

98.2 

13.2 

13.4 

13.4 

3.1 

3.4 

15  min. 

95.9 

30  min. 

93.2 

39.3 

8.2 

17.3 

11.9 

4.0 

1  day 

92.1 

61.7 

19.2 

4.9 

4.5 

1.4 

4  days 

81.5 

53.7 

17.1 

4.7 

4.2 

1.4 

8  days 

76.6 

50.2 

16.2 

7.4 

3.3 

1.4 

Including  blood. 


72 


HOWARD  BOROUGHS  AND  DELLA  F.  REID 


Biological  fractionation  of  Sr90-Y°° 

Three  fish  were  injected  with  Sr90-Y90  and  killed  five  minutes,  30  minutes,  and 
one  day  later.  Since  the  amount  of  separation  of  the  two  isotopes  by  the  glassware 
was  unknown,  it  is  not  possible  to  draw  a  curve  showing  the  rate  of  decay  of  the 
radioactivity  in  the  various  organs  that  would  be  a  true  measure  of  the  decay  due 
to  the  fractionation  by  the  organs  themselves.  The  planchettes  were  counted  one 
day  after  the  fish  were  killed,  and  this  value  was  taken  as  a  base  line.  They  were 
then  counted  until  secular  equilibrium  had  been  re-established.  Table  IV  shows 
the  percentage  increase  or  decrease  in  radioactivity  in  the  various  organs  with 
respect  to  the  radioactivity  present  at  one  day. 

TABLE  IV" 

Fractionation  of  intracard tally  injected  5V90-]'90  by  organs  and 
tissues  of  Tilapia  mossambica 


Organ  or  tissue  sample 

%  Decrease  of  activity 
1  day  to  secular  equilibrium 

Organ  or  tissue  sample 

f"c  Increase  of  activity 
1  day  to  secular  equilibrium 

Liver 

53.1 

Gills 

73.8 

Gall  bladder 

42.5 

Stomach 

22.3 

Heart 

38.6 

Brain 

18.3 

Kidney 
Spleen 
Gonads 

18.8 
10.6 
8.5 

Muscle 
Intestine 
Eyes 
Urinary  Bladder 
Skin 

16.3 
10.4 
6.9 
5.6 

5.0 

Urine 

28.5 

Blood  clots 
Scales 
Fat 

26.8 
14.1 
11.4 

Feces 

2.1 

It  can  be  seen  that  the  first  two  columns  represent  the  organs  which  concen- 
trated Y90  more  than  they  did  Sr90,  while  the  last  two  columns  represent  organs 
that  favored  the  Sr.  In  general,  the  more  vascular  organs  and  tissues  preferred 
yttrium. 

SUMMARY 

1.  Blood  can  be   easily   removed   without  clotting  from   the  heart   or   kidney 
sinus  of  fishes  if  the  fish  are  handled  gently  and  their  opercula  are  kept  immersed. 

2.  Blood  so  removed  has  a  uniform  number  of  red  blood  cells/mm3. 

3.  The  mixing  time  of  Sr90-Y90  injected  in  the  ventricle  of  Tilapia  mossambica, 
a  teleost  fish,  is  approximately  30  minutes. 

4.  Sr90-Y90  rapidly  disappears  from   the  blood.     At   24  hours,   only   between 
0.8  and  1.6  per  cent  of  the  injected  dose  remains  in  the  blood. 

5.  The  disappearance  of  radioactivity  from  the  blood  depends  on  more  than  a 
single  process. 

6.  Almost  all  of  the  Sr90  in  whole  blood  is  carried  by  the  plasma. 
7 '.  Very  little  Sr1'0  is  found  either  in  the  cells  or  on  the  cell  walls. 
8.  Yttrium90,  on  the  other  hand,  is  present  in  the  stroma. 


Sr^-Y90  IN  TELEOST  FISH  BLOOD 

9.  The  pattern  of  internal  distribution  of  intravascularly  injected   Sr90-Y90  is 
the  same  as  that  which  was  found  for  either  intramuscular  or  oral  administration 
in  the  same  species. 

10.  Vascularized  tissues  have  a  greater  avidity  for  Ym)  than  they  have  for  Sr 


90 


LITERATURE  CITED 

BOROUGHS,  H.,  S.  J.  TOWNSLEY  AND  R.  W.  HIATT,  1956.     The  metabolism  of  radionuclides  by 

marine   organisms.     I.   The   uptake,   accumulation,   and   loss   of   strontiums9   by   marine 

fishes.     Biol.  Bull,  111:  336-351. 
HALL,  F.  G.,  I.  E.  GRAY  AND  S.  LEPKOVSKY,  1926.     The  influence  of  asphyxiation  on  the  blood 

constituents  of  marine  fishes.     /.  Biol.  Chem.,  67 :  550. 
MARTIN,  A.  W.,  1950.     Some  remarks  on  the  blood  volume  of  fish.     Studies  Honoring  Trevor 

Kincaid.     Univ.  of  Washington  Press,  pp.   125-140. 
THOMAS,  R.  O.,  T.  A.  LITOVITZ,  M.  I.  RUBIN  AND  C.  F.  GESCHICKTER,  1950.     Dynamics  of 

calcium    distribution.     Time    distribution    of    intravenously    administered    radiocalcium. 

Amer.  J.  Physiol.,  169:  568-575. 
YOUNG,  R.  T.,  1949.     Variations  in  the  blood  cell  volume  of  individual  fish.     Copeia.  Sept.   15, 

1949,  No.  3. 


DISPERSAL  OF  THE  GELATINOUS  COAT  MATERIAL  OF  MELLITA 
QUINQUIESPERFORATA  EGGS   BY   HOMOLOGOUS    SPERM 

AND  SPERM  EXTRACTS1 

JOHN  W.  BROOKBANK 

Department  of  Biology,   University  of  Florida,   Gainesville,  Florida 

Live  sperm  or  sperm  extracts  of  a  number  of  animal  species  have  been  found 
to  possess  the  property  of  solubilizing  or  dispersing  the  secondary  and  tertiary 
envelopes  normally  surrounding  the  unfertilized  eggs  of  these  species,  thus  fa- 
cilitating the  approach  of  the  sperm  to  the  egg  surface.  Groups  in  which  this 
phenomenon  has  been  demonstrated  include  amphibians  (Hibbard,  1928;  Wintre- 
bert.  1929  and  1933),  mammals  (see  reviews  by  Duran-Reynals,  1942;  Meyer, 
1947;  Meyer  and  Rapport,  1952),  gastropods  (Tyler,  1939  and  1948;  von  Medem, 
1942),  and  bivalves  (Berg,  1949).  In  addition,  a  number  of  \vorkers  have  de- 
scribed the  solubilization  of  the  gelatinous  coat  material  (fertilizin)  of  echinoid 
eggs  by  live  sperm  or  sperm  extracts.  Hartmann  ct  al.  (1940)  extracted  the 
residue  of  methanol-precipitated  seminal  fluid  of  Arbacia  pushdosa  with  sea  water 
and  reported  that  the  resulting  solution  was  capable  of  dispersing  the  gelatinous 
coat  material  of  unfertilized  Arbacia  eggs.  This  extract  was  also  capable  of 
neutralizing  the  sperm  agglutinating  property  of  Arbacia  fertilizin,  and  thus  pos- 
sessed antifertilizin  activity.  Monroy  and  Ruffo  (1947)  described  an  acid  extract 
of  sea  urchin  sperm  which  was  reported  as  acting  to  dissolve  the  fertilizin  of 
unfertilized  eggs.  Others  have  described  a  decreased  viscosity  of  fertilizin  solu- 
tions in  the  presence  of  live  sperm  or  sperm  extracts  (Lundblad  and  Monroy. 
1950;  Vasseur,  1951 ;  Monroy  and  Tosi,  1952;  Monroy  et  al.,  1954).  It  has  been 
emphasized  (Tyler  and  O'Melveny,  1941  ;  Krauss.  1950;  Monroy  and  Tosi,  1952; 
Monroy  et  al.,  1954)  that  apparent  dispersal  of  the  gelatinous  coat  of  unfertilized 
eggs  by  sperm  or  sperm  extracts,  as  well  as  the  decrease  in  viscosity  observed  when 
live  sperm  or  extracts  are  added  to  fertilizin  solutions,  can  be  accounted  for  by 
precipitation  of  fertilizin  by  antifertilizin  present  in  the  extracts  or  on  the  surface 
of  the  live  sperm.  Therefore,  any  investigation  of  supposed  lytic  or  dispersing 
agents  from  sperm  must  include  experiments  which  demonstrate  that  the  activity 
of  the  agent  is  separable  from  the  activity  of  antifertilizin.  Ishida  (1954)  has 
presented  evidence  that  a  fertilizin-dissolving  factor  is  released  at  fertilization  from 
the  sperm  of  Heiniccntrotus  pulchcrrimus.  Treatment  of  the  sperm  with  fertilizin, 
which  rendered  the  sperm  non-fertilizing,  did  not  prevent  the  solution  of  the 
fertilizin  coat  of  the  eggs  by  these  sperm.  This  latter  observation  tends  to  eliminate 
antifertilizin  as  the  agent  responsible  for  removing  the  fertilizin  from  the  eggs. 
However,  though  the  sperm  concentration  employed  in  the  experiments  was  not 
stated,  sperm  carbon  dioxide  might  have  been  responsible  for  the  solubilizing  action 

1  This  investigation  was  supported  in  part  by  a  research  gfant   (RG  4659s)    from  the  Na- 
tional Institutes  of  Health  of  the  Puhlic  Health  Service. 

74 


GELATINOUS  COAT  DISPERSAL  BY  SPERM  75 

of  the  sperm.  Heated  (100°  C.  for  one  minute)  sperm  failed  to  cause  dispersal 
of  the  fertilizin  coat.  This  failure  of  heated  sperm  to  cause  dispersal  of  the 
gelatinous  coat  has  been  ascribed,  by  Ishida,  to  the  clenaturation  of  a  dispersing 
agent  on  the  sperm.  Alternatively,  the  denaturation  of  respiratory  enzymes,  re- 
sulting in  loss  of  motility  and  decreased  carbon  dioxide  production,  could  account 
for  the  failure  of  heated  sperm  to  solubilize  the  fertilizin  of  the  eggs. 

The  present  report  bears  evidence  that  a  factor,  which  is  distinct  from  anti- 
fertilizin  and  which  is  capable  of  causing  the  dispersal  of  the  gelatinous  coat  of 
unfertilized  eggs,  is  present  in  sperm  extracts  and  on  the  surface  of  live  sperm 
and  of  the  sand  dollar,  Mellita  quinquiesperforata. 

MATERIALS  AND  METHODS 

Animals  were  collected  by  dredging  on  the  shallow  banks  surrounding  the 
University  of  Florida  Marine  Laboratory  at  Sea  Horse  Key.  The  animals  were 
transported  to  Gainesville  and  kept  in  the  laboratory  at  12°  C.  in  sea  water 
supplied  with  a  continuous  Mow  of  washed,  compressed  air.  Under  these  con- 
ditions the  sand  dollars  remained  alive  for  approximately  two  weeks.  Eggs  and 
sperm  were  obtained  by  injecting  the  animals  with  an  isotonic  KC1  solution 
(Tyler,  1949). 

For  use  in  experiments  in  which  live  sperm  were  employed,  the  "dry"  sperm 
were  diluted  to  a  concentration  of  5(/f  with  filtered  sea  water.  Where  separation 
of  sperm  from  the  surrounding  Muid  was  desired,  the  suspensions  were  centrifuged 
at  2900  X  gravity  in  a  Sen-all  SS-1  centrifuge  for  10  minutes.  The  supernatant 
Muids  were  collected  and  tested  for  dispersing  activity  on  the  gelatinous  coat  of  fresh 
unfertilized  eggs.  The  sedimented  sperm  were  suspended  in  the  original  volume  of 
fresh  sea  water  and  tested  for  their  ability  to  disperse  the  gelatinous  coat  of  the  eggs. 

Sperm  extracts  were  prepared  from  washed  sperm  in  the  following  manner. 
Two  volumes  of  sea  water  were  added  to  the  sperm  following  centrifugation  and  re- 
moval of  seminal  Muid,  and  the  resulting  30%  suspension  was  frozen  at  --  20°  C.  for 
2  to  12  hours.  The  frozen  suspension  was  then  homogenized  in  an  ice  bath,  using 
a  Potter  homogenizer  with  a  motor-driven  pestle.  After  homogenization,  the  sus- 
pension was  centrifuged  at  11,000  X  gravity  for  15  minutes  in  a  Servall  SS-1  centri- 
fuge. This  procedure  yielded  a  gray  precipitate,  which  was  discarded,  and  an  opal- 
escent supernatant  Muid,  which  was  used  as  the  final  sperm  extract  preparation. 

In  assaying  for  the  dispersing  action  of  sperm  and  sperm  extracts  on  the  gelati- 
nous coat  of  the  eggs,  advantage  was  taken  of  the  presence  of  echinochrome  granules 
in  the  gelatinous  coat.  As  can  be  seen  in  Figure  la,  where  the  outer  boundary  of 
the  coat  has  been  outlined  with  antifertilizin,  the  granules  normally  have  a  rather 
regular  position  in  the  gelatinous  coat.  The  locus  of  this  position  could  be  described 
as  a  spherical  shell  lying  midway  between  the  outer  surface  of  the  gelatinous  coat 
and  the  surface  of  the  egg.  In  practice,  a  small  number  of  freshly  shed  eggs  were 
transferred  with  a  pipette  from  the  vessel  in  which  they  had  been  allowed  to  settle,  to 
fresh  sea  water,  and  used  in  the  various  tests.  Dispersal  of  the  egg  coat  could  be 
followed  by  noting  the  length  of  time  required  for  the  echinochrome  granules  to  fall 
to  the  bottom  of  the  culture  dish,  due  to  the  dispersal  of  the  gelatinous  matrix  in 
which  they  were  embedded.  The  time  at  which  the  granules  were  released  repre- 
sented the  time  at  which  approximately  half  the  gelatinous  coat  had  been  dispersed. 


76  JOHN  W.  BROOKBANK 

and  was  taken  as  the  end-point  of  the  reaction.  Naturally,  every  effort  was  made  to 
insure  that  the  eggs  used  in  the  experiments  possessed  comparable  amounts  of  gelati- 
nous coat  material  outside  the  layer  of  granules.  In  practice,  this  was  not  particu- 
larly troublesome  since  the  egg  coat  of  Mellita  eggs  is  quite  rigid  and  not  readily 
soluble  in  sea  water,  and  since  handling  of  the  eggs  was  restricted  to  a  single  transfer 
to  fresh  sea  water  following  shedding. 

la 


•  » 


Ib 


* 
ft 


FIGURE  la.  Unfertilized  egg  of  Mellita  treated  with  heated  (70°  C.  for  10  minutes)  sperm 
extract.  Final  magnification:  175  X. 

FIGURE  Ib.  Unfertilized  egg  of  Alellita  treated  with  heated  sperm  extract  for  30  minutes, 
followed  by  treatment  with  unheated  extract  for  15  minutes.  Final  magnification:  175  X. 

RESULTS 

When  one  drop  of  5%  sperm  suspension  was  added  to  one  drop  of  egg  suspen- 
sion (containing  about  200  eggs),  the  gelatinous  coat  was  dispersed  in  approxi- 
mately 10  minutes.  Elevation  of  the  fertilization  membrane  did  not  result  in  the 
dispersal  of  the  gelatinous  coat  of  control  eggs  which  were  washed  and  removed  to 
sea  wrater  following  exposure  to  the  sperm  suspension.  When  the  above  sperm  sus- 
pension was  centrifuged  at  2900  X  gravity  for  10  minutes,  and  the  supernatant  fluid 
decanted  and  tested,  it  was  found  to  be  inactive  (no  dispersal  of  the  gelatinous  coat 
occurred  even  after  12  hours  of  exposure  to  the  sperm  supernatant).  The  sedi- 
mented  sperm,  on  the  other  hand,  remained  able  to  disperse  the  gelatinous  coat  ma- 
terial after  being  diluted  to  the  original  concentration  with  sea  water. 


GELATINOUS  COAT  DISPERSAL  BY  SPERM 


77 


Acidification  of  a  5%  sperm  suspension  to  pH  4,  followed  by  return  to  pH  8  after 
2-4  minutes,  with  subsequent  centrifugation  at  2900  X  gravity  to  recover  sperm  and 
supernatant  fluid,  resulted  in  the  loss  of  the  capacity  of  the  sperm  to  disperse  the 
fertilizin  coat.  The  sperm,  in  most  instances,  remained  motile  following  the  treat- 
ment. The  supernatant  fluid  under  these  conditions  occasionally  showed  slight  anti- 
fertilizin  activity,  as  evidenced  by  the  formation  of  a  slight  precipitation  membrane 
on  the  gelatinous  coat,  but  remained  inactive  with  respect  to  the  dispersal  of  the 
gelatinous  coat.  The  fertilizing  capacity  of  such  acid-treated  sperm  was  reduced, 
perhaps  due  to  the  loss  of  antifertilizin  from  the  sperm  surface  (Tyler  and  O'Mel- 
veny,  1941),  or  to  the  loss  of  the  ability  to  disperse  the  gelatinous  coat,  or  both. 

Sperm  extracts,  prepared  as  described  in  the  previous  section,  were  also  ca- 
pable of  dispersing  the  gelatinous  coat  of  unfertilized  eggs.  The  final  extracts,  pre- 

TABLE  I 

The  effect  of  temperature  and  pH  on  dispersing  activity  of  Mellita  sperm 
extract.     Activity  of  extracts  assayed  at  25°  C. 


Date  of  preparation 
of  extract 

Exposed  to  pH 

Tested  at  pH 

Dispersal  time 

Antifertilizin  activity 

Dec.  13 

7 

7 

30' 

± 

Dec.  I') 

7 

7 

30' 

± 

Dec.    5 

4 

8 

— 

± 

fan.  17 

4 

8 

— 

± 

Dec.     5 

9 

8 

30' 

± 

fan.     3 

9 

8 

30' 

± 

Jan.  17 

8 

8 

15' 

± 

Dec.     5 

8 

8 

15' 

± 

Jan.     3 

8 

8 

10' 

± 

Dec.     5* 

8 

8 

— 

+  +  + 

Dec.  19* 

8 

8 

— 

+  +  + 

Dec.  19* 

8 

8 

— 

+  +  + 

*  Indicates  heated  extract  (70°  C.  for  two  minutes). 
-  Indicates  no  dispersing  activity  evident. 

pared  from  frozen-thawed  sperm,  had  a  pH  varying  between  6.9  and  7.1,  depending 
on  the  particular  preparation.  Extracts  at  this  pH  range  were  capable  of  dispersing 
the  gelatinous  coat  in  30  minutes  at  25°  C.  Control  eggs  in  sea  water  at  pH  6.9 
showed  no  release  of  echinochrome  granules  for  6  hours  or  more.  Exposure  of 
active  sperm  extracts  to  pH  4  for  3-5  minutes,  followed  by  return  to  the  original 
}>H,  inactivated  the  dispersing  factor.  The  optimum  pH  for  dispersing  activity  ap- 
peared to  be  8,  since  dispersal  occurred  in  approximately  10  minutes  at  this  pH. 
Alkalinization  to  pH  9,  followed  by  return  to  pH  8  after  3-5  minutes,  partially  in- 
activated the  dispersing  factor,  dispersal  occurring  after  30  minutes  in  these  prepa- 
rations. Heating  to  70°  C.  for  two  minutes  in  a  water  bath  completely  inactivated 
the  dispersing  factor.  The  heated  extracts  possessed  definite  antifertilizin  activity 
(Table  I).  The  heat  stability  of  antifertilizin  from  sperm  of  sea  urchins  (Frank, 
1939),  and  the  key-hole  limpet  (Tyler,  1939)  has  been  previously  described. 
Weak  antifertilizin  activity  was  also  evident  in  untreated  extracts  at  pH  8.  A  faint 
precipitation  membrane  appeared  on  the  surface  of  the  fertilizin  coat  about  two 


78  JOHN  W.  BROOKBANK 

minutes  after  the  addition  of  extract,  but  disappeared  after  5  minutes  under  the  in- 
fluence of  the  dispersing  factor.  This  point  was  further  illustrated  by  experiments 
with  heated  extracts  possessing  stronger  antifertilizin  activity.  The  heated  ex- 
tracts (70°  C.  for  5-10  minutes)  formed  definite  precipitation  membranes  on  the 
fertilizin  coat  after  two  minutes  (Fig.  la).  The  precipitation  membrane  so  formed 
did  not  contract  to  the  egg  surface,  but  remained  in  the  position  in  which  it  was 
originally  formed  for  a*-  long  as  24  hours  (with  no  dispersal  of  the  egg  coat).  Ad- 
dition of  unheated  sperm  extract  at  pH  8  caused  the  disappearance  of  the  precipita- 
tion membrane,  and,  after  10-15  minutes,  the  dispersal  of  the  gelatinous  coat 
(Fig.  Ib). 

Experiments  involving  the  addition  of  extract  to  eggs  up  to  five  minutes  prior 
to  the  addition  of  sperm  showed  that  fertilization  is  not  enhanced  by  this  treatment. 
The  fertilization  membrane  was  elevated  within  three  minutes  regardless  of  the 
presence  or  absence  of  the  extract.  The  sperm  are  apparently  supplied  with  enough 
dispersing  factor  to  make  their  way  through  the  gelatinous  coat  material.  More- 
over, the  gelatinous  coat  is  not  dispersed  by  the  dilute  suspensions  usually  employed 
for  insemination.  In  this  respect,  the  situation  parallels  that  of  hyaluronidase  of 
mammalian  sperm,  in  that  hyaluronidase  added  to  inseminates  does  not  enhance 
fertilization  (Chang,  1947;  Leonard  et  al.,  1947). 

The  Mellita  sperm  extracts  were  not  tested  on  eggs  of  other  species,  and  the  de- 
gree of  specificity  of  the  dispersing  agent  is  therefore  not  known  at  this  time.  An 
extract  of  frozen  Arbacia  piinctulata  sperm  (5%  suspension)  failed  to  cause  the  re- 
lease of  the  echinochrome  granules  of  Mellita  eggs,  but  showed  strong  antifertilizin 
activity  (the  precipitation  membrane  with  enclosed  granules  contracted  to  the  egg 
surface  in  5  minutes).  Addition  of  extract  of  Mellita  sperm,  at  pH  8.0,  caused  the 
gradual  disappearance  of  the  precipitation  membrane  formed  by  the  Arbacia  anti- 
fertilizin, and  release  of  the  echinochrome  granules  following  dispersal  of  the  fer- 
tilizin coat.  In  this  connection,  it  is  of  interest  that  a  fresh  suspension  of  Mellita 
sperm  was  capable  of  fertilizing  eggs  treated  with  Arbacia  antifertilizin.  in  the 
presence  or  absence  of  extract  of  Mellita  sperm,  indicating  the  ability  of  live  sperm 
to  penetrate  precipitation  membranes. 

Further  experiments  were  performed  in  the  hope  of  discovering  the  means  by 
which  the  Mellita  sperm  extract  accomplished  the  dispersal  of  the  gelatinous  coat. 
A  fertilizin  solution,  prepared  by  acid  (pH  4)  treatment  of  Mellita  eggs,  with  a 
sperm-agglutination  titer  of  1  :  1000,  was  gently  shaken  with  an  equal  volume  of 
sperm  extract  at  pH  8  for  60  minutes  at  25°  C.  No  decrease  in  titer  of  the  fertilizin 
solution  was  evident  at  the  end  of  the  experiment.  The  sperm  extract  used  in  this 
experiment  had  been  previously  shown  to  disperse  the  gelatinous  coat  in  10  minutes, 
and  showed  no  sperm-agglutinating  property.  The  experiment  indicated  that  no 
degradation  of  the  fertilizin  molecule  resulting  in  loss  of  agglutinating  activity  oc- 
curred in  the  presence  of  the  extract.  Most  probably,  the  dispersal  of  the  gelatinous 
coat  by  sperm  extract  is  accomplished  by  depolymerization  of  the  gelatinous  coat 
material,  and  not  by  splitting  of  individual  fertilizin  molecules. 

DISCUSSION 

Since  the  dispersing  factor  is  heat-labile  under  conditions  where  antifertilizin  is 
stable,  it  seems  reasonable  to  consider  them  to  be  separate  substances.  This  con- 


GELATINOUS  COAT  DISPERSAL  BY  SPERM  79 

elusion  is  supported  by  the  failure  of  strong  antifertilizin  solutions  to  cause  dis- 
persal of  the  gelatinous  coat,  even  though  a  precipitation  membrane  forms  and,  in 
some  instances,  contracts  to  the  surface.  One  can  distinguish,  therefore,  between 
the  dispersal  of  the  gelatinous  coat  and  its  precipitation.  Further,  acid  treatment  of 
sperm  or  sperm  extracts,  a  procedure  not  infrequently  used  for  antifertilizin  extrac- 
tion from  whole  sperm  (Tyler  and  O'Melveny,  1941),  results  in  the  inactivation  of 
the  dispersing  factor,  again  indicating  that  the  dispersing  factor  and  antifertilizin  are 
separate  substances.  Since  the  respiration  of  such  acid-treated  sperm  is  most 
probably  normal  (Tyler  and  O'Melveny,  1941),  carbon  dioxide  is  probably  not 
involved  in  the  dispersal  of  the  gelatinous  coat.  The  observed  temperature  and 
pH  sensitivity  of  the  dispersing  factor  suggest  that  it  is  protein  in  nature,  possibly  an 
enzyme. 

The  dispersing  factor  of  Mellita  sperm  apparently  does  not  act  on  fertilizin  in 
solution,  but  only  serves  to  liquify  or  disperse  the  gelatinous  coat.  If  the  fertilizin, 
in  the  gel  state,  is  bound  by  cross  linkages  involving  the  area  of  the  molecule  ca- 
pable of  combining  with  antifertilizin,  as  Tyler  (1948)  suggests,  the  dispersing 
factor  may  operate  by  breaking  such  cross  linkages,  thereby  releasing  fertilizin  from 
the  gel.  Further,  the  dissolution  of  fertilizin-antifertilizin  precipitation  membranes 
by  extracts  containing  the  dispersing  factor  may  be  due  to  the  breaking  of  linkages  at 
the  fertilizin-antifertilizin  combining  site.  Further  experiments  are  necessary  be- 
fore the  relationship  of  the  dispersing  factor  to  the  fertilizin-antifertilizin  reaction  can 
be  stated  with  certainty. 

SUMMARY 

1.  A  factor  causing  the  dispersal  of  the  gelatinous  coat  of  Mellita  eggs  was  shown 
to  be  present  on  the  surface  of  Mellita  sperm  and  in  frozen-thawed  extracts  of  sperm 
suspensions. 

2.  The  factor  was  separable  from  antifertilizin  on  the  basis  of  temperature  and 
pH  sensitivity. 

3.  The  factor  did  not  degrade  fertilizin  in  solution,  but  released  this  substance 
from  the  gel  surrounding  the  egg. 

4.  Active  extracts  were  capable  of  dissolving  fertilizin-antifertilizin  precipitation 
membranes,  formed  on  the  surface  of  the  fertilizin  coat  of  unfertilized  eggs  in  the 
presence  of  Arbacia  or  Mellita  antifertilizin. 

LITERATURE  CITED 

BERG,  W.  E.,  1949.     Some  effects  of  sperm  extracts  on  the  eggs  of  Mytilus.     Amer.  Nat.,  83: 

221-226. 

CHANG,  M.  C,  1947.     Effects  of  testis  hyaluronidase  and  seminal  fluids  on  the  fertilizing  ca- 
pacity of  rabbit  spermatozoa.     Proc.  Soc.  Exp.  Biol.  Med.,  66:  51-54. 
DURAN-REYNALS,  F.,  1942.     Tissue  permeability  and  the  spreading  factors  in  infection.     Bact. 

Rev.,  6:  197-252. 
FRANK,  J.  A.,  1939.     Some  properties  of  sperm  extracts  and  their  relationship  to  the  fertilization 

reaction  in  Arbacia  punctnlata.     Biol.  Bull.,  76:  190-216. 
HARTMANN,  M.,  O.   SCHARTAU  AND  K.  WALLENFELS,   1940.     Uber   die   Wechselwirkung  von 

Gyno-  und  Andro-Gamonen  bei  der  Befruchtung  der  Eier  des   Seeigels.     Naturwiss., 

28:  144. 
HIBBARD,  H.,  1928.     Contribution  a  1'etude  de  1'ovogenese,  de  la  fecondation  et  de  1'histogenese 

chez  Discoglossus  pictits.     Otth.  Arch.  Biol.,  38:  251-326. 


80  JOHN  W.  BROOKBANK 

ISHIDA,   J.,    1954.     Jelly-dissolving   principle   released   from    sea-urchin    sperm   at   the   time    of 

fertilization.     /.  Fac.  Sci.  Tokyo,  Sect.  IV ,  Zoology,  7 :  53-59. 
KRAUSS,  M.,  1950.     On  the  question  of  hyaluronidase  in  sea  urchin  spermatozoa.     Science,  112: 

759. 
LEONARD,  S.  L.,  P.  L.  PERLMAN  AND  R.  KURZROK,  1947.     Relation  between  time  of  fertilization 

and  follicle  cell  dispersal  in  rat  ova.     Proc.  Soc.  Exp.  Biol.  Med.,  66:  517-518. 
LUNDBLAD,    G.,    AND   A.    MoNROY,    1950.     Mucopolysaccharase   activity   of    sea-urchin    sperms. 

Ark.  f.  Kemi,  2:  343-347. 
VON  MEDEM,  F.  G.,  1942.     Beitrage  zur  Frage  der  Befruchtungs-stoffe  bei  marinen  Mollusken. 

Biol.  Zentr.,  62  :  431-446. 
MEYER,  K.,  1947.     The  biological  significance  of  hyaluronic  acid  and  hyaluronidase.     Physiol. 

Rev.,  27 :  335-359. 

MEYER,  K.,  AND  M.  M.  RAPPORT,  1952.     Hyaluronidases.     Adv.  in  Ensymol.,  13 :  199-236. 
MONROY,  A.,  AND  A.  RUFFO,  1947.     Hyaluronidase  in  sea-urchin  sperm.     Nature,  159 :  603. 
MONROY,  A.,  AND  L.  Tosi,   1952.     A  note  on  the  jelly-coat-sperm   interaction   in  sea  urchins. 

Experientia,  8:   393-394. 

MONROY,  A.,  L.  Tosi,  G.  GIARDINA  AND  R.  MAGGIO,  1954.     Further  investigations  on  the  inter- 
action between  sperm  and  jelly-coat  in  the  fertilization  of  the  sea  urchin  egg.     Biol. 

Bull.,  106:  169-177. 

TYLER,  A.,  1939.     Extraction  of  an  egg  membrane  lysin  from  sperm  of  the  giant  key-hole  lim- 
pet (Mcgathura  crenulata).     Proc.  Nat.  Acad.  Sci.,  25:  317-323. 
TYLER,  A.,  1948.     Fertilization  and  immunity.     Physiol.  Rev.,  28:  180-219. 
TYLER,  A.,  1949.     A  simple,  non-injurious  method  for  inducing  repeated  spawning  of  sea  urchins 

and  sand  dollars.     Coll.  Net,  19:  19-20. 
TYLER,  A.,  and  K.  O'MELVENY,  1941.     The  role  of  antifertilizin  in  the  fertilization  of  sea  urchin 

eggs.     Biol.  Bull,  81 :  364-374. 
VASSEUR,  E.,  1951.     Demonstration  of  a  jelly-splitting  enzyme  at  the  surface  of  the  sea-urchin 

spermatozoon.     Exp.  Cell  Res.,  2 :  144-146. 
WINTREBERT,  P.,  1929.     La  digestion  de  1'enveloppe  tubaire  interne  de  1'oeuf  par  des  ferments 

issus  des  spermatozoides,  et  de  1'ovule,  chez  Discoglossus  pictus.     Otth.  C.  R.  Acad. 

Sci.  Paris,  188 :  97-100. 

WINTREBERT,  P.,  1933.     La  fonction  enzymatique  de  1'acrosome  spermien  du  Discoglosse.     C.  R. 
r.  Biol.,  122 :  1636-1640. 


AN  EXOGENOUS  REFERENCE-CLOCK  FOR  PERSISTENT, 

TEMPERATURE-INDEPENDENT,  LABILE, 

BIOLOGICAL  RHYTHMS  *• 2 

FRANK  A.  BROWN,  JR. 

Department  of  Biological  Sciences,  Northwestern  University,  Evanston,  Illinois 

The  phenomenon  of  persistent  rhythmicity  of  one  or  more  of  their  vital  processes 
is  widespread  among  animals  and  plants.  By  persistent  rhythmicity  is  meant  that 
the  rhythm  still  continues  when  conditions  are  held  constant  with  respect  to  all  fac- 
tors generally  conceded  to  influence  the  organisms. 

Reviews  of  this  subject  have  included  those  by  Biinning  (1936,  1956a,  1956b), 
Jores  (1937),  Kalmus  (1938),  Welsh  (1938),  Park  (1940),  Kleitman  (1949), 
Calhoun  (1944,  1945-46),  Korringa  (1947),  Webb  (1950),  Caspers  (1951), 
Cloudsley-Thompson  (1953),  Bruce  and  Pittendrigh  (1957),  and  Brown  (1957d, 
1958).  The  broad  distribution  of  such  rhythmicity  is  suggestive  of  an  hypothesis 
that  all  living  things  have  potentially  the  means  of  persistent  rhythmicity  provided 
it  has  a  period  close  to  that  of  one  of  the  natural  geophysical  rhythms.  The  organis- 
mic  rhythms  usually  are  essentially  temperature-independent  in  their  frequencies, 
whether  the  periods  are  solar-daily,  lunar  or  annual. 

Most  of  the  observed  rhythms  are  clearly  endogenous,  and  are  labilely  adaptable 
in  form  and  phase  relationships  to  the  needs  of  the  organism.  Much  has  been 
learned,  particularly  in  recent  years,  as  to  the  properties,  including  modifiability,  of 
this  endogenous  rhythmicity.  The  fundamental  problem,  however,  that  of  the  tim- 
ing mechanism  of  the  rhythmic  periods,  has  largely  eluded  any  eminently  reasonable 
hypotheses  in  terms  of  cell  physiology  or  biochemistry.  The  problem  was  already 
a  difficult  one  when  only  solar-daily  cyclicity  was  under  consideration,  but  especially 
in  recent  years  it  has  been  found  that  one  and  the  same  organism  may  simultaneously 
possess  overt  daily  and  lunar  tidal  cycles  of  two  bodily  processes.  Further,  the 
possession  of  persistent  lunar  monthly  (Brown,  Bennett  and  Webb,  1958)  and 
even  annual  cycles  (Biinning  and  Miissle,  1951 ;  Biinning  and  Bauer,  1952;  Brown, 
1957c)  in  constant  conditions  has  emphasized  the  magnitude  and  complexity  of  this 
basic  problem. 

Added  to  the  property,  temperature-independence,  in  indicating  the  unconven- 
tional character  of  the  rhythm-timing  mechanisms,  are  the  repeated  demonstrations 
of  the  immunity  of  the  frequency-determining  mechanism  to  most  metabolic  poisons. 

Recently,  evidence  has  been  rapidly  accumulating  pointing  to  the  possession  by 
living  organisms  of  basic  metabolic  cycles  of  the  natural  geophysical  frequencies, 

1  This  study  was  aided  by  a  contract  between  the  Office  of  Naval  Research,  Department 
of  the  Navy,  and  Northwestern  University,  NONR-122803. 

2  The  author  wishes  to  express  his  appreciation  to  the  several  students  who  worked  many 
hours  in  assisting  to  obtain  and  process  the  data  used  here.     Thanks  are  especially  due  to 
Messrs.  W.  D.  Korte,  who  handled  the  carrot  experiments,  E.   F.   Lutsch,   F.   H.   Barnwell, 
E.  J.  Macey,  H.  Gibson,  Jr.,  and  Misses  J.  Strunk  and  B.  Getting. 

81 


82  FRANK  A.  BROWN,  JR. 

produced  in  the  organism  by  an  external  cyclic  stimulus  still  operative  in  so-called 
laboratory  constant  conditions  (Brown,  1957;  Brown,  Shriner  and  Webb,  1957; 
and  Brown,  Webb  and  Macey,  1957).  These  cycles  are  not  phase-  or  form-labile. 
The  problem  of  a  common  explanation  for  persistent  rhythmicities  of  all  the  well- 
known  natural  frequencies  including  the  year  becomes  at  once  more  susceptible 
to  reasonable  working  hypotheses  as  to  their  mechanism  when  it  is  firmly  estab- 
lished that  protoplasm  in  "constant  conditions"  is,  fundamentally,  exogenously 
rhythmic. 

For  the  study  to  be  reported  here,  the  potato  and  carrot  were  selected  as  organ- 
isms neither  of  which  appears  to  possess  any  obscuring,  labile,  endogenous  rhythms. 
It  was  considered  that  such  organisms  would  reveal  most  readily  any  extant  basic 
protoplasmic  cyclicities  and  also  permit  easier  analysis  of  any  mechanisms  they 
involved. 

On  the  basis  of  this  hypothesis,  of  an  exogenous  reference  clock  providing  the 
timing  of  cyclic  periods,  the  often-described  endogenous  rhythms  would  be  con- 
sidered a  consequence  of  the  evolution  by  the  organism  of  adaptive  labile  cyclic 
changes,  utilizing  the  basic  exogenous  cycle-timing  mechanism.  The  endogenous 
mechanisms  could  be  inherited.  The  only  inherited  aspect  of  the  exogenous 
cyclicity  would  be  the  fundamental  protoplasmic  responsive  systems  which  are 
involved. 

MATERIALS  AND  METHODS 

The  potatoes,  Solanum  tuberosum,  were  of  the  Idaho  variety  and  were  pur- 
chased from  local  grocery  stores.  The  carrots,  Daucus  carota,  were  similarly  pur- 
chased from  local  stores.  Using  a  cork-borer,  small  cylinders,  2.2  cm.  in  diameter 
and  about  1.5  cm.  tall,  were  prepared  from  the  potatoes  in  such  a  manner  that  each 
carried  an  eye  on  the  center  of  its  upper  surface.  These  were  permitted  to  heal 
their  cut  surfaces  before  being  set,  in  shallow  water,  in  respirometer  vessels  where 
the  same  individual  organisms  were  retained  up  to  three  or  more  months.  These 
always  gave  rise  to  sprouts  and  usually  also  to  a  root  system,  and  in  some  instances 
even  developed  new  tubers  up  to  a  centimeter  or  more  in  diameter  during  their 
sojourn  in  the  respirometers.  For  the  carrots,  short  cylindrical  sections,  about  the 
size  of  the  potato-cylinders,  were  cut  and  allowed  to  heal  over  before  being  placed  in 
respirometers. 

The  respirometers  have  been  described  earlier.  These  were  originally  designed 
by  Brown  (1954)  and  later  modified  (Brown,  1957a)  to  permit  maintenance  of 
constant  pressure. 

Five  independent  barostat-respirometer  ensembles,  each  with  4  respirometers 
recording  as  a  unit,  were  in  nearly  continuous  operation  during  the  period  of  study, 
Feb.  1,  1956  through  Feb.  28,  1958.  The  potatoes  in  the  respirometers  were  in 
constant  illumination  (estimated  at  0.05  ft.  c.  at  the  site  of  the  plants)  from  incan- 
descent lamps  supplied  by  a  voltage-regulated  line.  The  temperature,  20°  C.,  was 
maintained  constant  by  the  respirometers  being  immersed  in  a  large  non-stirred, 
copper  water-bath  (the  barostat)  deeply  immersed  in  an  outer,  stirred,  steel  (55-gal. 
drum)  water  bath,  with  the  latter  cycling  with  a  few-minute  period  within  a  ±  0.05° 
C.  range.  The  pressure  was  kept  constant,  28.5  in.  Hg,  through  hermetically  seal- 
ing the  respirometer-recorder-containing  barostat  and  then  aspirating  the  system  to 
this  level.  Oxygen  and  CO2  tensions  were  maintained  essentially  constant  through 


EXOGENOUS  BIOLOGICAL  RHYTHMICITY  83 

use  of  the  principle  of  continuous  CX-replacement  together  with  CO2  absorbents, 
and  there  were  clearly  no  regularly  cyclic  fluctuations  in  these  substances.  Also, 
the  sealed,  water-included  systems  allowed  for  no  changes  in  humidity. 

With  a  single  exception  (12  days)  the  copper  tanks,  or  barostats,  remained 
sealed  for  periods  ranging  from  2  to  8  days,  with  an  average  of  4.46  days.  At  these 
intervals  the  organisms  were  exposed  for  15-20  minutes  to  laboratory  conditions 
which  were  relatively  constant  over  the  year.  No  work  was  done  within  15  feet 
of  outside  windows ;  the  laboratory  fluorescent  illumination  at  table  top  was  about 
45  ft.  c.  (The  carrot  study  was  carried  out  wholly  in  a  dark-room  without  any 
natural  illumination.)  The  room  temperature  was  relatively  constant,  about  75° 
F.,  except  for  slightly  higher  values  during  the  summer  months.  The  barostats 
were  opened  at  various  hours  of  the  day  from  8  AM  to  10  PM.  Excluding  those 
days  the  respirometers  were  opened  to  renew  the  O2  and  the  CO2  absorbent,  a  total 
of  2485  uninterrupted  calendar  days  of  data  were  obtained. 

The  recording  systems  of  the  respirometers  possessed  two  points  of  slight  me- 
chanical frictional  resistance,  a)  a  two-point  pivotal,  spring-scale  bearing,  and  b) 
the  point  of  contact  of  the  ink-writing  pen  with  the  slowly  moving  paper.  These 
resulted  in  random,  spurious  apparent  intra-hour  fluctuations  in  rate  of  CX-con- 
sumption.  Since  the  principle  of  operation  of  the  recorder  was  one  with  which  the 
hourly  values  of  O2-consumption  were  obtained  by  calculating  the  differences  be- 
tween consecutive  hourly  markers  on  a  continuing  trend-line  denoting  cumulative 
0.,-consumption,  these  spurious  fluctuations  in  apparent  rate  could,  and  undoubtedly 
did,  produce  larger  hour-to-hour  differences  than  bore  any  significance.  Hence, 
time  units  of  less  than  three  hours  (three-hour  "moving  means")  were  never  used 
in  determining  the  mean  rates  centered  on  any  given  hour.  By  this  means  the 
random  mechanically  induced  error  was  reduced  to  about  one-third  its  single-hour 
influence.  For  most  of  the  study  reported  here,  a  weighted  (1:2:3:3:3:2:  1) 
seven-hour  "moving  mean"  was  used.  This  reduced  by  essentially  90%  the  ran- 
dom fluctuations  while  retaining  all  the  precision  of  measurement  of  average,  actual, 
O2-consumption  for  this  longer  interval,  as  modified  by  its  weighted  character. 
The  shorter  period,  three-hour,  means  were  found  necessary,  however,  to  expose  the 
relationship  between  day-by-day  6  AM  deviations  in  CX-consumption  from  daily 
linear  trends  and  the  concurrent  day-by-day  mean  rates  of  barometric  pressure 
change  for  the  2-6  AM  interval.  Although  some  clearly  significant  short-period 
fluctuations  were  obscured,  therefore,  by  the  seven-hour  weighted  "moving  means," 
these  were  considered  superior  to  the  shorter  periods  for  the  accurate  description  of 
the  general  characters  of  the  longer-period,  daily  and  annual  cycles  to  be  described 
herein. 

The  records  for  the  five  completely  independent,  respirometer-recording  systems 
were  first  dealt  with  individually  and  three-hour  and  weighted  seven-hour  "moving 
means"  were  prepared  month  by  month  for  the  period  of  study.  From  the  latter 
values  were  calculated  the  mean  daily  rates  of  O2-consumption  and  the  data  were 
then  converted  into  hourly  deviations  from  the  solar  daily  means.  The  number 
of  uninterrupted  days  of  data  from  the  24  months  of  study  ranged  from  93  to  129 
each  month.  The  hourly  deviations  for  all  the  respirometers  operating  were  aver- 
aged for  each  calendar  day,  and  these  average  daily  cycles  then  converted  to  hourly 
deviations  from  a  1  AM  to  12  midnight  linear  trend-line.  This  will  be  referred  to 
as  the  deviations  from  linear  dailv  trend.  From  these  data  the  forms  of  the  mean 


84 


FRANK  A.  BROWN,  JR. 


daily  cycles  for  each  month  were  obtained.  The  slope  of  this  linear  trend-line  it- 
self shows  apparent  monthly  and  annual  periodisms  which  have  been  treated  else- 
where (Brown,  1957c;  Brown,  Bennett  and  Webb,  1958).  The  trend  involved  a 
mean  daily  increase  during  the  two-year  study  of  6.7%,  and  included,  as  a  large 


o\° 


§*< 

LJ 

cr 


+3 


O 

cc 


LJ 
Q 

+1 

o\°0 

Z 

LJ 


O 


LJ 

Q 


+  1 


6PM 


o  o 


HOUR 


12 
OF    DAY 


18 


24 


FIGURE  1.  A.  The  mean  solar-day  cycle  of  Oo-consumption  in  the  potato  (solid  line)  with 
standard  errors  for  selected  hours.  This  is  expressed  as  %  deviations  from  linear  daily  trend. 
The  dashed  curve  is  the  cycle  for  the  first  year  of  study,  the  dotted  curve,  for  the  second.  B. 
The  mean  apparent  sidereal-day  cycle  of  the  potato  for  the  two-year  period  of  study. 

component,  the  apparent  smoothly  gradual  recovery  over  a  3-  to  5-day  period,  from 
the  inhibitory  influence  of  the  room-illumination  intensity.  The  mechanical  re- 
cording system,  itself,  departed  from  linearity  over  its  total  range  by  10%,  departing 
in  such  a  direction  that  there  would  be  expected  on  this  basis  an  average  of  about 
2%  increase  per  day. 


EXOGENOUS  BIOLOGICAL  RHYTHMICITY 


85 


An  entirely  independent  and  parallel  study  was  made  of  Go-consumption  of  the 
sections  of  the  carrots,  for  the  8-month  period  Oct.  1,  1956  through  May  31,  1957. 
Two  respirometer-barostat  ensembles  were  employed  for  the  first  three  months,  and 
four  for  the  remaining  five  months.  These  were  maintained  in  darkness  in  a  photo- 
graphic darkroom  about  60  feet  away  from  the  place  of  the  potato  study,  but  simi- 
larly on  the  ground  floor  of  Cresap  Biological  Laboratory,  a  three-story  steel  and 
mortar  building.  The  respirometers  were  maintained  and  the  data  processed  by 
a  person  not  involved  until  the  termination  of  the  carrot  study  in  the  paralleling  and 
continuing  potato  study. 


M      A 
1956 


M 


A      S 


N      D       J        F       M      A       M 

MONTHS        1957 


O       N      D 


J       F 

1958 


FIGURE  2.  The  relationship  between  average  %  noon  deviation  in  O2-consumption  in  the 
potato  from  linear  daily  trend  and  month  of  year  during  the  25-month  study.  Standard  errors 
of  means  are  shown. 


RESULTS 

During  the  period,  Feb.  1,  1956  through  Feb. 
the  only  days  omitted  were  May  25,  the  month 
July,  1956,  and  October  4,  1957. 

The  form  of  the  mean  daily  deviation  from 
the  daily  mean  rate,  is  shown,  with  the  standard 
values,  in  the  solid-line  curve  of  Figure  1,  A. 
quite  comparable  in  size.     Superimposed  on  this 
two  years  separately:  Feb.  1,  1956  through  Jan 


28,  1958,  in  the  study  of  the  potato, 
of  June  and  the  first  three  days  of 

trend,  expressed  as  percentage  of 
errors  of  arbitrarily  selected  mean 

The  errors  of  the  other  values  are 
are  the  mean  cycles  for  each  of  the 

.  31,  1957  (the  dashed  curve)  and 


86 


FRANK  A.  BROWN,  JR. 


FEB 


MAR 


APR 


JUNE 


JUL 


SEPT 


OCT 


DEC 


JAN 


6AM  12 

HOUR    OF    DAY 


6PM 


FIGURE  3.     The  forms  of  the  average  daily  cycles  for  each  month  of  the  year  obtained  in  the  two- 
year  study.     The  ordinate  values  are  deviations  in  comparable  arbitrary  units. 


EXOGENOUS  BIOLOGICAL  RHYTHMICITY 


87 


Feb.  1,  1957  through  Jan.  31,  1958  (the  dotted  curve).  The  average  amplitude  of 
the  daily  cycle  was  clearly  quite  reproducible  for  the  two  years  at  about  3.7%. 
There  was  also  clear  suggestion,  in  the  skewed  cycle  form,  of  a  bimodality  with 
morning  and  afternoon  maxima,  a  condition  more  conspicuous  for  the  second  than 
for  the  first  year  of  study.  The  mean  sidereal-day  cycle  (23  hours,  56.07  minutes) 
for  the  two-year  period  is  shown  in  Figure  1,  B.  This  was  obtained  by  displacing 


M 


M 


MONTHS 


FIGURE  4.  The  relationship  between  the  noon  deviations  in  Da-consumption  from  linear  daily 
trend  and  calendar  month  in  the  carrot  during  an  8-month  study.  The  standard  errors  are 
depicted. 

the  consecutive  mean  monthly  solar-day  cycles  each  by  two  hours  to  the  right  dur- 
ing the  two-year  period,  to  bring  into  reasonably  close  synchrony  (—1  hour)  the 
hours  of  the  sidereal  day.  The  numbered  hours  are  fixed  by  the  solar-day  hours 
of  the  first  month,  February,  1956.  This  process  also  randomizes  daily  trend. 

The  form  and  amplitude  of  the  solar  daily  cycle  showed  differences  from  month 
to  month  which  revealed  that  it  was  undergoing  a  modulation  of  an  annual  fre- 
quency. This  was  quite  evident  when  one  used,  for  example,  the  parameter  of 
average  monthly  noon  deviation,  in  percentage,  from  1  AM  to  midnight  daily 


88 


FRANK  A.  BROWN,  JR. 


linear  trend.  The  deviations,  month-by-month,  for  the  period  Feb.  1,  1956 
through  Feb.  28,  1958,  together  with  their  standard  errors,  are  depicted  in  Figure  2. 
These  indicate  minimum  annual  values,  involving  often  even  apparent  cycle  inver- 
sion, during  the  coldest  months  of  the  year  and  a  major  maximum  in  the  month 
of  October.  A  lesser,  or  incipient,  maximum  occurred  in  April-May.  The  maxi- 
mum range  is  seen  to  extend  from  --  3.4%  to  +  14.2%. 

An  annual  cycle  in  over-all  form  of  the  mean  daily  cycles  for  the  months  of  the 
year  is  evident  in  Figure  3,  where  twelve  average  cycles,  the  means  for  two  years, 
have  been  plotted  in  terms  of  average  deviation  in  arbitrary  units  from  linear-trend. 


6AM  12 

HOUR    OF    DAY 


6PM 


6AM  12 

HOUR    OF    DAY 


6   PM 


FIGURE  5.  A  comparison,  for  the  same  8-month  period  of  study,  of  the  forms  of  the  mean 
daily  cycles  for  carrots  (A)  and  potatoes  (B).  Solid  curves  show  the  mean  cycles  for  the  whole 
8-month  period.  The  dashed  curves  show  the  average  cycles  for  October,  November,  April,  and 
May.  The  dotted  curves  show  the  average  cycles  for  December,  January,  February,  and 
March.  Standard  errors  of  selected  times  of  day  are  shown. 

Although  these  data  are  not  expressed  as  percentage  deviations,  they  do  illustrate  the 
gradually-changing  form  of  the  cycles  from  unimodality  with  essential  inversion  in 
February,  but  with  a  7  PM  maximum,  through  a  period  of  bimodality  with  the  two 
daily  maxima  gradually  converging  towards  noon  to  reach  unimodality  \vith  a 
maximum  at  11  AM  in  October.  Thereafter,  bimodality  reappears  and  continues, 
becoming  only  feebly  evident  as  an  apparent  residual  in  the  essentially  unimodal  in- 
verted cycle  of  January  which  like  the  succeeding  month,  February,  has  a  7  PM 
maximum. 

The  study  of  the  carrot  revealed  striking  similarity  of  its  major  mean  cycles 
with  those  of  the  potato.  Figure  4  shows  the  mean  %  noon  deviation  from  linear 
trend  for  each  of  the  eight  months.  Like  the  results  obtained  with  the  potato  for 


EXOGENOUS  BIOLOGICAL  RHYTHMICITY 


89 


the  same  calendar  period,  this  passed  from  an  early-fall  higher  value,  through  a 
winter  minimum  and  back  to  a  higher  spring  value.  Fewer  data  were  available  dur- 
ing the  first  three  months,  hence  the  errors  were  larger.  The  range  was  less  than 
for  the  potatoes.  Figure  5,  A  and  B  solid  curves,  compares  the  mean  8-month  daily 
cycles  for  the  carrot  and  potato,  and  the  average  cycles  for  the  two  fall  and  two 
spring  months  (dashed  curves)  as  compared  with  those  for  the  four  intervening 
colder  months  (dotted  curves),  The  similarities  of  these  two  widely  different 
kinds  of  plants  and  plant  portions  (roots  vs.  stems)  for  the  same  periods,  in  the  % 


z 
o 

H 
Q_    10 


Z 
O 

o 

Icvj 


id 
I- 
< 

ct 


0 


5 


J 


M 


M  J  J 

MONTHS 


A 


O 


N 


D 


FIGURE  6.  The  relationship  between  mean  rates  of  Oo-consumption  and  each  of  the  24 
months  studied  during  1956,  1957,  and  1958,  and  time  of  year.  Standard  errors  of  the  means 
are  indicated. 

amplitudes  of  the  fluctuations,  in  the  times  and  the  changing  times  with  time  of  year 
of  the  primary  maxima,  and  in  the  times  of  secondary,  or  incipient,  maxima,  are 
strikingly  apparent  from  the  figures. 

A  second  kind  of  annual  cycle  appears  also  present  in  the  data.  This  is  in  the 
mean  daily  metabolic  rates.  In  Figure  6,  are  to  be  found  the  mean  monthly  rates 
of  Oo-consumption,  in  arbitrary  units,  for  each  of  the  24  months  of  study,  together 
with  their  standard  errors.  Two  conclusions  are  evident  from  the  figure :  ( 1 ) 
The  maximum  rate  of  Oo-consumption  occurs  in  the  April-May  period  of  the  year 
and  minimum  rate  in  October-November.  The  rate  for  the  former  period  ap- 


90 


FRANK  A.  BROWN,  JR. 


TABLE  I 

Signs  of  the  average  monthly  correlations  of  the  6  A  M  deviations  from  linear 
trend  with  the  mean  2-6  AM  rate  of  barometric  pressure  change 


Jan. 

Feb. 

Mar. 

Apr. 

May 

June 

July 

Aug. 

Sept. 

Oct. 

Nov. 

Dec. 

Year  1 

— 

— 

+ 

+ 

+ 

+ 

+ 

— 

— 

Year  2 

— 

— 

+ 

+ 

+ 

+ 

+ 

-f 

+ 

+ 

— 

— 

Year  3 

preaches  twice  that  of  the  latter.  (2)  The  mean  rates  for  corresponding  months  of 
the  two  consecutive  years  may  be  quite  significantly  different  from  one  another,  sug- 
gestive of  a  specific,  time-environmental  factor  involved  in  an  exogenous  regulation. 
There  is  nothing  in  these  data  to  suggest  other  than  that  the  mean  form  of  this 
annual  cycle  will  ultimately  be  found  essentially  sinusoidal. 

In  view  of  the  correlations  highly  significantly  different  from  zero  earlier  re- 
ported (Brown,  1957a)  to  exist  between  the  5-6—7  AM  mean  deviations  (without 
sign)  in  O2-consumption  from  the  daily  means  and  the  mean  2-6  AAI  rates  of  baro- 
metric pressure  change,  this  relationship  was  examined  for  the  two-year  period 
involved  here.  Three-hour  values  of  (X-consumption  centered  on  6  AM  were  re- 
corded as  deviations  from  linear  daily  trend,  and  three-day  moving  means  calcu- 
lated. These  were  correlated  with  comparable  three-day  moving  means  of  the  av- 


0          +1         +2 
2-6  A.M.    B.  R  C. 


o\o-8 


-16 


85 


FIGURE  7.  A.  Solid  line  :  An  outline  of  the  general  form  of  the  scatterplot  between  mean 
2-6  AM  rate  of  barometric  pressure  change  and  the  5-6-7  AM  mean  deviation  of  rate  CL-con- 
sutnption  from  daily  trend  for  the  same  day  during  the  "colder"  months  (see  text).  Broken 
line :  The  same  for  the  "warmer"  months.  Data  for  both  involve  three-day  moving  means. 
The  two  patterns  together  include  98%  of  all  points.  B.  An  outline  of  the  form  of  the  scatter- 
plot (97%  of  all  points)  between  %  noon  deviations  from  linear  trend  in  potatoes  in  constant 
conditions  and  concurrent  outdoor  air  temperature,  taken  from  data  of  149  non-overlapping 
three-day  averages. 


EXOGENOUS  BIOLOGICAL  RHYTHMICITY 


91 


erage  2-6  AM  rate  of  barometric  pressure  change,  for  the  corresponding  days.  It 
should  be  emphasized  that  only  a  single  value  was  used  for  each  day  for  each  phe- 
nomenon ;  hence,  this  did  not  involve  a  correlation  of  parallel  daily  cyclic  trends.  A 
positive  coefficient,  highly  significantly  different  from  zero,  was  obtained.  This 
correlation,  as  one  would  anticipate  in  view  of  the  essentially  aperiodic,  large  cli- 
matic barometric  pressure  changes,  rapidly  drops  to  insignificance  as  one  correlates 


±.6 


Q 

U  £4 

cr 

h- 


Q 

5.» 


i 

CD 

•   +.4 


+.2 


0 


B 


O 


-I 


0  +1  +  2  +3 

2-6  A.M.    BAR.    PRESS.  CHANGE 


FIGURE  8.  The  regressional  relationship  of  the  average  three-hour  rate  of  O^-consumption 
of  the  potato  centered  on  6  AM,  and  expressed  as  deviation  from  linear  daily  trend,  on  the 
average  rate  of  barometric  pressure  change  during  the  2-6  AM  interval  f  jr  the  same  morning 
for  the  colder  months  cf  the  year  (see  Table  I ).  P  <  0.005.  B.  The  relationship  comparable  to 
that  in  A.  but  for  the  warmer  months  of  the  year  (see  Table  I).  P<  0.001. 


92 


FRANK  A.  BROWN,  JR. 


in  increasing  lag  on  lead  relationships  up  to  two  to  three  days  (Brown,  1957a)  indi- 
cating a  direct  response  of  the  organisms  to  some  pressure-change-correlated  ex- 
ternal variable.  But  this  relationship  was  found  to  contain  a  characteristic  sign- 
change  twice  a  year  as  seen  in  Table  I.  In  this  table,  a  dash  indicates  those  months 
in  which  there  was  a  negative  correlation  between  the  rate  of  the  2-6  AM  baro- 
metric pressure  change  and  the  6  AM  deviation,  without  sign,  from  linear  trend. 
The  form  of  the  scatterplot  relationship  for  the  299  days  of  this  negative  period  is 
outlined  by  the  solid  curve  (encloses  91  %  of  the  points)  in  Figure  7,  A.  The  re- 
gressional  relationship  of  the  deviation  in  CX-consumption,  without  sign,  upon  pres- 
sure change  is  seen  in  Figure  8,  A.  During  the  +  months,  on  the  other  hand,  there 
was  a  positive  correlation  between  the  rate  of  2-6  AM  barometric  pressure  change 
and  the  6  AM  deviation  in  O2-consumption  from  linear  trend.  Ninety-one  per  cent 
of  the  389  days  in  a  scatterplot  of  the  relationship  for  these  months  fell  within  the 
broken  curve  of  Figure  7,  A  (98%  of  all  688  daily  points  +  or  —  months  fell  in  the 
areas  prescribed  by  both  the  solid  or  broken  lines).  The  regressional  relationship 
of  O2-consumption  on  pressure  for  the  +  months,  with  sign,  is  seen  in  Figure  8,  B. 

TABLE  II 
Signs  of  the  average  monthly  2-6  PM  change  in  barometric  pressure 


Jan. 

Feb. 

Mar. 

Apr. 

May 

June 

July 

Aug. 

Sept. 

Oct. 

Nov. 

Dec. 

Year  1 

+ 

+ 

+ 

— 

— 

— 

— 

— 

— 

+ 

+ 

Year  2 

+ 

— 

+ 

— 

— 

— 

— 

— 

— 

— 

+ 

+ 

Year  3 

+ 

Since  in  the  warmer,  positive,  months  of  the  year,  the  overwhelming  mass  of  the 
deviations  was  +,  it  was  not  possible  to  find  any  real  difference  between  the  corre- 
lations whether  the  deviations  were  treated  with,  or  without,  sign.  In  the  colder, 
negative,  months,  on  the  other  hand,  about  half  of  the  deviations  were  negative,  and 
the  range  of  the  latter  as  great  as  for  positive  deviations. 

In  view  of  the  earlier  report  (Brown,  1957a)  of  comparable  correlations  be- 
tween the  6  PM  deviation  in  CX-consumption  from  daily  mean  values  in  potatoes, 
and  the  afternoon  rate  of  barometric  pressure  change,  and  also  correlations  with 
the  mean  daily  pressure  of  the  second  day  thereafter  (Brown,  Webb  and  Macey, 
1957),  the  former  relationship  including  signs,  it  is  of  interest  to  compare  the  an- 
nual cycle  in  the  sign  of  the  average  2—6  PM  barometric  pressure  change.  These 
are  seen  in  Table  II. 

The  similarities  between  Tables  I  and  II  suggest  that  this  aspect  of  organismic 
annual  cyclicity,  involving  the  mean  forms  of  daily  cycles,  might  in  some  manner 
be  caused  by  a  factor  whose  daily  fluctuation  reflects  the  annual  cycle  in  form  of  the 
well-known  mean  daily  tidal  atmospheric  pressure  cycles.  In  these  daily  pressure 
cycles,  the  time  of  the  morning  maximum  remains  relatively  fixed  throughout  the 
year  at  9-10  AM,  but  the  afternoon,  major  minimum  of  the  day  gradually  shifts  from 
about  2  PM  in  winter  to  about  7  PM  in  summer.  This  last  is  the  basis  for  the  sign 
changes  in  Table  II.  Thus,  any  pressure-correlated  effective  external  physical 
factor  could  provide  such  an  annual  cycle  in  the  daily  cycles  as  that  described  herein. 

Another  clearly  evident  correlation  is  seen  in  the  relationship  of  the  %  noon  de- 


EXOGENOUS  BIOLOGICAL  RHYTHMICITY  93 

viation  from  linear  trend  in  the  potato  to  the  concurrent  outside  mean  daily  air 
temperatures.3  A  two-year  study  of  the  comparison  of  non-overlapping  three-day 
periods  of  air-temperature  and  of  noon  deviation  of  the  daily  cycles  in  constant  con- 
ditions yields  a  scatter  plot  relationship  as  illustrated  in  Figure  7,  B.  The  line  in- 
cludes 97%  of  the  149  values.  The  regressional  relationship  of  noon-deviation  of 
the  potatoes  on  temperature  (using  5°  F.  class  intervals)  is  illustrated  in  Figure  9. 
The  relationship  seems  adequately  described  as  a  linear  one,  but  with  a  sign  change 
near  57.5°  F.  Calculation  of  the  coefficient  of  correlation  for  noon  deviation  in 


Q 

Z     8 

LJ 

CC. 

r- 


4  o 


O 
cr 


LJ 
Q 


.-4 


O 

o 


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o 


0 


-7.5  2.5  12.5         22.5        32.5        42.5          52.5         62.5         725         82.5 

OUTSIDE   AIR    TEMP.      °E 

FIGURE  9.  The  regressional  relationship  of  the  noon  deviation  in  O^-consumption  in  the 
potato,  expressed  as  %  deviation  from  daily  linear  trend  on  simultaneous  outdoor  air 
temperature. 

O, -consumption  with  the  ±  deviation  in  temperature  from  57.5°  F.,  yielded  a  value 
of  -  0.51  ±  0.049.  This  clearly  indicates  that  the  external  factor  responsible  for 
the  24.0-hour  cycles  of  metabolism  is  correlated  in  its  fluctuations  with  air  tempera- 
ture, resulting  in  a  condition  where  a  spurious  organismic  Q10  of  cycle  amplitude  of 
more  than  3  could  be  apparent  (e.g.,  in  range  32.5°  to  52.5°  F.).  This  provides 
another  piece  of  information  which  will  probably  lead  eventually  to  identification 
of  the  still  unknown  external  factor  responsible  for  the  organismic  basic  periodisms. 
That  the  relationship  to  temperature  is  rather  substantial,  is  given  further  sup- 
port in  that  the  regressional  relationship  of  noon  deviation  on  temperature  exhibited 
a  sign  change  about  57°  F.  in  the  first  year,  1956-57,  just  as  it  did  again  in  the  sec- 
ond. 1957-58,  despite  the  fact  that  in  1956  there  was  no  clear  absolute  summer  de- 

3  These  data  were  generously  provided  to  me  by  the  Chicago  Office  of  the  U.  S.  Weather 
Bureau. 


94  FRANK  A.  BROWN,  JR. 

cline  in  amplitude  of  the  mean  monthly  cycles  (Fig.  2)  as  was  found  in  1957. 
Also,  during  the  winter  months  the  correlations  are  observed  (Fig.  9)  to  continue 
in  the  same  linear  relationship  even  at  temperatures  lower  than  the  lowest  mean 
monthly  ones  in  the  two  years  involved  in  this  correlation  (29.4°,  28.7°,  18.8°,  27.3°, 
and  26.3°  F.)  which  averaged  about  26°  F.  These  last  facts  suggested  intra-month 
significant  temperature  correlations  which  were  borne  out  by  investigation  of  the 
correlations  using  the  data  of  the  five  coldest  months  now  expressed  as  deviations 
from  monthly  means.  The  correlations  continued  in  temperature  ranges  well  ex- 
ceeding any  mean  month-to-month  difference. 

Again,  using  the  data  for  both  of  the  two  years,  the  transitional  months,  April, 
May,  October,  with  a  mean  temperature  of  54.8°  F.  (47.2,  60.5,  60.8,  49.5.  58.8, 
52.0),  expressed  as  deviations  from  monthly  means,  the  critical  temperature  for 
sign  change  was  again  quite  apparent.  Finally,  employing  the  warmest  months  of 
the  year,  June,  July,  August,  and  September  with  a  mean  temperature  of  71.1°  F., 
(72.9,  74.1,  65.0,  71.2,  76.4,  73.4,  64.2),  there  was  a  suggestion  of  the  existence  of 
a  second  sign  change  with  again  a  positive  correlation  at  the  higher  temperature. 
The  number  of  high-temperature  days  was  insufficient,  however,  to  enable  resolu- 
tion of  this  last  point. 

DISCUSSION 

From  the  foregoing  results  it  is  evident  that  potatoes,  and  apparently  the  car- 
rots too,  display  a  quite  reproducible  mean  solar  daily  cycle  provided  adequately 
long  periods  of  time  are  used  to  render  random  the  influences  of  such  modulating 
longer  cycles  as  a  lunar  day  (Brown,  Freeland  and  Ralph,  1955),  a  synodic  month 
(Brown,  Freeland  and  Ralph,  1955 ;  Brown,  Bennett  and  Webb,  1958),  and,  in  this 
report,  a  low  amplitude  apparent  sidereal  day,  and  an  annual  cycle.  These  described 
mean  solar-day  cycles  are  obviously  of  quite  precise  24.0-hour  frequency,  and  ade- 
quate evidence  is  at  hand  to  be  assured,  beyond  all  reasonable  doubt,  that  these 
have  their  frequency  exogenously  determined.  This  last  conclusion  is  assured 
in  part  through  the  well-known  knowledge  that  there  are  solar-day  tidal  rhythms  of 
atmospheric  pressure,  together  with  the  fact  that  the  living  organism  has  access  to 
information  of  them  through  its  responses  to  the  day-to-day,  essentially  random, 
weather-induced,  disturbances  in  their  regularity.  That  the  factor  influencing  the 
organism  is  not  pressure  itself,  is  evident  from  the  fact  that  these  and  other  experi- 
ments have  involved  organisms  maintained  for  long  periods  in  constant  pressure. 
The  external  factor  which  is  involved  appears  to  have  its  primary  action  upon  the 
organism  at  the  times  correlated  with  the  early-morning  rise  in  barometric  pressure 
and  the  afternoon  fall.  These  would  presumably  be  the  times  of  most  rapid  change 
in  physical  factors  fluctuating  with  the  day-night  cycle,  and  hence  be  the  times  of 
their  maximal  stimulative  effectiveness. 

As  pointed  out  earlier  in  this  report,  the  presence  of  the  well-known  annual 
change  in  the  form  of  the  daily,  tidal,  barometric  pressure  cycles,  and  the  de- 
scribed response  of  the  organism  in  the  late  afternoon  to  a  pressure-correlated  ex- 
ternal variable  would  have  led  to  the  prediction  of  the  occurrence  of  an  annual  cycle 
in  the  form  of  the  daily  cycles.  Such  a  prediction  has  been  fulfilled  in  this  study. 
This  adds  still  further,  therefore,  to  the  assurance  that  the  forms  of  the  daily  basic 
metabolic  oscillations  in  living  organisms  are  exogenous. 


EXOGENOUS  BIOLOGICAL  RHYTHMICITY  95 

Since  background  radiation,  too,  possesses  good  mean  solar-day  cyclicity,  and 
the  organism  follows  the  essentially  random  fluctuations  in  its  cycle  amplitudes  from 
day-to-day  (Brown,  Shriner  and  Webb,  1957)  very  safely  beyond  what  would  be 
expected  through  chance,  when  and  only  when  contemporary  data  are  correlated, 
this  constitutes  a  third  line  of  evidence  for  exogenous  origin  of  mean  daily  metabolic 
cycles. 

The  existence  of  an  annual  cycle  in  the  potato  in  constant  illumination,  tempera- 
ture, pressure,  etc.,  was  reported  earlier  for  fluctuations  in  linear  daily  trend 
(Brown,  1957c),  as  were  also  synodic  monthly  cycles  of  this  parameter  (Brown, 
Bennett  and  Webb,  1958).  In  this  paper  there  is  described  an  apparent  annual 
cycle  in  basic  metabolic  rate,  a  cycle  which  appears  to  be  of  simple  sinusoidal  char- 
acter with  maximum  in  April-May  and  minimum  in  October-November.  This 
cycle  involves  an  approximate  doubling  of  rate  in  passing  from  minimum  to  maxi- 
mum values  in  the  annual  cycle.  Comparable,  synodic  monthly,  cycles  in  metabolic 
rate  in  potatoes  (Brown,  Bennett  and  Webb,  1958)  involved,  as  the  average  during 
a  year  of  study,  about  a  15%  increase  from  minimum  (new  moon)  to  maximum 
(third  quarter)  values.  By  further  comparison,  the  amplitude  of  the  daily  cycles, 
though  undoubtedly  artificially  depressed  through  the  use  of  the  seven-hour  weighted 
moving  means,  displayed  about  a  3.7%  increase  from  midnight  minimum  to  6  PM 
maximum  values. 

The  regressional  relationship  of  amplitude  of  the  daily  cycles  on  mean  daily 
temperature  for  three-day  periods  (Fig.  9),  with  its  coefficient  of  determination  of 
about  0.26,  and  its  critical  temperature  for  sign-reversal,  together  with  the  earlier 
barometric-pressure-change  reversing  correlation,  suggests  again  the  exogenous 
origin  of  this  daily  cycle  period,  and,  at  least  in  large  measure,  also  cycle  form. 
This  is  especially  true,  since  the  relationship  to  temperature  seems  to  persist  into 
the  weather-correlated,  intra-month,  temperature  fluctuations. 

In  examining  Figure  9  and  noting  the  relationship  of  cycle  amplitude  to  tem- 
perature, and  recalling  that  the  mean  daily  temperature  range  is  about  16°  F.,  with 
not  very  uncommonly  single  days  with  ranges  up  to  25°  to  30°  F.,  one  is  tempted 
to  postulate  that  the  factor  that  is  responsible  for  transmitting  to  the  organism  in 
"constant  conditions"  information  on  outside  air-temperature,  is,  through  its  tempera- 
ture-correlated fluctuations  alone,  contributing  importantly  to  the  24-hour  periodic 
metabolic  fluctuations  themselves.  In  support  of  this  hypothesis  is  the  rough  simi- 
larity in  the  average  forms  of  the  annual  fluctuations  in  the  amplitudes  of  ground- 
level  daily  temperature  change  and  metabolic  cycles.  Both,  as  average  for  the  two 
years,  showed  lowest  values  in  the  coldest  winter  months  and  highest  values  in  late 
spring  and  late  summer  to  early  fall,  with  a  summer  amplitude  reduction.  The  re- 
lationship between  these  two  phenomena  is  seen  in  Figure  10.  The  October  peak, 
so  conspicuous  for  the  metabolic  cycles,  is  much  less  evident  for  the  temperature 
changes. 

For  each  year  this  relationship  between  these  two  phenomena  appeared  to  trace 
out  general  ovoid  form.  The  two-year  mean  month-by-month  relationship  is  shown 
by  the  numerals  1  (January)  through  12  (December),  and  the  dotted  ovoid  curve 
roughly  traces  their  course.  It  is  interesting  to  speculate  that  this  difference  be- 
tween the  organismic  and  temperature  annual  cycles  may  find  its  explanation  in  the 
changing  natural  smog  content  of  the  atmosphere.  The  terpenes,  volatilized  from 


96  FRANK  A.  BROWN,  JR. 

plants,  polymerized  by  the  ultra  violet  light  from  sun,  reach  a  maximum  in  October 
(personal  communication  from  Professor  F.  W.  Went).  This  smog  may,  through 
influencing  the  amount  of  heat  absorption  from  sunlight,  produce  in  October  the 
highest  amplitude  daily  temperature  changes  of  the  year  at  levels  in  the  atmosphere 
where  temperature  changes  produce  greatest  influence  upon  the  factor  directly  af- 
fecting the  organism.  One  process,  known  to  be  temperature-dependent,  is  the  rate 
of  spontaneous  decay  of  cosmic-ray-derived  mesons.  The  larger  the  atmospheric 
depth  involved  in  the  temperature  change,  in  this  instance,  the  larger  would  be  ex- 
pected the  temperature  influence. 


O  16 

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8  10          12  14          16          18         20 

DAILY     TEMP.    RANGE     °F 


22 


24 


FIGURE  10.  The  relationship  between  mean  monthly  daily  temperature  ranges  and  the 
mean  monthly  noon  deviations  in  Oo-consumption  from  daily  trend.  The  Arabic  numerals  in- 
dicate the  mean  monthly  relationships  for  both  of  the  years,  with  January  as  1,  February  as  2, 
March  as  3,  etc. 

Now  that  it  appears  true  beyond  reasonable  doubt  that  the  living  organism,  as 
part  of  its  cyclic  geophysical  environment,  is  exhibiting  metabolic  cycles  with  the 
natural  solar  day,  lunar,  and  annual  periods,  the  question  arises  as  to  what  the  re- 
lationship of  these  metabolic  cycles  might  be  to  the  well-known  endogenous  physio- 
logical and  behavior  cycles  which  have  been  abundantly  described,  especially  dur- 
ing the  past  fifty  years  for  species  representing  the  gamut  of  the  animal  and  plant 
kingdoms.  Those  studied  experimentally  have  included  especially  the  sleep-move- 
ments of  plants,  the  spontaneous  motor  activity  of  numerous  animals,  physical  and 
chemical  adaptation  of  compound  eyes,  integumentary  color  changes,  eclosion  in 
flies,  learned  periodic  behavior  in  insects  and  lunar-tidal  rhythms  of  color-change 
and  of  motor  activity.  These  cycles  are  clearly  endogenous  in  most  instances  since 
the  forms  and  phase  relationships  of  the  cycles  relative  to  external  physical  ones 


EXOGENOUS  BIOLOGICAL  RHYTHMICITY  97 

may  be  modified  by  appropriate  treatment  of  the  organism  and  will  persist  for  a 
few,  and  often  many,  cycles.  The  endogenous  cycles  also  may  gradually  drift  away 
from  their  initial  phase  relationships  when  placed  in  constant  conditions  away  from 
cyclic  light  and  temperature  changes.  The  rate  of  drift  is  characteristically  a  func- 
tion of  the  constant  illumination,  or  constant-temperature,  level ;  brighter  light  be- 
haves usually  like  higher  temperature.  But  in  some  other  instances  the  cycles  show 
no  measurable  drift  over  long  periods,  behaving  in  their  precision,  therefore,  more 
like  the  mean  metabolic  cycles.  Also,  cycles  of  quite  other  periods  than  the  natural 
daily  ones  may  be  directly  induced  by  light,  for  example,  but  the  organism  there- 
after placed  in  constant  conditions  reverts  at  once  to  daily  cycles. 

The  characteristic  of  essential  temperature-independence  of  the  cycle-frequencies, 
whether  one  deals  with  solar-day,  lunar,  or  annual  ones,  made  it  rather  improbable 
that  the  basic  frequency-determining  mechanism  was  endogenous.  However,  one 
hypothesis  that  has  been  rather  widely  entertained  bears  the  assumption  of  the  pos- 
session by  the  organisms  of  accurate,  fully  autonomous,  endogenous  biological  clocks 
timing  all  the  natural  period  lengths.  Although  the  majority  of  investigators  have 
always  cautiously  dissociated  the  possible  clock  mechanism  from  the  endogenous 
physiological  cycles  studied  and  whose  frequencies  they  appeared  to  regulate,  some  of 
the  more  recent  supporters  of  this  hypothesis  of  an  autonomous  endogenous  clock 
have  uncritically  identified  the  fundamental  clock  system  with  each  of  the  various 
observed  endogenous  and  labile  physiological  cycles  studied.  On  the  basis  of  this 
quite  unjustified  assumption,  the  postulated  clock  is  then  usually  considered  to  have 
all  the  properties  demonstrable  for  these  clock-regulated,  and  undoubtedly  endoge- 
nous, cycles. 

None  of  the  several  hypotheses  suggested  for  the  independent  biological  clock, 
however,  have  satisfactorily  resolved  the  problem  of  the  determination,  in  an  es- 
sentially temperature-independent  manner,  the  exact  intervals  of  the  turning  points 
in  the  persisting  cycles.  So-called  endogenous  rhythms  with  single  cycles  ranging 
from  24  hours  to  a  year  in  length  cannot  readily  be  conceived  in  terms  of  any  fully 
autonomous  mechanism  based  upon  the  reaction  kinetics  of  any  biochemical,  or 
biophysico-chemical,  systems  with  which  we  are  now  familiar. 

An  alternative  hypothesis,  advanced  by  Brown  (1957b),  proposed  that  the  basic 
mechanism  of  temperature-independence  of  the  frequency  of  biological  rhythms  in 
"constant  conditions"  involves  the  operation  of  a  cyclic  exogenous  stimulus  operat- 
ing upon  a  responsive  protoplasmic  system,  and  giving  rise  in  all  cells  to  systematic 
fluctuations  containing  all  the  major  natural  periodisms  of  the  external  environment. 
These  could  then  be  readily  used  by  the  organism  in  timing  its  endogenous  cycles. 
The  latter  could  be  considered  as  bearing  any  pre-set,  fixed  lag  or  lead  relationship, 
or  even  possess  a  smoothly  and  continuously  drifting  relationship,  thus  providing 
endogenous  cycles  of  frequencies  differing  slightly  in  lengths  from  24  hours.  The 
often-reported  small  influence  of  light  and  temperature  level  on  cycle  length  in 
constant  conditions  can  well  operate  through  influencing  the  coupling  mechanism 
between  the  exogenous  basic  clocks  and  the  endogenous  organismic  rhythms. 

We  still  do  not  know  whether  the  exogenous  daily  clocks  operate  on  a  universal 
or  local-time  basis.  It  is  even  quite  possible  that  both  kinds  of  cyclic  elements  are 
present,  each  through  correlation  with  other  geophysical  factors  possessing  one  or 
other  of  these  two  cyclic  characters,  e.g.  atmospheric  electrical  potential  change  (uni- 


98  FRANK  A.  BROWN,  JR. 

versal  time)  ;  tides  of  atmospheric  pressure  (local  time).  The  existence,  however, 
of  exogenous  daily  cycles  has  been  demonstrated  by  means  of  local-time-related, 
aperiodic  phenomena,  which  are  in  good  measure  superimposed  on  the  local-time- 
related  tides  of  the  atmosphere. 

It  is  evident  that  the  solar  daily  cycle  described  in  this  research  is  clearly  of  solar- 
day  rather  than  sidereal-day  length.  The  possible  existence  of  a  cycle  of  the  latter 
period-length  was  investigated.  The  existence  of  one  was  suggested  by  the  annual 
cycle  involving  a  positive  to  negative  deviation  about  1  AM-to-midnight  linear  daily 
trend  (Fig.  2),  and  by  the  apparent  gradual  shifting  of  the  solar-day  maximum 
across  the  day  (Fig.  3).  This  day,  being  3.93  minutes  shorter  than  the  solar  day, 
would  be  expected  to  scan  the  solar  day  almost  exactly  once  each  year.  Similarities 
in  the  form  of  mean-metabolic  cycles  with  cosmic  radiation  ones  (Brown,  Webb 
and  Bennett,  1958)  and  of  metabolic  cycle  amplitude  with  background  radiation 
cycle  amplitude  (about  half  of  this  radiation  is  thought  to  be  of  cosmic-ray-origin), 
both  give  further  likelihood  for  the  existence  of  such  a  period-length  in  the  living 
organism.  The  mean,  apparent  sidereal-day  cycle  which  was  found  showed  an  am- 
plitude of  less  than  2%.  However,  in  view  of  the  extreme  closeness  of  the  period 
of  the  year  to  the  length  of  the  cycle  of  periodic  reinforcement  of  solar-day  with 
sidereal-day  cycles,  many  years  of  data  would  obviously  be  required  to  resolve  the 
problem  of  the  relative  roles  of  the  sidereal  day  and  annual  cyclicities  in  the  produc- 
tion of  the  cycle  depicted  in  Figure  1,  B. 

It  should  be  emphasized  that  the  low  correlations  obtained  in  this  study  in  no 
manner  imply  that  the  correlations  with  the  effective  agent  would  be  similarly  small. 
Correlations  between  consecutive  values  in  parallel  cycles  of  the  same  frequencies 
would  be  expected  to  be,  and  are,  much  higher.  The  latter,  however,  could  not  be 
used  to  demonstrate  the  dependence  of  one  cycle  upon  another  or  even  on  a  factor 
correlated  with  the  second.  The  cycles  of  the  effective  factor  may  be  substantially 
more  regular,  and  its  correlations  with  organismic  metabolism  of  a  much  higher 
order. 

SUMMARY 

1.  Oxygen-consumption  was  monitored  almost  continuously   in  potatoes,   So- 
latium tuberosum,  in  constant  conditions,  including  pressure,  for  more  than  two 
years.     A  paralleling  8-month  study  of  O2-consumption  in  carrots,  Dancus  carota, 
was  also  made. 

2.  The  potatoes  showed  an  essentially  bimodal  mean  daily  cycle  with  an  average 
amplitude  of  the  major  maximum  of  3.7%.     The  cycles  for  the  two  years  taken 
separately  were  very  closely  similar. 

3.  The  daily  cycle  exhibited  an  annual  cycle  of  form  change,  with  cycles  uni- 
modal,  inverted,  with  a  7  PM  maximum  in  February  and  unimodal  with  an  11  AM 
maximum    in    October.     The    intervening    months    yielded    bimodal    cycles,    with 
graded  transitional  forms. 

4.  The  daily  cycle  and  its  annual  fluctuation  in  the  carrot  resembled  in  great  de- 
tail those  obtained  concurrently  for  the  potato. 

5.  An  annual  cycle  in  average  daily  rate  of  O2-consumption  was  found  in  the 
potato.     The  cycle  was  essentially  sinusoidal  with  minimum  in  October-November 
and  maximum,  the  rate  about  doubled,  in  April-May. 


EXOGENOUS  BIOLOGICAL  RHYTHMICITY  99 

6.  Throughout  the  two  years  the  5-6-7  AM  deviation  in  (X-consuniption  from 
linear  daily  trend  was  always  correlated  with  the  2-6  AM  mean  rate  of  barometric 
pressure  change  for  the  same  morning.     The  sign  of  this  correlation  exhibited  a 
characteristic  change  twice  each  year,  once  in  the  spring  and  again  in  the  fall. 

7 .  For  the  two-year  period  of  study  the  amplitude  of  the  daily  cycles  showed  a 
linear  correlation  with  the  concurrent  outside  air-temperature,  with  the  sign  of  the 
correlation  reversing  about  57.5°  F.     With  temperature  expressed  as  deviation  from 
57.5°  F.,  the  coefficient  of  correlation  was  —  0.51  ±  0.049. 

8.  The  data  suggest  the  existence  of  a  rhythmic  component  of  sidereal-day  length 
in  the  potatoes.     Problems  in  its  final  resolution  are  discussed. 

9.  The  evidence  points  quite  conclusively  to  the  possession  by  the  organism,  even 
in  so-called  "constant  conditions,"  of  environmentally  imposed  oscillations  of  the 
natural,  daily  and  annual  periods. 

10.  The  fluctuations  in  the  still  unidentified,  external  effective  factor  appear 
importantly  influenced  by,  and  may  possibly  even  in  some  measure  determine,  me- 
teorologic  changes  of  temperature  and  pressure. 

11.  The  significance  of  these  findings  for  the  problem  of  the  mechanism  of  the 
basic  daily  and  annual  biological  "clock"  regulating  in  "constant  conditions"  the 
well-known  endogenous  organismic  rhythms  is  discussed  at  some  length. 

LITERATURE  CITED 

BROWN,    F.    A.,    JR.,    1954.     Simple,    automatic    continuous-recording    respirometer.     Rev.    Sci. 

Instr.,  25:  415^17. 
BROWN,  F.  A.,  JR.,  1957a.     Response  of  a  living  organism,  under  "constant  conditions"  including 

pressure,  to  a  barometric-pressure-correlated,  cyclic,  external  variable.     Biol.  Bull.,  112: 

288-304. 

BROWN,  F.  A.,  JR.,  1957b.     Biological  chronometry.     Amer.  Nat.,  91 :  129-133. 
BROWN,  F.  A.,  JR.,  19S7c.     An  annual  metabolic  cycle  in  an  organism  in  constant  conditions, 

including  pressure.     Anat.  Rec.,  128:  528-529. 
BROWN,   F.  A.,  JR.,    1957d.     The   rhythmic   nature  of   life.     Recent   Advances   in    Invertebrate 

Physiology ;  pp.  287-304.     Univ.  of  Oregon  Press. 

BROWN,  F.  A.,  JR.,  1958.     Physiological  rhythms.     Physiology  of  Crustacea,  Ed.  by  T.  H.  Wa- 
terman.    Academic  Press,  New  York  (in  press). 
BROWN,  F.  A.,  JR.,  M.  F.  BENNETT  and  H.  M.  WEBB,  1958.     Monthly  cycles  in  an  organism  in 

constant  conditions  during  1956  and  1957.     Proc.  Nat.  Acad.  Sci.,  44:  290-296. 
BROWN,  F.  A.,  JR.,  R.  O.  FREELAND  AND  C.  L.  RALPH,  1955.     Persistent  rhythms  of  O2-consump- 

tion  in  potatoes,  carrots,  and  the  seaweed,  Fucus.     Plant  Physiol.,  30 :  280-292. 
BROWN,  F.  A.,  JR.,  J.  SHRINER  AND  H.  M.  WEBB,  1957.     Similarities  between  daily  fluctuations 

in  background  radiation  and  O  -consumption  in  the  living  organism.     Biol.  Bull.,  113: 

103-111. 
BROWN,  F.  A.,  JR.,  H.  M.  WEBB  AND  M.  F.  BENNETT,  1958.     Comparisons  of  some  fluctuations 

in  cosmic  radiation  and  in  organismic  activity  during  1954,  1955,  and  1956.     Amcr.  J. 

Physiol.  (in  press). 
BROWN,  F.  A.,  JR.,  H.  M.  WEBB  AND  E.  J.  MACEY,  1957.     Lag-lead  correlations  of  barometric 

pressure  and  biological  activity.     Biol.  Bull.,  113:  112-119. 

BRUCE,  V.  G.,  AND  C.  S.  PITTENDRIGH,   1957.     Endogenous   rhythms  in  insects  and  microor- 
ganisms.   Amer.  Nat.,  91 :  179-195. 
BUNNING,    E.,    1936.     Die   Entstehung   der    Variationsbewegungen   bei    den    Pflanzen.     Ergeb. 

Biol,  13 :  235-347. 

BUNNING,  E.,  1956a.     Endogenous  rhythms  in  plants.     Ann.  Rev.  Plant  Physiol.,  7 :  71-90. 
BUNNING,  E.,  1956b.     Endogene  Aktivitatsrhythmen.     Encycl.  Plant  Physiol.,  Berlin,  2  :  878- 

907. 


100  FRANK  A.  BROWN,  JR. 

BUNNING,    E.,    AND    E.    W.    BAUER,    1952.     t)ber    die    Ersachen    endogener    Keimfahigkeits- 

schwankungen  in  Samen.     Zeitschr.  Bot.,  40:  67-76. 
BUNKING,  E.,  AND  L.  MUSSLE,  1951.     Der  Verlauf  der  endogenen  Jahresrhythmik  in   Samen 

unter   dem    Einfluss    verscheidenartiger    Aussenfaktoren.     Zeitschr.    Natnrforsch.,    6b : 

108-112. 
CALHOUN,    J.    B.,    1944.     Twenty-four    hour    periodicities    in    the    animal    kingdom.     Part    I. 

The  Invertebrates.     /.  Tenn.  Acad.  Sci.,  19:  179-200;  252-262. 
CALHOUN,  J.  B.,  1945-1946.     Twenty-four  hour  periodicities  in  the  animal  kingdom.     Part  II. 

The  Vertebrates.     /.  Tenn.  Acad.  Sci..  20:  228-232;  291-308;  373-378.     21:  208-216, 

281-282. 
CASPERS,   H.,   1951.     Rhythmische   Erscheinungen   in   der    Fortpflanzung    von    Clunio    marinus 

(Dipt.   Chiron.)    und   das   Problem   der   lunaren   Periodizitat   bei    Organismen.     Arch. 

Hydrobiol,  Suppl,  18:  415-594. 

CLOUDSLEY-THOMPSON,  J.  L.,  1953.     Diurnal  rhythms  in  animals.     Sci.  News,  28:  76-98. 
JORES,  A.,  1937.     Die  24-Stunden-Periodik  in  der  Biologic.     Tab.  Bio!.,  14:  17-109. 
KALMUS,  H.,   1938.     Periodizitat  und  Autochronie  als  Zeitregelnde   Eigenschaften   bei   Mensch 

und  Tier.     Tab.  Biol.,  26  :  60-109. 

KLEITMAN,  N.,  1949.     Biological  rhythms  and  cycles.     Physiol.  Rev.,  29 :  1-30. 
KORRINGA,  P.,  1947.     Relations  between  the  moon  and  periodicity  in  the  breeding  of  marine 

animal.     Ecol.  Monogr.,  17:  347-381. 

PARK,  O.,  1940.     Nocturnalism — The  development  of  a  problem.    Ecol.  Monogr.,  10 :  487-536. 
WEBB,  H.  M.,  1950.     Diurnal  variations  of  response  to  light  in  the  fiddler  crab,  Uca.     Physiol. 

Zodl,  23 :  316-337. 
WELSH,  J.  H.,  1938.     Diurnal  rhythms.     Quart.  Rev.  Biol,  13 :  123-139. 


SULFURIC  ACID  IN  DESMARESTIA 

RICHARD  W.  EPPLEY  L  "-  AND  CARLTON  R.  BOVELL  a,  * 
Hopkins   Marine   Station    of   Stanford    University,   Pacific    Grove,    California 

Several  species  of  Desmarestia  accumulate  acid  in  their  tissues  (Blinks,  1951). 
Kylin  (1938)  first  considered  malic  acid  to  be  responsible  for  the  low  pH  values 
of  expressed  Desmarestia  sap.  But  Wirth  and  Rigg  (1937)  and  Meeuse  (1956) 
identified  the  acid  as  sulfuric.  The  titration  curve  of  Desmarestia  sap  is  that  of  a 
strong  acid ;  the  pH  may  be  as  low  as  0.78  and  sulfate  ions  are  present  in  high  con- 
centration. 

In  a  review,  Blinks  (1951)  pointed  out  a  correlation  between  the  thickness  and 
relative  surface  area  of  three  California  species  of  Desmarestia  and  their  acid  content. 
The  pH  of  the  sap  of  Desmarestia  mnnda,  a  species  with  thick  blades,  is  one  or  less; 
D.  herbacea  has  somewhat  narrower  and  thinner  blades  and  its  sap  has  a  pH  of  two 
to  three ;  and  D.  latifrons,  the  most  delicately  branched  of  the  three  species,  is  only 
weakly  acid  with  sap  of  approximately  pH  five.  Blinks  also  stated  that  the  locality 
of  the  acid  within  the  algal  tissues  is  unknown,  and  suggested  that  it  may  occur  in 
the  cell  vacuoles.  He  found  that  the  outer  cell  membranes  were  injured  by  acid, 
and  thus  it  seems  unlikely  that  the  acid  is  free  in  the  cytoplasm. 

Kylin  (1938)  stained  Desmarestia  cells  with  the  vital  dye,  brilliant  cresyl  blue, 
and  observed  that  the  vacuoles  appeared  purple.  He  concluded  that  the  vacuolar 
contents  were  alkaline,  because  the  dye  changes  from  blue  to  reddish-violet  from 
pH  7.0  to  7.5.  He  apparently  overlooked  that  fact  that  the  dye  also  changes  from 
blue  to  purple  from  pH  1.0  to  pH  0.7.  So  his  conclusion  that  the  vacuoles  are  alka- 
line may  be  questioned.  It  seems  more  likely  that  the  vacuoles  are  strongly  acidic. 
This  report  provides  additional  evidence  that  hydrogen  ion  accounts  for  a  large  part 
of  the  vacuolar  cation  content  of  the  acid-accumulating  species  of  Desmarestia. 

METHODS  AND  MATERIALS 

Desmarestia  mitnda  and  D.  herbacca  were  collected  at  Pebble  Beach,  California. 
For  comparison,  two  non  acid-accumulating  brown  algae,  Egregia  laei'igata  and 
Dictyoneurum  calif  ornictim,  were  also  collected.  The  plants  were  kept  in  running 
sea  water  in  the  laboratory  before  use. 

The  blades  were  washed  free  of  sea  water  in  0.6  M  sucrose  for  five  minutes  and 
extracted  with  five  per  cent  trichloracetic  acid.  Sodium  and  potassium  contents  of 
the  extracts  were  determined  by  flame  photometry.  Total  acidity  was  determined 
by  titration  of  hot  water-extracts  of  the  algae. 

1  Present  address :  Department  of  Biology,  University  of  Southern  California,  University 
Park,  Los  Angeles  7,  California. 

-  Public  Health  Service  Research  Fellow  of  the  National  Heart  Institute. 

3  Present  address :  Department  of  Biology,  University  of  California,  Riverside,  California. 

4  National  Science  Foundation  Post-Doctoral  Fellow. 

101 


102 


RICHARD  W.  EPPLEY  AND  CARLTON  R.  BOVELL 


Amounts  of  adsorbed  cations  were  assumed  to  be  the  same  for  living  and  dead 
tissues,  hence  the  values  determined  for  killed  tissues  were  subtracted  from  those  for 
living  tissues.  The  difference  may  represent  the  intracellular  cation  content.  The 
apparent  osmotic  volume  of  the  tissues  (Briggs  and  Robertson,  1957)  was  estimated 
as  the  difference  in  sucrose  apparent  free  space  of  living  and  killed  tissues  (Eppley 
and  Blinks,  1957).  The  tissues  were  killed  by  boiling  or  by  soaking  in  fifty  per 
cent  ethanol.  Ion  concentrations  are  expressed  on  the  basis  of  the  estimated  osmotic 
volumes. 

Tissues  were  placed  in  a  Waring  Blendor  in  0.8  M  "tris"  buffer  (tris  hydroxy- 
methyl  amino  methane-HCl),  initial  pH  approximately  8.5,  blended  for  five  minutes, 
and  filtered  through  cheesecloth  for  use  in  methylene  blue  reduction  experiments. 

TABLE  I 

The,  effects  of  inhibitors  of  selective  permeability  on  the  rate  of  acid-loss  into  the  medium  of  Desmarestia 
munda  tissues  as  determined  by  the  time  required  for  color  change  of  methyl  orange. 

Tissues  in  aerated  sea  water 


Expt.  1 

Minutes  for 
color  change 

Expt.  2 

Minutes  for 
color  change 

no  inhibitor 

>60 

no  inhibitor 

>60 

p-chloromercuribenzoate 

12 

p-chloromercuribenzoate 

14 

(0.0005  M) 

16 

(0.0005  Af) 

17 

NaCX  (0.001  J/) 

10 

12 

iodoacetate  (0.002  M) 

2 

1 

RESULTS 

Several  experiments  indicate  that  the  acid  is  within  the  cells,  yet  not  free  in 
the  cytoplasm.  Tissue  extracts  were  capable  of  reducing  methylene  blue  with  a 
variety  of  substrates,  under  nitrogen,  only  if  buffered  near  neutrality.  No  activity 
was  noted  in  preparations  in  which  the  pH  of  the  extract  was  less  than  five  or  if  the 
tissues  were  homogenized  in  unbuffered  sea  water.  Rates  of  dye  reduction  were 
somewhat  greater  in  the  presence  of  ribose  and  aspartate  than  with  other  substrates. 
Some  activity  was  also  present  in  buffered  extracts  with  no  substrate  added,  but 
quantitative  studies  were  not  made. 

In  other  experiments,  discs  of  the  thalli,  cut  out  with  a  two-cm,  cork  borer,  were 
tested  for  acid  loss  in  sea  water  in  the  presence  and  absence  of  inhibitors.  Samples 
of  five  or  ten  discs  were  placed  in  ten  ml.  of  sea  water  containing  a  drop  of  methyl 
orange,  and  the  time  required  for  a  color  change  of  the  indicator  was  recorded.  The 
rate  of  acid  loss  was  much  greater  in  neutralized  sea  water  in  the  presence  of 
0.0005  M  p-chloromercuribenzoate,  0.001  M  sodium  cyanide,  and  0.002  M  iodo- 
acetate than  in  sea  water  alone  (Table  I). 

Rates  of  acid  loss  in  dinitrophenol  (0.005  M)  were  determined  by  measuring  the 
pH  of  the  solution.     The  curve  resulting  from  a  plot  of  pH  against  time  (Fig.  1) 
suggests  an  autocatalytic  reaction.     This  autocatalytic  injury  is  implicit  in  Blinks' 
(1951)  description  of  the  rates  of  carotenoid  color  change  in  Desmarestia  as  the 
alga  dies. 


SULFURIC  ACID  IN  DESMARESTIA 


103 


8 


PH 


Control 


DNP 


20 


40 
MINUTES 


60 


FIGURE  1.     Acid  release  by  Desmarestia  munda  tissues  in  sea  water  (control)  and  in  sea  water 

containing  0.0005  M  dinitrophenol   (DNP). 

On  accumulation  of  the  dye,  brilliant  cresyl  blue,  the  vacuoles  of  D.  munda  and 
D.  herbacea  are  stained  purple  in  confirmation  of  Kylin's  results  (1938).  How- 
ever, we  feel  the  color  to  be  indicative  of  the  change  at  pH  1.0-0.7,  rather  than  7.0— 
7.5. 

If  the  acid  is  localized  within  the  vacuoles,  one  might  expect  the  cations  normally 
found  in  the  vacuoles  of  brown  algae  to  be  replaced  by  hydrogen  ions.  In  Egregia 
laevigata  and  Dictyoneurum  californicum  (Table  II)  potassium  is  the  most  abun- 
dant cellular  cation  measured.  It  occurs  at  a  concentration  approximately  isotonic 
with  sea  water.  In  D.  munda  about  75  per  cent  of  the  potassium  is  replaced  by  hy- 

TABLE  II 

Potassium,  sodium,  and  hydrogen  ion  contents  of  Desmarestia  munda,  D.  herbacea,  and  two  non-acid- 
accumulating  species  of  brown  algae.      Values  are  corrected  for  the  ion  contents  of  killed 
tissues  and  represent  averages  of  four  determinations.      Units  milli-equivalents /liter 

estimated  cell  osmotic  volume 


Alga 

est.  cell. 

H 

K 

Na 

Sum 

osm.  vol. 

Desmarestia  munda 

84% 

438 

148 

— 

586 

Desmarestia  herbacea 

69% 

254 

234 

13 

501 

Dictyoneurum  californicum 

63% 

— 

523 

21 

544 

Egregia  laevigata 

71% 

— 

542 

45 

587 

104 


RICHARD  W.  EPPLEY  AND  CARLTON  R.  BOVELL 


drogen,  and  about  50  per  cent  is  replaced  in  D.  hcrbacea  (Fig.  2).  The  reciprocity 
of  potassium  and  hydrogen  ion  concentrations  agrees  with  the  above  mentioned  ex- 
pectation. The  approximation  of  the  total  cation  concentration  among  the  four 
brown  algal  species  to  that  of  sea  water  suggests  that  most  of  the  cation  content  is 
accounted  for,  although  magnesium  and  calcium  were  not  measured  and  may  be 
present. 


100 


UJ 
CD 
O 

cr 

Q 


50 


0        D.  mundo 


Ov     D.  herbaceo 


other 
browns 


50 


100 


POTASSIUM 


FIGURE  2.  Hydrogen  and  potassium  ion  contents  of  Desmarestia  inunda,  D.  herbacea,  and 
two  other  brown  algae :  Egregia  laevigata  and  Dictyoneumm  califoniicum.  Units  :  per  cent  of 
total  cation  content  determined. 

The  binding  of  large  amounts  of  sodium  by  dead  tissues  was  detected.  This 
may  represent  adsorption  of  the  cation  to  the  carboxyl  groups  of  alginic  acid,  a 
structural  polysaccharide  of  the  brown  algae  (Wasserman,  1949). 


DISCUSSION 

The  vacuoles  of  Desmarestia  contain  sulfuric  acid  in  amounts  up  to  0.44  N,  in 
D.  inunda.  Direct  evidence  for  this  view  is  the  purple  color  of  brilliant  cresyl  blue 
accumulated  by  the  vacuoles  of  D.  inunda  and  D.  herbacea.  Indirect  supporting 
evidence  includes  the  following :  1 )  The  acid  is  lost  more  rapidly  on  exposure  of 
tissues  to  inhibitors  which  abolish  selective  membrane  permeability  than  it  is  in  the 
absence  of  such  inhibitors.  In  this  group  are  sodium  cyanide,  iodoacetate,  p-chloro- 


SULFURIC  ACID  IN  DESMARESTIA  105 

mercuribenzoate,  and  dinitrophenol.  2)  The  autocatalytic  release  of  acid  in  the 
presence  of  dinitrophenol  suggests  that  extra-vacuolar  acid  injures  the  cells,  caus- 
ing an  increasing  rate  of  acid  release.  3)  Oxidative  metabolism  is  sensitive  to  high 
hydrogen  ion  concentrations  as  evidenced  by  the  inability  of  tissue  extracts  to  re- 
duce methylene  blue  in  unbuffered  suspensions.  4)  The  reciprocity  of  potassium 
and  hydrogen  ion  concentrations  among  the  brown  algae  tested  suggests  that  hy- 
drogen replaces  potassium  as  the  most  abundant  cellular  cation  in  D.  munda,  and 
that  about  one-half  of  the  potassium  is  replaced  in  D.  herbacea. 

The  tonoplasts  of  Desmarestia  cells  must  be  quite  unique  in  their  resistance  to 
acid  injury,  and  in  their  permeability  characteristics.  A  hydrogen  ion  concentration 
gradient  of  about  107  is  apparently  maintained  between  the  vacuolar  sap  and  sea 
water.  However,  the  sea  water  is  probably  not  the  "substrate"  for  hydrogen  ion  ac- 
cumulation. Metabolically  produced  hydrogen  in  the  cytoplasm  may  well  be  the 
source  for  vacuolar  accumulation.  Efforts  to  leach  the  acid  from  the  cells  so  that 
the  progress  of  acid  reaccumulation  could  be  studied  have  not  been  successful.  The 
cells  are  killed  as  the  acid  is  released. 

The  production  of  hydrogen  ion  due  to  anaerobic  conditions  in  the  interior  cells 
of  massive  species  of  Desmarestia  may  explain  Blinks'  (1951)  observation  of  a  cor- 
relation between  tissue  massiveness  and  acid  content.  The  interior  cells  of  D. 
munda  are  much  larger,  contain  fewrer  plastids,  and  show  a  greater  percentage  of 
purple  vacuoles,  on  staining  with  brilliant  cresyl  blue,  than  the  peripheral  cells  or 
the  cells  of  D.  herbacea. 

The  high  acidity  of  Desmarestia  cells  may  limit  the  vertical  distribution  of  the 
alga  in  the  intertidal  zone.  Because  injury  spreads  so  rapidly  when  water  circula- 
tion is  poor,  it  seems  reasonable  that  the  acid-accumulating  species  must  be  confined 
to  regions  of  constant  water  circulation.  Desmarestia  herbacea  occurs  below  the 
lowest-lower-low-water  tide  mark  (Doty,  1946)  and  D.  munda  is  limited  to  the 
lower  portion  of  the  intertidal  zone  (Smith,  1944). 

SUMMARY 

1.  Brilliant  cresyl  blue  accumulates  in  the  vacuoles  of  Desmarestia  munda  and 
D.  herbacea  and  the  accumulated  dye  appears  purple,  indicating  that  the  pH  of  the 
vacuolar  sap  is  less  than  1.0  or  greater  than  7.5.     However,  the  expressed  saps  of 
these  two  brown  algae  have  pH  1.0  or  less  and  about  2.0,  respectively.     The  outer 
cell  membranes  are  injured  by  the  low  pH  of  the  sap  and  methylene  blue  is  not  re- 
duced by  tissue  homogenates  at  such  low  pH  values. 

2.  Sodium  cyanide,  dinitrophenol,  iodoacetate,  and  p-chloromercuribenzoate  in- 
duce the  release  of  acid  from  the  cells,  in  which  potassium,  normally  the  cation  most 
abundant  in  brown  algal  cells,  is  largely  replaced  by  hydrogen.     In  D.  munda  hy- 
drogen accounts  for  75  per  cent  of  the  intracellular  cation  content.     Tissue  sodium 
is  largely  bound  and  contributes  little  to  the  cellular  cation  content. 

3.  The  simplest  interpretation  of  these  data  is  that  the  acid  is  localized  within 
the  vacuoles  of  Desmarestia  cells. 

LITERATURE  CITED 

BLINKS,  L.  R.,  1951.     Physiology  and  biochemistry  of  algae.     In:  Manual  of  Phycology  (G.  M. 
Smith,  editor).     Chronica  Botanica  Co.,  Waltham.  Mass.;  pp.  263-91. 


106  RICHARD  W.  EPPLEY  AND  CARLTON  R.  BOVELL 

BRIGGS,  G.  E.,  AND  R.  N.  ROBERTSON,  1957.     Apparent  free  space.     Ann.  Rev.  Plant  Physio!., 

8:  11-30. 
DOTY,  MAXWELL,  1946.     Critical  tide  factors  that  are  correlated  with  the  vertical  distribution 

of  marine  algae  and  other  organisms  along  the  Pacific  Coast.     Ecology,  27:  315-328. 
EPPLEY,  R.  W.,  AND  L.  R.  BLINKS,  1957.     Cell  space  and  apparent  free  space  in  the  red  alga, 

Porphyra  pcrforata.     Plant  PhysioL,  32 :  63-64. 
KYLIN,  HARALD,  1938.     t)ber  die  Konzentration  der  Wasserstofinonen  in  den  Vakuolen  einiger 

Meeresalgan.     I'drh.  Kgl.  Fysiograf.  Sallsk.  Lund,  8 :  194-204. 
MEEUSE,  B.  J.  D.,  1956.     Free  sulfuric  acid  in  the  brown  alga,  Dcsmarcstia.     Biochhn.  Biophys. 

A  eta,  19:  372-374. 

SMITH,  G.  M.,  1944.     Marine  Algae  of  the  Monterey  Peninsula.     Stanford  Univ.  Press,  Stan- 
ford, Calif. ;  622  pp. 
WASSERMAN,  A.,  1949.     Cation  adsorption  by  brown  algae.     The  mode  of  occurrence  of  alginic 

acid.     Annals  Bot.,  13 :  79-88. 
WIRTH,  H.  E.,  AND  G.  B.  RIGG,  1937.     The  acidity  of  the  juice  of  Dcsmarcstia.     .hncr.  J.  Bot., 

24 :  68-70. 


THE  SENSITIVITY  OF  ECHOLOCATION  IN  THE  FRUIT  BAT, 

ROUSETTUS 

D.  R.  GRIFFIN,  A.  NOVICK  1  AND  M.  KORNFIELD  2 
Biological  Laboratories,  Harvard  University,  Cambridge  38,  Massachusetts 

Moehres  and  Kulzer  (1956b)  have  reported  that  among  the  Megachiroptera 
(Old  World  fruit  bats  and  flying  foxes)  the  genus  Pier  opus  orient  visually  while 
Rousettus  aegypticus  orient  visually  but  also  echolocate.  Six  additional  mega- 
chiropteran  genera,  Eidolon,  Cynopterus,  Ptenochirus,  Lissonycteris,  Eonycteris, 
and  Macroglossus,  have  all  proved,  like  Pteropus,  to  orient  visually  and  not  acousti- 
cally. Observations  of  two  additional  species  of  Rousettus,  R.  amplexicaudatus  and 
R.  seminudus  as  well  as  R.  aegypticus,  have  confirmed  Moehres  and  Kulzer's  con- 
clusions (Novick,  1958).  Rousettus  generate  clicks  by  movements  of  the  tongue 
and  emit  these  through  the  open  corners  of  the  mouth  (Kulzer,  1956)  rather  than 
producing  sounds  laryngeally  as  do  the  Microchiroptera  (Griffin,  1946,  1952;  No- 
vick, 1955;  Griffin,  1958). 

As  far  as  is  known  at  present  all  of  the  Megachiroptera  except  Rousettus  are 
helpless  in  total  darkness.  Rousettus  apparently  make  use  of  vision  and/or  echolo- 
cation  depending  upon  the  light  conditions,  the  difficulty  of  their  flight  path,  and 
the  type  of  flight  required  (take-offs  and  landings,  for  example).  The  echolocation 
system  used  by  Rousettus  has  almost  surely  evolved  independently  of  the  system 
employed  by  the  Microchiroptera.  Furthermore,  it  resembles  in  design  the  system 
serving  much  the  same  purpose  in  the  cave-dwelling  birds,  Steatornis  and  Collocalia. 
The  isolation  of  these  three  natural  sonars  in  single  genera,  their  simple  designs, 
and  their  facultative  employment  (all  three  genera  orient  visually  in  adequate  light) 
make  it  seem  likely  that  they  are  recent  developments  compared  with  undoubtedly 
ancient  microchiropteran  echolocation  systems.  There  is,  therefore,  considerable 
interest  in  comparing  the  effectiveness  of  the  echolocation  system  of  Rousettus  in  the 
detection  of  small  objects  with  that  achieved  by  the  Microchiroptera,  especially  some 
carefully  studied  species  of  the  families  Vespertilionidae  and  Phyllostomatidae 
(Curtis,  1952;  Griffin  and  Novick,  1955;  Grinnell  and  Griffin,  1958). 

Since  the  orientation  clicks  of  Rousettus,  Steatornis,  and  Collocalia  are  clearly 
audible  to  man,  they  obviously  contain  more  energy  at  frequencies  below  20  kc  than 
do  the  orientation  pulses  of  most  of  the  Microchiroptera.  The  principal  compo- 
nent in  Rousettus  clicks  is  between  12  and  18  kc,  depending  upon  the  species  and 
the  individual,  but  overtones  and  harmonics  are  present  to  a  considerable  degree 
(Novick,  1958).  Saccopteryx  and  Taphosous  (Emballonuridae)  and  some  species 
of  Tadarida  (Molossidae)  emit  partly  audible  orientation  cries.  Rhino poma  also 
emit  orientation  pulses  with  audible  components  (Moehres  and  Kulzer,  1956a). 
Rousettus,  Steatornis,  and  Collocalia,  though,  unlike  all  of  the  Microchiroptera, 

1  Present  address:  Osborn  Zoological  Laboratory,  Yale  University,  New  Haven  11, 
Connecticut. 

-  Present  address :  New  York  University-Bellevue  Medical  Center,  New  York,  N.  Y. 

107 


108  D.  R.  GRIFFIN,  A.  NOVICK  AND  M.  KORNFIELD 

produce  clicks  with  relatively  little  energy  above  20  kc.  Thus,  it  appeared  that  only 
relatively  long  wave-lengths  would  be  available  for  echolocation  and  that  Rouscttus 
and  the  two  cave-dwelling  birds  might  be  unable  to  detect  obstacles  as  small  as  the 
wires  that  had  been  used  as  standardized  test  objects  for  the  Microchiroptera 
(Hahn,  1908;  Griffin  and  Galambos,  1941 ;  Griffin  and  Novick,  1955;  and  Grinnell 
and  Griffin,  1958). 

A  single  male  Rouscttus  acgypticus,  captured  in  a  dimly  lighted  cave  at  Eaux 
Chaudes,  Katana,  Kivu  Province,  Belgian  Congo  in  July,  1956,  was  brought  to 
Harvard  University  in  good  health  in  August,  1956.  This  bat  survived  for  nine 
months  on  a  diet  of  bananas  and,  after  a  short  period  of  recuperation  from  its  jour- 
ney and  its  restriction  to  a  small  cage,  flew  skillfully  in  an  experimental  flight  room 
32'  long,  12'  wide,  and  8'  high.  Its  ability  to  avoid  a  variety  of  cylindrical  test  ob- 
stacles arranged  in  a  row  across  the  center  of  this  room  was  tested  by  methods  di- 
rectly comparable  with  those  previously  used  to  measure  obstacle-avoiding  skill  in 
the  Microchiroptera.  This  Rouscttus  proved  able  to  avoid  surprisingly  small  wires 
even  in  total  darkness.  Its  skill  is  here  compared  with  that,  measured  previously, 
of  the  vespertilionid,  Myotis  I.  liicifiigus  (Curtis,  1952). 

This  work  was  partly  sponsored  by  the  Office  of  Naval  Research,  the  United 
States  Public  Health  Service,  and  the  Belgian  American  Education  Foundation. 
During  this  time,  Novick  held  a  Post-doctoral  Fellowship  of  the  National  Institute 
of  Neurological  Diseases  and  Blindness.  We  are  grateful  to  the  personnel  of  the 
Institut  pour  la  Recherche  Scientifique  en  Afriquc  Ccntrale,  Lwiro,  Belgian  Congo 
for  their  help  in  capturing  the  experimental  subject.  Reproduction  of  this  paper 
in  whole  or  in  part  is  permitted  for  any  purpose  of  the  United  States  government. 

METHODS 

After  the  bat  had  become  accustomed  to  the  problems  of  flight  both  in  light  and 
in  total  darkness  in  the  flight  room,  and  to  the  task  of  dodging  between  vertical  ob- 
stacles suspended  from  the  ceiling  across  the  middle  of  the  room,  we  tested  its  ability 
to  avoid  cylindrical  obstacles,  spaced  53  cm.  apart,  varying  in  size  from  cardboard 
tubes  5  cm.  in  diameter  to  bare  metal  wires  0.28  mm.  in  diameter.  In  each  case 
these  obstacles  were  suspended  in  a  movable  frame  in  a  plane  parallel  to  the  end  walls 
of  the  room.  This  plane  had  to  be  crossed  by  the  bat  in  flying  from  its  roost  at  one 
end  to  its  roost  at  the  other  end.  We  forced  such  flights  by  agitating  the  roost 
which  was  a  loosely  suspended  horizontal  bar  of  wood.  The  bat  would  take  off 
and  fly  the  length  of  the  room  to  the  opposite  roost  or  would,  on  occasion,  make  sev- 
eral flights  back  and  forth  before  landing.  In  each  of  the  tests  considered  below, 
the  frame  holding  the  obstacles  was  shifted  horizontally  in  the  dark  just  before  each 
flight  so  that  the  absolute  position  of  the  obstacles  and  their  location  relative  to  the 
walls  were  unknown  to  the  bat,  though  their  position  relative  to  one  another  was 
constant.  Thus,  the  bat  could  not  learn  the  location  of  the  open  spaces  nor  could 
it  depend  on  following  the  walls  because  the  space  adjacent  to  the  walls  was  fre- 
quently and  randomly  too  narrow  to  permit  passage.  The  room  was  totally  dark 
during  all  these  observations,  but  we  often  noticed  by  listening  to  the  bat's  audible 
clicks  or  to  its  wingbeats  that  it  hesitated  in  front  of  the  obstacles  and  executed 
dodging  maneuvers  to  pass  between  them. 


ECHOLOCATION  IN  ROUSETTUS 


109 


The  Rousettus  was  thus  required  to  fly  through  an  obstacle  plane  and  its  ac- 
curacy of  echolocation  was  evaluated  in  terms  of  its  ability  to  avoid  the  obstacles. 
One  must  consider  whether  it  was  constantly  and  equally  motivated  to  avoid  col- 
lisions and  whether  its  physical  agility  was  sufficient  for  it  to  make  the  maximum 
use  of  its  orientation  system.  The  flights  were  scored  simply  as  hits  or  misses  by 
means  of  the  sound  of  hits  or  in  doubtful  cases  by  inspecting  the  obstacles  in  light 
switched  on  immediately  after  the  bat's  passage.  A  hit  always  caused  a  clearly  visi- 
ble, sustained  vibration  of  the  obstacles  as  they  were  suspended  from  rubber  bands. 
All  hits  were  considered  equal  although  some  undoubtedly  represented  the  bat's 

TABLE  I 

Comparison  of  the  obstacle  avoidance  scores  of  a  Rousettus  aegypticus  with  those  of  Myotis  I.  lucifugus 

(Curtis,  1952}.      The  wires  or  other  cylindrical  obstacles  were  arranged  vertically  and 

spaced  53  cm.  apart  for  Rousettus  and  30  cm.  apart  for  Myotis 


Diameter  of  obstacle 

(mm.) 

Myotis  I.  lucifugus 

Rousettus  aegypticus 

No.  trials 

%  misses 

No.  trials 

%  misses 

Cardboard  tubes 

25 



— 

109 

76% 

Rubber  tubing 

19 

— 

— 

161 

78% 

Rubber  tubing 

13 

— 

— 

100 

77% 

Rubber  tubing 

6 

— 

— 

50 

80% 

Metal  rods 

4.76 

140 

85% 

— 

— 

Insulated  metal  wires    3 

— 

— 

442 

85% 

Bare  metal  wire 

1.5 

— 

— 

200 

77% 

Bare  metal  wire 

1.21 

3820 

82% 

— 

— 

Bare  metal  wire 

1.07 

— 

— 

280 

68% 

Bare  metal  wire 

0.68 

480 

77% 

— 

— 

Bare  metal  wire 

0.65 

— 

— 

225 

58% 

Bare  metal  wire 

0.46 

— 

— 

134 

45% 

Bare  metal  wire 

0.35 

660 

72% 

— 

— 

Bare  metal  wire 

0.28 

— 

— 

50 

18% 

Bare  metal  wire 

0.26 

600 

52% 

— 

— 

Bare  metal  wire 

0.12 

530 

38% 

— 

— 

Bare  metal  wire 

0.07 

460 

36% 

— 

— 

inability  to  maneuver  successfully  even  though  it  had  detected  the  obstacle,  and 
some  represented  light  touches  by  the  wingtips  which  may  have  been  sufficiently 
painless  to  call  for  no  great  effort  to  avoid  their  occurrence.  Unlike  the  Micro- 
chiroptera,  this  Rousettus  rarely  turned  back  from  the  obstacles.  Its  position  and 
attitude  in  passing  through  the  obstacle  plane  were  recorded  on  about  40  flights 
with  a  camera  and  electronic  flash.  All  wing  positions  from  completely  spread  to 
considerably  folded  were  photographed  both  just  before  and  just  after  passage 
through  the  barrier,  but  we  could  not  determine  whether  the  bat  was  reducing  its 
potential  collision  diameter  just  at  the  obstacle  plane.  Its  maximum  wingspread 
was  about  75  cm.,  and  while  we  cannot  accurately  estimate  its  mean  wingspread 
this  must  have  been  at  least  45  cm.  or  very  little  below  the  spacing  between  the  wires. 
Finally,  the  possibility  that  the  bat  would  detect  the  presence  of  the  obstacles  by 
their  fastenings  to  the  ceiling  and/or  floor  and  learn  that  they  were  suspended  ver- 


110 


D.  R.  GRIFFIN,  A.  NOVICK  AND  M.  KORNFIELD 


tically  between  these  two  points  was  excluded  by  framing  the  obstacle  plane  with 
uniform  fiberboard  so  that  only  the  obstacles  themselves  and  not  their  fastenings 
were  exposed  to  acoustic  or  visual  inspection.  As  a  last  precaution,  lest  the  bat 
learn  to  recognize  the  position  of  the  obstacles  by  listening  to  the  movement  of  the 
frame  between  flights,  the  readjustment  was  covered  with  loud  noise.  The  nature 
and  size  of  the  obstacles  used  are  shown  in  Table  I. 


TABLE  II 

Experiments  with  a  captive  Rousettus  exposed  to  thermal  noise  while  flying  through  a  row  of  vertical 

wires,  3  mm.  in  diameter  spaced  53  cm.  apart.     All  flights  in  total  darkness  except  as  noted.     The 

noise  was  filtered  with  high  pass  (HP)  or  low  pass  (LP)  electronic  filters  as  noted 


Date 

Conditions  of  test 

No.  misses/No,  trials 

Per  cent 
misses 

Remarks 

Apr.  23 

Quiet 

30/40 

75% 

Noise,  25  kc  HP 

0/10 

0 

Totally  disoriented 

Quiet 

17/20 

85% 

Noise,  25  kc  HP  with 

lights  on 

9/10 

90% 

Flew  normally 

Noise,  15  kc  LP 

4/10 

40% 

Somewhat  disoriented 

Quiet 

7/10 

70% 

Noise,  15  kc  LP 

3/10 

30% 

Disoriented,   but    less   so 

than  at  25  kc  HP 

Quiet 

10/10 

100% 

Apr.  26 

Quiet 

10/10 

100% 

Reluctant  to  fly- 

Noise,  25  kc  HP 

0/8 

0 

Badly  disoriented 

Quiet 

6/6 

100% 

Very  tired 

Apr.  28 

Quiet 

8/10 

80% 

Noise,  15  kc  LP 

1/10 

10% 

Badly  disoriented 

Quiet 

4/10 

40% 

Tired 

May  3 

Died 

Averages 

Quiet 

93/116 

79% 

of  all 

Noise,  25  kc  HP 

0/18 

0 

tests 

Noise,  25  kc  HP  with 

lights  on 

9/10 

90% 

Noise,  15  kc  LP 

8/30 

27% 

RESULTS 

The  results  are  presented  in  tabular  form.  The  only  data  excluded  from  con- 
sideration are  those  which  were  obtained  when  the  bat  was  clearly  fatigued  or  in 
poor  condition  near  the  end  of  a  long  series  of  trials  or  after  many  days  of  inac- 
tivity. The  data  are  compared  directly  in  Table  I  with  similar  data  obtained  by 
Curtis  (1952)  with  Myotis  I.  lucifugus. 

A  short  series  of  experiments  was  carried  out  to  compare  the  resistance  of 
Rousettus  to  interference  with  its  echolocation  by  thermal  noise  but  before  further 
studies  could  be  completed  the  bat  died,  possibly  of  injuries  sustained  in  these  ex- 
perimental flights.  The  data  are  shown  in  Table  II,  because  they  indicate  a  mark- 


ECHOLOCATION  IN  ROUSETTUS  111 

edly  greater  vulnerability  to  interference  by  noise  than  occurs  with  the  Vespertilioni- 
dae  (Griffin,  1958).  Thermal  noise  was  generated  in  20  electrostatic  loudspeakers 
adjacent  to  the  plane  of  obstacles.  This  noise  was  limited  in  frequency  band,  by 
electronic  niters,  in  one  of  two  ways.  Either  the  filter  was  set  at  15  kc  high  pass  so 
that  frequencies  above  15  kc  were  generated  at  a  high  level  while  lower  frequencies 
were  attenuated  progressively  at  24  db  per  octave,  or  else  a  25  kc  low  pass  filter  was 
used  to  transmit  audio  frequencies  while  attenuating  ultrasonic  components  of  the 
noise,  also  at  24  db  per  octave.  Without  noise,  the  bat  avoided  3  mm.  wires  79% 
of  the  time  in  the  dark.  In  the  light,  and  with  the  noise,  in  a  very  short  series,  it 
avoided  the  wires  90%  of  the  time.  But  in  the  dark  the  bat  was  incapable  of  avoid- 
ing these  wires  at  all  in  intense  noise  above  25  kc.  In  noise  below  15  kc,  it  scored 
27%  misses.  The  bat's  total  inability  to  avoid  large  wires  in  noise  above  25  kc  and 
its  very  poor  performance  in  noise  below  15  kc  suggest  several  hypotheses.  If  we 
assume  that  the  poor  performance  was  due  to  unfavorable  signal-noise  ratio  at  the 
same  frequencies,  then  we  have  evidence  that  Rouscttns  depends  upon  a  wide  range 
(from  less  than  15  kc  to  more  than  25  kc)  of  frequencies  in  echolocation.  But  al- 
ternatively the  analytical  ability  of  Rouscttus'  ears  may  not  suffice  for  distinguish- 
ing a  14  kc  echo  from  either  type  of  noise  tested,  that  is,  we  may  simply  have 
shown  that  the  accuracy  of  acoustic  orientation  in  Rouscttus  can  be  reduced  (even 
totally)  by  noise.  The  results  may  also  have  been  complicated  by  the  bat's  panic, 
discomfort,  loss  of  motivation,  or  confusion  in  an  unusual  situation  aside  from  its 
ability  to  perceive  echoes  in  a  noisy  environment. 

DISCUSSION 

In  these  experiments,  the  wires  were  less  widely  spaced  relative  to  the  wing- 
spread  of  Rouscttus  than  in  Curtis'  experiments  with  M \otis,  but  Roiiscttns  almost 
always  approached  the  plane  of  the  obstacles  perpendicularly  while  Myotis  often 
approached  obliquely.  Our  flight  room  was  also  considerably  larger  than  the 
15'  X  9'  X  ()'  room  used  by  Curtis.  The  percentage  of  misses  for  relatively  large 
obstacles  was,  nevertheless,  almost  exactly  the  same — 85.0%  for  Myotis  with  4.76- 
mm.  rods  and  84.5%  for  Rouscttus  dodging  3 -mm.  wires.  Rouscttus  was  slightly 
less  successful  at  avoiding  even  larger  obstacles  (cardboard  and  rubber  tubes)  but 
these  tests  were  conducted  early  in  the  bat's  experience  in  the  exacting  task  of  flying 
in  a  dark  room  (with  its  multiplicity  of  echoing  surfaces). 

This  Rouscttus  was  able  to  detect  and  avoid,  with  a  considerable  degree  of  suc- 
cess, wires  as  small  as  1.07  mm.  in  diameter.  Only  when  confronted  with  wires  of 
less  than  1  mm.  did  its  skill  fall  seriously  below  its  own  standards  as  well  as  those 
of  Myotis.  Rouscttus'  score  decreased  rather  gradually.  If  we  consider  its  poor 
performance  (T8f,Y  misses)  against  0.28-mm.  wires  as  due  to  chance,  then 
Rouscttus  was  clearly  detecting  0.46-mm.  wires  against  which  it  scored  45%  misses. 
Even  18%'  misses  against  0.28-mm.  wires  may  have  represented  some  degree  of 
echolocation  for.  when  flying  in  a  noise  field,  this  bat  did  even  more  poorly  (100% 
hits)  against  3-nim.  wires.  It  seems  reasonable  that  the  ease  with  which  a  small 
object  is  echolocated  depends  upon  its  position  relative  to  the  angle  of  sound  emis- 
sion and  its  beaming  and  the  angle  of  sound  reception.  Thus  there  is  likely  to  be  an 
optimal  angle  of  approach  (probably,  but  not  necessarily,  straight  ahead)  where 
the  maximum  echo  will  be  received  and  less  easilv  detected  obstacles  will  be  echo- 


112  D.  R.  GRIFFIN,  A.  NOVICK  AND  M.  KORXFIFLD 

located.  Obstacles  which  lie  less  optimally  relative  to  the  bat  will  have  to  have  more 
effectively  echoing  surfaces  to  be  detectable.  Thus  the  bat  might  well  succeed  in 
avoiding  a  0.46-mm.  or  0.65-mm.  wire  only  if  it  chanced  to  approach  it  favorably 
and  so  its  score  when  working  against  obstacles  of  marginal  size  would  be  an  aver- 
age of  chance  misses,  active  misses,  and  "blind"  hits.  One  of  the  limiting  factors  in 
exploring  the  threshold  of  echolocation  is  the  danger  of  serious  injury  to  the  bat 
every  time  it  collides  with  an  obstacle.  Such  collisions  may  be  major  accidents  or 
simply  touches.  Collisions  with  small  wires  tend  to  be  more  serious  than  those 
with  large  obstacles.  Roiiscttns'  performance  varied  considerably  from  trial  to 
trial.  Whenever  possible  we  ran  long  series  of  tests  and  interspersed  tests  with 
3-mm.  wire  between  those  with  smaller  sizes.  The  results  were  consistent  with  the 
average  scores  listed  in  Table  I. 

The  design  of  Myotis  orientation  pulses  is  very  different  from  that  of 
Rouse  ft  its  clicks.  Mvotis  pulses  are  produced  laryngeally  and  emitted  through 
the  open  mouth.  They  have  a  frequency  modulated  pattern  with  a  gradually  fall- 
ing frequency  starting  on  the  average  at  about  80  kc  and  ending  at  about  40  kc  but 
with  beginnings  ranging  from  at  least  60  to  120  kc.  Similar  variety  among  terminal 
frequencies  also  occurs.  Thus  Myofis  in  single  pulses  and  in  consecutive  pulses 
produce  prominent  frequencies  covering  about  two  octaves  (Griffin,  1958;  Xovick. 
1955).  Furthermore,  harmonics  also  occur  in  Myotis  pulses  and  represent  a  sec- 
ond octave  sweep  within  the  pulses  in  which  they  occur.  The  importance  of  the 
harmonics  as  components  of  the  outgoing  pulses  and  the  returning  echoes  and  in  the 
carrying  of  information  about  the  environment  to  the  bats  has  not  been  evaluated. 
In  Ronsettns.  the  pulses  are  produced  by  tongue  clicks  and  are  impure  in  frequency 
and  irregular  in  frequency  pattern.  The  bulk  of  the  energy,  however,  appears  to  be 
in  the  range  of  about  12  to  18  kc.  Additional  energy  is  scattered  from  6.5  to  over 
100  kc  with  a  second  maximum  at  about  20  to  40  kc  (  Moehres  and  Kulzer,  1956a; 
Kulzer,  1956;  Novick.  1958). 

SUMMARY 

1.  The  ability  of  a  single  specimen  of  the  fruit  bat,  Roiiscttns  aegypticus,  to  avoid 
test  obstacles  of  various  sizes  by  echolocation  in  total  darkness  was  tested.     This  bat 
avoided  vertically  placed  3-mm.  metallic  wires  85%  of  the  time.     Its  success  de- 
clined gradually  as  the  wires  were  reduced  in  size  but  the  bat  displayed  considerable 
success  (68%  misses)  against  1.07-mm.  wire  and  did  significantly  better  than  chance 
(45%  misses)  against  wires  0.46  mm.  in  diameter. 

2.  These  results  have  been  compared  with  those  of  Curtis   (1952)  who  studied 
the  vespertilionid,  Al  \otis  I.  In  din;/ its. 

3.  Roiiscttns'  success  at  echolocation  was  considerably  reduced  when  it  was  forced 
to  fly  in  a  field  of  intense  thermal  noise. 

LITERATURE  CITED 

CURTIS,  W.  E.,  1952.  Quantitative  studies  of  echolocation  in  bats  (Myotis  I.  lucifugus)  ;  stud- 
ies of  vision  in  bats  (Myotis  I.  Iucifn(jus  and  Eptcsicus  f.  fuse  us)  ;  and  quantitative 
studies  of  vision  of  owls  (Tyto  alba  pratincola) .  Thesis  deposited  in  the  library  of 
Cornell  University,  Ithaca,  New  York. 

GRIFFIN,  D.  R.,  1946.  The  mechanism  by  which  bats  produce  supersonic  sounds.  Anat.  Rec., 
96:  519. 


ECHOLOCATION  IN  ROUSKTTUS  113 

GRIFFIN,  D.  R.,  1952.     Mechanisms  in  the  bat  larynx  for  production  of  ultrasonic  sounds.     l;cd. 

Proc..  11:  59. 

GRIFFIN,  D.  R.,  1958.     Listening  in  the  Dark.     New  Haven,  Yale  University  Press. 
GRIFFIN,  D.  R.,  AND  R.  GALAMBOS,   1941.     The  sensory  basis  of  obstacle  avoidance  by   flying 

bats.     /.  £.r/>.  Zooi,  86:  481-506. 
GRIFFIN,  D.  R.,  AND  A.  NOVICK,  1955.     Acoustic  orientation  of  neotropical  bats.     /.  E.rp.  Zoo/.. 

130:  251-300. 
GRINNELL,   A.   D.,   AND   D.   R.   GRIFFIN,   1958.     The   sensitivity   of   echolocation   in   bats.     Biol. 

Bull.,  114:  10-22. 
HAHN,  W.  L.,  1908.     Some  habits  and  sensory  adaptations  of  cave-inhabiting  bats.     I  and  II. 

Biol.  Bull.,  15:  135-193. 
KULXER,   E.,   1956.     Flughunde  erzeugen   Orientierungslaute   durch    Zungenschlag.     Naturzviss., 

43:  117-118. 
MOKIIKKS,  I".  P.,  AND  K.  KuLZEK,  1956a.     Untersuchungen  iiber  die  Ultraschallorientierung  von 

vier   afrikanischen    Fledermausfamilien.     I'crli.    dtsch.    zool.    Gcs.    in    Erlangcn,    Zool. 

.Inzciticr  Siifplemcntlnind.  19:  59-65. 
MOEHRES,  F.   P.,  AND  F.  KULZER,   1956b.     tjber  die  Orientierung   der  Flughunde    ( Chiroptera- 

Pteropodidae).     Zcitschr.  f.  rcrt/l.  Pliysiol.,  38:  1-29. 
NOVICK,  A.,   1955.     Laryngeal  muscles  of  the  bat  and  production  of  ultrasonic  sounds.     Aincr. 

J.  Physiol..  183':  648. 
N'uvicK.    A.,    1958.     Orientation    in    palaeotropical    bats.     II.    Megachiroptera.     /.    E.\-p.    Zool., 

137   ( in  press). 


ELECTROPHYSIOLOGICAL  STUDIES  OF  ARTHROPOD  CHEMO- 

RECEPTION.     III.  CHEMORECEPTORS  OF  TERRESTRIAL 

AND  FRESH-WATER  ARTHROPODS1 

EDWARD  S.  HODGSON 

Department  of  Zoology,  Columbia   University,  New   York  27,  A'.    )'..  and  Mountain   Lake  Bio- 

logical Station,  r 


While  an  extensive  literature  documents  the  role  of  chemoreceptors  in  the  be- 
havior of  invertebrates  (Hodgson,  1955),  the  small  size  of  chemoreceptor  cells  is  a 
major  handicap  in  any  attempt  to  study  their  functions  using  conventional  electro- 
physiological  procedures  (  Chapman  and  Craig,  1953  ;  Roys,  1954).  Barber  (  1956) 
recorded  afferent  impulses  from  neurons  which  supply  the  gnathobase  chemore- 
ceptors of  Lunnlns  and  noted  an  increase  in  nerve  activity  when  aqueous  extracts 
of  marine  bivalves  were  applied  to  the  gnathobase.  Use  of  microelectrodes  enabled 
Schneider  (1957)  to  record  afferent  impulses  from  groups  of  antennal  chemore- 
ceptors in  male  silkmoths  (Bomb\.\-)  during  stimulation  with  extracts  of  the  scent 
glands  from  female  moths.  Possible  synaptic  effects  between  receptor  cells  and 
nerves  supplying  them,  or  the  unpredictable  numbers  of  cells  represented  in  most 
recordings,  make  it  difficult,  however,  to  interpret  the  results  in  terms  of  single  unit 
activity  of  the  actual  chemoreceptor  cells. 

A  relatively  simpler  technique  is  that  of  recording  the  afferent  impulses  from 
primary  chemoreceptor  cells  through  the  same  fluid  which  is  applied  as  a  stimulus 
(Hodgson,  Lettvin  and  Roeder,  1955).  This  method  has  thus  far  been  applied 
only  in  studying  contact  chemoreceptors  of  two  animals:  labellar  chemoreceptors  of 
the  blowfly  Phornria  (Hodgson  and  Roeder,  1956;  Wolbarsht.  1957)  and  tarsal 
chemoreceptors  of  the  butterfly  J  \iucssa  (  Morita  ct  al.,  1957).  The  conclusions 
from  studies  of  these  two  preparations  point  to  a  number  of  unexpected  properties 
of  primary  chemoreceptor  cells. 

With  both  Phonnia  and  1'ancssa,  it  was  found  that  different  chemoreceptor  cells 
were  specialized  to  respond,  not  to  the  different  modalities  of  stimuli  generally  held 
to  be  effective  for  contact  chemoreceptors  of  vertebrates  (e.g.  Beidler,  1952),  but 
either  to  sugars  or  to  various  non-sugars,  with  the  presence  of  a  water-specific  re- 
ceptor also  strongly  indicated  in  1'ancssa  (Morita  ct  al.,  1957).  Seemingly  at  vari- 
ance with  the  usual  concept  of  single  specificities  of  receptor  cells  (Granit,  1955), 
a  single  primary  receptor  cell  of  Phonnia  may  respond  to  chemical,  tactile,  and 
thermal  stimuli  within  normal  physiological  ranges  (Hodgson  and  Roeder,  1956). 
Unfortunately,  information  on  this  point  is  not  available  for  1'ancssa. 

In  view  of  these  unexpected  results,  and  the  lack  of  any  comparable  electrophysi- 
ological  data  on  primary  chemoreceptors  of  other  invertebrates,  it  seemed  desirable 
that  the  method  of  recording  through  fluid-filled,  externally  applied  electrodes 

1  This  investigation  was  aided  by  Public  Health  Service  Grant  No.  E-1010,  and  by  the 
Higgins  Fund  of  Columbia  University.  The  field  work  was  aided  by  a  National  Science 
Foundation  Grant  to  the  Mountain  Lake  Biological  Station. 

114 


ARTHROPOD  CHEMORECEPTION  115 

should  be  tried  on  chemoreceptors  of  a  wider  variety  of  animals,  in  order  to  de- 
termine how  generally  the  characteristics  found  in  Plwnnia  and  Vanessa  receptors 
may  apply  to  the  functions  of  other  primary  chemoreceptor  cells.  For  technical 
reasons,  this  method  is  best  adapted  to  recording  from  chemoreceptors  in  arthropods 
(Hodgson,  Lettvin  and  Roecler,  1955).  The  object  of  the  present  paper  is  to  report 
the  results  of  tests  conducted  using  this  method  upon  the  chemoreceptors  of  some 
terrestrial  and  fresh-water  arthropods.  In  each  case  where  the  method  could  be 
successfully  applied,  answers  to  the  following  questions  were  sought :  (  1 )  Does  the 
same  receptor  cell  respond  to  chemical,  tactile,  and  thermal  stimuli  within  normal 
physiological  ranges?  (2)  What  modalities  of  chemical  stimuli  excite  the  indi- 
vidual primary  chemoreceptor  cells?  (3)  Does  the  relationship  between  the  reac- 
tion of  the  animal  to  chemicals  and  the  range  of  sensitivity  of  its  chemoreceptors 
indicate  a  peripheral  discrimination  mechanism,  such  as  found  in  PJwrmia? 

METHODS 

Thirty-seven  species,  representing  the  major  classes  of  arthropods  and  eight  or- 
ders of  insects,  were  tested.  These  species  are  arranged  according  to  taxonomic 
status  below.  All  specimens  were  collected  in  the  field  and  tested  within  12  hours 
after  capture.  The  animals  were  allowed  to  drink  water  to  repletion,  but  no  attempt 
was  made  to  control  their  diet  prior  to  testing.  At  least  three  individuals,  usually 
more,  belonging  to  each  species  were  studied. 

The  technique  of  recording  action  potentials  from  chemoreceptors  using  ex- 
ternally applied,  fluid-filled  electrodes  has  been  described  in  detail  elsewhere  (Hodg- 
son, Lettvin  and  Roeder,  1955;  Hodgson  and  Roeder,  1956).  This  technique  was 
used  with  only  such  minor  modifications  as  were  necessary  to  manipulate  the  va- 
riety of  receptor-bearing  appendages  tested.  All  experiments  were  tape  recorded 
and  photographs  made  from  the  tape  recordings,  beginning  one-half  second  after 
the  stimulus  was  applied,  thus  avoiding  the  base-line  fluctuations  which  commonly 
accompany  the  stimulus  artifact. 

The  species  tested  were  as  follows,  with  each  group  and  each  species  yielding 
potentials  from  chemoreceptors  designated  by  an  asterisk.  (Except  as  otherwise 
noted,  identifications  were  checked  through  the  courtesy  of  Dr.  R.  E.  Blackwelder 
of  the  U.  S.  National  Museum.)  Class:  Crustacea* — Cambarus  bartomi  scioten- 
sis*  (Det.  H.  H.  Hobbs,  Jr.)  ;  Class:  Arachnida — Latrodectus  mactans  (black  widow 
spider).  Theridion  tepidariorum  (house  spider)  ;  Class:  Diphpoda* — -Pseudotremis 
sp.  (Det.  H.  F.  Loomis),  Pseudopolydesmus  serratus*  (Det.  M.  Walton)  ;  Class: 
Insccta;  Order:  Odonata — Aeschna  constricta.  Libellula  pulchella,  Progomphus 
obscurus ;  Order:  Ortlwptera* — Acheta  assimilis  (common  field  cricket),  Ceu- 
thophilus  gracilipes*  (cave  cricket),  Crytocercus  punctulatus  (wood-eating  roach) 
(Det.  L.  R.  Cleveland).  Hadenoecus  putaneus*  (cave  cricket).  Scudderia  furcata 
(katydid)  ;  Order:  Heiuiptera — Arilus  cristatus,  Oncopeltus  fasciatus  (large  milk- 
weed bug)  ;  Order:  Coleoptera — Cicindela  sexguttata  (six-spotted  tiger  beetle), 
Dineutes  americanus  (whirligig  beetle),  Dytiscus  fasciventris  (large  diving  beetle), 
Laccophilus  maculosus  (common  pond  beetle),  Nicrophorus  tomentosus  (carrion- 
beetle),  Phymatodes  dimidiatus  ( longhorn  beetle),  Saperda  Candida  (apple  tree 
borer),  Silpha  americana  (carrion  beetle),  Tropisternus  lateralis  (keeled  water 
beetle)  ;  Order:  Megaloptera — Carydalus  cornutus  (dobsonfly)  ;  Order:  Nenrop- 


116  EDWARD  S.  HODGSON 

tcra — Chrysopa  sp.  (golden  eyed  lacewing)  ;  Order:  Diptera* — -Amoebaleria  de- 
fessa*  (cave  fly)  (Det.  C.  H.  Curron).  Tipula  trivittata  (crane  fly)  ;  Order:  Lef>i- 
doptera* — Atlides  halesus  (purple  hairstreak)  ;  Epargyreus  clarus*  (silver  spotted 
.skipper),  Limenitis  arthemis  astyanax*  (red  spotted  purple),  Papilio  marcellus* 
( zebra  swallowtail),  Papilio  philenor*  (pipe  vine  swallowtail),  Protoparce  quin- 
quemaculata  (  five-spotted  hawk  moth),  Speyeria  cybele*  (great  spangled  britillary), 
Tropaea  luna  (luna  moth),  Vanessa  atalanta*  (red  admiral). 

The  chemicals  tested  were  sodium  chloride,  sucrose,  d-levulose,  glycine.  DL 
glutamic  acid,  citric  acid,  oil  of  citronella  and  oil  of  wintergreen.  Sodium  chloride 
was  tested  as  a  0.25  molar  aqueous  solution.  Oils  of  citronella  and  wintergreen  were 
tested  by  bringing  swabs  soaked  in  these  chemicals  to  within  an  inch  of  the  sensory 
structure.  Although  quantitative  control  of  stimulus  concentration  was  not  obtained 
by  this  method,  the  results  obtained  with  these  two  oils  were  quite  reproducible. 
All  of  the  other  chemicals  were  mixed  with  sodium  chloride  so  that  the  final  test 
solution  was  an  unbuffered  aqueous  solution  containing  0.1  molar  XaCl  and  a  0.25 
molar  concentration  of  the  test  chemical.  Results  were  compared  with  activity  re- 
corded when  0.1  molar  XaCl  was  applied  alone. 

Temperatures  were  measured  with  a  thermistor  implanted  just  under  the  cuticle 
near  the  receptor  being  studied.  The  temperature  was  changed  by  bringing  a  warm 
glass  rod  or  small  ice-pack  near  the  preparation.  Spike  potentials  from  mechano- 
receptors  were  recorded  by  bending  sensilla  or  whole  appendages  with  needles. 
Certain  departures  from  the  usual  tests  are  described  at  appropriate  points  below. 

RESULTS 

All  of  the  preparations  yielded  numerous  spike  potentials  originating  from  tac- 
tile receptors,  thus  providing  assurance  that  the  preparations  were  alive  when 
studied.  In  only  five  orders  of  the  arthropods  tested,  however,  was  it  possible  to 
obtain  unequivocal  recordings  from  chemoreceptors.  These  five  groups  are  desig- 
nated by  asterisks  above.  The  several  factors  believed  to  be  responsible  for  failure 
to  record  action  potentials  in  all  of  the  tested  species  are  considered  in  the  discussion, 
and  a  complete  description  of  the  results  will  be  presented  only  for  those  forms 
in  which  chemoreceptors  could  be  studied  using  fluid-filled  electrodes. 

1.  DECAPODA  Cainbants  bartonii  sciotensis  (16  individuals) 

This  large  crayfish  proved  to  be  an  exceptionally  interesting  experimental  ani- 
mal. Recordings  could  be  made  with  the  usual  0.1  molar  NaCl  conducting  solution 
in  the  electrode,  or  else  by  using  distilled  water  or  pond  water  as  a  solvent  for  the 
chemicals.  Although  the  results  showed  few  differences  whichever  solvent  was 
used,  all  of  the  tests  were  run  with  chemicals  dissolved  in  distilled  water,  thus  avoid- 
ing any  possible  complications  of  the  sodium  chloride. 

The  antennae  and  the  lateral  branches  of  the  antennules  were  alike  in  yielding 
only  records  of  mechanoreceptors  at  low  amplitudes  (30  /iV).  From  the  entire 
medial  branch  of  the  antennule,  however,  it  was  possible  to  record  a  variety  of  spike 
potentials  ranging  in  amplitude  from  30  /Ar  to  500  /tY .  The  large-amplitude  spikes 
(200  fjiV  to  500  p.\7)  were  recorded  only  when  the  antennule  was  bent.  Conse- 
quently, the  cells  giving  rise  to  these  potentials,  which  are  relatively  few  in  this 


ARTHROPOD  CHEMORECEPTION 


117 


A 


FIGURE  1.  Typical  spike  potentials  from  arthropod  chemoreceptors.  A,  response  of 
medial  branch  of  Cainhanis  antennule  to  glutamic  acid;  Bl,  single  sensillum  on  Cambarus  walk- 
ing leg,  tested  with  distilled  water  ;  B2,  same  as  Bl,  except  glycine  test  solution ;  Cl,  Pscudo- 
polydcsmus  tarsus,  NaCl  control:  C"_',  same  as  Cl,  except  sucrose  test  solution;  Dl,  spontaneous 
activity,  Hadcnoccits  tibia;  I~>2,  same  as  Dl,  except  exposed  to  citronella  vapor;  E,  single  tarsal 
sensillum  of  Eparyyreus,  control  NaCl  solution ;  /•",  same  as  E,  except  sucrose  test  solution ;  G, 
antenna  of  Amocbalcria,  distilled  water  in  electrode;  H,  same  as  G,  except  exposure  of  antenna 
to  oil  of  citronella  vapor.  Time  bases  for  all  records,  100  cycles  per  second.  Consult  text  for 
additional  details. 


118  EDWARD  S.  HODGSON 

branch  of  the  antennule,  are  mechanoreceptors.  The  majority  of  the  spikes  have 
amplitudes  of  30  ^V  to  50  ^V.  These  respond  to  the  application  of  glycine  and 
glutamic  acid,  of  the  test  series  of  chemicals  used.  Because  a  number  of  different 
amplitudes  of  spikes  were  recorded  even  with  the  smallest  practicable  areas  of  elec- 
trode contact,  it  was  not  possible  to  determine  whether  identical  cells  were  respond- 
ing to  both  chemical  and  tactile  stimuli.  Record  A  of  Figure  1  is  taken  from  an 
experiment  in  which  a  test  solution  of  glutamic  acid  was  allowed  to  flow  around  the 
medial  branch  of  an  antennule  without  changing  electrode  contact.  Activity  re- 
corded when  the  antennule  is  in  distilled  water  (on  the  left  of  the  large  stimulus 
artifact)  is  negligible,  but  many  small-amplitude  spike  potentials  follow  the  intro- 
duction of  the  glutamic  acid.  The  frequency  of  firing  during  chemical  stimulation 
was  not  influenced  by  temperature  changes  within  the  range  tested — -five  degrees 
(C.)  above  or  below  the  room  temperature  of  25  degrees. 

Chemoreceptors  were  also  found  on  the  first  two  pairs  of  walking  legs.  The 
chemoreceptors  wrere  located  on  the  chelae  and,  to  a  lesser  extent,  elsewhere  on  the 
protopodites  of  those  legs.  The  external  chemosensory  structures  can  be  recog- 
nized in  C.  barton ii  as  tufts  of  setae,  numbering  ten  to  twenty  setae  per  tuft,  each 
such  tuft  arising  from  a  circular  depression  in  the  cuticle.  Contact  of  the  electrodes 
with  other  parts  of  the  cuticle  failed  to  terminate  the  open  circuit  condition  between 
the  indifferent  electrode  inside  and  the  recording  electrode  outside  the  cuticle. 
The  best  records  were  obtained  after  the  claw  had  been  allowed  to  dry  at  room 
temperature  for  thirty  minutes  following  its  removal.  This  prevented  short  circuits 
between  the  recording  and  indifferent  electrodes.  By  teasing  apart  the  hairs  of  a 
single  tuft,  the  tip  of  an  electrode  could  then  be  positioned  over  a  single  sensory 
hair.  In  this  way  the  firing  of  a  single  chemoreceptor  cell  could  be  studied.  The 
spike  potentials  recorded  from  different  sensilla  ranged  from  30  to  60  /A/r  in  ampli- 
tude. It  was  found  that  these  receptors  resemble  chemoreceptors  on  the  antennule 
in  being  insensitive  to  test  chemicals  other  than  amino  acids  of  the  series  used. 
(Records  Bl  and  B2  of  Figure  1  illustrate  typical  results  during  applications  of  a 
control  NaCl  solution,  and  the  test  mixture  of  NaCl  and  glycine,  respectively.) 
The  chemoreceptors  on  the  first  two  walking  legs  were  never  observed  to  respond 
to  mechanical  movement  of  the  sensory  hairs  during  recordings.  The  small  size  of 
the  hairs  (about  20  microns  in  length)  and  their  position  surrounded  by  rigid  cuticle 
would  appear  to  render  their  usefulness  as  tactile  receptors  unlikely.  The  insensi- 
tivity  of  these  receptors  to  temperature  changes  within  the  range  tested  resembles 
that  of  the  receptors  on  the  antennule.  Impulses  from  chemoreceptors  were  not 
detected  from  the  chelipeds,  third  maxillae,  or  elsewhere  on  the  body  of  the  crayfish 
using  the  present  method. 

Behavioral  experiments  were  run  to  check  the  possibility  of  a  peripheral  dis- 
crimination for  amino  acids.  Ablations  of  antennae,  antennules,  or  the  first  two 
pairs  of  walking  legs,  and  combinations  of  these  operations,  were  performed  on  thirty 
crayfish.  The  results  were  difficult  to  interpret  in  many  cases  because  of  abnormal 
behavior  of  operated  animals.  It  was  easy  to  demonstate,  however,  that  the  animals 
can  locate  food  using  the  first  two  pairs  of  walking  legs,  even  when  antennae,  anten- 
nules, and  maxillipeds  are  removed.  Activity  resembles  that  during  normal  feed- 
ing and  can  be  initiated  by  injecting  0.25  molar  solutions  of  glycine  and  glutamic 
acid  into  the  water,  while  even  intact  animals  fail  to  give  clear-cut  responses  to  the 
other  test  solutions.  Thus  there  seems  to  be  a  clear  correlation  between  the  elec- 


ARTHROPOD  CHEMORECEPTION  119 

trophysiological  data  and  the  behavioral  results.  Attempts  to  determine  by  be- 
havioral tests  whether  the  antennae  and  lateral  branches  of  the  antennules  bear 
chemoreceptors  yielded  results  which  could  not  be  unequivocally  interpreted. 
Doflein  (1910),  on  the  basis  of  behavioral  tests,  has  reported  that  the  antennules 
of  decapods  contained  chemoreceptors,  and  Luther  (1930),  using  similar  methods, 
reported  chemoreceptors  on  mouth  parts,  walking  legs,  and  pincers  of  brachyurans. 

2.  DIPLOPODA  Pseudotremis  sp.    (4  individuals)  ;  Pseudopolydesmus  scrratus 

(4  individuals) 

In  both  Pseudotremis  and  Pseudopolydesmus  many  action  potentials  could  be 
recorded  from  the  tips  of  the  antennae  and  from  the  tips  of  the  legs  when  an  elec- 
trode filled  with  0.1  molar  NaCl  was  applied  to  those  parts.  In  Pseudotremis.  the 
smaller  species,  the  action  potentials  were  never  more  than  40  /zV  in  amplitude,  and 
all  clearly  responded  to  mechanical  bending  of  the  antenna  or  leg.  In  Pseudopoly- 
desmus the  largest  spikes  from  the  antenna  were  about  60  //.V  in  amplitude,  and  those 
from  the  tarsus  were  about  80  //.V.  All  of  the  larger  spikes  increased  in  frequency 
during  bending  of  the  appendages  being  tested,  and  it  was  therefore  assumed  that 
these  spikes  represented  the  afferent  impulses  from  mechanoreceptors.  Spike  po- 
tentials of  smaller  amplitude  (30-50  ju,V)  from  tarsi  of  Pseudopolydesmus  occurred 
with  increased  frequency  when  the  tarsi  were  bent,  or  sugars  applied.  (See  Fig. 
1C.)  They  did  not  change  during  application  of  other  test  solutions  or  during  tem- 
perature changes  within  five  degrees  (C.)  of  the  room  temperature  of  25  degrees. 
No  significant  changes  in  the  frequency  or  pattern  of  impulses  were  noted  in  re- 
cordings from  the  antennae  of  the  two  species  when  chemical  stimuli  were  applied. 

The  small  trichoid  sensilla  which  probably  enclose  the  actual  chemosensory  cells 
on  the  tarsi  of  Pseudopolydesmus  are  too  closely  spaced  to  make  possible  a  re- 
striction of  the  area  of  electrode  contact  to  a  single  sensillum.  Attempts  to  record 
activity  using  electrodes  filled  with  distilled  water  were  likewise  unsuccessful.  In 
view  of  the  smaller  size  of  the  mechanoreceptor  spikes  recorded  from  Pseudotremis, 
and  the  generally  smaller  size  of  action  potentials  from  chemoreceptors  as  compared 
with  mechanoreceptors,  it  would  hardly  be  expected  that  chemoreceptor  spikes  from 
Pseudotremis  would  be  detectable  above  the  inherent  "noise  level"  of  the  apparatus 
Behavioral  test  showed  that  sucrose  or  levulose,  placed  in  contact  with  the  tarsi, 
initiated  feeding  responses  even  after  the  antennae  wrere  removed.  Tarsal  contact 
with  citric  acid  caused  the  animals  to  move  away  from  the  test  solution,  but  this 
was  the  only  test  solution,  other  than  the  sugars,  which  elicited  a  behavioral  re- 
sponse. With  the  exception  of  citric  acid,  receptors  for  which  could  not  be  de- 
tected electrophysiologically,  the  behavioral  and  electrophysiological  results  suggest 
the  existence  of  a  peripheral  discrimination  mechanism. 

3.  ORTHOPTERA    Ceuthophilns    c/racilipcs    (7    individuals)  ;    Hadcnoccus    pn- 

tancus  (3  individuals) 

The  orthopterans  tested  showed  considerable  variation,  some  of  which  appears 
to  be  related  to  habitat.  Cryptocercus,  a  wood-eating  roach,  was  completely  refrac- 
tory to  the  recording  method,  except  for  a  few  mechanoreceptors  in  the  antennae  and 
palpi.  A  larger  number  of  tactile  receptors  were  recorded  from  the  antennae  and 


120  EDWARD  S.  HODGSON 

palpi  of  the  katydid,  Scuddcria,  and  the  field  cricket.  Acheta.  Hest  results,  how- 
ever, were  obtained  with  the  cave  crickets  Ceuthophilus  and  Hadenoecus,  which 
have  antennae  elongated  to  many  times  the  length  of  the  body  and  also  have  un- 
usually long  legs  and  palpi.  The  data  support  the  generally  expressed  assumption 
that  these  anatomical  modifications  are  associated  with  hypertrophy  of  tactile  and 
chemical  senses  which  would  presumably  be  of  selective  value  in  dark  subterranean 
environments. 

In  tests  of  seven  adult  specimens  of  Ceuthophilus  and  three  of  Hadenoecus,  the 
antennae  were  found  to  contain  spontaneously  active  and  quick-adapting  mechano- 
receptors  (spike  amplitudes  50-80  yuV)  along  with  spontaneously  active,  relatively 
non-adapting  chemoreceptors  (spike  amplitude  20—40  /tV).  The  latter  were  seen 
in  one  antennal  preparation  of  Ceuthophilus  and  all  three  preparations  of  Hade- 
noecus. The  frequency  of  the  small  spikes  did  not  change  during  application  of 
any  of  the  test  chemicals  in  solution,  or  during  temperature  changes  between  20  and 
30  degrees  C..  but  did  increase  when  swabs  soaked  in  citronella  or  wintergreen  were 
brought  near  the  region  of  the  antenna  in  contact  with  the  electrode.  Essentially 
similar  results  were  obtained  from  recordings  of  the  receptor  activity  in  both  the 
maxillary  and  labial  palpi  and  the  tarsi  of  Ceuthophilus  and  Hadenoecus.  In  addi- 
tion, small  spikes  (30-50  ^V)  were  recorded  from  the  trochanter  and  tibia  of  the 
prothoracic  and  mesothoracic  legs  of  Hadenoecus,  in  six  out  of  eight  preparations 
when  the  legs  were  exposed  to  vapors  of  wintergreen  or  citronella.  Mechanical 
bending  of  sensilla  on  the  trochanter  and  tibia  also  increased  the  frequency  of  these 
same  spike  potentials.  Record  Dl  of  Figure  1  shows  the  spontaneous  activity  of 
receptors  in  the  tibia  of  a  prothoracic  leg  of  Hadenoecus,  and  record  D2  shows  the 
increase  in  frequency  of  spikes  during  application  of  citronella  vapor.  Xo  effects 
of  the  test  chemicals  in  solution  could  be  detected  in  either  Ceuthophilus  or  Hade- 
noecus, and  chemoreceptor  activity  could  not  be  recorded  from  the  cerci,  ovipositor, 
general  body  surface,  or  the  larger  spines  on  the  legs  of  either  species.  Ceuthophilus 
did  not  give  any  clear-cut  behavioral  response  to  citronella  or  wintergreen  in  tests  of 
the  intact  animals,  but  Hadenoecus  gave  intense  avoidance  reactions,  moving  quickly 
away  from  these  stimuli.  Removal  of  the  antennae  and  palpi  did  not  abolish  this 
reaction  in  Hadenoecus,  which  always  responded  most  strongly  when  stimuli  were 
near  the  legs. 

4.  LEPIDOPTERA 

Nine  species  of  Lepidoptera  were  tested.  Only  a  few  impulses  associated  with 
tactile  stimulation  could  lie  recorded  from  the  antennae  of  any  of  these  species,  even 
when  vapors  were  applied.  In  all  six  species  of  butterflies  tested,  records  were  ob- 
tained from  the  tarsal  receptors  (described  by  Minnich,  1921).  Tests  upon  the 
tarsal  sensilla  of  Eparyyrcus  and  Limcnitis  revealed  that  each  sensillum  had  a  few- 
receptor  systems  functioning  similarly  to  that  in  the  labellar  hairs  of  Phonnia. 
(Compare  the  records  E  and  F  of  Figure  1.  taken  from  tests  of  a  single  tarsal 
sensillum  of  Epargyreus,  and  note  that  the  small  spike  potentials  predominate  only 
in  record  F  when  sugar  is  present  in  the  electrode.)  The  maximum  number  of  re- 
ceptors represented  in  recordings  from  single  sensilla  of  these  two  species  is  four, 
and  the  minimum  two.  Variations  within  these  limits  were  commonly  encountered 
in  comparisons  of  the  records  from  several  hairs,  even  on  the  same  tarsus.  The 


ARTHROPOD  CHEMORKCKPTION  121 

variations  characteristically  occurred  in  the  smaller  spike  potentials,  but  under  the 
conditions  of  these  tests  all  of  the  smaller  spikes  increased  in  frequency  during 
stimulation  with  sugars,  and  the  largest  spike  responded  with  increased  frequencies 
during  application  of  any  of  the  non-sugar  solutions.  These  receptors  were  not 
observed  to  respond  to  vapors  of  citronella  or  wintergreen. 

With  the  other  species  of  butterflies  tested,  there  appeared  to  be  as  many  as  12 
different  receptors  associated  with  each  tarsal  sensillum  and  the  records  were  too 
complex  for  analysis  of  the  functions  of  any  single  receptor  cells.  Responses  to 
tactile  stimulation  were  obtained  in  tests  with  tarsal  hairs  of  all  the  butterflies  used ; 
in  those  preparations  involving  only  a  few  fibers  it  was  clear  that  all  fibers  responded 
to  bending  of  the  tarsal  hair,  and  probably  this  was  the  case  with  the  many-fiber 
preparations  also,  but  this  could  not  be  determined  with  certainty  because  of  the 
complexity  of  the  records.  The  frequency  of  impulses  recorded  during  continuous 
stimulation  of  single  sensory  hairs  of  Eparc/yrcns  and  Li  men  it  is  was  increased  by 
temperature  rises  of  as  little  as  1.2  degrees  C.  These  particular  tarsal  receptors,, 
then,  bear  a  greater  resemblance  to  the  labellar  chemoreceptors  of  flies  than  do  any 
of  the  other  preparations  (excluding  the  labellar  chemoreceptors  of  Amoebalcria] 
encountered  in  this  survey.  Feeding  responses  (proboscis  extensions)  in  butter- 
flies are  known  to  be  elicited  by  sugars,  with  negative  responses  being  elicited  by 
other  types  of  chemicals  (Dethier,  1953  ;  Minnich,  1921 ).  A  peripheral  mechanism 
for  discrimination  of  acceptable  and  unacceptable  chemicals  is  thus  indicated  by 
both  the  behavioral  and  electrophysiological  results  with  butterflies. 

Tarsal  chemoreceptors  were  not  detected  in  any  of  the  three  species  of  moths. 
No  impulses  could  be  recorded  from  the  trichoid  sensilla  described  by  Frings  and 
Frings  (1949)  on  the  proboscis  of  lepidopterans.  The  characteristics  of  the  records 
obtained  from  such  tests  indicated,  however,  that  a  short-circuit  between  the  record- 
ing and  indifferent  electrodes,  established  through  the  fluids  in  the  proboscis,  prob- 
ably accounted  for  the  lack  of  any  spike  potentials  detected  through  an  active 
electrode  near  the  tip  of  the  proboscis. 

5.  DIPTERA  Amocbalcria  dcfcssa  (7  individuals)  ;  Tipnla  tririttata   (3  individu- 
als) 

Studies  on  four  genera  of  Diptera  having  been  previously  reported  (Hodgson 
and  Roeder,  1956),  the  present  work  was  confined  to  two  types  in  which  the  chemo- 
receptors might  be  expected  to  be  of  special  interest.  The  helomyzid  fly  Amoe- 
balcria  was  tested  because  of  its  occurrence  in  caves,  a  habitat  often  associated  with 
hypertrophy  of  chemical  or  tactile  senses  (Hodgson,  1955),  and  the  crane  fly  Tipitla 
was  tested  because  the  branching  structure  of  its  antennae  suggested  that  recordings 
might  be  made  from  one  or  a  few  antennal  receptors  in  a  single  antennal  branch. 
Only  Ainoebalcria  yielded  results  of  interest,  however. 

The  labellar  chemoreceptors  and  chemoreceptors  within  the  tarsal  hairs  of 
Amocbolcria  proved  to  function  similarly  to  those  in  Plionnict,  in  that  they  exhibited 
L  and  S  spikes  when  stimulated  by  sugars  or  non-sugars,  and  showed  comparable 
responses  to  tactile  and  temperature  stimulation.  Some  data  on  olfactory  receptors 
were  obtained  in  recordings  from  the  antennae  of  Amocbalcria.  A  typical  result, 
obtained  by  placing  a  fluid-filled  electrode  on  the  antenna,  is  shown  in  record  G  of 
Figure  1 .  Distilled  water  is  adequate  in  the  electrode,  and  the  results  are  essentially 


122  EDWARD  S.  HODGSON 

the  same  whether  contact  is  made  with  the  distal  tip  of  the  antenna  or  the  enlarged 
third  segment  near  the  hase  of  the  antenna.  Ablation  experiments  show  that  most 
of  the  activity  recorded  originates  in  the  third  segment  of  the  antenna  in  either 
case.  The  abundant  spikes  which  seem  to  represent  the  basal  level  of  receptor  ac- 
tivity in  the  absence  of  externally  applied  stimulation  are  not  affected  by  any  of  the 
test  solutions  applied,  but  are  decreased  in  frequency  by  vapors  of  wintergreen,  or 
citronella  (see  record  H  of  Figure  1).  This  result  was  so  contrary  to  anticipated 
findings  that  tests  were  run  with  benzene,  toluene,  and  carbon  tetrachloride  vapors, 
all  of  which  produced  similar  reversible  decreases  in  amount  of  receptor  activity. 
Unfortunately,  so  little  is  known  of  the  natural  historv  of  this  fly  that  it  is  im- 
possible to  say  what  might  constitute  the  normal  olfactory  stimuli. 

Tactile  effects  upon  the  antennal  receptors  were  observed  only  when  the  surface 
of  the  antenna  was  prodded  or  bent  in  excess  of  any  amount  of  stimulation  which  the 
antenna  would  encounter  in  flight.  Blowing  upon  the  antenna  during  a  recording  or 
varying  the  temperature  from  20  to  28  degrees  C.  produced  no  discernible  effect 
upon  the  frequencv  or  pattern  of  the  impulses  recorded.  Attempts  to  make  similar 
antennal  recordings  using  other  species  of  flies  have  yielded  only  negative  results. 

DISCUSSION 

In  view  of  the  considerable  differences  in  chemoreceptors  which  have  already 
been  reported  from  electrophysiological  studies  of  mammals  (Beidler.  Fishman  and 
Hardiman,  1955)  it  is  not  surprising  that  much  greater  differences  should  be  found 
among  members  of  such  a  heterogeneous  group  as  the  arthropods.  It  seems  clear 
that  sensitivities  to  tactile  and  temperature  stimuli  within  the  normal  physiological 
range  are  not  essential  characteristics  of  primary  chemoreceptor  cells,  even  among 
the  arthropods,  because  several  exceptions  to  this  situation  were  found  as  soon  as 
tests  were  made  of  chemoreceptors  other  than  those  on  the  fly  labellum.  Yet  it 
would  probably  be  incorrect  to  regard  the  labellar  receptors  as  primitive  or  unspecial- 
ized  receptor  cells.  Their  similarity  to  receptors  in  the  tarsal  sensilla  of  at  least 
two  of  the  butterflies  tested  suggests  that  a  sensitivity  of  the  same  cell  to  more  than 
one  type  of  energy  in  the  environment  may  have  a  high  selective  value  in  cases 
where  only  a  relatively  small  number  of  receptors  contact  a  substrate,  man}'  fea- 
tures of  which  are  significant  for  the  animal's  behavior.  This  certainly  would  be 
the  case  with  receptors  on  the  tarsus  or  proboscis  of  a  fly  or  butterfly,  or  on  the  tips 
of  the  tarsi  of  a  millipede.  The  demonstrated  multiple  sensitivities  of  single  receptor 
cells  in  those  locations  may.  therefore,  be  one  of  the  solutions  which  evolution  has 
produced  for  the  problem  of  obtaining  a  variety  of  information  about  the  environ- 
ment when  only  very  small  areas  of  the  body  are  actually  in  contact  with  the  en- 
vironment. Whether  the  several  types  of  stimuli  all  eventually  affect  the  same 
excitatory  process  within  a  single  receptor  cell  will  have  to  be  determined  by  further 
investigations.  Cases  of  double  specificities  of  receptors  in  vertebrates,  such  as  the 
temperature-touch  receptors  of  the  rattlesnake  facial  pit  (Bullock  and  Diecke,  1956), 
have  been  reported  but  it  is  very  doubtful  that  more  than  one  type  of  stimulation 
normally  acts  upon  the  same  receptor  units,  and  even  if  this  were  true  these  would 
have  to  be  considered  exceptions  to  the  general  rule  of  single  specificities  for  single 
receptors  (Granit,  1955). 

Several  correlations  might  be  noted  between  receptor  distribution  or  function 


ARTHROPOD  CHEMORECEPTION  123 

and  the  natural  history  of  the  particular  animals  concerned.  Of  the  two  cave 
crickets  providing  favorable  receptor  preparations,  Hadenoecits,  with  the  more  ex- 
tensively distributed  chemoreceptors  on  the  legs,  is  reported  to  be  more  strictly  lim- 
ited to  caves  than  CcutJiophilus  (Giovannoli.  1933).  The  selective  advantage  of 
highly  developed  chemical  senses  in  a  totally  dark  environment  is  obvious.  The 
sensitivity  of  the  chemoreceptors  of  Cainbarus  to  amino  acids  is  undoubtedly  related 
to  a  diet  of  decaying  meat,  and  the  absence  of  any  response  of  its  receptors  to  sugars 
can  be  correlated  with  the  lack  of  any  behavioral  response  to  sugars  by  this  species. 
The  results  with  butterflies  likewise  indicate  the  existence  of  a  peripheral  discrimina- 
tion mechanism  for  the  chemicals  constituting  the  normal  food  in  this  case,  sugars. 

All  of  the  spike  potentials  recorded  from  chemoreceptors  were  smaller  in  am- 
plitude than  the  spikes  from  mechanoreceptors  of  the  same  animal,  unless  the  same 
receptor  cell  responded  to  both  types  of  stimuli.  This  is  in  accord  with  the  usual 
assumption  that  chemoreceptor  fibers  are  smaller  than  mechanoreceptor  fibers 
(Dethier,  1953;  Hodgson,  1955).  The  fact  that  many  receptors  in  Cainbarus, 
Hadcnoccus,  and  Aiuoebaleria  showed  spontaneous  activity  supports  another  idea 
believed  to  be  of  some  general  applicability — that  spontaneous  activity  is  widespread 
among  sensory  cells,  and  that  anv  changes  in  the  frequency  or  pattern  of  the  spon- 
taneous activity  (  rather  than  the  mere  presence  of  impulses )  may  constitute  the  af- 
ferent ''message"  from  the  sense  organs  (Roeder,  1955).  The  antennal  receptors 
of  Amoebaleria,  showing  decreased  numbers  of  impulses  during  administration  of 
vapors,  may  illustrate  a  less  common  direction  of  change  in  spontaneous  activity 
which  serves  as  the  afferent  message. 

The  present  experiments  resolve  a  discrepancy  between  the  earlier  work  on  the 
labellar  chemoreceptors  of  the  blowfly  (Hodgson,  Lettvin  and  Roeder,  1955)  and 
the  results  obtained  by  Morita  ct  al.  (1957,  and  personal  communication)  using  the 
butterfly.  I'ancssa.  The  polarity  of  the  spike  potentials  recorded  from  Phonnia 
was  previously  reported  as  negative,  using  the  present  recording  method,  but  posi- 
tive under  similar  conditions  in  Vanessa.  All  the  spike  potentials  recorded  from 
chemoreceptors  in  the  present  studv  resulted  from  an  increase  in  positivity  at  the 
distal  tip  of  the  sensory  hairs  (position  of  the  recording  electrode)  relative  to  the 
base  of  the  same  hairs  (position  of  the  indifferent  electrode),  and  the  contrary  po- 
larity reported  in  Phonnia  was  subsequently  traced  to  an  error  in  instrumentation. 
A  precise  explanation  for  the  positive  spike  potentials  obtained  by  this  method  can- 
not be  given  at  the  present  time,  but  might  possibly  be  explained  by  generation  of 
the  main  negative  spike  potential  at  the  cell  body  region  of  the  receptor,  which 
would  leave  the  actual  chemosensory  area  with  a  relatively  positive  charge.  Ex- 
periments to  localize  the  main  impulse  generating  area  within  the  receptor  are  now 
underway. 

The  failure  to  record  potentials  from  chemoreceptors  in  a  large  majority  of  the 
arthropods  tested  could  result  from  a  real  absence  of  these  receptors  in  the  ap- 
pendages tested  or  from  limitations  of  the  technique.  The  latter  is  the  more 
probable  explanation  in  most  cases.  Particularly  unfortunate  is  the  apparent  inap- 
plicability of  the  technique  to  recordings  from  the  antennae  of  most  insects.  Unavoid- 
able short  circuits  between  indifferent  and  recording  electrodes  explain  some  nega- 
tive results,  as  noted  above,  but  inability  to  position  the  recording  electrode  over  one 
or  a  few  receptor  sensilla  and  the  small  size  of  the  spike  potentials  from  the  chemo- 


124  EDWARD  S.  HODGSON 

receptors  undoubtedly  account  for  most  of  the  failures.  The  optimum  preparation 
for  use  with  this  technique  appears  to  he  an  elongated  sensillum,  well  isolated  from 
surrounding  sensilla,  and  containing  very  few  receptor  cells — an  ideal  approached 
more  conveniently  in  the  lahellar  chemoreceptors  of  flies  than  with  any  other  ar- 
thropod preparations  }et  tested.  A  similar  survey  of  the  chemnreceptors  of  marine 
arthropods  is  planned. 

It  is  a  pleasure  to  acknowledge  the  courtesy  of  Dr.  Horton  H.  Hobhs,  Jr.,  Di- 
rector of  the  Mountain  Lake  Biological  Station,  who  facilitated  the  held  work  in 
many  ways.  Mr.  David  Bardack  assisted  in  collecting  the  animals.  Drs.  V.  G. 
Dethier  and  K.  D.  Roeder  have  been  most  helpful  in  critically  reading  the 
manuscript. 

SUMMARY 

1.  Electrophysiological  tests  with  externally  applied,  fluid-filled  electrodes  were 
performed  upon  thirty-seven  species  representing  four  classes  of  arthropods.     Af- 
ferent chemoreceptor  impulses  were  recorded  in  animals  of  five  types:  a  crayfish 
( Caiubants ) ,  a   millipede    (Pseudopolydesmus},   two   orthopterans    (Ceuthophilus 
and  Hadenoccus) ,  a  helomyzid  fly  (Ainocbalcria),  and  six  species  of  butterflies. 

2.  Receptors  sensitive  to  chemical,  tactile,  and  temperature  stimuli  within  nor- 
mal physiological  ranges  are  found  in  certain  Lepidoptera   (Epargyreus  and  Linic- 
nitis)   and  Diptera   (Aiuocbaleria] .     Receptors  with  a  dual  sensitivity  to  at  least 
two  of  the  above  types  of  stimulation  are  found  in  Pscitdopolydesinus.  Ccitthophilns, 
and  Hadenoccus.     It  is  concluded  that  multiple  sensitivities  of  receptors  are  not 
exceptional  in  arthropods. 

3.  Chemoreceptors  sensitive  to  amino  acids,  but  insensitive  to  tactile  and  tem- 
perature stimuli,  are  found  on  the  chelae  and  protopodites  of  the  first  two  walking 
legs  of  Caiubants  bartonii  sciotcnsis. 

4.  \Yith  the  present  recording  method,  spike  potentials  from   chemoreceptors 
represent  increases  in  positivity  at  the  distal  tip  of  the  receptor  cell,  relative  to  the  cell 
body. 

5.  Relationships  between  functional  characteristics  of  chemoreceptors  and  the 
natural  history  of  the  animals  are  discussed. 

LITERATURE  CITED 

BARBER,    S.    B.,    1956.     Chemoreception   and   proprioception   in    Limulus.     /.    P..rp.    Zonl..    131  : 

51-73. 

BEIDLER,  L.  M.,  1952.     Our  taste  receptors.     Sci.  Monthly,  75:  343-349. 

BEIDLER,  L.  M.,  I.  Y.  FISHMAX  AND  C.  \Y.  HARDIMAN,  1955.     Species  differences  in  taste  re- 
sponses.    Amer.  J.  Physiol..  181  :  235-239. 
BULLOCK,  T.  H.,  AND  F.  P.  J.  DIECKE,  1956.     Properties  of  an  infra-red  receptor.     /.  Phvsiol.. 

134 :  47-87. 
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MORPHOLOGY  OF  MAIN  AND  ACCESSORY  ELECTRIC 

ORGANS  OF  NARCINE  BRASILIENSIS  (OLFERS)  AND 

SOME  CORRELATIONS  WITH  THEIR  ELECTRO- 

PHYSIOLOGICAL  PROPERTIES 

ROBERT    MATHEWSON,1    ALEXANDER   MAURO,-    ERNEST    AMATNIEK 

AND  HARRY  GRUNDFEST  3 

Department  of  Neurology,  (-  ollajc  of  Physicians  and  Surgeons,  Columbia  University,  Ncv.'  }'ork, 
and   Marineland   Research    Laboratory,   St.    Aitf/iistine, 


Like  other  Torpedinidae  (  Bigelow  and  Schroeder,  1953).  Xarcine  brasiliensis 
(Olfers)  possesses  electric  organs.  While  they  have  previously  been  studied 
physiologically  (Chagas  ct  <?/.,  1953;  Cox  and  Breder,  1943)  no  anatomical  descrip- 
tions seem  to  have  been  published  of  the  organs  and  their  innervation,  presumably 
because  these  have  been  considered  to  be  identical  with  the  findings  in  Torpedo. 
In  the  course  of  a  new  study  (unpublished  data  from  this  laboratory)  of  the  electro- 
physiology  of  Narcine  electric  organs  with  intracellular  recording,  certain  dis- 
crepancies were  observed  which  indicated  differences  between  the  structure  of 
Torpedo  organs  and  those  of  Narcine.  A  hitherto  undescribed.  smaller,  accessory 
organ  was  also  found,  distinct  in  its  electrophysiological  properties  from  that  which 
will  now  be  called  the  main  electric  organ.  Anatomical  data  to  be  reported  here 
show  that  the  accessory  organ  also  differs  from  the  major  organ  in  structure,  in- 
nervation, and  in  the  size  and  orientation  of  the  electroplaques.  The  present  paper 
reports  chiefly  the  gross  anatomical  and  general  histological  findings,  relating  these 
to  the  functional  properties  of  electric  organs.  Data  on  the  embryology  and  on  the 
fine  structure  of  the  electroplaques,  including  detailed  studies  of  electron  microscopic 
preparations,  will  be  reported  elsewhere  (  Mathewson  and  Lehrer.  and  Mathewson 
and  Wachtel,  unpublished  data  )  . 

MATERIALS  AND  METHODS 

Narcine  brasilicnsis  inhabits  the  inshore  waters  of  the  Atlantic  Ocean  from 
Brazil  to  North  Carolina  (Bigelow  and  Schroeder,  1953).  It  is  one  of  the  smallest 
of  the  Torpedinidae,  the  42  adult  specimens  brought  into  the  Marineland  Research 
Laboratory  for  the  present  study  ranging  from  20  to  45  cm.  in  length.  In  contrast, 
the  larger  of  the  T.  occidental-is  dissected  by  Hunter  (  rf.  Keynes,  1956)  measured 

1  Staten  Island  Institute  of  Arts  and  Sciences. 

2  Department  of  Physiology,  Yale  University  Medical  School. 

3  This  work  was   supported  in  part  by  grants  from   Muscular   Dystrophy  Associations  of 
America,  National  Institutes  of  Health   (  B-389  C2),  National  Science  Foundation,  and  United 
Cerebral  Palsy  Research  Foundation. 

4  We  express  our  thanks  to  the  Trustees  and  staff  of  the  Marineland  Research  Laboratory, 
where  the  initial  phases  of  the  work  were  carried  out.     To  Mr.  F.  G.  Wood,  Jr.,  Curator  of 
the   Laboratory,   we   are   especially   indebted   for   his   hospitality   during   our   stay   and   for   his 
continued  cooperation. 

126 


ELECTRIC  ORGANS  OF  N.  BRASILIENS1S  127 

about  1.25  m.  Although  it  seems  that  specimens  of  this  size  have  now  hecome 
rare  (Bigelow  and  Schroeder,  1953),  a  fish  almost  0.75  m.  long  was  brought  into 
the  Marine  Biological  Laboratory  at  Woods  Hole  in  1956  (Grundfest,  1957a). 

Many  of  the  fish  available  to  us  were  used  chiefly  for  electrophysiological  work, 
and  dissection  at  this  time  was  confined  to  a  minimum.  The  electrophysiological 
data  provided  clues  to  probable  anatomical  structures  which  were  then  sought  for 
in  other  specimens,  studied  in  fresh  dissection,  or  in  preserved  preparations.  Histo- 
logical  material  was  prepared  with  a  number  of  techniques  for  different  purposes. 
The  details  of  these  methods  will  be  given  in  later  publications. 

RESULTS 
A.  The  Main  Organs 

Gross  anatomy.  Superficially  the  paired,  kidney-shaped  main  electric  organs 
of  N.  brasilicnsis  (Fig.  1)  resemble  closely  those  of  the  other  Torpedinidae 
(Bigelow  and  Schroeder,  1953;  Fritsch,  1890).  They  comprise  about  twenty  per 
cent  of  the  total  weight  of  the  fish.  Both  dorsally  and  ventrally  the  main  electric 
organs  are  in  close  contact  with  the  skin  and  their  surface  area  is  clearly  outlined. 
However,  the  patterned  pigmentation  of  the  dorsal  skin  surface  of  Narcinc  partially 
obscures  this  outline.  On  the  ventral  surface,  not  only  is  the  total  outline  of  the 
organ  clearly  visible,  but  also  the  honeycomb-like  arrangement  of  the  columns  of 
electroplaques  is  seen  through  the  skin.  The  main  organ  of  each  side  extends 
laterally  from  the  outer  surface  of  the  gill  sacs  to  a  clearly  demarcated  line  near  the 
edge  of  the  pectoral  fin.  Rostrocaudally  the  organ  starts  slightly  anterior  to  the 
eyes  and  extends  back  to  the  lines  of  the  pectoral  girdle. 

Columns  of  electroplaques.  As  in  other  Torpedinidae  the  main  organ  is  made 
up  of  a  number  of  closely  packed  vertical  columns,  each  column  composed  of 
electroplaques  stacked  one  atop  the  other  like  a  roll  of  coins.  The  packing  of  the 
columns  leads  to  the  honeycomb  pattern  mentioned  above,  but  each  column  takes 
on  an  irregular  rather  than  a  hexagonal  shape.  In  10  adult  specimens  the  number 
of  columns  ranged  from  386  to  452  (average  419,  Table  I).  This  figure  falls 
within  the  range  previously  given  for  this  species  (Bigelow  and  Schroeder,  1953; 
Cox  and  Breder,  1943;  Fritsch,  1890).  In  late  embryos  and  new-born  specimens 
examined,  the  number  of  columns  in  the  main  organ  was  appreciably  less  than  in 
the  adults  (Table  I).  This  matter,  important  in  the  theoretical  aspects  of  electric 
organ  development,  will  be  dealt  with  in  more  detail  in  the  Discussion. 

The  arrav  of  electro  plaques  in  a  column.  As  in  other  Torpedinidae,  the  electro- 
plaques  in  the  main  organ  of  Ar.  brasilicnsis  are  innervated  only  at  their  ventral 
surfaces.  This  was  established  both  electrophysiologically  (unpublished  data  from 
this  laboratory)  and  anatomically.  Electrophysiological  data,  employing  successive 
penetration  of  the  cells  in  a  column  with  a  microelectrode,  indicated  that  the  electro- 
plaques  were  exceedingly  thin.  This  was  confirmed  on  histological  preparations, 
the  average  thickness  of  the  electroplaques  being  about  7  //,  (cf.  Fig.  3),  although 
the  surface  they  present  is  large  (1.5  to  2  mm.  in  diameter,  cf.  Fig.  2).  As  with 
many  other  types  of  electroplaques,  however,  there  is  considerable  extracellular 
material  (Ballowitz.  1938:  Ellis,  1913 ;  Grundfest,  1957a ;  Luft.  1956;  Szabo,  1956). 
Thus,  there  were  onlv  486  to  541  cells  (average  495)  in  the  thickest  (ca.  2  cm.) 


128       R.  MATHEWSON.  A.  MAURO,  E.  AMATXIEK  AND  H.  GRUXDFEST 


TRIGEMINAL 


-FACIAL 


GLOSSO- 
PHARYNGEAL 


VAGUS 


MAIN  ORGAN 


ACCESSORY  ORGAN 


FIGURE   1.     Electric   organs  and   their   innervation   in   N.   brasiliensis. 
Dorsal  view,  partly  schematic. 

TABLE  I 
Number  of  electroplaque  columns  in  main  organ 


Type  of  specimen 

Number  of  specimens 

Number  of  columns* 

Average  number 

Adult 

10 

386,  381,  394,  400, 

430,  440,  447,  450, 

419 

452 

New-born 

3 

269,  287,  348 

301 

Embryo** 

5 

277,  297,  300,  348,  351 

315 

*  In  each  fish  the  columns  of  one  organ  were  counted  three  times.     The  figures  given  are 
averages. 


N  The  embryos  were  all  in  a  very  late  stage  of  development. 


ELECTRIC  ORGANS  OF  N.  BRASILIENSIS 


129 


P 


**f 

\ 


»     k 


• 


r: 


FIGURE  2.  Innervation  of  individual  electroplaque.  Arrows  point  to  four  nerve  fibers 
\\liich  become  unmyelinated  and  disperse  profusely  over  innervated  surface.  Fiber  in  lower 
left  quadrant  probably  was  cut  parallel  to  its  axis.  Largest  diameter  of  fiber  is  2  //. 

portions  of  the  main  organ.     In  the  shorter  columns,  nearest  the  outer  edge  of  the 
fish,  the  average  number  of  cells  was  314. 

B.  Accessory  Organs 

Caudad  to  each  main  organ  in  Narcine  there  lies  a  smaller  structure  that  both 
by  electrophysiological  and  anatomical  criteria  has  now  been  identified  as  another, 
hitherto  undescribed,  electric  organ.  This  differs  significantly  in  many  respects 
from  the  main  organ  and  therefore  is  termed  an  accessory  electric  organ. 

Gross  anatom\.  The  accessory  organs  arise  dorsally  in  the  articulation  of  the 
cartilaginous  scapular  process  of  each  side.  They  run  obliquely  ventral  and  slightly 


130       R.  MATHEWSON.  A.  MAURO,  E.  AMATNIEK  AND  H.  GRUNDFEST 


1  VI 


FIGURE  3.  Electron  micrograph  of  a  slightly  oblique  cross  section  of  electroplaque.  Index 
(lower  left)  is  1  M.  Innervated  surface,  seen  diagonally,  at  lower  left,  shows  nerve  fiber  follow- 
ing involuted  contour  of  cell  membranes.  Ring-like  structures  are  sections  of  infolded  fingers 
of  innervated  membrane.  Interior  of  electroplaque  shows  fibrous  material.  Non-innervated 
face  of  cell  (upper  right)  has  complex  system  of  interconnected  canaliculi.  Collagen  fibers  in 
extracellular  space.  Outlines  of  membranes  not  clear  m  this  section  which  was  cut  thick 
(0.1  M)  to  show  other  structures. 


ELECTRIC  ORGANS  OF  N.  BRASILIENSIS  131 

rostral,  toward  the  inidline,  terminating  in  contact  with  the  ventral  skin  immedi- 
ately behind  the  main  organs  (Fig.  1 ).  Each  is  about  1  cm.  in  diameter  and  about 
2  cm.  long  in  30-cm.-long  adult  fish.  It  is  separated  from  the  main  organ  by  a 
distinct  layer  of  connective  tissue. 

Columns  of  cells.  On  the  average,  only  10  columns  are  found  in  the  accessory 
organ.  At  the  ventral  surface  the  columns  are  tightly  compressed  and  assume  a 
honeycomb  appearance  which  is  also  visible  through  the  ventral  skin.  The  cells 
in  each  are  2  mm.  or  slightly  more  in  diameter.  The  area  of  each  cell  thus  is  not 
much  larger  than  in  the  main  organ  and  the  anterior  ventral  margin  of  the  accessory 
organ  is  not  clearly  differentiated  from  the  adjacent  main  organ. 

Cellular  array  in  column.  A  striking  feature,  further  differentiating  the  ac- 
cessory organ,  is  the  thickness  of  the  individual  cells,  which  is  20-30  p..  or  3  to  4 
times  greater  than  in  the  main  organ.  The  number  of  cells  in  series  in  a  single 
column  is  about  200,  indicative  of  the  large  proportion  of  extracellular  material 
also  in  this  structure.  Another  difference  between  the  main  and  accessory  organs 
is  the  orientation  of  the  innervation  of  the  electroplaques.  In  the  accessory  organ, 
the  dorsal  surfaces  of  the  electroplaques  are  innervated,  the  discharge  of  this  organ 
producing  positivity  at  the  ventral  surface  of  the  fish  (unpublished  data  from  this 
laboratory)  whereas  discharge  of  the  main  organ  results  in  negativity  at  the  ventral 
surface  (cf.  Grundfest,  1957a).  Responses  to  a  single  stimulation  of  the  accessory 
organ  produce  only  0.5  to  1  volt,  in  comparison  with  the  25  to  35  volts  generated 
during  the  discharge  of  the  main  organ. 

C.  Ncrrc  Supply  to  the  Electric  Or;/ans 

Gross  dissections  of  !\'arcine,  as  well  as  histological  preparations,  disclose  some 
differences  in  the  neural  anatomy  between  this  form  and  Torpedo.-' 

Gross  anatomv.  In  Xarcine  the  facial  (VII).  glossopharyngeal  (IX),  and 
vagus  (X)  nerves  supply  the  electric  organ  (Fig.  1).  The  fifth  cranial  nerve 
(trigeminal )  does  not  enter  the  electric  organ,  although  in  Torpedo  it  is  reported 
to  participate  in  the  innervation  (  Fritsch.  1890;  Szabo,  1955). 

The  facial,  immediately  after  emerging  from  the  skull,  bifurcates  into  two  large 
trunks.  The  cranial  branch  passes  around  the  anterior  periphery  of  the  main 
electric  organ  and  thence  radiates  throughout  the  head  and  pectoral  fin.  The 
remaining  branch  enters  the  electric  organ  immediately  anterior  to  the  first  gill 
sac.  The  glossopharyngeal  nerve,  which  emerges  from  the  brain  closely  associated 
with  the  auditory  nerve  (VIII),  leaves  the  skull  ventral  to  the  otic  capsule. 
Passing  between  the  first  and  second  gill  sacs,  it  immediately  enters  the  electric 
organ.  Of  the  total  of  four  branches  which  comprise  the  vagus  nerve,  the  first 
two  pass  between  the  second  and  third,  and  the  third  and  fourth  gill  sacs,  re- 
spectively, then  entering  the  main  electric  organ.  The  third  branch  passes  between 
the  fourth  and  fifth  gill  sacs,  runs  along  the  anterior  edge  of  the  scapular  process 
and  enters  the  accessory  electric  organ.  The  fourth  branch  of  the  nerve  runs 
parallel  to  the  vertebral  column  back  to  a  point  near  the  pectoral  girdle,  then 
disappears  into  the  musculature  of  this  area. 

5  But  one  specimen  of  Torpedo  occllata  was  available  for  comparison.  However,  published 
descriptions  of  Torpedo  neuroanatomy  are  detailed  (Bigelow  and  Schroeder,  1953;  Fritsch, 
1890;  Szabo,  1955). 


132       R.  MATHEWSON,  A.  MAURO,  E.  AMATXIKK  AND  H.  GRUNDFEST 

Innervation  within  the  electric  organ.  Once  having  entered  the  electric  organs, 
the  nerves  branch  profusely  in  the  connective  tissue,  as  has  been  described  for 
Torpedo  by  Fritsch  (1890).  Individual  electroplaques  are,  in  general,  each  sup- 
plied by  four  single  myelinated  fibers  (Fig.  2).  The  fibers  lose  their  myelin 
sheath  close  to  the  inn  ?rvated  surface,  and  their  branching  is  extremely  profuse 
(Mathewson  and  Wachtel,  unpublished  data).  The  synaptic  contacts  are  dis- 
persed over  the  innervated  electroplaque  surface,  which  is  ventral  in  the  main 
organ  and  dorsal  in  the  accessory. 

Detailed  electron  microscopic  and  histochemical  studies  on  the  K archie  electric 
organs  will  be  reported  elsewhere  (Mathewson  and  Lehrer.  unpublished  data). 
Electron  micrographs  indicate  a  considerable  degree  of  gross  differentiation  be- 
tween the  innervated  and  non-innervated  faces  of  the  electroplaques  (Fig.  3).  The 
nerves  running  along  the  innervated  surface  often  make  intimate  contact  with 
finger-like  inpocketings  of  this  membrane.  Vesicular  structures  (de  Robertis  and 
Bennett,  1953;  Robertson,  1957)  are  seen  in  the  cytoplasm  of  the  nerve  terminals. 
At  the  non-innervated  face  the  cell  is  profusely  riddled  by  interconnected- canaliculi. 
some  of  which  appear  to  extend  up  to  the  tubules  made  by  the  inpocketing  of  the 
innervated  surface. 

DISCUSSION 

Distinctions  between  the  main  and  the  accessory  electric  organs.  Several  dif- 
ferences in  electrophysiological  properties  have  already  been  indicated.  A  marked 
distinction  is  the  reverse  polarity  of  the  activity  in  the  accessory  organ.  The 
voltage  produced  by  a  single  stimulation  of  the  accessory  organ  is  small,  but  grows 
rapidly  upon  repetitive  stimulation  (unpublished  data  from  this  laboratory).  The 
large  facilitation  is  in  marked  contrast  to  the  behavior  of  the  main  organ  and  indi- 
cates important  differences  between  the  functional  synaptic  connections  of  the 
organs,  which  may  be  more  clearly  revealed  after  further  histochemical  and 
electron  microscopic  studies. 

The  anatomical  distinctions  are  also  marked,  particularly  the  reversed  surface 
of  innervation  and  the  greater  thickness  of  the  electroplaques  of  the  accessory  organ. 
The  different  orientation  of  this  organ,  with  the  columns  running  obliquely  rather 
than  dorsoventrally  as  in  the  main  organ,  and  the  delineation  of  the  accessory  organ 
by  a  distinct  investment  of  connective  tissue  suggest  that  the  muscles  from  which 
this  organ  is  derived  are  different  from  those  which  are  precursors  of  the  main 
organ  (Fritsch,  1890).  Embryological  material  has  not  yet  been  studied  sufficiently 
to  reveal  that  origin. 

Number  of  columns  and  clcctroplaqnes.  Our  counts  of  the  number  of  columns 
in  adult  fish  closely  approximate  other  data  in  the  literature.  However,  the  embryos 
and  new-born  fish  had  a  significantly  smaller  number  of  columns,  although  the 
number  of  cells  in  each  of  these  columns  was  comparable  with  that  in  adults.  This 
finding  appears  to  contradict  the  delle  Chiaie-Babuchin  rule  ( du  Bois-Reymond, 
1881  ;  Grundfest,  1957a)  which  states  that  the  total  number  of  electroplaques  laid 
down  is  fixed  early  in  development  and  does  not  increase  thenceforth.  It  seems, 
rather,  that  the  transformation  of  muscles  (or  of  their  anlagen)  to  electroplaques 
proceeds  at  unequal  rates,  more  rapidly  in  the  central  portions  of  the  main  organ 
than  at  its  periphery.  It  has  been  noted  (Grundfest,  1957a)  that  regeneration  of 


ELECTRIC  ORGANS  OF  N.  BRASILIKNSIS  133 

tissue  takes  place  in  knifefishes  (Ellis,  1913)  and  this  apparent  contradiction  of  the 
dell   Chiaie-Bahuchin   rule  has  been  confirmed   in   this   laboratory    (unpublished). 

In  three  adult  Narcinc  Cox  and  Breder  (1943  )  obtained  an  average  count  of  380 
columns  (range  343^-16).  The  average  in  three  embryos  was  286  (range  264— 
340).  These  authors  also  concluded  that  the  number  of  columns  increased  with 
growth  of  the  fish.  In  two  embryos,  they  also  counted  the  number  of  cells  in  a 
column,  finding  an  average  of  305  in  one  and  482  in  the  other.  The  last,  which  is 
close  to  the  value  obtained  in  the  present  work  from  adults  and  embryos  ( 500 ) ,  sup- 
ports the  conclusion  that  the  number  of  electroplaques  in  a  column  does  not  increase. 

The  higher  values  obtained  in  the  present  work  help  to  remove  a  puzzling  diffi- 
culty in  electrophysiological  data  ( Grundfest,  1957a).  On  the  basis  of  the  older  fig- 
ure for  the  series  elements  and  of  reported  maximal  discharge  voltage,  it  had  been 
estimated  that  the  EMF  of  a  single  electroplaque  of  Narcinc  was  about  120  mv. 
However,  these  cells  do  not  produce  spikes  but  only  postsynaptic  potentials 
(p.s.p.'s)  (Grundfest,  1957a;  and  unpublished  data  from  this  laboratory).  Un- 
like spikes  which  in  skeletal  muscle  fibers  and  in  eel  electroplaques  attain  amplitudes 
of  about  150  mv.  the  known  varieties  of  depolarizing  p.s.p.'s  do  not  exceed  the 
resting  potential,  which  is  generally  some  60-80  mv.  The  maximal  discharges  in 
the  fish  used  in  the  present  experiments  were  about  35  volts  in  amplitude  and  this 
indicates  that  each  electroplaque  was  capable  of  a  maximal  response  of  about  70  mv. 
This  calculation  approximates  the  resting  potentials  obtained  in  the  cells,  and  ac- 
cords also  with  direct  observations  on  the  responses  of  single  electroplaques  (un- 
published data  from  this  laboratory).  Calculation  for  the  e.m.f.'s  of  single  electro- 
plaques  of  Torpedo  inaniiorata  (Grundfest,  1957a)  also  gave  very  high  values.  It 
is  likely  that  Fritsch  (  1890)  also  underestimated  the  number  of  cells  arrayed  in 
series  in  each  column  in  these  fish.  Torpedo  electroplaques  are  about  20  ^  thick, 
but  are  densely  packed  (Luft,  1956,  Fig.  4).  It  is  very  unlikely,  therefore,  that  in 
these  larger  fish  the  series  array  (given  as  400)  is  smaller  than  that  found  in 
Narcinc  (  500). 

Inncrrations  of  the  electric  organs.  It  has  already  been  noted  that  the  trigeminal 
nerve  does  not  supply  the  electric  organs  in  Narcinc.  According  to  Fritsch  ( 1890) 
the  Torpedo  organs  are  innervated  by  the  trigeminal  and  vagus  nerves.  However, 
he  details  an  extensive  controversy  regarding  this  question.  Other  authors  (cf.  also 
Rosenberg,  1928)  included  the  facial  and  hypoglossal  in  the  nerve  supply  of  the  or- 
gans. Hunter's  famous  preparation  (reproduced  in  Rosenberg,  1928)  shows  the 
electric  organ  of  T.  occidental!*  supplied  only  by  the  nerves  that  would  appear  to 
be  the  glossopharyngeal  and  the  first  two  branches  of  the  vagus  nerve. 

Another  difference  between  Torpedo  and  Narcinc  is  the  finding  that  the  innerva- 
tion  of  single  electroplaques  in  Narcinc,  while  as  regular  as  is  that  pictured  for 
Torpedo  (Fritsch,  1890;  Grundfest,  1957a),  does  not  occur  at  six,  but  at  four  points 
<»f  the  cell  surface.  It  seems  likely  that  Fritsch  (1890)  overemphasized  the  hex- 
agonal configuration  in  Torpedo  and,  indeed,  in  a  number  of  illustrations  of  his 
monograph  he  shows  surface  views  of  the  columns  with  shapes  that  are  far  from 
being  regular  or  hexagonal. 

Some  functional  correlations  "a'itli  the  anatomical  data.  The  dense  synaptic  in- 
nervation  of  the  individual  electroplaques  in  Narcinc  is  in  agreement  with  the  simi- 
lar data  reported  for  Torpedo  and  Raia  ( Ballowitz,  1938;  Grundfest,  1957a). 


134       R.  MATHEWSON.  A.  MAURO,  E.  AMATNIEK  AND  H.  GRUNDFEST 

This  would  appear  to  have  an  important  functional  value.  The  cells  generate  only 
p.s.p.'s  which  are  not  electrically  excitable  and  therefore  do  not  propagate  (Grund- 
fest,  1957b).  A  maximal  discharge  of  the  organ  would  be  produced  only  by 
simultaneous  activation  of  a  large  proportion  of  the  membrane.  This  result  is 
achieved  by  dense  synaptic  terminations. 

The  usefulness  of  this  densely  innervated  surface  for  a  number  of  experimental 
purposes  (Grundfest,  1957a)  is  augmented  by  the  finding  that  the  accessory  organ 
has  different  kinetics  of  responsiveness  to  its  neural  stimuli.  As  noted  above,  this 
will  provide  a  comparison  material  in  the  same  preparations,  not  only  for  electro- 
physiological  studies,  but  also  for  correlation  of  structure  and  function. 

The  preliminary  electron  microscopic  data  also  suggest  some  possible  correla- 
tions between  function  and  structure.  The  current  theories  of  muscle  structure  and 
of  the  contractile  process  (cf.  Huxley,  1957)  seek  to  account  for  transfer  of  activity 
from  the  electrically  excitable  membrane  to  the  contractile  elements  by  a  special 
membrane  extending  into  the  muscle  fiber  and  lying  perhaps  at  the  electrically  ex.- 
ritnblr  mrmbrnnr  tn  tin  i  i  ml  rnrt  ilr  i"1rnmit  i  by  n  ;-;nrrirrr-mrmbrni1r  i — ITU d inn;  into 


fiber  nt-L/L-U4nrr-|tf^Uaps  it  the  Z  lines.     The  dense  canaliculi  seen  in  the 


non-innervated  aspect  of  the  Narcine  electroplaques  might  be  the  remnants  of  these 
structures,  tubules  which  represent  extensions  of  the  membrane.  The  inpocketings 
of  the  opposite,  synaptically  excitable  membrane  may  be  continuous  with  the  seg- 
ments of  the  canalicular  structures.  A  rather  similar,  but  not  so  dense  system  is  also 
found  in  eel  electroplaques  (Grundfest,  1957a;  Luft,  1956)  and  in  other  types  of 
electric  organ  (personal  communication  from  Dr.  Luft).  Examination  of  electric 
organs  in  their  various  embryological  states  therefore  may  furnish  important  clues 
to  the  nature  of  the  transfer  from  conductile  to  contractile  activity. 

Narcine  electric  organ  also  may  be  a  favorable  object  to  test  current  views  (del 
Castillo  and  Katz.  1956;  de  Robertis  and  Bennett,  1953;  Robertson,  1957)  that  the 
vesicles  in  the  presynaptic  terminals  may  represent  concentrations  of  transmitter 
agent.  Repetitive  stimulation  of  the  electric  nerve  of  Narcine  (Chagas  et  al..  1953  ) 
rapidly  blocks  the  response  of  the  organ.  With  the  period  of  unresponsiveness  is 
correlated  a  large  fall  in  the  concentration  of  acetylcholine  in  the  organ.  The  grad- 
ual return  of  responsiveness  is  also  associated  with  a  rise  in  the  acetylcholine  concen- 
tration. If  the  vesicles  are  the  sites  of  transmitter  storage,  they  should  be  sub- 
jected to  marked  changes  upon  repetitive  stimulation  and  during  subsequent  re- 
covery of  responsiveness.6 

LITERATURE  CITED 

BALLOWITZ,  E.,  1938.     Elektrische  Organe.     Hdbch.  d.  -ccnjl.  Anatomic.  5:  657-682. 
BIGELOW,  H.  B.,  AND  W.  C.  ScHROEDER,  1953.     Fishes  of  the  Western  North  Atlantic.     No.   1. 

part  2.     Yale  University  Press. 
DU  BOIS-REYMOND,  E.,  1881.     Dr.   Carl   Sachs:   Untersuchungen  am   Zitteraal   Gyinnntus  clcc- 

tricus.     Leipzig,  Veit,  446  pp. 
DEL  CASTILLO,  J.,  AND  B.  KATZ,  1956.     Biophysical  aspects  of  neuromuscular  transmission.     In: 

Progress  in  Biophysics,  Vol.  6.     London,  Pergamon  Press. 
CHAGAS,  C.,  L.  SOLLERO  AND  M.  MIRANDA,  1953.     On  the  utilization  of  acetylcholine  during  the 

electric  discharge  of  Narcine  hrasiliensis  (Otters).     An.  Acad.  Bras.  Cicuc..  25:  319- 

325. 

6  For  the  use  of  the  electron  microscope  and  other  facilities  we  are  indebted  to  Drs.  Leonard 
Ornstein  and  Allen  Wachtel  of  the  Cell  Research  Laboratory,  Mt.  Sinai  Hospital,  New  York. 
Thanks  are  also  due  to  Miss  Ruby  Tamura  for  preparing  much  of  the  tissue  used  in  this  study. 


ELECTRIC  ORGANS  OF  N.  BRASILIENSIS  135 

Cox,  R.  T.,  AND  C.  M.  BREDER,  1943.  Observations  on  the  electric  discharge  of  Narcinc  bmsili- 
ensis  (Olfers).  Zooloi/ica,  8 :  45-51. 

ELLIS,  M.  M.,  1913.  The  Gymnotid  Eels  of  Tropical  America.  Mem.  Museum  Carnegie  In- 
stitute, 6,  No.  3.  Pittsburgh. 

FRITSCH,  G.,   1890.     Die  Elektrischen  Fische :   II  Abteilung:   Die  Torpedineen.     Leipzig,   Veit. 

GRUNDFEST,  H.,  1957a.  The  mechanisms  of  discharge  of  the  electric  organ  in  relation  to  gen- 
eral and  comparative  electrophysiology.  Pp.  1-85  in  :  Progress  in  Biophysics,  Vol.  7. 
London,  Pergamon  Press. 

GRUNDFEST,  H.,  1957b.  Electrical  inexcitahility  of  synapses  and  some  consequences  in  the  cen- 
tral nervous  system.  Physiol.  Revs..  37:  337-361. 

HUXLEY,  A.  F.,  1957.  Muscle  structure  and  theories  of  contractions.  Pp.  255-318  in :  Progress 
in  Biophysics,  Vol.  7.  London,  Pergamon  Press. 

KEYNES,  R.  D.,  1956.     The  generation  of  electricity  in  fishes.     Endeavour,  15:  215-222. 

LUFT,  J.  H.,  1956.  The  fine  structure  of  the  electric  organ  of  the  electric  eel  and  Torpedo  ray. 
/.  Biophys.  Biochcm.  L'ytol.  Suppl.,  2:  279-321. 

DE  ROBERTIS,  E.  D.  P.,  AND  H.  S.  BENNETT,  1953.  Some  features  of  the  submicroscopic  mor- 
phology of  synapses  in  frog  and  earthworm.  /.  Biophys.  Biochcm.  Cytol..  1  :  47-58. 

ROBERTSON,  J.  D.,  1957.  Some  aspects  of  the  ultrastructure  of  double  membranes.  In:  Ultra- 
structure  and  Cellular  Chemistry  of  Neural  Tissue.  New  York,  Hoeber-Harper. 

ROSENBERG,  H.,  1928.  Die  elektrischen  Organe.  Handb.  d.  normalen  u.  pathologischen  Physiol. 
Berlin,  Springer. 

SZABO,  T.,  1955.  Quelques  precisions  sur  le  noyau  de  commande  centrale  de  la  decharge 
electrique  chez  le  Raie  (  Ruin  clat '<//<;).  /.  dc  Physiol.,  47  :  283. 

SZABO,  T.,  1956.  Sur  la  structure  et  le  type  d'innervation  de  1'electroplaque  d'un  Mormyre, 
Gnathonemus  seneqalensis  elongatus.  t.  7^.  Acad.  .\ci..  242:  2045-2048. 


THE  OXIDAT1VE  METABOLISM  OF  EGGS  OF  URECHIS  CAUPO  : 

LORD  ROTHSCHILD  =  AND  ALBERT  TYLER 
Division  of  Bioloi/v,  California  Institute  of  Technology,  Pasadena,  Lalifoniiii 

The  cytochrome  system  is  of  such  widespread  occurrence  in  cells  of  aerobic  or- 
ganisms that  reports  of  its  absence  in  any  particular  case  are  of  special  interest. 

There  have  been  several  instances  in  which  spectroscopic  detection  of  absorp- 
tion bands  of  the  cytochromes  was  at  first  reported  to  be  negative  and  later  shown  to 
be  positive  when  improved  methods  were  employed.  Thus  in  eggs  of  sea  urchins 
Brachet  (1934),  Linclahl  (1936).  Krahl.  Keltch  and  Clowes  (1939)  and  Ball  and 
Meyerhof  (  1940)  reported  that  the  cytochrome  bands  did  not  show  up  spectroscopi- 
cally,  although  early  evidence  of  inhibition  of  O.,-uptake  by  cyanide  and  by  carbon 
monoxide  (Runnstrom,  1930)  indicated  the  operation  of  the  cytochrome  system,  at 
least  in  the  fertilized  eggs.  Later,  Rothschild  (1949),  Borei  (1951)  and  Yeas 
(1954),  using  the  method  of  Keilin  and  Hartree  (1939,  1949)  of  intensifying  cy- 
tochrome bands  by  cooling  the  material  in  liquid  air.  were  able  to  demonstrate  the 
bands  of  cytochromes  a  and  h. 

./ 

It  was  also  thought,  at  one  time,  that  the  respiratory  system  differed  qualitatively 
in  unfertilized  and  fertilized  sea  urchin  eggs,  the  cytochrome  system  being  inopera- 
tive in  the  former  and  brought  into  play  upon  fertilization  (Korr,  1939).  This  was 
based  on  evidence  of  insensitivity  of  the  respiration  of  unfertilized  eggs  to  inhibi- 
tion by  cyanide  and  carbon  monoxide  (Runnstrom,  1930;  Lindahl,  1939;  Korr, 
1937),  and  reported  differences  in  the  effect  of  temperature  on  the  respiration  of 
unfertilized  and  fertilized  eggs  ( Rubenstein  and  Gerard,  1934;  Korr,  1937). 
However,  this  evidence  has  now  been  largely  contradicted.  Thus,  Robbie  ( 1946b) 
showed  that  the  C), -uptake  of  unfertilized  sea  urchin  eggs  could  be  almost  com- 
pletely inhibited  by  low  concentrations  of  cyanide  when  the  precaution  is  taken  of 
preventing  the  distillation  of  cyanide  from  the  egg  suspension  to  the  center-well  of 
the  manometer  vessel,  by  the  use  of  appropriate  Ca(CN)2-Ca(OH)2  mixtures  in 
the  center  well.  In  regard  to  the  effect  of  carbon  monoxide  on  unfertilized  sea 
urchin  eggs,  Rothschild  (1949)  was  able  to  demonstrate  a  photo-reversible  inhibi- 
tion of  O,-uptake  when  account  was  taken  of  a  CO-induced  stimulation  and  a  light- 
induced  inhibition  of  respiration.  Concerning  the  effect  of  temperature  on  respira- 
tory rates,  further  measurements  (Tyler  and  Humason,  1937;  Borei  and  Lybing, 
1949)  have  shown  no  significant  differences  between  unfertilized  and  fertilized  sea 
'rchin  eggs. 

Tn  eggs  of  the  echiuroid  worm  UrccJiis  canpo,  a  failure  to  detect  the  absorption 

of  cytochrome  was  reported   by   Horowitz   and   Baumberger    (1941).     In 

thes          '-s  there  is  a  reversibly  autoxidizable  pigment  which  Horowitz    (1940a) 

ivestigation  was   supported  by  a  research  grant    (C-2302-C3)    from   the   National 
Cancer  Institute   of  the  National  Institutes  of  Health,  U.  S.  Public  Health  Service. 

-  Permanent    address :     Department    of    Zoology,    University    of    Cambridge,    Cambridge, 
England. 

136 


METABOLISM  OF  URECHIS   K(iGS  137 

called  urechrome.  From  the  facts  that  both  the  oxidized  and  reduced  states  are 
observed  naturally  in  the  eggs,  depending  upon  the  presence  or  absence  of  oxygen, 
that  the  pigment  is  reducible  by  the  cells,  and  that  it  autoxidizes  in  the  physiological 
range  of  pH,  he  concluded  that  it  was  probably  involved  in  the  cellular  respiration. 
Upon  further  characterization  Horowitz  and  Baumberger  (1941)  suggested  that 
the  pigment  was  related  to  the  hemins.  Its  chemical  constitution  has  not  as  yet 
been  determined. 

For  these  and  other  reasons  we  decided  to  examine  the  CX-uptake  of  fertilized 
eggs  of  U.  caupo  in  the  presence  of  cyanide  and  of  carbon  monoxide,  and  to  examine 
the'  eggs  spectroscopically  using  the  lo\v  temperature  method  of  Keilin  and  Hartree 
(1939).  Previous  measurements  of  the  respiration  of  this  material  were  made  for 
other  purposes  (Tyler,  1936;  Tyler  and  Humason,  1937  ;  Tyler  and  Horowitz,  1938; 
Horowitz,  1940b)  and  it  was  noted  that  the  fertilized  eggs  have  rather  consistent 
values  for  their  absolute  rate  of  O ..-uptake,  although  that  of  the  unfertilized  eggs  may 
vary  greatly  from  one  batch  to  another.  Some  preliminary  experiments  with  cy- 
anide and  azide  were  mentioned  in  the  report  of  a  seminar  talk  (Tyler,  1937). 
These  were  for  the  purpose  of  investigating  possible  correlations  between  cleavage 
retardation  and  respiratory  inhibition  and  were  done  before  the  introduction  by  Rob- 
bie (1946a)  of  the  Ca(CN)2-Ca(OH).,  center- well  mixtures  for  preventing  loss 
of  cyanide  from  cell-suspensions  in  the  manometer  vessels.  The  preliminary  ex- 
periments indicated,  however,  that  inhibition  of  respiration  by  cyanide  was  obtain- 
able in  these  eggs. 

MATERIAL  AND  METHODS 

General  manometric  procedure.  Eggs  of  U .  caupo  were  inseminated  and  washed 
in  sea  water  buffered  at  pH  8  with  0.01  M  glycylglycine,  which  Tyler  and  Horo- 
witz (1937)  showed  to  be  a  suitable  non-injurious  agent  to  replace  the  bicarbonate 
system  of  ordinary  sea  water.  The  latter  system  does  not  provide  satisfactory 
buffering  because  the  absorption  of  CO._,  by  the  alkali  in  the  manometer  vessels  oc- 
casions a  rise  in  pH  of  the  sea  water  which  is  only  partially  and  variably  compen- 
sated by  the  CO,  production  of  the  respiring  cells. 

After  examination  to  check  that  fertilization  had  been  successful,  3-  or  4-ml.  ali- 
quots  of  egg-suspension  were  transferred  to  standard  Warburg-Barcroft  manometer 
flasks  whose  calibration  volumes  were  around  20  ml.  Readings  were  taken  after 
a  30-minute  equilibration  period  in  the  water-bath,  the  shaker  speed  being  95  c.p.m. 
at  4  cm.  stroke.  The  temperature  was  20°  C. 

Cyanide  experiments.  Robbie's  (T946a)  Ca(CN)2-Ca(OH)2  mixtures,  in 
0.6-ml.  quantity,  were  used  in  the  center-wells  of  the  manometer  vessels  in  order 
to  establish  and  maintain  known  concentrations  of  cyanide  in  the  egg  suspensions, 
and  provide  sufficient  alkali  to  absorb  the  respiratory  CO...  Fluted  filter  papers 
were  used  in  the  center-wells  to  increase  the  absorbing  surface.  A  stock  1.32  M 
calcium  cyanide  solution  was  prepared  according  to  Robbie  and  Leinfelder  (1945) 
and  this  was  diluted  with  lO^r  Ca(OH)2  according  to  Robbie's  (1946a)  figures  to 
provide  center-well  mixtures  establishing  the  following  concentrations  of  HCN  in 
the  experimental  fluid  at  20°  C. 

HCN  molarity  10^  \Q->  5X10  10-'  5X1Q-" 

Molarity  of  Ca(CN)2  in  10%  Ca(OH)2         (U8  0.046          0.023         0.0054         0.0028 


138 


LORD  ROTHSCHILD  AND  ALBERT  TYLER 


In  some  experiments  an  appropriate  quantity  of  XaCX  was  added  to  the  egg-sus- 
pensions in  the  manometer  flasks  just  before  the  beginning  of  the  experiment.  In 
the  latter  case  the  equilibration-time  was  reduced  from  thirty  to  fifteen  minutes. 
Manometer  flasks  and  other  vessels  containing  cyanide  solutions  were  kept  stoppered 
at  all  times  except  when  eggs  were  added  and  the  flasks  were  put  on  the  manometers. 
CO  experiments.  The  gas  phase  of  the  manometers  was  filled  with  95%  CO 
in  O2  (95%  CO/Oo),  after  flushing  out  the  air.  Xinety-five  per  cent  XL,/O2  and 
air  controls  were  run  at  the  same  time.  The  center  wells  contained  0.3  ml.  X/l 
KOH  and  filter  papers.  Equilibration  was  in  the  dark  for  ten  minutes. 

RESULTS 

Cyanide  experiments.  The  results  of  three  sets  of  experiments  were  clear-cut  in 
the  sense  that,  even  at  low  concentrations,  cyanide  inhibited  the  respiration  of  fer- 
tilized eggs.  Data  from  one  of  these  are  plotted  in  Figure  1.  The  lines  labelled 

TABLK   1 

The  effect  of  cyanide,  added  20  to  25  minutes  after  fertilization,  on  the  percentage  development 
of  eggs  of  Urechis  caitpo,  examined  at  3  hours.      The  sen  water  contained  0.0 1   M 

glycyl  glycine,  pH  8.0, 


Cone.  HCN 

1  "ncleaved 

2-cell 

4-cell 

8-cell 

16-32  cell 

Unfertilized 

10~4  M 
5-10-5  M 
IfT5  M 

99  \ 
99jP°la 

49 

r  bodies 
30 

20 

1 
1 
1 

5-10-6  M 

2 

1 

4 

46 

4ft 

1 

0 

3 

96 

1 

O.KOH  and  O,Ca(OH)o  were  controls  to  compare  the  CO2-absorptive  powers  of 
10%  KOH  and  Ca(OH),  in  the  center-wells  of  the  manometer  flasks.  As  this 
and  other  tests  showed,  the  Ca(OH)2  proved  as  effective  as  the  KOH  in  absorbing 
CO2  under  the  conditions  of  these  experiments. 

Table  I  shows  the  effects  of  the  different  concentrations  of  cyanide  on  the  de- 
velopment of  the  eggs  when  examined  at  the  end  of  the  experiment. 

Carbon  monoxide  experiments.  The  results  of  an  experiment  in  which  just- 
fertilized  eggs  were  subjected  to  95%  CO/Oo  and  95%  N2/O,  are  shown  in  Figure 
2,  in  which  periods  of  illumination  and  darkness  are  indicated  by  black  and  white 
blocks  along  the  time  axis.  If  the  rate  of  Oo-consumption  "  in  the  curve  labelled 
CO/Oo  is  examined  by  itself,  it  is  clear  that  it  rises  upon  illumination  and  falls  in 
darkness  in  the  manner  considered  characteristic  of  cytochrome-catalyzed  respira- 
tion. When,  however,  comparison  is  made  between  the  curve  labelled  CO/O2  and 
the  control  labelled  N2/O2,  it  is  equally  clear  that,  in  the  light,  CO  also  stimulates 
the  gas-uptake  of  these  eggs.  Illumination  had  no  inhibitory  effect  on  the  O2- 
uptake  of  eggs  in  equilibrium  with  air. 

Table  II  shows  the  effect  of  CO  in  this  experiment  on  egg  development.     The 

3  The  use  of  the  terms  Oo-consumption,  O.-uptake,  and  respiration  in  the  description  and 
discussion  of  the  CO-experiments  is  subject  to  the  qualification  that  there  is  the  possibility  (see 
Discussion)  that  some  of  the  gas  consumed  might  be  CO. 


METABOLISM  OF  URECHIS  EGGS 


139 


inhibition  is  not  so  marked  as  in  the  cyanide  experiments,  but  it  might,  of  course,  be 
more  dramatic  if  higher  CO  tensions  were  used. 

The  results  of  six  sets  of  experiments  with  95%  CO/CX  and  95%  N2/O2  are 
presented  in  Table  III.  The  last  two  columns  of  the  table  give  a  measure  of  the 
effect  of  CO  on  the  respiratory  rate,  in  the  dark  and  in  the  light,  based  on  lateral 


60 
time,  minutes 


90 


FIGURE  1.     The  respiration  of  eggs  of  Urcchis  caiipo  in  the  presence  of  HCN.     For  further 

details  see  text. 


140 


LORD  ROTHSCHILD  AND  ALBERT  TYLER 


20  40         80         120 

time,  minutes 


200 


FIGURE  2.  The  oxygen  uptake  of  eggs  of  Vrcchis  canpo  in  the  presence  of  95%  CO  in  O2 
and  of  95%  N«  in  O,.  The  black  and  white  blocks  along  the  time  axis  correspond  to  periods  of 
darkness  and  illumination.  For  further  details  see  text. 


METABOLISM  OF  URECHIS  EGGS 


141 


TABLE   1 1 

The  effect  of  95%  CO  in  O*  and  95%  N2  in  Oi  on  the  percentage  development  of  eggs  of  Urechis  caupo, 
exposed  at  j  hour  and  examined  at  5  hours  after  fertilization.      The  sea  water  contained 

0.01  M  glycyl  glycine,  pH  8.0,  T°  C.  20 


Gas 


I'ncleaved 


64-cell 


128-cell 


Air 

15 

50 

35 

N2 

15 

50 

35 

CO 

15 

85 

TABLE  1 1 1 

Effect  of  carbon  monoxide  on  the  respiration  of  eggs  of  L'rechis  caupo  in  the  light  and  in  the  dark 
(All  experiments  started  about  40  minutes  after  fertilization.      Temp.  20°  C.) 


Experiment 

Respiration 
period 

Cu.mm.  O?  per  hr.  per  10-'  eggs 

Resp.  in  95%  CO-5%  O2 

rv  '  1  1  Irt1^ 

'    resp.  in  95%  N2-5%  Oz 

95%  CO-5%  02 

95%  N2-5%  02 

Dark 

Light 

1 

0'-15' 

dark 

7.6 

9.7 

0.78 

15'-30' 

light 

14.6 

9.7 

1.59 

30'-45' 

light 

13.5 

8.2 

1.65 

45'-60' 

dark 

5.9 

7.7 

0.77 

60'-75' 

dark 

4.9 

6.7 

0.73 

2 

0'-20' 

light 

15.7 

9.8 

1.60 

20'-60' 

dark 

7.1 

9.1 

0.78 

60'~100' 

light 

19.5 

9.3 

1.88 

100'-  140' 

dark 

7.8 

8.8 

0.89 

140'-160' 

light 

15.7 

9.8 

1.60 

3 

0'-21' 

dark- 

7.5,     7.6 

9.4,    8.7 

0.83 

20'-40' 

light 

17.1,  18.3 

8.0,  10.1 

1.95 

40'-80' 

dark 

6.8,    6.8 

7.2,     6.1                  1.02 

80'-120' 

light 

13.5,  15.1 

6.9,     8.4 

1.87 

120'™  140' 

dark 

6.9,    6.6 

8.0,     7.5 

0.87 

4 

0'-30' 

dark 

8.7 

11.8,  10.8 

0.77 

30'-60' 

dark 

9.6 

11.1,  14.7 

0.75 

60'-90' 

light 

18.4 

8.2,    9.3 

2.10 

90'-120' 

light 

17.6 

8.8,    8.5 

2.02 

120'-240' 

light 

15.2 

8.9,    8.9 

1.71 

240'-270' 

light 

14.4 

8.9,    8.5 

1.66 

270'-300' 

light 

15.2 

10.4,  10.0 

1.49 

5 

0'-90' 

light 

15.8 

6.4 

2.47 

90'-  150' 

light 

13.0 

5.7 

2.28 

150'-180' 

light 

14.1 

5.7 

2.47 

180'-240' 

light 

13.8 

6.5 

2.12 

6 

0'-60' 

light 

13.5 

9.4 

1.44 

60'-210' 

light 

15.4 

10.9 

1.41 

210'-240' 

light 

13.5 

7.4 

1.82 

240'-270' 

light 

11.8 

5.7 

2.07 

270'-300' 

light 

15.2 

9.9 

1.53 

142  LORD  ROTHSCHILD  AND  ALBERT  TYLER 

comparisons  (i.e.,  of  different  vessels  run  in  parallel  with  aliquots  of  the  same  egg- 
suspension).  In  the  dark  the  respiratory  rate  in  95%  CO/O2  is  consistently  lower 
than  in  95%  N2/O2.  Rigid  statistical  treatment  would  be  complicated  because  of 
the  differences  in  times  of  readings,  magnitude  of  respiration,  etc.,  in  the  different 
experiments.  However,  a  simple  averaging  of  the  percentage  decrease  (with  double 
and  quadruple  weights  for  experiments  4  and  3,  respectively)  gives  a  15  per  cent 
inhibition  of  respiratory  rate  in  95%  CO/O2  in  the  dark. 

Similarly  calculated,  there  is  in  these  experiments,  in  the  light,  an  85  per  cent 
average  increase  in  respiratory  rate  of  the  eggs  in  95%  CO/O2  over  that  of  the 
parallel  controls  in  95^  N2/O,.  The  figures  in  Table  III  also  show,  for  individual 
manometer  vessels,  the  great  effect  of  alternate  light  and  dark  periods  on  the  respira- 
tion of  the  eggs  in  95%  CO/O2  and  the  lack  of  significant  effect  of  light  and  dark 
periods  on  the  respiration  of  the  eggs  in  95%  N../O.,. 

Spectroscopic  examination  of  eggs.  We  have  examined  the  unfertilized  eggs 
of  Urechis  with  a  narrow-dispersion  hand  spectroscope  (Keilin,  1925)  at  the  tem- 
perature of  liquid  nitrogen,  the  eggs  being  suspended  in  5Q%  glycerol  (v/v)  with 
sodium  dithionite  added  (Keilin  and  Hartree,  1939,  1949,  1955).  A  double  ab- 
sorption band  at  551  m^,  which  is  in  the  region  of  the  a-band  of  cytochrome  c, 
could  be  clearly  seen.  A  further,  faint,  absorption  band  at  580-590  m/x  (cyto- 
chrome a)  was  also  seen.  The  presence  of  these  absorption  bands  was  confirmed 
by  Professor  D.  Keilin  and  Dr.  R.  Hill. 

Reduced  cytochrome  c  was  rapidly  oxidized  by  egg  brei  in  phosphate  buffer. 
A  peculiar  phenomenon  was  observed  during  examination  of  the  oxidation  of  cyto- 
chrome r  by  egg  brei.  When  the  oxidized  cytochrome  c  and  egg  brei  was  kept  in 
comparative  darkness  and  then  illuminated  through  the  microscope  sub-stage  con- 
denser (which  automatically  occurs  during  spectroscopic  examination),  the  absorp- 
tion bands  of  reduced  cytochrome  c  gradually  reappeared.  This  also  was  con- 
firmed by  Professor  D.  Keilin  and  Dr.  R.  Hill.  The  most  probable  interpretation  is 
that  in  the  presence  of  light,  some  reducing  substance  is  produced  by  the  eggs,  caus- 
ing the  reduction  of  cytochrome  c.  This  phenomenon  may  have  some  connection 
with  the  inhibitory  action  of  light  on  the  respiration  of  sea  urchin  eggs  (Rothschild, 
1949),  though,  as  mentioned  above,  we  have  not  observed  any  comparable  light- 
inhibition  of  respiration  in  Urechis  eggs.  Certain  dyes  are  affected  by  light  in  ways 
which  would  be  consistent  with  the  observed  reduction  of  cytochrome  c  in  light, 
which  raises  the  possibility  that  urechrome  may  be  concerned  in  the  phenomenon. 
For  example,  Equ.  (3)  in  Clare's  article  in  Hollaender's  Radiation  Biology,  Vol. 
Ill  (1956) 

DHL,  +  O,  ->  H,O,  +  D. 
if  written  in  the  form 

DH2  +  2  cyt.c3*  ->  2  cyt.c2-  +  D  +  2H+ 
is  suggestive  in  this  connection. 

DISCUSSION 

In  the  introduction  to  this  paper,  reference  was  made  to  Horowitz's  (1940a) 
view  that  urechrome  and  not  cytochrome  catalyzed  the  respiration  of  Urechis  eggs ; 


METABOLISM  OF  URECHIS  EGGS  143 

this  opinion  was  based  on  the  facts  that  urechrome  is  reversibly  autoxidizable  and 
that  no  absorption  bands  of  cytochrome  were  observed.  We  have  now  shown  that 
the  absorption  bands  of  cytochrome  are  present  in  these  eggs  and  that  an  egg  brei 
can  oxidize  reduced  cytochrome  c.  Moreover,  the  inhibition  studies  with  cyanide 
and  carbon  monoxide  support  the  view  that  the  respiration  of  these  eggs  is  cyto- 
chrome-catalyzed.  Just  where  urechrome  fits  into  the  picture  is,  at  present,  un- 
certain. The  effects  of  CO  and  cyanide  on  this  pigment  have  not,  as  yet,  been 
studied. 

The  stimulating  effect  of  carbon  monoxide  on  respiration  has  been  noted  in  many 
experiments  with  eggs  and  other  tissues.  The  following  citations  from  the  litera- 
ture on  this  subject  will  serve  to  illustrate  the  widespread  occurrence  of  the 
phenomenon. 

Runnstrom  ( 1930)  found  that  the  respiration  of  unfertilized  eggs  of  Paracentro- 
tus  and  Arbacia  was  either  not  inhibited  or  somewhat  higher  in  carbon  monoxide- 
oxygen  mixtures  than  in  air,  while  that  of  the  fertilized  eggs  was  greatly  inhibited. 
Presumably,  although  not  explicitly  stated,  these  experiments  were  run  in  the  dark. 

Lindahl  (1939)  obtained  a  44%  stimulation  of  the  respiration  of  unfertilized 
eggs  of  Paracentrotus  by  75%  CO/O2  in  the  dark,  and  this  increased  (to  ca.  100%) 
upon  illumination.  With  decrease  in  oxygen  tension  to  5%  (+  15%  N2  and  75% 
CO)  the  stimulation  decreased.  For  freshly  fertilized  eggs  in  the  dark  he  obtained  a 
slight  stimulation  in  75%  CO/O2  and  a  marked  inhibition  in  95%  CO/O2.  In  the 
light  the  fertilized  eggs  showed  marked  stimulation  by  75%  CO/O2  and  this  effect 
decreased  as  the  O2  concentration  was  dropped  to  5%  at  constant  CO. 

Rothschild  (1949)  measured  the  respiration  of  unfertilized  eggs  of  Psam- 
mechinus  miliaris  in  various  CO-O,  mixtures.  In  14  comparisons  of  the  effect  of 
95%  CO/O2  with  95%  N2/O2  in  the  dark  there  was  no  difference  in  two,  an  11% 
decrease  in  three  and  a  14%  increase  in  nine.  Twenty-four  comparisons  of  the 
effect  of  95%  CO/O2  in  dark  with  that  in  light  showed  a  44%  increase  in  the  light. 
At  the  same  time  he  found  an  inhibitory  effect  of  light  on  the  respiration  of  the  un- 
fertilized eggs  in  air.  This  averaged  38%  in  44  experiments.  With  80%  CO/O2 
in  the  dark  there  was  an  average  of  55%  increase  in  respiration  above  that  in  80% 
N,/O2,  and  no  significant  change  upon  illumination. 

In  the  ascidian  Phalhisia  mamniillata  Minganti  (1957)  found  an  increase  in 
respiration  of  the  unfertilized  eggs  in  95%  CO/O2  in  the  dark  and  a  further  in- 
crease in  the  light.  The  fertilized  eggs  showed  a  14%  to  20%  decrease  in  the  dark, 
which  is  about  the  same  degree  of  inhibition  as  in  the  present  experiments,  and  an 
increase  (up  to  40%)  in  the  light. 

Bodine  and  Boell  (1934)  obtained  CO-stimulation  of  respiration  of  diapause 
embryos  of  the  grasshopper  Mclanoplus  differentialis  and  no  significant  effect  of 
light.  A  similar  stimulation  by  CO  was  found  by  Wolsky  (1941)  in  a  bivoltine 
race  of  the  silkworm  Bombyx  inori,  but  not  (Wolsky,  1938)  in  pupae  of  Drosophila 
inclanogaster.  Wolsky  (1938)  attributes  this  difference  to  the  pupal  stage  being 
one  of  great  activity  as  compared  with  diapause.  Schneiderman  and  Williams 
(1954)  found  that  the  respiration  of  diapausing  pupae  of  the  Cecropia  silkworm  was 
but  slightly  affected  by  high  concentrations  of  carbon  monoxide ;  further  experiments 
(Harvey  and  Williams,  1958)  demonstrated  that  a  cytochrome  system  functioned  in 
this  material,  the  resistance  to  CO  being  accounted  for  by  cytochrome  oxiclase  being 
present  in  great  excess  relative  to  cytochrome  c. 


144  LORD  ROTHSCHILD  AND  ALBERT  TYLER 

In  non-embryonic  tissue  the  most  extensively  studied  examples  of  CO-stimula- 
tion  of  respiration  were  those  first  reported  by  Fenn  and  Cobb  (1932a,  1932b)  in 
skeletal  and  heart  muscle  of  frog  and  rat.  This  stimulation  occurs  in  the  dark  or 
diffuse  daylight  and,  as  shown  by  Schmitt  and  Scott  (1934),  is  increased  by  strong 
illumination.  Fenn  and  Cobb  (1932b)  adduced  evidence  to  show  that  the  CO  was 
oxidized  to  CO2  and  this  has  been  further  substantiated  by  Clark,  Stannard  and 
Fenn  (1950)  by  the  use  of  isotopically  labelled  CO.  The  latter  investigators  (1949) 
also  reported  such  oxidation  of  CO  by  the  intact  animal  (turtles  and  mice). 

In  plants  Daly  (1954)  obtained  increases  of  about  20%  to  30%,  in  95%  to  97% 
CO,  with  leaf  tissue  of  the  wild  plum,  Primus  americana,  in  the  dark.  From  the  re- 
sults of  experiments  with  labelled  CO  he  concluded  that  the  increased  gas-uptake  by 
the  tissue  represents  a  real  stimulation  of  respiration  rather  than  oxidation  of  CO 
to  CO2.  He  also  found  a  rather  high  R.Q.  (up  to  1.33)  for  the  extra  gas  con- 
sumed and  therefore  suggested  that  aerobic  glycolysis  was  increased  by  CO  to  a 
greater  degree  than  O2-uptake.  He  cited  cases  of  such  stimulation  of  aerobic  gly- 
colysis by  CO  which  have  been  reported  in  spinach  (Ducet  and  Rosenberg,  1952  4), 
carrot  (Marsh  and  Goddard,  1939),  and  rat  retina  and  mouse4  chorion  (Laser, 
1937). 

The  above-mentioned  investigations  indicate  that  the  stimulating  action  of  CO 
on  respiration  is  of  wide  incidence  in  cells  and  tissues  of  animals  and  plants.  In 
some  cases  (skeletal  and  heart  muscle  of  frog  and  rat)  there  is  strong  evidence  that 
the  extra  gas-uptake  is  due  to  the  oxidation  of  CO.  In  others  (plum  leaves)  it  ap- 
pears to  be  due  to  the  stimulation  of  endogenous  respiration.  In  the  case  of  the 
fertilized  Urechis  eggs,  and  the  other  cases  that  have  been  cited  above,  the  mecha- 
nism of  the  stimulating  action  of  CO  is,  as  yet,  unknown  and  would  constitute  an  in- 
teresting area  of  further  investigation.  For  the  present  purpose  the  demonstration 
of  a  light-sensitive  action  of  CO  on  the  gas-uptake  of  the  Urechis  eggs  serves  to  sup- 
port the  other  evidence  presented  that  a  cytochrome  system  is  operative  in  this 
material. 

One  of  us  (R.)  is  indebted  to  the  Biology  Division,  the  California  Institute  of 
Technology,  for  their  hospitality  during  the  course  of  these  experiments.  We  are 
indebted  to  Miss  Mary  Jones  for  technical  assistance. 

SUMMARY 

1.  The  respiration  and  normal  development  of  fertilized  eggs  of  Urechis  caupo 
are  inhibited  by  low  concentrtaions  of  HCN,  5  X  10~6  M.     Known  concentrations  of 
HCN   were   established  within  the  manometer   flasks   by  the   use  of   Ca(CN)2- 
Ca(OH)2  mixtures  in  the  center- wells,  with  and  without  the  appropriate  amounts 
of  NaCN  in  the  egg  suspensions. 

2.  The  respiration  of  fertilized  eggs  was  photo-reversibly  inhibited  by  95%  CO 
in  O2.     The  inhibition  of  development  was  not  so  marked  at  this  tension  as  in  the 
cyanide  experiments. 

3.  CO  markedly  stimulated  the  respiration  of  the  eggs  in  the  light.     The  oc- 
currence of  a  similar  action  in  the  dark  is  presumed  to  account  for  the  moderate  de- 
gree of  depression  of  respiration  by  CO  in  the  dark. 

*  Daly  (1954)  cited  a  1951  paper  instead  of  the  1952  paper  listed  here;  also  he  referred  to 
chicken  chorion  whereas  Laser  (1937)  refers  to  mouse  chorion. 


METABOLISM  OF  URECHIS  EGGS  145 

4.  Spectroscopic  examination  of  the  eggs  at  the  temperature  of  liquid  nitrogen 
revealed  absorption  bands  at  551  m/i  and  580-590  niju.     Absorption  bands  at  these 
wave-lengths  are  associated  with  the  presence  of  cytochromes  c  and  a. 

5.  An  egg  brei  rapidly  oxidized  reduced  cytochrome  c,  but  intense  illumination  of 
the  system  reversed  the  process. 

6.  It  is  concluded  that  the  respiration  of  Urechis  eggs  is  cytochrome-catalyzed. 

LITERATURE  CITED 

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succinic  dehydrogenase  in  marine  organisms  possessing  the  copper  blood  pigment 
hemocyanin.  /.  Biol.  Chew.,  134:  483-493. 

BODINE,  J.  H.,  AND  E.  J.  BOELL,  1934.  Carbon  monoxide  and  respiration.  Action  of  carbon 
monoxide  on  respiration  of  normal  and  blocked  embryonic  cells  (Orthoptera).  /. 
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BOREI,  H.,  1951.     Cytochrome  c  in  sea  urchin  eggs.     Ada  Chem.  Scand.,  4:  1607-1608. 

BOREI,  H.,  AND  S.  LYBING,  1949.  Temperature  coefficients  of  respiration  in  Psammechimis  eggs. 
Biol.  Bull.,  96:  93-116. 

BRACKET,  J.,  1934.  fitude  du  metabolisme  de  1'oeuf  de  Grenouille  (Rana  fusca)  au  cours  du 
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CLARE,  N.  T.,  1956.  Photodynamic  action  and  its  pathological  effects.  Chapter  15,  pp.  693- 
723  in  Radiation  Biology,  Vol.  Ill,  ed.  by  A.  Hollaender.  McGraw  Hill  Book  Co., 
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CLARK,  R.  T.,  J.  N.  STANNARD  AND  W.  O.  FENN,  1949.  Evidence  for  the  conversion  of  carbon 
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DUCET,  G.,  AND  A.  J.  ROSENBERG,  1952.  Action  d'oxyde  de  carbone  sur  la  respiration  des  feuilles 
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HOROWITZ,  N.  H.,  1940a.  A  respiratory  pigment  from  the  eggs  of  a  marine  worm.  Proc.  Nat. 
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HOROWITZ,  N.  H.,  1940b.  The  respiratory  metabolism  of  the  developing  eggs  of  Urechis  caupo. 
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HOROWITZ,  N.  H.,  AND  J.  P.  BAUMBERGER,  1941.  Studies  on  the  respiratory  pigment  of 
Urechis  eggs.  /.  Biol.  Chem.,  141 :  407^15. 

KEILIN,  D.,  1925.  On  cytochrome,  a  respiratory  pigment,  common  to  animals,  yeasts,  and 
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KEILIN,  D.,  AND  E.  F.  HARTREE,  1939.  Cytochrome  and  cytochrome  oxidase.  Proc.  Roy. 
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KEILIN,  D.,  AND  E.  F.  HARTREE,  1949.  Effect  of  low  temperature  on  the  absorption  spectra  of 
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146  LORD  ROTHSCHILD  AND  ALBERT  TYLER 

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208-217. 


REGENERATION  OF  BUDS  IN  BOTRYLLUS  x 

MARGARET  J.  WATKINS 

Department  of  Zoology,  University  of  Minnesota,  Minneapolis,  Minnesota 

The  process  of  budding  in  the  colonial  ascidian,  Botryllus  schlosseri,  has  been 
carefully  analyzed  by  N.  J.  Berrill  (1941a)  and  recently  by  Sabbadin  (1955). 
The  new  buds  (Z.,)  arise  from  the  atrial  epithelium  and  epidermis  of  large  buds 
(Z2)  in  which  internal  structure  is  nearly  complete  but  which  are  still  attached  to 
the  parent  (ZJ.  The  disc-like  thickening  of  the  atrial  epithelium  increases  in  cell 
number  and  area  until  a  certain  size,  called  the  maximum  disc,  is  reached.  It  then 
folds  out  into  a  hemisphere  and  finally  to  a  closed  sphere  attached  to  the  large  bud 
by  a  stalk.  Three  generations  are  thus  present  and  connected  together  at  one  time. 
The  sphere  then  goes  through  a  process  of  expansion,  folding,  and  evagination  to 
form  the  internal  structure  of  the  new  zooid.  The  bud  continues  to  grow  until  it 
reaches  a  size  nearly  equal  to  the  parent,  at  which  time  the  latter  degenerates  and  the 
bud  becomes  functional.  There  is  considerable  variation  among  colonies  in  the 
number  of  buds  formed  and  the  number  which  reach  maturity. 

Berrill  (1941a,  1941b,  1945)  has  shown  that  in  young  colonies  the  diameters  of 
the  maximum  disc  and  sphere  stages  are  less  than  half  those  of  older  colonies  and 
that  they  gradually  increase  with  each  successive  generation.  The  size  of  the  adult 
zooid  is  closely  related  to  the  size  of  the  bud  and  hence  to  the  number  of  cells  initially 
present.  A  sphere  with  a  diameter  of  0.035  mm.,  for  example,  becomes  a  zooid  with 
a  length  of  1.1  mm.,  while  a  sphere  of  0.080  mm.  becomes  a  zooid  of  2.6  mm. 
Sabbadin  (1956a,  1956b)  showed  that  the  growth  of  the  bud  is  conditioned  not  only 
by  its  initial  dimensions,  but  also  by  the  quantity  of  food  made  available  to  it  by  the 
regression  of  the  parent  zooid  and  the  duration  of  its  growth  period.  In  his  ex- 
periments all  but  one  bud  was  removed  from  each  zooid  in  the  experimental  colo- 
nies. These  buds  and  the  zooids  from  them  attained  greater  maximum  length  than 
corresponding  buds  and  zooids  in  control  colonies.  Sabbadin  concludes  that  the 
buds  on  one  zooid  compete  for  food  made  available  by  the  parent  as  it  regresses. 
This  does  not  explain,  however,  the  gradual  increase  in  size  of  the  zooids  with  each 
generation.  It  was  deemed  of  interest  to  determine  whether  the  size  of  the  zooid 
depends  directly  on  the  number  of  cells  present  in  the  early  bud  or  is  determined  in 
some  other  manner  by  the  parent.  In  order  to  study  this  problem,  buds  were 
damaged  at  an  early  stage  and  the  amount  of  regeneration  as  shown  by  the  final 
size  was  noted. 

This  degeneration  of  the  parent  zooid  has  generally  been  thought  to  be  due  to  the 
increasing  need  of  the  bud  for  space  and  nourishment  (Berrill,  1935).  To  test 

1  This  work  was  done  while  the  author  held  an  Anderson  Summer  Fellowship  from  the 
University  of  Minnesota  Graduate  School  as  a  part  of  the  Embryology  Course  given  at  the 
Marine  Biological  Laboratory,  Woods  Hole,  Massachusetts.  The  author  wishes  to  thank  Dr. 
Mac  V.  Edds,  Jr.  for  his  help  with  both  the  research  and  the  manuscript.  Dr.  N.  J.  Berrill 
read  the  manuscript  and  offered  valuable  advice. 

147 


148  MARGARET  J.  WATKINS 

whether  this  is  true  the  buds  were  removed  in  an  attempt  to  prolong  the  life  of  the 
zooid. 

MATERIALS  AND  METHODS 

Adult  colonies  of  Botryllus  were  collected  from  the  dock  in  Eel  Pond  at  Woods 
Hole,  Massachusetts,  and  placed  in  finger  bowls  on  a  table  of  running  sea  water. 
Each  day  a  few  of  the  tadpole  larvae  were  released  from  the  colonies  and  these  were 
collected  and  placed  in  Syracuse  dishes  for  approximately  12  hours.  After  the  tad- 
poles had  attached  to  the  glass,  the  Syracuse  dishes  were  inverted  in  wooden  racks 
placed  in  tanks  of  running  sea  water.  The  tadpoles  metamorphosed  in  less  than 
one  day,  forming  oozoids  with  large  right  buds  which  became  the  first  blastozooids. 
Under  these  conditions  at  22.5  ±  0.5°  C.  the  adult  zooid  persisted  for  from  4  to  6 
days,  with  as  much  as  24  hours'  variation  between  two  colonies  in  the  same  dish. 
The  experimental  colonies  were  examined  every  day  or  two  with  a  binocular  dis- 
secting microscope  and  rough  sketches  were  made  to  follow  the  fate  of  individual 
zooids  and  buds. 

Buds  were  removed  by  cutting  through  the  stalk  with  a  needle  sharpened  to  a 
blade.  With  care  this  could  be  done  with  very  little  damage  to  the  parent,  but 
sometimes  the  latter  was  damaged  severely  and  disappeared.  To  determine  whether 
the  new  buds  originated  from  the  same  area  as  the  destroyed  buds  or  from  bud  pri- 
mordia  posterior  to  it,  all  the  small  buds  (Z,)  were  cut  off  220  large  buds  (Z,)  in 
31  colonies.  The  site  of  formation  of  the  new  buds  was  then  observed.  In  5  col- 
onies an  attempt  was  made  to  keep  the  parent  zooids  from  degenerating  by  con- 
stantly removing  new  buds  as  they  appeared. 

Buds  were  damaged  with  sharpened  steel  needles  inserted  through  the  tunic. 
An  effort  was  made  to  destroy  half  or  more  of  the  forming  bud.  Although  the 
amount  of  actual  damage  varied  from  bud  to  bud,  in  most  cases  at  least  half  of  the 
bud  was  destroyed.  Frequently,  part  of  the  bud  was  torn  away  and  could  be  seen 
sticking  to  the  needle.  In  preliminary  experiments  on  21  colonies,  buds  ranging 
from  the  sphere  stage  to  those  with  some  internal  structure  present  were  damaged. 
These  were  watched  to  see  whether  they  reached  maturity,  but  no  measurements  were 
made  to  determine  if  they  were  full  size.  In  order  to  examine  the  effect  of  destroy- 
ing approximately  half  of  the  cells  at  a  stage  before  the  closed  sphere,  both  right 
and  left  buds  were  damaged  when  the  atrial  epithelium  had  begun  to  fold  out  into  a 
hemisphere  (between  stages  2  and  3  of  Berrill,  stage  2  Sabbadin).  The  length  of 
the  zooid  which  formed  these  buds  was  then  measured  with  an  ocular  micrometer 
and  compared  with  the  length  of  undamaged  zooids.  The  width  of  the  zooids  varied 
in  different  colonies  of  the  same  age  and  seemed  to  decrease  as  the  number  of  zooids 
around  the  cloaca  increased ;  therefore,  no  measurements  of  width  were  made. 

RESULTS 
a)   Degeneration  of  parent  sooids 

In  no  case  observed  did  an  adult  zooid  persist  beyond  24  hours  of  the  time  of 
degeneration  of  other  zooids  of  the  same  age.  When  all  the  buds  were  removed 
from  a  zooid,  that  zooid  degenerated  at  the  same  time  as  the  rest  of  the  zooids  in 
that  colonv  or  in  other  colonies,  whether  new  buds  formed  or  not.  The  five  col- 


REGENERATION  OF  BUDS  IN  BOTRYLLUS 


149 


onies  in  which  new  buds  were  constantly  removed  as  they  appeared  degenerated 
and  disappeared  within  24  hours  of  the  time  of  degeneration  of  control  colonies. 

b)  Formation  of  new  buds 

If  the  large  bud  which  normally  occurred  on  the  right  side  of  an  oozoid  was  re- 
moved, a  bud  then  appeared  in  four  cases  out  of  five  on  the  left  side  of  the  oozoid  and 
became  a  normal  blastozooid.  In  later  generations,  if  all  the  large  buds  (Z2)  with 
complete  internal  structure  were  cut  off  (4  colonies),  the  colony  degenerated.  If, 
however,  only  approximately  half  of  the  larger  buds  were  cut  off  (5  colonies), 
those  remaining  proceeded  to  maturity,  and  in  addition  a  few  new  buds  appeared. 
When  all  the  small  buds  (Z3)  at  the  sphere  stage  were  cut  off  220  large  buds  (Z2) 
in  31  colonies,  a  total  of  72  new  buds  appeared,  an  average  of  one  bud  for  every 
three  parents.  There  was  a  great  deal  of  variation  among  the  colonies,  with  any- 
where from  zero  to  seven  buds  produced  by  the  six  or  seven  parents.  Of  the  new 
buds,  26  appeared  on  the  left  side  of  the  blastozooids  and  29  on  the  right  side.  Of 
the  latter  6  definitely  were  from  the  same  area  as  the  destroyed  bud,  18  were  prob- 
ably from  this  area,  and  5  appeared  posterior  to  the  destroyed  bud.  The  origin 
of  the  other  17  buds  was  impossible  to  determine.  Most  of  these  were  first  seen  in 
the  midst  of  a  degenerating  colony  quite  separate  from  any  blastozooid. 

c)  Bud  regeneration  after  damage 

In  preliminary  experiments  in  which  107  buds  ranging  from  the  sphere  stage 
to  those  with  some  internal  structure  present  were  damaged,  55  reached  maturity. 
The  rest  of  the  buds  became  progressively  smaller  and  eventually  disappeared.  In 
36  colonies  in  which  361  hemispheres  were  damaged,  40%  reached  maturity  as  com- 
pared to  78%  in  8  control  colonies  with  124  hemisphere  stages. 

TABLE  I 

Size  regulation  of  zooids  in  partially  damaged  colonies 


Colony 
number 

No.  of  zooids 
in  colony 

No.  of  zooids 
damaged 

Average  length 
of  all  zooids  in 
colony,  in 
mm.zfcS.D. 

Average  length 
of  damaged  zooids 
in  colony,  in 
mm.  ±S.D. 

Average  length 
of  undamaged  zooids 
in  mm.  ±S.D. 

A12c 

12 

5 

1.5  ±  .1 

1.6  ±  .1 

1.5  ±  .1 

A12e 

1 

3 

1.8  ±  .1 

1.8  ±  .2 

1.9  ±  .1 

A12h 

21 

8 

1.9  ±  .1 

2.0  ±  .1 

1.8  ±  .1 

A12k 

13 

6 

1.6  ±  .1 

1.6  ±  .1 

1.6  ±  .1 

A121 

17 

4 

1.4  ±  .1 

1.5  ±  .1 

1.4  ±  .1 

B2d 

25 

4 

1.8  ±  .2 

1.8  ±  .1 

1.8  ±  .2 

A5a 

8 

3 

1.8  ±  .1 

1.8  ±  .2 

1.9  ±  .1 

A5e 

15 

13 

1.6  ±  .1 

1.6  ±  .1 

1.6  ±  .1 

A5i 

7 

6 

1.8  ±  .1 

1.8  ±  .1 

1.9 

Ala 

16 

16 

1.8  ±  .2 

A5b 

6 

0 

1.4  ±  .1 

A5g 
A5h 

13 
13 

0 
0 

1.6  ±  .1 
1.7  ±  .1 

A6a 

7 

0 

1.8  ±  .1 

A6b 

11 

0 

1.7  ±  .1 

150 


MARGARET  J.  W ATKINS 


TABLE  II 

Size  regulation  of  zooids  in  experimental  and  control  colonies 


Colony 
number 

No.  of 
Zi 

Length  of  Zi  in 

mm.iS.D.  at 
time  of  experi- 
ment 

No.  of 
Zi 

Length  of  Zi  in 
mm.iS.D.  1-2 
days  after  reach- 
ing maturity 

No.  of 
Z3 

Length  of  Zs  in 
mm.iS.D.  1-2 
days  after  reach- 
ing maturity 

Length  of  Z*  in 
mm.iS.D.  3-4 
days  after  reach- 
ing maturity 

Experimental  Colonies 


A3e 

9 

1.7  ±  .1 

8 

2.0  ±  .2 

12 

2.0  ±  .2 

2.4  ±  .3 

A7a 

5 

1.8  ±  .1 

8 

2.0  ±  .1 

12 

2.2  ±.1 

2.8  ±  .1 

A8a 

7 

1.7  ±  .1 

4 

1.7  ±  .1 

4 

1.9  ±  .1 

2.4  ±  .1 

AlOa 

4 

1.6  ±  .1 

4 

— 

8 

2.2  ±  .1 

— 

AlOe 

— 

— 

6 

1.7  ±  .1 

10 

1.8  ±  .1 

— 

Total 

25 

1.7  ±.1 

30 

1.9  ±  .2 

46 

2.1  ±  .2 

2.6  ±  .3 

Control  Colonies 


A3d 

6 

1.8  ±  .1 

8 

1.8  ±  .2 

13 

1.8  ±  .1 



A7b 

5 

1.6  ±  .2 

9 

1.8  ±  .1 

15 

1.9  ±  .1 

2.3  ±  .2 

A8a 

7 

1.8  ±  .1 

10 

2.0  ±  .1 

17 

2.0  ±  .1 

2.6  ±  .1 

AlOb 

5 

1.8  ±  .1 

5 

1.8  ±  .1 

11 

2.1  ±  .1 

— 

AlOf 

— 

— 

5 

1.7  ±  .1 

9 

1.8  ±  .2 

— 

Total 

23 

1.7  ±  .1 

37 

1.8  ±  .2 

65 

1.9  ±  .2 

2.4  ±  .2 

d)   Size  regulation  in  damaged  buds 

In  the  first  experiments,  only  some  of  the  hemispherical  buds  in  each  colony 
were  damaged  with  the  idea  of  using  the  others  as  controls.  The  data  for  15  such 
colonies  are  given  in  Table  I.  In  colonies  A12h  and  A121  the  damaged  left  buds 
did  not  survive,  so  the  right  buds  measured  received  the  full  food  supply  from  the 
parents.  In  all  other  colonies  as  many  damaged  left  buds  survived  as  undamaged, 
so  the  supply  of  food  did  not  affect  the  results.  The  colonies  are  not  all  of  the  same 
size  or  age  at  the  time  of  the  experiments,  so  the  average  length  for  different  col- 
onies cannot  be  directly  compared,  but  the  average  lengths  of  damaged  and  un- 
damaged zooids  in  the  same  colony  show  no  significant  difference. 

In  later  experiments,  all  the  hemisphere  stages  in  a  colony  were  damaged  and 
these  colonies  were  compared  with  control  colonies.  Three  generations  were  pres- 
ent at  the  time  of  the  experiment:  the  parent  zooids  (ZJ,  the  large  buds  (Z2),  and 
the  hemispherical  buds  (Z3) .  Each  of  these  was  measured  as  it  in  turn  reached  ma- 
turity. In  both  experimental  and  control  colonies,  most  of  the  left  buds  reached 
maturity,  so  the  food  supply  was  about  the  same  for  all  buds.  The  data  for  these 
experiments  are  given  in  Table  II.  No  significant  difference  can  be  seen  between 
the  experimental  and  control  colonies. 

DISCUSSION 

If  the  degeneration  of  the  adult  zooid  is  due  only  to  the  increasing  need  of  the 
growing  buds  for  space  and  nourishment,  removal  of  all  the  buds  in  a  colony  ought 


REGENERATION  OF  BUDS  IN  BOTRYLLUS  151 

to  have  prolonged  the  life  of  the  zooids.  In  this  study,  any  attempt  to  postpone  de- 
generation of  the  zooid  in  this  way  met  with  failure.  No  zooid  was  observed  to  per- 
sist more  than  24  hours  longer  than  other  zooids  of  the  same  age  even  if  its  buds 
were  continually  removed.  Sabbadin  (1956b),  however,  found  that  when  all  but 
one  bud  was  removed  from  each  zooid,  that  zooid  had  a  prolonged  stage  of  func- 
tional maturity.  Perhaps  removal  of  all  buds  was  a  shock  to  the  zooid  and  partially 
caused  its  regression ;  however,  it  seems  likely  that  adult  regression  will  occur  with- 
out the  presence  of  buds.  At  the  same  time  the  buds  may  play  an  important  part 
in  the  process  by  their  increasing  need  for  nourishment. 

There  is  some  question  as  to  the  origin  of  new  buds  after  removal  of  these  already 
growing.  Blastozooids  have  two  potential  budding  areas,  one  on  the  right  side  and 
one  on  the  left,  although  frequently  only  the  bud  on  the  right  side  reaches  ma- 
turity. At  times,  a  third  bud  may  be  formed  posterior  to  the  bud  normally  found 
on  the  right  side  (see  Watterson,  1945,  and  Sabbadin,  1956a,  for  a  discussion  of  the 
number  of  buds  usually  formed).  It  would  appear  that  a  certain  amount  of  atrial 
epithelium  is  set  aside  for  bud  formation ;  after  that  is  used  no  more  buds  can  be 
formed.  Frequently  in  these  experiments,  when  buds  were  removed  from  the 
blastozooid,  new  buds  were  formed  at  the  same  area  as  the  buds  were  destroyed. 
Sabbadin  (1956a)  reports  that  he  never  observed  buds  arising  "de  novo"  after  re- 
moval of  buds  present.  Sometimes,  however,  after  he  had  removed  buds  in  the 
hemisphere  stage,  he  saw  fragments  adhering  to  the  atrial  side  of  the  parent  zooid, 
and  these  fragments  formed  new  buds.  This  is  a  possible  explanation  of  the  pres- 
ent results  although  every  effort  was  made  to  remove  the  entire  bud  intact.  In 
these  experiments  all  the  buds  removed  were  at  least  in  the  closed  sphere  stage  and 
many  were  quite  large  and  visibly  separated  from  the  parent  though  still  attached 
by  the  stalk.  It  would  take  considerable  powers  of  regeneration  for  fragments  of 
such  buds  to  form  a  whole  new  zooid. 

The  17  buds  whose  origin  it  was  impossible  to  determine  might  possibly  be  cases 
of  vascular  budding  (Oka  and  Watanabe,  1957).  They  arose  during  or  after  the 
regression  of  the  adult  zooids,  so  a  vascular  origin  seems  likely.  They  were  not 
observed,  however,  until  they  were  large  enough  to  obscure  their  point  of  origin. 

Although  development  of  ascidians  from  egg  to  tadpole  is  determinate  (Conk- 
lin,  1905),  the  adults  have  remarkable  powers  of  regeneration  (Berrill,  1951). 
Zhinken  (1939)  has  shown  that  while  tadpoles  have  little  ability  to  replace  lost  parts, 
the  oozooid  has  acquired  considerable  regulative  powers.  The  present  study  would 
indicate  that  buds  also  have  the  ability  to  regenerate  lost  tissues  from  the  earliest 
stages  onward. 

Berrill  has  shown  (1941b,  1941c,  1945)  that  the  size  of  the  maximum  disc  and 
sphere  stages  increases  with  succeeding  generations  and  that  the  size  of  the  adult 
zooid  is  clearly  related  to  the  number  of  cells  or  the  diameter  of  the  maximum  disc 
and  sphere.  This  might  suggest  that  the  parent  determines  the  size  of  the  new  zooid 
by  the  number  of  cells  which  are  initially  incorporated  into  the  early  stages  of  the 
bud.  If  this  were  true,  then  destroying  some  of  these  cells  would  have  resulted  in 
smaller  adult  zooids.  However,  using  length  as  an  index  of  zooid  size,  it  was  found 
that  there  was  no  decrease  in  size  of  the  zooids  damaged  at  the  hemisphere  stage. 
After  a  bud  was  damaged  it  either  disappeared  completely  or  reached  the  predeter- 
mined size.  Thus  the  size  of  the  adult  does  not  appear  to  depend  directly  on  the 
number  of  cells  originally  present  since  these  cells  can  be  replaced.  Either  the 


152  MARGARET  J.  WATKINS 

parent  zooid  retains  control  over  the  growth  of  the  bud  or  the  bud  has  "received 
instructions"  as  to  the  size  it  should  attain  and  follows  them  by  regenerating  lost 
tissue  and  then  continuing  to  grow.  Sabbadin  (1956c)  has  shown  that  zooids  with 
the  position  of  the  digestive  tube  reversed  may  appear  if  the  growth  of  the  bud  is 
delayed  at  an  early  stage.  The  buds  on  these  abnormal  zooids  showed  a  marked 
tendency  to  be  the  same  as  their  parents  unless  the  parent  has  started  to  regress  be- 
fore organogenesis  is  complete.  This  would  indicate  that  the  parents  do  retain  con- 
trol over  the  growth  and  organogenesis  of  their  buds. 

SUMMARY 

1.  The  degeneration  of  the  adult  zooid  of  Botryllus  schlosscri,  which  normally 
occurs  when  the  buds  become  functional,  occurred  even  after  all  buds  were  removed. 

2.  All  stages  of  the  growing  buds  of  Botryllus  have  considerable  regenerative 
ability. 

3.  Buds  damaged  in  the  hemisphere  stage  became  adult  zooids  with  the  same 
length  as  undamaged  zooids  of  the  same  age.     Control  over  the  size  of  the  adult 
zooid  appears  to  be  maintained  during  the  growth  of  the  bud. 

LITERATURE  CITED 

BERRILL,    N.    J.,    1935.     Studies    in    tunicate    development.     IV.    Asexual    reproduction.     Phil. 

Trans.  Roy.  Soc.  London,  Ser.  B,  225 :  327-379. 

BERRILL,  N.  J.,  1941a.     The  development  of  the  bud  in  Botryllus.     Biol.  Bull.,  80 :  169-184. 
BERRILL,  N.  J.,  1941b.     Size  and  morphogenesis  in  the  bud  of  Botryllus.     Biol.  Bull.,  80 :  185-193. 
BERRILL,  N.  J.,  1941c.     Spatial  and  temporal  growth  patterns  in  colonial  organisms.     Growth, 

51  (Supplement)  :  89-111. 
BERRILL,  N.  J.,  1945.     Size  and  organization  in  the  development  of  ascidians.     In:    Essays  on 

Growth  and  Form.     Ed.  by  W.  E.  Le  Gros  Clark  and  P.  B.  Medawar.     Oxford  Univ. 

Press ;  pp.  231-263. 

BERRILL,  N.  J.,  1951.     Regeneration  and  budding  in  tunicates.     Biol.  Rev.,  26:  456-475. 
CONKLIN,  E.  G.,  1905.     Mosaic  development  in  ascidian  eggs.     /.  E.rp.  Zoo/.,  2 :  145-223. 
OKA,  HIDEMITI,  AND  HIROSHI  WATANABE,  1957.     Vascular  budding,  a  new  type  of  budding  in 

Botryllus.     Biol.  Bull,  112:  225-240. 
SABBADIN,  ARMANDO,   1955.     Osservazioni   sullo   suiluppo,   1'accrescimento  e   la   riproduzione   d. 

Botryllus  schlosseri  (Pallas),  in  condizioni  di  laboratorio.     Boll.  d.  Zoo/.,  22:  243-263. 
SABBADIN,  ARMANDO,  1956a.     Studio  sperimentale  della  gemmazione  in  "Botryllus  schlosscri" 

(Pallas).     Rend.  d'Accademia  Nasionalc  dei  Lincei,  20:  380-385. 
SABBADIN,  ARMANDO,   1956b.     Osservazioni   suH'accrescimento  delle  gemme   e  degli   zooidi   di 

"Botryllus  schlosscri"    (Pallas)     (Ascidiacea),   in   condizioni   normal!    e    sperimentali. 

Rend.  d'Accademia  Nasionale  dei  Lincei,  20:  485-491. 
SABBADIN,     ARMANDO,     1956c.     "Situs     inversus     viscerum"     provocato     sperimentalmente     in 

"Botryllus  schlosscri"  (Pallas)   (Ascidiacea).     Rend.  d'Accademia  Nasionale  dei  Lined, 

20 :  659-666. 
WATTERSON,  RAY  L.,  1945.     Asexual  reproduction  in  the  colonial  tunicate,  Botryllus  schlosseri 

(Pallus)   Savigny,  with  special  reference  to  the  developmental  history  of  inter siphonal 

bands  of  pigment  cells.     Biol.  Bull.,  88 :  71-103. 

ZHINKEN,  L.,  1939.     Alteration  of  regenerative  power  of  the  larvae  of  ascidea  during   meta- 
morphoses.    C.  R.  Acad.  Sci.  Moscozv  (N.S.),  24:  623-625. 


Vol.  115,  No.  2  \AV     MA£*       /CV/  October,  1958 

THE 

BIOLOGICAL  BULLETIN 

PUBLISHED  BY  THE  MARINE  BIOLOGICAL  LABORATORY 


THE  OXIDATION  OF  CARBON  MONOXIDE  BY  FERTILIZED  EGGS 
OF  URECHIS  CAUPO  SHOWN  BY  USE  OF  A  C13  LABEL1 

ROBERT  E.  BLACK,2  SAMUEL  EPSTEIN3  AND   ALBERT   TYLER 
Division  of  Biology,  California  Institute  of  Technology,  Pasadena,  California 

In  some  previous  experiments  (Rothschild  and  Tyler,  1958)  with  eggs  of 
Urecliis,  it  was  found  that  the  rate  of  respiration  in  the  presence  of  carbon  monoxide 
(95%  CO:  5%  OL,)  in  the  light  was  greatly  increased  above  that  of  the  con- 
trols (95%  N2:5%  O2).  The  average  increase  amounted  to  85  per  cent.  In 
the  dark  there  was  a  slight  decrease,  averaging  about  15  per  cent. 

In  many  earlier  investigations  on  eggs  and  other  tissues  of  various  animals  and 
plants  there  have  been  reports  of  a  stimulating  action  of  CO  on  respiratory  rate. 
Examples  of  this  are  found  in  experiments  on  eggs  of  sea  urchins  by  Runnstrom 
(1930),  Lindahl  (1939)  and  Rothschild  (1949);  on  ascidian  eggs  by  Minganti 
(1957)  ;  on  diapausing  grasshopper-  and  silkworm-embryos  by  Bodine  and  Boell 
(1934)  and  by  Wolsky  (1941)  ;  on  skeletal  and  heart  muscle  of  frog  and  rat  by 
Fenn  and  Cob'b  (1932a,  1932b),  Schmitt  and  Scott  (1934),  and  Clark.  Stannard 
and  Fenn  (1950)  ;  on  leaf  tissue  of  the  wild  plum  by  Daly  (1954). 

In  the  experiments  on  vertebrate  muscle  tissues,  Fenn  and  Cobb  (1932b)  and 
Clark,  Stannard  and  Fenn  (1950)  obtained  evidence  that  CO  is  oxidized  to  CO2. 
Clark  ct  al.  (1949)  also  reported  that  intact  whole  turtles  and  mice  could  effect 
such  oxidation  of  CO  when  this  was  administered  at  very  low  tensions.  In  the 
experiments  on  plum-leaves,  on  the  other  hand,  Daly  (1954)  found  that  the  in- 
creased gas-uptake  in  the  presence  of  CO  represents  a  stimulation  of  ordinary 
respiration  rather  than  an  oxidation  of  the  CO.  The  question  of  whether  or  not 
the  stimulation  of  respiration  in  eggs  of  sea  urchins  and  ascidians  is  due  to  oxida- 
tion of  the  CO  was  considered  by  Lindahl  (1939),  Minganti  (1957)  and  Roths- 
child (1949).  The  former  two  investigators  rejected  this  view  while  the  latter 
considered  it  to  be  the  most  probable  explanation  of  the  increased  respiration.  In 
a  review  of  various  experiments  Runnstrom  (1956)  concludes  that  the  evidence  is 
against  the  possibility  of  oxidation  of  CO  by  sea  urchin  eggs.  However,  there  has 
as  yet  been  no  direct  test  of  this  proposition,  such  as  would  be  provided  by  the  use 
of  isotopically  labelled  CO. 

1  This  investigation  was  supported  by  a  research  grant   (C-2302)   from  the  National  Cancer 
Institute  of  the  National  Institutes  of  Health,  Public  Health  Service. 
-  Postdoctoral  Research  Fellow  of  the  U.  S.  Public  Health  Service. 
3  Division  of  Geological  Sciences,  California  Institute  of  Technology. 

153 
Copyright  ©  1958,  by  the  Marine  Biological  Laboratory 


154  R.  E.  BLACK,  S.  EPSTEIN  AND  A.  TYLER 

In  the  present  experiments  C13-labelled  CO  was  employed  in  an  investigation 
of  the  possibility  of  its  oxidation  by  eggs  of  UrccJiis.  The  results  show  that  such 
oxidation  occurs  and  that  it  accounts  for  all  of  the  extra  gas-uptake  of  the  eggs  in 
the  light.  The  data  also  show  that  an  oxidation  of  CO  occurs  in  darkness,  but 
at  a  lower  rate. 

MATERIAL  AND  METHODS 

Eggs  of  the  gephyrean  worm  Urcchis  canpo  were  employed  in  these  experi- 
ments. They  were  inseminated  in  sea  water  and  washed  in  sea  water  buffered  at 
pH  8  with  0.01  M  glycylglycine  (Tyler  and  Horowitz,  1937). 

Gas-uptake  was  measured  with  Warburg-Barcroft  manometers  using  vessels 
whose  calibration  volumes  ranged  around  25  ml.  The  vessels  generally  contained 

3  ml.   of  egg  suspension  and  0.3   ml.   of  M/\   KOH    (low   in   CO,).     In   some 
experiments  in  which  CO2  was  to  be  released  from  the  egg  suspension  as  well  as 
from  the  alkali,  magnetically  held  cups  were  employed,  one  for  the  alkali  and  one 
containing  0.3  ml.  of  6  M  H2SO4.     The  contents  of  these  could  be   separately 
tipped  into  the  egg  suspension  at  the  desired  time  by  removal  of  an  externally 
supported  magnet.     The  KOH  used  in  the  alkali-wells  of  the  manometer  vessels 
was  prepared  from  a  saturated  solution,  in  which  K2CO3  is  largely  insoluble,  and 
diluted  with  CO2-free  double-distilled  water  under  CO2-free  air.     An  analysis  of 
the  alkali  prepared  in  this  manner  gave  0.8  X  10~G  mole  of  total  carbonate  per 
0.3  ml.     In  filling  the  manometer  vessels  the  alkali  was  introduced  last. 

The  use  of  C13  offers  some  advantages  over  C14  for  these  experiments.  Use 
of  C1 1  would  involve  precipitating  and  weighing  very  small  quantities  ( less  than 

4  mg.  as  BaCO3)  of  the  carbonate  derived  from  the  respired  CO2  in  the  alkali  well 
of  the  usual  manometer  vessels.     This  is  unnecessary  for  the  mass  spectrometric 
measurement  of  C13  which  provides  the  required  quantitative  data  in  the  form  of 
the  ratio  of  C13  to  O~  in  the  sample.     It  also  avoids  such  uncertainties  as  are  en- 
tailed by  the  self-absorption  of  radiation  in  the  measurement  of  C14.     In  addition, 
use  of  C13  eliminates  possible  health-hazards  and  possible  effects  of  radiation  on 
the  system  under  investigation. 

The  labelled  carbon  monoxide  was  prepared  from  barium  carbonate  containing 
3.85%  C13.  This  was  obtained  from  the  Stable  Isotopes  Division  of  the  Oak 
Ridge  National  Laboratories.  The  method  employed  was  essentially  similar  to 
the  continuous  flow  technique  described  by  Bernstein  and  Taylor  (1947).  The 
apparatus  consisted  of  a  CO2  generator  connected  to  a  Pyrex  combustion  tube 
(8  mm.  i.d.),  containing  about  50  grams  of  zinc-dust-asbestos  fiber  (95:  5),  within 
a  combustion-furnace  of  18  cm.  length,  and  leading  through  a  three-way  stopcock 
to  the  top  of  a  storage  bulb.  The  latter  was  provided  also  with  a  bottom  stop- 
cock leading  to  a  levelling  bottle  containing  N/10  NaOH.  At  the  start  of  the 
preparation  the  storage  bulb  was  filled  with  the  alkali  up  to  the  three-way  stop- 
cock. A  weighed  amount  of  the  C13-enriched  BaCO3  wras  placed  in  the  generator, 
and  the  generator  and  combustion  tube,  up  to  the  three-way  stopcock,  were  flushed 
with  unlabelled  CO.  The  furnace  was  set  at  520°  C.  Hydrochloric  acid  was 
introduced  into  the  generator  at  a  rate  producing  about  25  to  50  cc.  of  CO2  per 
minute.  Measurements  of  the  volume  of  fluid  displaced  in  the  storage  bulb  showed 


OXIDATION  OF  CO  BY  EGGS  OF  URECHIS  155 

that  the  amount  of  CO  obtained  in  this  system  was  close  to  that  expected.  After 
CO2  generation  had  stopped,  the  gas  remaining  in  the  generator  and  combustion 
tube  was  flushed  into  the  storage  bulb  with  enough  unlabelled  CO  to  make  a  final 
volume  of  one  liter.  The  relative  volumes  of  labelled  and  unlabelled  CO  were 
362  to  638  for  the  preparation,  giving  a  C13  content  of  2.14%.  Relative  to  a  C13 
content  of  1.17%  found  for  the  CO2  from  Urechis  eggs  respiring  in  air,  this  gives 
82.9%  for  the  atom  percentage  excess  C13  of  the  preparation.  The  labelled  CO  was 
stored  over  alkali  for  at  least  one  day  prior  to  use.  Storage  over  alkali  for  several 
weeks  showed  no  change  in  gas  volume,  indicating  no  significant  contamination 
by  acidic  gases. 

After  attachment  of  the  Warburg  vessels  to  their  manometers  they  were  flushed 
with  one  liter  or  more  of  oxygen.  They  were  then  attached  to  a  Toepler  pump  and 
evacuated  to  one-fifth  of  the  original  pressure,  precautions  being  taken,  by  stopper- 
ing the  open  end  of  the  manometers  and  closing-ofr"  the  bottom  rubber  well  with 
a  clamp,  to  avoid  drawing  the  Brodie's  fluid  out  of  the  manometers.  The  C13- 
labelled  CO  was  then  introduced  through  the  three-way  stopcock  at  the  top  of  the 
manometers,  after  a  preliminary  flushing  of  connecting  tubes.  By  this  procedure 
the  CO-O2  ratio  could  be  fixed  with  considerable  accuracy  to  the  desired  value, 
which  was  4:1  in  the  present  experiments.  About  15  minutes  were  required  for 
these  procedures  and  10  minutes  were  allowed  for  equilibration  in  the  temperature 
bath.  The  control  vessels  were  left  open  to  air  during  the  gassing  of  the  experi- 
mental vessels.  The  experiments  were  run  at  20°  C.  Shaker  speed  was  95  c.p.m. 
at  3-cm.  stroke.  Illumination  was  provided  by  a  bank  of  30-watt  reflector-type 
G-E  incandescent  lamps  located  below  a  glass  shelf  of  the  water  bath.  This  sup- 
plied 1100  to  1200  foot-candles  at  the  level  of  the  egg  suspensions  in  the  Warburg 
vessels. 

The  C13  determinations  were  made  with  a  Nier  mass  spectrometer  (Nier,  1947) 
modified  for  detection  of  relatively  small  enrichments  by  McKinney  at  al.  (1950). 
The  sensitivity  of  the  instrument  is  such  that  differences  of  two  parts  in  ten  thou- 
sand in  the  CI3-to-C12  ratios  can  be  readily  detected.  For  introduction  of  the 
respired  CO2  into  the  mass  spectrometer,  the  procedure  followed  in  two  of  the 
experiments  (No.  1  and  No.  2)  was  to  transfer  the  alkali  from  the  center  well  of 
the  Warburg  vessel  quantitatively,  with  CO2-free  water  and  with  precautions  to 
avoid  contamination  with  atmospheric  CO2,  to  a  reaction  vessel  wherein  the  CO2 
could  be  liberated  by  tipping-in  concentrated  H3PO4  from  a  side-arm  (McCrea, 
1950).  This  was  attached  to  the  vacuum-line  of  the  mass  spectrometer.  In  one 
of  the  experiments  (No.  3),  the  CO2  was  liberated  within  the  Warburg  vessels 
by  tipping  acid  from  one  of  the  contained  insert-wells  into  the  egg  suspension  and 
the  alkali.  After  measurement  of  their  amounts  the  CO,  samples  were  transferred 
to  the  reaction  vessels  by  means  of  the  Toepler  pump.  In  two  of  the  experiments 
(No.  2  and  No.  3)  a  measured  amount  of  NaHCO3  was  added  to  the  reaction 
vessel  in  order  to  decrease  the  C13  enrichment  to  values  within  the  range  best 
suited  for  the  mass  spectrometer.  The  measurements  are  corrected  for  the  dilution 
factor. 


156 


R.  E.  BLACK,  S.  EPSTEIN  AND  A.  TYLER 


RESULTS 
Effect  of  CO  on  gas-uptake  of  eggs  of  Ureehis 

The  relevant  respiration-data  for  three  experiments  are  presented  in  Table  I. 
The  first  two  are  for  eggs  run  in  the  light  starting  shortly  after  fertilization,  and 
the  third  is  a  dark-experiment  with  eggs  at  a  similar  period  of  development. 

The  increase  in  gas-uptake  reported  by  Rothschild  and  Tyler  (1958)  for  freshly 
fertilized  eggs  of  Ureehis  in  the  light  in  95%  CO/O,  is  shown  also  in  the  present 
experiments  (No.  1  and  No.  2)  with  80%  CO/CX.  Likewise,  the  lack  of  appre- 
ciable inhibition  in  the  dark  is  shown  in  the  results  of  experiment  No.  3.  Exami- 
nation of  the  eggs  at  the  end  of  the  respiration  runs  in  experiments  No.  1  and  No.  2 
showed  no  significant  difference  in  rate  of  development  between  those  in  80% 
CO/CX  and  those  in  air.  The  eggs  from  experiment  No.  3  were  not  available  for 
examination  because  of  the  acidification,  but  separate  experiments  on  eggs  run  in 
the  dark  in  CO-O2  mixtures  show  only  a  small  amount  of  inhibition  of  develop- 
ment, as  reported  previously  (Rothschild  and  Tyler,  1958). 

The  data  in  Table  I  present  amounts  of  gas-uptake  calculated  as  if  the  total  gas 
were  oxygen.  Part  of  the  gas-uptake  of  the  eggs  in  the  CO-O2  mixture  could 
(and,  as  later  shown,  does)  represent  disappearance  of  CO.  However,  calcula- 
tions using  the  solubility  of  CO  instead  of  O2  in  the  usual  formula  for  converting 
the  manometric  pressure  difference  into  volume  of  gas  would  change  these  figures 
by  only  0.1%,  since  the  solubility  coefficients  of  the  two  gases  are  of  the  same 
order  of  magnitude  and  this  factor  contributes  relatively  little  to  the  vessel  constant. 
This  difference  is  negligible  here. 

Experiments  No.  1  and  No.  2  give  values  of  154  and  130  mm3.,  respectively, 
for  the  excess  gas  uptake.  Assuming  that  this  is  due  to  the  oxidation  of  CO 
(  2  CO  +  O2  — »  2  CO2),  then  %  of  these  quantities  represent  the  amounts  of  CO 
oxidized  and  the  corresponding  amounts  of  CO2  produced  therefrom;  namely,  102 
and  87  mm3.,  respectively.  The  corresponding  control  vessels  yield  318  and  305 
mm3,  of  CO2,  respectively,  on  the  basis  of  an  R.Q.  of  unity  (Horowitz,  1940).  The 
percentage  of  the  CO2  derived  from  oxidation  of  CO  would  therefore  be  24.3  for 
experiment  No.  1  and  23.4  for  experiment  No.  2.  These  are  entered  in  the  last 
column  of  Table  II  as  expected  values,  and  involve  also  the  assumption  that  in  the 
light  there  is  no  inhibition  of  the  ordinary  respiration. 


TABLE  I 
Respiration-data  for  eggs  of  Ureehis  used  in  Cn-labelled  CO  experiments 


(1) 

Experiment 

(2) 

Number  of  eggs 
per  flask 

(3) 

Time  interval 
of  experiment 
in  hours  after 
fertilization 

(4) 

(5) 

(6) 

Excess  gas- 
uptake  in 
80%  CO/O2 

(mm.3) 

Total  gas-uptake 

In  air 
(mm.3) 

In  80  "c  CO/O2 
(mm.3) 

1    (light) 
2  (light) 
3  (dark) 

389,000 
622,000 
421,000 

l*-8* 

ii-6* 

1-10 

318 
305 
408 

472 

435 
394 

+  154 
+  130 
-14 

OXIDATION  OF  CO  BY  EGGS  OF  URECHIS 


157 


TABLE  II 

Percentage  of  respired  COz  derived  from  oxidation  of  CO,  as  determined  from  'measurements 

of  C13  in  mass  spectrometer  and  as  calculated  on  the  assumption  that  such  oxidation 

accounts  for  all  excess  gas-uptake  in  CO-Oz  mixtures  in  the  light 


(1) 

Experiment  No. 

(2) 

Atom  %  excess 
C13  in  CO  used 
in  gas  space 
of  manometer 
vessels 

(3) 

(4) 

(5) 

(6) 

(7) 

Expected 
percentage 
of  total  CO* 
derived  from  CO 
as  calculated 
from  excess 
gas-uptake 
in  light 

Mass  spectrometer  data 

Atom  %  excess  C13  in  respired  COs 

Percentage 
of  CC>2  de- 
rived from 
oxidation 
of  CO 

With  reference 
to  standard  CO2 

With  reference 
to  control 

Experimental 
vessel 

Control 

vessel 

Experimental 
vessel 

1    (light) 

2  (light) 
3  (dark) 

82.9 
82.9 
82.9 

19.92 
20.28 
16.23 

0.61 
0 
1.35 

19.31 
20.38 
14.88 

23.3 
24.5 
18.0 

24.3 
23.4 

A  calculation  of  expected  CO-oxidation  cannot  be  made  in  this  way  for  experi- 
ment No.  3  which  was  run  in  the  dark,  wherein  both  inhibition  of  ordinary  respira- 
tion and  oxidation  of  the  CO  might  well  take  place. 

Moss  spectrometer  data  relating  to  oxidation  of  CO 

The  results  of  determinations  of  C13  abundance  in  the  respired  CO,  of  the  above 
three  experiments  are  presented  in  Table  II.  The  atom  percentage  excess  C13 
in  the  CO  used  in  these  experiments  is  listed  in  the  second  column  of  the  table. 
These  figures  also  represent  the  excess  that  would  be  expected  if  all  of  the  respired 
CO,  were  derived  from  oxidation  of  CO.  The  values  obtained  from  the  mass 
spectrometer  measurements  for  the  excess  C13  in  the  CO,  from  experimental,  rela- 
tive to  that  from  control  vessels,  are  given  in  the  fifth  column  of  the  table.  Division 
of  these  figures  by  the  corresponding  ones  of  column  two  gives  the  percentages 
(column  6)  of  the  CO,  derived  from  CO-oxidation  in  these  three  experiments. 
Comparison  with  the  expected  percentages  (column  7)  calculated  from  the 
manometrically  determined  extra  gas-uptake,  on  the  assumption  that  all  of  this 
surplus  in  the  light  is  derived  from  CO-oxidation,  shows  close  agreement  in 
experiments  No.  1  and  No.  2. 

This  closeness  of  agreement  may,  however,  be  largely  fortuitous  as  the  follow- 
ing considerations  of  further  details  of  the  experiments  indicate.  In  experiment 
No.  1  the  control  was  an  aliquot  of  the  same  egg  suspension  respiring  in  air.  The 
alkali  from  both  experimental  and  control  flasks  was  transferred  quantitatively  to 
the  reaction  vessels  and  no  carrier  NaHCO3  added.  The  respective  percentages 
of  excess  C13,  relative  to  the  standard  used  in  the  instrument,  are  given  in  columns 
3  and  4  of  the  table.  The  air-control  shows  a  small  excess  of  C13  relative  to  the 
standard  source.  This  simply  reflects  variation  in  C13/C12  ratios  of  living  and 
non-living  materials  from  various  sources  (rf.  Craig,  1953).  Since  the  carbon 
of  the  respired  CO,  of  the  air-control  is  all  derived  from  the  eggs  this  indicates 


158  R.  E.  BLACK,  S.  EPSTEIN  AND  A.  TYLER 

a  higher  C13  content  in  the  eggs  than  in  the  standard.  In  the  absence  of  other 
information  the  best  method  of  applying  a  correction  for  the  control  is  uncertain, 
but  it  seemed  most  reasonable  to  us  simply  to  subtract  it  from  the  value  for  the 
experimental  flask.  In  any  case  this  correction  has  relatively  little  effect  on  the 
calculations  of  CO-oxidation. 

In  experiment  No.  2  the  respired  CO2  from  the  air-control  vessel  was  not  sub- 
jected to  C13  analysis.  Instead,  a  second  type  of  control  was  investigated.  This 
consisted  of  a  preparation  of  lyophilized  eggs  that  was  run  along  with  the  experi- 
mental flask  in  the  80%  labelled  CO-20%  O2  atmosphere  in  the  light.  This 
preparation  showed  a  negligible  amount  of  gas-uptake,  and  was  employed  to  test 
for  possible  exchange  of  carbon  atoms  between  CO2  and  the  labelled  CO.  For 
this  purpose  about  300  mm3,  of  CO2  were  introduced  into  the  Warburg  flask. 
The  analysis  of  the  CO2  in  the  alkali  of  this  flask  showed  no  difference  in  C13  con- 
tent from  that  of  the  standard.  This  indicates  that  no  significant  exchange  of 
carbon  atoms  between  the  CO  and  CO2  occurs  in  this  system. 

The  determined  value  for  atom  percentage  excess  C13  in  the  CO2  of  the  experi- 
mental flask  of  experiment  No.  2  was  not  corrected  for  any  possible  contribution 
from  ordinary  respiration  since  the  air  control  in  this  experiment  was  not  analyzed 
for  C13.  A  correction  of  the  same  order  as  in  experiment  No.  1  would  lower  very 
little  the  calculated  percentage  of  CO2  derived  from  oxidation  of  CO  (column  6). 

The  principal  source  of  uncertainty  in  these  two  experiments  is  CO2-retention 
in  the  egg  suspensions  of  the  Warburg  flasks.  As  shown  in  later  experiments  the 
egg  suspensions  may  contain  considerable  amounts  of  bicarbonate  at  the  beginning 
of  the  experiments,  despite  the  normal  precautions  to  keep  this  at  a  low  value. 
This  unlabelled  bicarbonate  would  presumably  form  a  common  pool  during  the 
run  with  bicarbonate  derived  both  from  ordinary  respiration  and  from  the  oxida- 
tion of  labelled  CO.  The  CO2  collected  in  the  alkali  for  analysis  would  then  have 
been  diluted  with  the  unlabelled  CO2  present  in  the  egg  suspension  at  the  start  of 
the  experiment.  Also,  some  of  the  labelled  CO2  produced  during  the  experiment 
would  be  retained  in  the  suspension  at  the  end  of  the  run.  If  corrections  were 
made  for  the  above  effects,  the  values  calculated  in  column  6  for  CO-oxidation  in 
experiments  No.  1  and  No.  2  would  be  higher  than  those  presented.  In  other 
words,  the  value  used  for  atom  percentage  excess  C13  to  be  expected  if  only  CO- 
oxidation  took  place  would  be  lower  than  those  listed  in  column  2.  Therefore, 
the  calculated  percentages  of  COo  derived  from  CO-oxidation  in  these  two  experi- 
ments represent  minimum  values. 

It  should  be  noted  that  the  expected  percentages  of  CO2  produced  from  CO  by 
the  eggs,  as  calculated  from  excess  gas-uptake  (column  7),  also  represent  minimum 
values,  since  they  depend  on  the  assumptions  that  the  R.Q.  is  1.0,  and  that  there 
is  no  inhibition  of  ordinary  respiration  by  CO  in  the  light.  Lindahl  (1939)  has 
shown  that  in  75%  CO/O2  in  the  light,  the  eggs  of  the  sea  urchin  have  a  lower 
R.Q.  than  one  would  expect,  even  if  one  were  to  account  for  all  the  excess  gas- 
uptake  as  CO  oxidation.  This  could  be  due  to  an  inhibition  of  ordinary  respiration 
t>y  CO  in  the  light,  which  is  masked  by  the  utilization  of  CO.  In  the  present 
•experiments  if  an  R.Q.  of  0.67  instead  of  1.0  were  assumed  for  the  ordinary 
respiration,  as  well  as  the  CO-oxidation,  then  the  expected  percentages  of  CO, 
derived  from  CO-oxidation  (column  7)  would  be  32  and  30  for  experiments  No.  1 
and  No.  2,  respectively. 


OXIDATION  OF  CO  BY  EGGS  OF  URECHIS  159 

In  experiment  No.  3  the  bicarbonate  in  the  egg  suspension,  as  well  as  that  in 
the  alkali  well,  was  collected  for  analysis  of  C13  content  in  the  mass  spectrometer. 
A  control  flask  of  egg  suspension,  into  which  acid  was  tipped  at  the  time  of  the 
first  reading  of  the  manometers,  provided  a  measure  of  unlabelled  CO2  originally 
present.  The  retained,  as  well  as  the  respired,  CO2  was  determined  before  transfer 
to  the  reaction  vessel  of  the  mass  spectrometer,  as  described  in  Materials  and 
Methods.  The  total  amounts  of  CO2  (375  mm3,  in  experimental  and  384  mm3,  in 
control  flask)  were  diluted  with  0.5  ml.  of  carrier  0.04  M  NaHCO3  (480  mm3,  of 
CO2).  Initial  bicarbonate  content  of  the  CO/O2  blank  amounted  to  160  mm3. 
The  corresponding  dilution  factors  applied  to  the  mass  spectrometer  data  were 
therefore  (375  +  480) / (375  --  160)  and  (384  +  480)/384  for  experimental  and 
control  flasks,  respectively.  The  figures  entered  in  columns  3  and  4  of  Table  II  are 
corrected  for  the  dilution  factor. 

The  value  of  18  per  cent  for  the  CO2  derived  from  CO-oxidation  in  this  experi- 
ment is  then  not  subject  to  uncertainties  of  retention  and  can  be  considered  to 
represent  reasonably  closely  the  extent  of  CO-oxidation  occurring  in  the  dark. 
Since  there  is  about  3%  inhibition  of  gas-uptake  (Table  I)  in  this  experiment  and 
since  27%  (%  of  18%)  of  the  gas-uptake  represents  CO-oxidation,  then  there  is 
29%  inhibition  (100-97(0.73))  of  the  ordinary  respiration  by  the  CO  in 
the  dark. 

DISCUSSION 

The  results  show  that  eggs  of  Urcchis  can  oxidize  carbon  monoxide. This 
occurs  both  in  the  light  and  in  the  dark.  The  amount  of  carbon  monoxide  that  is 
oxidized  in  the  light  can  account  for  all  of  the  excess  gas-uptake  that  occurs  in  a 
CO-O2  mixture.  In  the  dark  the  percentage  of  CO2  derived  from  CO-oxidation 
is  somewhat  less  than  in  the  light,  according  to  the  present  data.  It  should  be 
noted  again  that  the  values  obtained  for  oxidation  of  CO  in  the  light  are  probably 
minimal.  In  other  words,  there  may  be  a  small  amount  of  inhibition  of  the 
"ordinary"  respiration  in  the  light  which  is  obscured  by  the  oxidation  of  CO. 

It  is  possible  that  in  the  dark  CO  may  be  inhibiting,  to  some  extent,  its  own 
oxidation.  Clark,  Stannard  and  Fenn  (1950)  found  that  sodium  azide  and 
hydroxylamine  completely  blocked  the  oxidation  of  CO  by  skeletal  muscle,  as 
measured  both  by  manometric  and  isotope  techniques. 

Information  available  from  the  literature  and  from  the  present  experiments 
does  not  permit  identification  of  the  enzymatic  system(s)  involved  in  the  oxidation 
of  CO.  It  seems  likely  that  a  haem  compound  is  involved  because  of  the  known 
affinity  of  CO  for  the  Fe++  of  such  substances.  Also,  it  may  well  go  through 
cytochrome  oxidase.  However,  tests  of  cytochrome  oxidase  preparations  from 
Urechis  and  sea  urchin  eggs  (to  be  reported  later)  gave  no  oxidation  of  CO. 

In  certain  bacteria  CO  can  serve  as  the  sole  carbon  source  (cf.  van  Niel,  1954). 
Fixation  of  CO  has  been  demonstrated  in  barley  leaves  (Krall  and  Tolbert,  1957), 
in  which  the  labelled  carbon  appears  initially  in  serine  and  choline.  This  fixation 
occurs  in  both  light  and  dark  but  the  rate  is  much  higher  in  the  light.  The  possi- 
bility of  fixation  of  CO  has  not,  as  yet,  been  examined  in  animals,  but  it  does  seem 
likely  that  some  of  the  CO2  produced  by  its  oxidation  would  be  assimilated. 

As  previously  reported  (Rothschild  and  Tyler,  1958)  and  as  noted  here,  the 
development  of  the  eggs  was  not  significantly  accelerated  or  retarded  in  the  CO-O2 


160  R.  E.  BLACK,  S.  EPSTEIN  AND  A.  TYLER 

mixtures  in  the  light.  It  might  appear,  then,  that  the  energy  released  by  the 
oxidation  of  the  CO  is  not  put  to  useful  developmental  work  in  this  system. 
However,  it  should  be  noted  that  the  CO-oxidation  would  provide  much  less  energy 
per  mole  of  carbon  than  the  oxidation  of  the  ordinary  substrates  of  the  cell.  So, 
even  if  the  energy  were  utilized,  the  increase  in  developmental  rate  might  be  too 
small  to  be  readily  detected  under  the  present  conditions  in  which  roughly  25  per 
cent  of  the  respiration  is  attributed  to  oxidation  of  CO.  Furthermore,  as  indicated 
above,  the  figure  of  25  per  cent  is  a  minimum  value.  Some  inhibition  of  ordinary 
respiration  could  be  occurring  in  the  light.  If,  for  example,  the  inhibition  amounted 
to  25  per  cent  and  if  it  is  assumed  that  oxidation  of  CO  supplies  half  as  much 
energy  per  mole  of  carbon  as  does  the  ordinary  respiration,  then  the  total  rate  of 
energy  supply  would  be  the  same  for  eggs  in  80%  CO/O2  in  the  light  as  for  eggs 
respiring  in  air.  It  is  then  possible  that  the  energy  released  by  oxidation  of  CO 
is  utilized  by  the  cell  for  developmental  work. 

SUMMARY 

1.  The  fertilized  eggs  of  Urechis  canpo  have  been  found  to  oxidize  CO  to  CO2 
both  in  the  light  and  in  the  dark.     This  has  been  shown  by  the  use  of  C13-labelled 
CO.     In  the  light  there  is  a  previously  described  increase  in  gas-uptake  in  80% 
CO/O2  as  compared  with  air.     All  of  this  excess  gas-uptake  can  be  attributed 
to  the  oxidation  of  CO. 

2.  In  the  dark  the  percentage  of  respiratory  CO2  derived  from  CO  is  less  than 
in  the  light.     If  the  oxidation  of  CO  is  subtracted  from  the  total  gas  uptake,  the 
"ordinary"  respiration  is  shown  to  be  inhibited  about  29%   in  the  dark  by  80% 
CO/O2. 

LITERATURE  CITED 

BERNSTEIN,  R.  B.,  and  T.  I.  TAYLOR,  1947.     Conversion  of  isotopically  enriched  CO2  to  CO. 

Science,  106:  498-499. 
BODINE,  J.  H.,  and  E.  J.  BOELL,   1Q34.     Carbon  monoxide  and  respiration.     Action  of  carbon 

monoxide  on  respiration  of  normal  and  blocked  embryonic  cells  (Orthoptera).     /.  Cell. 

Comp.  Physiol,  4:  475^82. 
CLARK,   R.   T.,   J.   N.    STANNARD  and   W.    O.   FENN,    1949.     Evidence   for   the   conversion   of 

carbon  monoxide  to  carbon  dioxide  by  the  intact  animal.     Science,  109:  615-616. 
CLARK,  R.  T.,  J.  N.  STANNARD  and  W.  O.  FENN,  1950.     The  burning  of  CO  to  CO2  by  isolated 

tissues  as  shown  by  the  use  of  radioactive  carbon.     Amer.  J.  Physiol.,  161  :  40-46. 
CRAIG,    H.,    1953.     The   geochemistry   of   the    stable   carbon    isotopes.     Gcochimica    et    Cosino- 

chimica  Acta,  3 :  53-92. 
DALY,  J.  M.,  1954.     Stimulation  of  respiration  by  carbon  monoxide.     Arch.  Biochcm.  Biophys., 

51:  24-29. 
FENN,    W.    O.,   and   D.    M.    COBB,    1932a.     The   stimulation   of   muscle   respiration   by   carbon 

monoxide.     Amer.  J.  Physiol.,  102  :  379-392. 
FENN,  W.  O.,  and  D.  M.  COBB,  1932b.     The  burning  of  carbon  monoxide  by  heart  and  skeletal 

muscle.     Amer.  J.   Physiol,   102:   393-401. 
HOROWITZ,  N.  H.,  1940.     The  respiratory  metabolism  of  the  developing  eggs  of  Urechis  caupo. 

J.  Cell.  Comp.  Physiol.,  15:  299-308. 
KRALL,  A.  R.,  and  N.  E.  TOLBERT,  1957.     A  comparison  of  the  light  dependent  metabolism  of 

carbon   monoxide   by   barley   leaves    with   that   of   formaldehyde,    formate   and   carbon 

dioxide.     Plant  Physiol.,  32:   321-326. 

LINDAHL,    P.    E.,    1939.     t)ber    die    biologische    Sauerstoffaktivierung    nach    Versuchen    mit 
Kohlenmonoxyd  an  Seeigeleiern  und  Keimen.     Zeitschr.  I'crgl.  Physiol.,  27  :    136-168. 


OXIDATION  OF  CO  BY  EGGS  OF  URECHIS  161 

McCREA,  J.  M.,   1950.     On  the  isotopic  chemisty  of  carbonates  and  a  paleotemperature  scale. 

/.  Chem.  Phys.,  18:  849-857. 

McKiNNEY,  C.  R.,  J.  M.  McCREA,  S.  EPSTEIN,  H.  A.  ALLEN  and  H.  C.  UREY,  1950.     Improve- 
ments   in   mass    spectrometers    for    the   measurement   of    small    differences    in   isotope 

abundance  ratios.     Rev.  Sci.  Instr.,  21 :   724-730. 
MINGANTI,  A.,  1957.     Experiments  on  the  respiration  of  Phallusia  eggs  and  embryos    (ascidi- 

ans).     Acta   Embryologiac  et  Morphologiae  Experimentalis,   1:    150-163. 
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398-411. 
ROTHSCHILD,  LORD,  1949.     The  metabolism  of  fertilized  and  unfertilized  sea-urchin  eggs.     The 

action  of  light  and  carbon  monoxide.     /.  E.rp.  Biol.,  26:    100-111. 
ROTHSCHILD,  LORD,  and  A.  TYLER,  1958.     The  oxidative  metabolism  of  eggs  of  Urechis  caupo. 

Biol.  Bull,   115:    136-146. 
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Protoplasma,  10:   106-173. 
RUNNSTROM,   J.,    1956.     Some   considerations   on   metabolic   changes   occurring   at   fertilization 

and  during  early  development  of  the  sea  urchin  egg.     Pubbl.  Staz.  Zoo/.  Napoli,  28  : 

315-340. 
SCHMITT,  F.  O.,  and  M.  G.  SCOTT,  1934.     The  effect  of  carbon  monoxide  on  tissue  respiration. 

Amer.  J.  Physiol.,   107:   85-93. 
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85-86. 
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biol.,  8:   105-132. 
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THE  SALT  GLAND  OF  THE  HERRING  GULL1 

R.  FANGE,2  K.  SCHMIDT-NIELSEN  AND  H.  OSAKI 

Department   of  Zooloi/y,   Duke    University,  Durham,   North    Carolina, 
and  Mount  Desert  Island  Biological  Laboratory,  Salisbury   Cove,  Maine 

The  long  known  fact  that  the  nasal  gland  is  conspicuously  larger  in  marine 
birds  than  in  terrestrial  species  has  recently  been  given  a  functional  explanation. 
It  has  been  found  that  in  birds  taking  their  food  from  the  ocean  the  nasal  gland 
is  developed  into  an  organ  whose  main  function  is  the  secretion  of  salt.  We  have, 
therefore,  in  our  publications  on  the  function  of  this  gland,  referred  to  it  as  the 
"salt  gland."  After  large  salt  intake,  due  to  ingestion  of  sea  water  or  marine 
invertebrate  organisms,  the  salt  gland  assists  the  kidney  in  the  excretion  of  excess 
of  sodium  chloride.  In  some  marine  birds  the  gland  is  more  important  than  the 
kidney  in  the  elimination  of  salt  from  the  organism  (Schmidt-Nielsen  and  Sladen, 
1958;  Schmidt-Nielsen  and  Fange.  1958b). 

The  anatomy  of  the  avian  nasal  gland  in  a  large  number  of  birds,  both  terres- 
trial and  marine,  was  described  in  a  monograph  by  Technau  (1936).  Although 
Technau  mainly  dealt  with  the  gross  anatomy  of  the  gland  he  also  made  histo- 
logical  observations.  Other  microscopical  observations  have  been  made  by  Mar- 
pies  (1932)  and  Mihalik  (1932).  and  the  embryology  has  been  studied  by 
Grewe  (1951). 

The  discovery  of  the  osmoregulatory  importance  of  the  salt  gland  of  marine 
birds  made  it  necessary  to  re-investigate  its  histology  in  the  light  of  the  present 
knowledge  of  its  function. 

MATERIALS  AND  METHODS 

The  material  consisted  of  young  specimens  of  the  herring  gull  (Larns  argen- 
tatus)  caught  at  the  Atlantic  coast  at  Beaufort,  North  Carolina,  and  at  Mount 
Desert  Island,  Maine. 

For  histological  examination  glands  were  fixed  in  Bouin's  fluid,  and  paraffin 
sections  were  stained  in  azan  (Romeis,  1924)  or  haematoxylin-eosin. 

The  main  structure  of  the  arterial  supply  to  the  gland  was  studied  by  injection 
of  methacrylate  plastic  into  the  carotid  arteries,  followed  by  maceration  of  the 
tissues  with  KOH.  The  detailed  vascularization  was  studied  in  preparations 
injected  with  India  ink  through  the  carotids,  fixed  in  Bouin's  fluid,  and  subse- 
quently cleared  in  benzyl  benzoate.  The  glandular  duct  system  was  studied  by 
injection  of  India  ink  or  methacrylate  plastic  into  the  lateral  duct  opening.  Paraf- 
fin sections  were  prepared  of  some  of  the  India  ink-injected  specimens. 

1  Supported  by  National  Institutes  of  Health,  Grant  No.  H-2228. 

2Present  address  :  Department  of  Zoophysiology,  University  of  Lund,  Lund,   Sweden. 

162 


SALT  GLAND  OF  THE  GULL 


163 


Gross  anatomy 

In  the  gull  the  large,  paired  salt  gland  is  situated  on  the  top  of  the  skull  in  the 
supraorbital  grooves  of  the  frontal  bone  (Fig.  1).  Strictly  speaking  each  gland 
consists  of  two  parts,  as  seen  from  the  fact  that  there  are  two  ducts  on  each  side 
of  the  head  leading  forwards  into  the  beak  (Technau,  1936).  However,  the  two 
parts  of  the  gland  have  a  similar  structure  and  are  joined  so  closely  together 
that  they  can  be  considered  as  one  functional  unit  and  may  be  regarded  as  one 
gland.  Thus,  the  glands  are  flat  and  crescent  shaped,  and  two  ducts  pass  from 
the  anterior  end  of  each  to  the  anterior  nasal  cavity  (vestibulum).  On  the  upper 
side  the  gland  is  covered  by  a  thin,  tough  connective  tissue  membrane.  The 
anterior  part  of  it  extends  somewhat  laterally  from  the  margin  of  the  frontal  bone 
and  forms  part  of  the  roof  of  the  orbit.  Blood  vessels  and  nerves  pass  from  the 
orbit  into  the  gland  through  holes  in  the  frontal  bone. 


FIGURE  1.  Skull  of  the  herring  gull  from  above,  showing  the  position  of  the  salt  gland. 

The  two  ducts  on  each  side  of  the  head  take  their  origin  from  the  lower  side 
of  the  anterior  part  of  the  gland  and  run  close  together  into  the  beak,  where  they 
open  at  the  posterior  end  of  the  vestibular  concha  (Marples,  1932;  Technau 
1936).  The  lateral  duct  opens  on  the  lower  median  side  of  the  vestibular  concha 
(pre-concha)  while  the  median  duct  has  its  opening  on  the  nasal  septum  close 
to  the  transverse  fold  separating  the  anterior  nasal  cavity  (vestibulum)  from  the 
upper  nasal  cavity.  The  openings  of  the  ducts  can  be  found  if  a  longitudinal 
incision  is  made  in  the  palate  somewhat  lateral  to  the  midline,  and  it  is  then  pos- 
sible to  cannulate  the  lateral  duct  opening  for  the  collection  of  secretion  in  living 
birds  (Fange,  Schmidt-Nielsen  and  Robinson,  1958),  or  for  injection  of  fluids 
into  the  duct.  For  some  reason  our  attempts  to  cannulate  the  median  duct  were 
unsuccessful. 

Marples  (1932)  found  in  Lams  ridibimdiis  (black-headed  gull)  that  the  ducts 
are  formed  at  an  early  embryonic  stage  as  outgrowths  from  the  nasal  cavity. 
Later  the  ducts  branch  above  the  frontal  bone,  forming  the  glandular  tissue. 
Corresponding  to  the  branches  of  the  embryonic  ducts,  the  gland  of  the  adult  is 
composed  of  tubes  or  lobes,  giving  it  a  characteristic  surface  structure  (Fig.  2). 
Most  of  the  gland  consists  of  long  lobes,  some  of  which  stretch  along  the  whole 
length  of  the  gland.  In  the  gland  of  Lams  argentatus  about  15  such  longitudinal 


164 


R.  FANGE,  K.  SCHMIDT-NIELSEN  AND  H.  OSAKI 


lobes  can  be  seen  in  a  transversal  section.     In  addition  to  these  longitudinal  lobes 
there  are  shorter  lobes  oriented  in  various  directions. 

In  our  material  the  combined  weight  of  the  two  salt  glands  varied  from  700  to 
900  mg.  The  weight  of  the  animals  was  700-1000  grams  (young  specimens). 
Technau  (1936)  found  in  the  herring  gull  a  gland  weight  (probably  unilateral), 
of  555  mg.,  but  in  the  related  common  gull.  L.  canus,  150  mg.,  and  in  the  black- 
headed  gull.  L.  ridibnndns,  only  50  mg.  Of  these  three  gulls,  the  herring  gull  is 
the  most  salt  water-bound  species  while  the  black-headed  gull  is,  to  a  large  extent, 
associated  with  fresh  water.  Thus,  there  is  a  good  correlation  between  the  size  of 
the  salt  gland  and  the  habitat  of  the  different  gull  species  (Schildmacher,  1932). 

GLANDULAR       DUCTS 


CENTRAL     CANAL 


!  i  • 
•  It! 

Ml. 
I  II  / 
I" 
I  "  I 


^     ANTERIOR 


LOBE 


POSTERIOR 

FIGURE  2.  Diagram  showing  the  gross  structure  of  the  salt  gland   (left  side). 


Microscopic  structure 

In  each  lobe  there  is  a  central  canal  (Fig.  2)  which  connects  with  the  lumen 
of  one  of  the  two  main  ducts  from  the  gland.  Branching  tubular  glands  radiate 
out  from  this  central  canal  which  is  surrounded  by  a  rather  voluminous  connective 
tissue  mass  (Figs.  3,  4).  Close  to  the  central  canal,  where  the  gland  tubules 
have  not  yet  branched  extensively,  the  tubules  are  round  in  transverse  section  and 
separated  by  the  connective  tissue.  In  the  periphery  of  the  lobe  the  tubules  are 
closely  packed  together  and  run  parallel  to  each  other,  separated  by  very  delicate 
connective  tissue  membranes  and  blood  capillaries.  In  tangential  sections  through 


SALT  GLAND  OF  THE  GULL 


165 


FIGURE  3.  Longitudinal  section  through  a  lobe.  Note  the  difference  in  stainability  between 
the  outer  and  the  inner  zone  of  tubules.  The  blue-stained  connective  tissue  is  dark,  due  to  use 
of  a  yellow  filter  when  taking  the  microphotograph.  (Bourn's  fluid,  azan.) 

FIGURE  4.  Transverse  section  through  the  central  part  of  a  lobe.  An  artery  (vertical 
in  the  figure)  passes  into  the  connective  tissue  around  the  central  canal.  (Bouin's  fluid,  azan, 
yellow  filter. ) 

FIGURE  5.  Tangential  section  through  a  lobe  half-way  between  the  surface  of  the  lobe  and 
the  central  canal.  The  capillaries  between  the  tubules  are  partly  filled  with  blood.  (Bouin's 
fluid,  azan,  yellow  filter.) 

FIGURE  6.  Transverse  section  through  a  lobe  halfway  between  the  surface  and  the  central 
canal.  India  ink  was  injected  into  the  lateral  duct  of  the  gland  before  fixation.  (Bouin's 
fluid,  azan.) 


166 


R.  FANGE,  K.  SCHMIDT-NIELSEN  AND  H.  OSAKI 


the  peripheral  parts  of  a  lobe  the  cross-sectioned  tubules  have  a  polygonal  outline 
and  form  a  honeycomb-like  pattern  (Fig.  5). 

The  tubules  branch  4-6  times  forming  different  "generations"  or  "orders"  of 
tubules.  In  the  center  of  the  lobe,  close  to  the  central  canal,  the  tubules  are  thick 
and  consist  of  cylindrical  epithelial  cells  with  the  approximate  dimensions  6  p  X  15- 
20  /z..  In  the  periphery  of  the  lobe  the  diameter  of  the  tubules  is  smaller,  and  here 
the  size  of  the  cell  is  6-9  X  6-9  .  The  cell  nuclei  are  about  the  same  size  in 


ARTERY 

TO   THE   BEAK 


ANASTOMOSIS 


A.OPHTH. 
INTERNA 


A.OPHTH. 
EXTERNA 


FIGURE  7.  The  arterial  supply  of  the  salt  gland.     The  sketch  shows  the  left  gland  from 
below.     Drawn  from  a  methacrylate  plastic  cast  of  the  vascular  system. 

the  central  and  the  peripheral  tubules.  Thus,  the  amount  of  cytoplasm  in  relation 
to  the  nuclear  volume  is  largest  in  the  central  portion  of  the  tubules,  possibly 
indicating  that  these  gland  cells  carry  out  more  work  than  those  of  the  peripheral 
portions. 

The  cytoplasm  of  the  cells  has  a  lamellated  or  striated  appearance.  The  stria- 
tion  is  not  limited  to  a  striated  border,  but  extends  through  Lhe  cells  from  the 
lumen  to  the  periphery,  where  the  cells  are  in  contact  with  blood  capillaries.  In 
sections  from  specimens  in  which  India  ink  had  been  injected  into  the  lateral 
duct,  the  lumen  of  the  tubules  had  an  irregular  shape,  indicating  the  presence  of 
secretory  intra-  or  intercellular  canaliculi  (Fig.  6). 


SALT  GLAND  OF  THE  GULL  167 

The  cytoplasm  takes  a  reddish  colour  in  azan  stain.  In  the  most  peripheral 
part  of  the  tubules  the  cytoplasm  is  less  heavily  stained  than  in  the  central  tubules 
(Fig.  3). 

The  central  canal  consists  of  2-4  layers  of  cuboidal  epithelium.  The  two  main 
ducts  passing  to  the  anterior  nasal  cavity  also  consist  of  a  multi-layered  epithelium. 
In  some  preparations  the  boundaries  between  the  epithelial  cells,  especially  those 
of  the  central  canal,  had  a  vacuolated  appearance  which  gave  the  illusion  of  a 
system  of  intercellular  canals.  This,  however,  could  be  a  fixation  artefact  due  to 
shrinkage  of  the  cells.  The  two  main  ducts  are  surrounded  by  the  same  con- 
nective tissue  which  surrounds  the  accompanying  blood  vessels  and  nerve  and  have 
no  connective  tissue  of  their  own.  No  smooth  muscle  cells  could  be  found  in  the 
walls  of  the  ducts.  Neither  was  it  possible  to  detect  any  smooth  muscle  in  the 
gland  except  that  of  the  arteries  supplying  the  lobes.  Neither  the  connective  tissue 
of  the  upper  side  of  the  gland,  the  interlobular  connective  tissue  mass,  the  con- 
nective tissue  membranes  around  the  tubules,  nor  the  central  connective  tissue 
mass  around  the  central  canal  contains  any  smooth  muscle. 

Vascularization 

The  blood  supply  of  the  nasal  glands  of  the  duck  has  been  described  by  Mar- 
pies  (1932)  and  earlier  authors  (Gadow,  1891).  According  to  our  observations 
in  Lams  argentatus  the  main  arterial  supply  comes  from  the  arteria  ophthalmica 
interna.  The  vessel  penetrates  the  wall  of  the  orbit  above  the  optic  nerve  and, 
passing  upwards  along  the  median  wall  of  the  orbit,  it  divides  into  two  branches 
to  the  salt  gland.  The  anterior  branch  gives  off  several  small  arteries  to  the  gland 
and  then  continues  into  the  beak  (Fig.  7).  The  posterior  branch  supplies  the 
posterior  part  of  the  glands.  Anastomosing  with  this  branch  another  artery  from 
the  posterior  wall  of  the  orbit  also  gives  blood  to  the  gland.  This  artery  probably 
corresponds  to  the  arteria  ophthalmica  externa  described  by  previous  authors 
(Gadow,  1891;  Slonaker,  1918).  Both  the  arteria  ophthalmica  interna  and  the 
arteria  ophthalmica  externa  are  branches  of  the  arteria  carotis  interna. 

The  arteries  reaching  the  salt  glands  are  among  the  largest  arteries  in  the  head 
of  the  gull.  The  arrangement  of  the  arteries  is  such  that,  in  spite  of  the  rich 
blood  supply,  the  blood  could  probably  bypass  the  gland  via  the  arterial  arch 
formed  by  the  anastomosis  between  the  anterior  and  posterior  branch  of  the  arteria 
ophthalmica  interna  (Fig.  7).  This  arrangement  may  permit  a  large  reduction 
in  glandular  blood  flow  without  reducing  the  blood  flow  to  the  upper  beak  when 
the  glands  are  not  functioning.  The  control  of  the  blood  flow  through  the  glands 
may  be  exerted  by  contractile  arterioles  in  the  glands. 

The  veins  from  the  salt  glands  follow  the  arteries  in  their  main  courses 
(Marples,  1932). 

Microscopic  distribution  of  blood  vessels 

The  connective  tissue  between  the  individual  gland  lobes  contains  a  large  num- 
ber of  branching  arteries  and  veins.  At  intervals  the  arteries  give  off  branches 
which  pass  into  the  lobes.  These  arteries  pass  straight  through  the  gland  tissue 
between  the  tubules  towards  the  central  canal  without  branching  (Figs.  4,  8),  but 
after  reaching  the  central  connective  tissue  mass  they  break  up  into  numerous 


168 


R.  FANGE,  K.  SCHMIDT-NIELSEN  AND  H.  OSAKI 


capillaries.  These  capillaries,  which  have  frequent  branchings  and  anastomoses, 
run  radially  out  towards  the  surface  of  the  lobes.  In  their  main  course  the  capil- 
laries are  parallel  to  the  tubules.  Tubules  and  capillaries  form  a  regular  pattern 
in  sections  cut  tangentially  through  a  lobe  (see  Fig.  5).  The  tubules  are  polygonal 
in  shape  and  the  capillaries  are  situated  at  the  corners  of  the  polygons,  each  tubule 
being  surrounded  by  5-7  capillaries.  The  regularity  of  the  arrangement  reminds 
of  the  rete  mirabile  of  the  fish  swimbladder,  or  the  regular  arrangement  of  tubules 
and  blood  vessels  in  the  medulla  of  the  mammalian  kidney.  At  the  surface  of  the 


ARTERY 


VEIN 


1  central    connective 
tissue 


interlobular 
connective     tissue 


FIGURE  8.  Diagram  of  a  transverse  section  through  a  lobe  of  the  salt  gland. 

lobe  the  capillaries  leave  the  tubules  and  pass  over  into  a  venous  plexus  drained 
by  veins  in  the  interlobular  connective  tissue  (Fig.  8).  No  veins  were  observed 
within  the  lobes.  No  lymph  vessels  could  be  observed  in  the  glands,  but  as  they 
may  be  difficult  to  detect  in  histological  sections,  we  hesitate  to  claim  that  there  are 
none  in  the  salt  gland.  A  diagrammatic  picture  of  the  blood  flow  in  the  gland 
is  shown  in  Figure  9. 

Innervation 

The  nasal  gland  of  birds  has  been  reported  to  be  innervated  from  a  para- 
sympathetic  ganglion  in  the  anterior  part  of  the  orbit  (Cords,  1904;  Webb,  1957). 
The  ganglion  has  connections  with  different  cranial  nerves  and  with  the  sympathetic 
system  (Cords,  1904).  The  nerve  supply  of  the  salt  gland  in  the  herring  gull 
will  be  described  in  another  publication  which  will  also  deal  with  the  physiological 
responses  of  the  gland  to  various  kinds  of  stimulation  (Fange,  Schmidt-Nielsen 
and  Robinson,  1958). 

Other  bird  species 

The  presence  of  salt  glands  has  been  demonstrated  in  birds  of  five  different 
orders  (Schmidt-Nielsen  and  Fange,  1958a).  We  have  undertaken  some  pre- 


SALT  GLAND  OF  THE  GULL 


169 


ARTERY 


VEIN 


INTERLOBULAR 
CONNECTIVE 
TISSUE 


SECRETORY 
TUBULES 


CENTRAL 

CONNECTIVE 

TISSUE 


CENTRAL 
CANAL 


FIGURE  9.  Diagram  of  the  circulation  showing  the  opposing  directions  of  the  flow  in  the 
gland  tubules  and  in  the  capillaries.  The  tubules  branch  repeatedly,  but  for  simplicity  only 
t\vo  ramifications  are  pictured. 


liminary  histological  studies  of  the  salt  glands  of  pelican  (Pclecanus*),  cormorant 
(Phalacrocora.v) ,  eider  duck  (Somateria),  petrel  (Occanodroma),  etc.  In  these 
birds  the  glands  have  essentially  the  same  histological  structure  as  in  the  gull,  and 
consists  of  lobes  with  tubular  glands  radially  arranged  around  a  central  canal. 
In  the  pelican  and  the  cormorant  the  lobes  are  not  tubiform  as  in  the  gull,  but 
rather  short  and  of  a  rounded  shape.  In  the  connective  tissue  of  the  salt  glands 
of  many  birds  black  pigment  cells  occur. 


170  R.  FANGE,  K.  SCHMIDT-NIELSEN  AND  H.  OSAKI 

DISCUSSION 

The  salt  gland  of  marine  birds  has  a  very  characteristic  structure  consisting  of 
closely  packed  secretory  tubules  with  blood  vessels  between  them.  The  tubules 
radiate  from  a  central  canal.  In  terrestrial  birds,  where  the  nasal  glands  have 
no  salt  excretory  function,  the  glands  contain  only  a  few  tubules  or  have  sac-shaped 
diverticula  instead  of  tubules  (Marples,  1932).  The  strictly  parallel  arrangement 
of  closely  packed,  glandular  tubules  may  be  necessary  for  the  osmotic  work  per- 
formed by  the  gland.  It  is  probable  that  the  manner  of  distribution  of  the  blood 
capillaries  within  the  gland  tissue  is  also  of  importance  in  this  respect.  It  may  be 
noted  that  the  arrangement  of  blood  vessels  within  the  lobe  is  such  that  the  capil- 
lary blood  flows  in  a  direction  opposite  to  that  of  the  secreted  fluid.  The  func- 
tional significance  of  this  counter-current  flow  in  the  salt  gland  is  not  clear.  The 
counter-current  principle,  although  manifested  in  a  different  way,  seems  to  play 
an  important  role  in  the  production  of  a  concentrated  urine  in  the  kidney  of  mam- 
mals and  birds  (Hargitay  and  Kuhn,  1951).  Although  the  structure  of  the  salt 
gland  in  marine  birds  and  of  the  mammalian  kidney  otherwise  are  entirely  different, 
it  is  striking  that  a  counter-current  flow  is  found  in  both  these  organs,  which  in 
higher  vertebrates  are  the  only  ones  known  to  produce  a  highly  hypertonic  secretion. 

The  counter-current  flow  in  the  salt  gland  cannot,  as  such,  explain  the  large 
osmotic  work  performed  by  the  gland.  Active  ionic  transport  can  be  assumed  to 
be  the  fundamental  cellular  process  responsible  for  the  osmotic  work.  The  striated 
or  lamellated  appearance  of  the  cytoplasm  of  the  gland  cells  and  the  presence  of 
secretory  canaliculi  indicate  a  highly  specialized  transport  function  of  the  cyto- 
plasm. A  more  detailed  study  of  the  microscopic  and  electron  microscopic  struc- 
ture of  the  cytoplasm  of  the  avian  salt  gland  cells  is  in  progress. 

SUMMARY 

1.  The  salt  gland  of  the  herring  gull    (Lanis  argcntatits)    is  a  large,  paired 
gland  on  top  of  the  skull.     On  each  side  twro  ducts  lead  to  the  anterior  nasal  cavity. 
When  the  gland  is  secreting,  its  discharge  comes  out  through  the  nares  and  drips 
off  from  the  tip  of  the  beak. 

2.  The  gland  has  long,  tubular  lobes,   each  with  a  central  canal.     Tubulous 
glands  radiate  from  the  central  canal.     The  gland  cells  have  a  striated  or  lamellated 
cytoplasm,  and  seem  to  have  secretory  canaliculi. 

3.  The  blood  supply  is  mainly  from  arteria  ophthalmica  interna.     Within  the 
gland  the  capillary  blood  flow  is  in  a  direction  opposite  to  that  of  the  secreted 
fluid.     The  innervation  of  the  gland  is  from  a  ganglion  of  predominantly  para- 
sympathetic  nature. 

4.  The  salt  glands  of  other  marine  birds  have  the  same  characteristic  structure 
with  the  secreting  tubules  radiating  out  from  a  central  canal. 

LITERATURE  CITED 

CORDS,   E.,   1904.     Beitrage   zur   Lehre   vom   Kopfnervensystem   der   Vogel.     Anat.   Heftc,    26: 

49-100. 
FANGE,  R.,  K.  SCHMIDT-NIELSEN  AND  MARYANNE  ROBINSON,  1958.     The  control  of  secretion 

from  the  avian  salt  gland.     Amcr.  J.  Physiol.   (in  press). 


SALT  GLAND  OF  THE  GULL  171 

GADOW,  H.,  1891.     Dr.  H.  G.  Bronn's  Klassen  und  Ordnungen  des  Thier-Reichs.     VI.  4  Abt. 

Vogel.     I.   Anatomischer    Theil.    767-785.        Das    Arteriensystem.     C.    F.    Winter'sche 

Verlagshandlung,   Leipzig. 
GREWE,    F.   J.,    1951.     Nieuwe   gegewens   aangaaende   die   ontogenese   van   die   neuskliere,   die 

orgaan  van  Jacobson  en  die  dekbene  van  die  schedel  by  die  benus  Anas.     Annals  Univ. 

Stellenbosch,  27 :  69-99. 
HARGITAY,   B.,   AND  W.   KUHN,   1951.     Das   Multiplikationsprinzip  als   Grundlage   der   Harn- 

konzentricrung   in   der   Niere.     Zcitschr.   f.   Elcktrochcm.   11.    angeiv.   physik.    Cheinic., 

55:   539-558. 
MARPLES,  B.  J.,  1932.     The  structure  and  development  of  the  nasal  glands  of  birds.       Proc. 

Zool.  Soc.  Land.,  829-844. 
AimALiK,  P.  V.,  1932.     Uber  die  Glandula  lateralis  nasi  der  Vogel.     Ergebn.  d.  Anat.  u.  Entw. 

Gesch.,  29:  399-448. 

ROMEIS,  B.,  1924.     Mikroskopische  Technik.     R.  Oldenbourg,  Mtinchen. 
SCHILDMACHER,    H.,    1932.     Uber    den    Einfluss    des    Salswassers    auf    die    Entwicklung    der 

Nasendrusen.     /.  /.   Ornitlwl.,  80:   293-299. 

SCHMIDT-NIELSEN,  K.,  AND  R.  FANGE,  1958a.     Extrarenal  salt  excretion.     Fed.  Proc.,  17:  142. 
SCHMIDT-NIELSEN,  K.,  AND  R.  FANGE,  1958b.     The  function  of  the  salt  gland  in  the  brown 

pelican.     The  Auk  (in  press). 
SCHMIDT-NIELSEN,  K.,  AND  W.  J.   L.   SLADEN,   1958.     Nasal  salt  secretion  in   the   Humboldt 

Penguin.     Nature,  181:   1217-1218. 
SLONAKER,  J.  R.,   1918.     A  physiological  study  of  the  anatomy  of  the  eye  and  its  accessory 

parts  of  the  English  Sparrow   (Passer  domesticus).     J.  MorphoL,  31:   351-459. 
TECHNAU,  G.,  1936.     Die  Nasendriise  der  Vogel.     /.  /.  Ornitlwl..  84:  511-617. 
WEBB,    M.,    1957.     The   ontology   of   the   cranial    bones,    cranial    peripheral   and   cranial    para- 
sympathetic  nerves,  together  with  a  study  of  the  visceral  muscles  of  Struthio.     Ada 

Zool,  Stockh.,  38:   1-203. 


THE  SWIMBLADDER  OF  THE  TOADFISH    (OPSANUS  TAU  L.) 

RAGNAR  FANGE  1  AND  JONATHAN  B.  WITTENBERG  2 

The  Marine  Biological  Laboratory,  Woods  Hole.  Massachusetts;    The  Department  of 

Zoophysiology,  University  of  Lund.  Lund,  Sweden;  and   The  Departments  of 

Physiology   and    Biochemistry,   Albert   Einstein    College    of   Medicine 

of    }'eshira    University.   AVtc-    York    61.    N.    V. 

The  swimbladder  of  the  toadfish  (Opsanus  tail  L.)  offers  a  particularly  favor- 
able object  for  the  experimental  study  of  gas  secretion.  To  provide  a  basis  for 
physiological  studies  we  describe  here  the  structure  of  the  swimbladder,  its  gas 
gland  and  its  vascular  supply.  In  addition,  some  physiological  observations  are 
presented.  Further  physiological  studies  of  this  species  are  reported  elsewhere 
(Wittenberg,  1958). 

Brief  anatomical  descriptions  of  the  swimbladder  of  the  toadfish  are  found  in 
Tower  (1908)  and  Rauther  (1945).  Greene  (1924a)  has  studied  a  related 
species,  Porichtliys.  Tracy  (1911)  presents  some  embryological  and  histological 
data.  Tracy  observed  that  the  posterior  chamber  of  the  embryonic  toadfish  de- 
velops from  the  pneumatic  duct,  which  secondarily  loses  its  connection  with  the  gut. 

MATERIAL  AND  METHODS 

Animals:  Toadfish  caught  at  Woods  Hole  were  maintained  in  a  shallow  live 
car  for  several  months  before  they  were  used. 

Histological:  After  fixation  in  Bouin's  fluid,  histological  sections  were  made 
and  stained  with  azan  (Romeis,  1948)  or  haematoxylin  and  eosin.  The  blood 
vessels  were  studied  by  injection  of  India  ink  into  the  coeliac  artery.  The  injected 
specimens  were  fixed  in  Bouin's  fluid  and  later  cleared  in  benzyl  benzoate. 

Gas  analyses:  These  were  by  the  method  of  Scholander  ct  al.  (1955). 

RESULTS 
The  swimbladder  gases 

In  contrast  to  the  majority  of  shallow-living  marine  fishes,  the  toadfish  nor- 
mally maintains  a  very  high  proportion  of  oxygen  in  the  swimbladder  gases.  The 
oxygen  ranges  from  40  to  80  per  cent  and  in  most  animals  is  about  50  per  cent  of 
the  total  gas.  Similar  high  oxygen  concentrations  (maximum  88  per  cent)  have 
previously  been  observed  in  a  related  species,  Porichthys  (Greene,  1924b). 

When  forced  experimentally  to  renew  repeatedly  the  gaseous  contents  of  the 
bladder,  the  toadfish  is  able  to  maintain  the  secretion  of  gas  undiminished  in  rate 
and  oxygen  content.  Thus  in  one  experimental  series  the  swimbladders  of  three 
animals  were  emptied  every  24  hours  for  six  days.  During  this  time,  each  animal 

1  Present  address :   Department  of  Zoophysiology,  University  of  Lund,   Lund,   Sweden. 
-This  investigation  was  supported  by  a   Senior  Research  Fellowship    (S.F.   57)    from   the 
Public  Health  Service,  and  by  a  research  grant  from  The  National  Science  Foundation. 

172 


SWIMBLADDER  OF  THE  TOADFISH 


173 


secreted  a  volume  of  gas  equivalent  to  six  times  the  volume  of  the  swimbladder. 
At  the  end  of  the  six-day  period,  the  rate  of  secretion  and  the  composition  of  the 
secreted  gas  remained  unchanged.  The  newly  secreted  gas  is  characterized  by  an 
extraordinarily  high  proportion  of  oxygen  which  averages  90  per  cent  and  may  be 
as  high  as  96  per  cent  of  the  total  gas.  The  proportion  of  carbon  dioxide  is  low, 
about  4  per  cent  (Wittenberg,  unpublished  data).  The  ratio,  argon  to  nitrogen, 
in  the  secreted  gas  is  very  high,  2.4  X  10~-  to  2.6  X  1O2,  and  approaches  the 
maximum  which  can  be  achieved  by  a  mechanism  of  inert  gas  secretion  proposed 
elsewhere  (Wittenberg,  1958).  These  properties  combine  to  indicate  a  very 
powerful  development  of  oxygen  transport  in  the  gas  gland  of  the  toadfish,  making 
this  an  animal  of  choice  for  experimental  studies  concerning  oxygen  transport. 

The  principal  layers  of  the  sivimbladdcr  wall 

The  external  appearance  of  the  swimbladder  is  shown  in  Figure  1.  It  is  of 
the  euphysoclist  type  (Rauther,  1922;  Fange,  1953).  The  wall  may  be  described 
as  formed  of  three  layers,  conveniently  called  tunica  externa,  submucosa  and 


Coeliac  artery 


Nerve 


::: 

' 


Nerve 


Sound  producing 
striated  muscles 


Swim  bladder^ 
artery 

Swim  bladder 
vein 


from  posterior  chamber 
DORSAL   VIEW 


Portal  vein  ^^SlS^ 
VENTRAL  VIEW 


FIGURE  1.  External  view  of  the  swimbladder  of  the  toadfish  seen  in  dorsal  and  ventral  view. 
The  nerve  shown  in  the  picture  is  the  motor  nerve  to  the  striated  sound-producing  muscle. 
According  to  Tracy  (1911)  it  is  a  branch  of  the  first  spinal  nerve. 

mucosa.  The  tunica  externa  is  a  tough,  somewhat  rigid  external  connective  tissue 
capsule.  Laterally  this  layer  includes  the  sound-producing  striated  muscle  masses 
(Figs.  1  and  2;  compare  with  Rauther,  1945). 

The  submucosa  consists  of  very  loose  fibrous  connective  tissue  which  allows  a 
limited  movement  of  the  mucosa  relative  to  the  tunica  externa.  In  fresh  specimens 
it  is  possible  to  take  advantage  of  the  loose  consistency  of  the  submucosa  to  dissect 
away  the  tunica  externa,  including  the  striated  muscle  masses.  The  mucosa  is 


174 


RAGNAR  FANGE  AND  JONATHAN  B.  WITTENBERG 


then  revealed  as  a  transparent,  richly  vascularized,  sac  composed  of  two  chambers 
separated  by  a  deep  transverse  constriction,  the  diaphragm  (Fig.  2).  The  lumina 
of  the  two  chambers  communicate  by  a  hole  in  the  diaphragm  (Fig.  2). 


Capillaries  of  the 
Resorbent  Mucosa 


Posterior 
chamber 


Anterior 
chamber 


Gas  Gland 
Capillaries 


Diaphragm 
Striated  muscles 


Rete  Mirabile 


FIGURE  2.  The  swimbladder  opened  dorsally.  Portions  of  the  secretory  mucosa  and  the 
resorbent  mucosa  are  shown  in  higher  magnification,  in  order  to  demonstrate  the  typical 
appearance  of  the  blood  vessels. 

The  anterior  chamber,  gas  gland  and  retia  mirabilia 

The  gas  gland  forms  the  epithelial  lining  of  the  floor  of  the  anterior  chamber 
and  to  a  lesser  extent  it  is  developed  on  the  anterior  face  of  the  diaphragm.  Periph- 


SWIMBLADDER  OF  THE  TOADFISH 


175 


erally  the  gas  gland  is  continuous  with  the  cuboidal,  apparently  non-glandular, 
epithelium  of  the  roof  of  the  anterior  chamber.  The  gas  gland  is  most  strongly 
developed  and  heavily  folded  within  a  few  millimeters  of  the  retia  mirabilia  (Fig, 
3).  At  a  distance  from  the  retia  the  degree  of  folding  dwindles  rapidly  and  the 
glandular  cells  become  smaller.  The  glandular  epithelium  is  everywhere  only  one 
cell  thick.  The  cells  are  columnar  with  a  dense  cytoplasm  stained  red  by  azan. 
An  interesting  feature  of  the  gas  gland  cells  is  the  position  of  the  cell  nuclei  (Fig. 
3).  These  are  situated  near  the  secretory  lumen  and  not  adjacent  to  the  basal 
blood  vessel  as  in  most  gland  cells.  This  peculiar  position  of  the  nuclei  has  been 
noted  by  Woodland  (1911)  in  the  gas  gland  of  the  eel  (Anguilla)  and  other 
species,  but  in  the  toadfish  the  nuclei  are  situated  far  more  apically  than  in  any  of 
the  fish  studied  by  Woodland. 


Gas  gland 

Vascular  bed 

Dense  connective  tissue 
Muscularis  mucosae 
Dense  connective  tissue 

Loose  connective  tissue 


FIGURE  3.  Partly  diagrammatic  drawing  of  a  section  through  the  secretory  mucosa. 
Blood  vessels  are  found  within  the  folds  of  the  secretory  epithelium. 

The  structure  of  the  retia  mirabilia  is  essentially  of  the  type  described  for  the 
eel  by  Woodland  (1911).  There  are  6-8  distinct  retia  ("red  bodies")  situated  in 
the  submucosa  at  the  junction  of  the  floor  of  the  anterior  chamber  and  the  dia- 
phragm. The  capillaries  emanating  from  the  retia  mirabilia  rejoin,  to  some  ex- 
tent, forming  arterioles  and  venules  which  go  to  the  gas  gland,  where  they  break 
up  into  capillaries  providing  a  very  rich  blood  supply  to  the  glandular  membrane. 
Every  fold  of  the  membrane  contains  blood  vessels  (Fig.  3),  and  it  is  probable 
that  each  gland  cell  has  access  to  a  blood  capillary  at  its  base  and  is  separated  from 
the  blood  only  by  a  very  thin  endothelium.  Capillary  connections  are  found 
between  arterioles  and  venules  emanating  from  the  same  rete  as  well  as  between 
blood  vessels  emanating  from  different  retia  (Fig.  2). 

The  capillaries  of  a  single  rete  mirabile  were  counted  in  a  histological  section. 
A  very  rough  calculation  indicated  that  the  total  number  of  capillaries  of  all  the 


176  RAGNAR  FANGE  AND  JONATHAN  B.  WITTENBERG 

retia  mirabilia  is  200.000-300,000,  which  is  of  the  order  of  magnitude  found  by 
Krogh   (1929)   in  the  eel. 

In  the  connective  tissue  surrounding  the  central  parts  of  the  retia  mirabilia 
there  are  numerous  nerves  and  ganglion  cells.  The  ganglion  cells  probably  give 
fibers  to  the  gas  gland  or  innervate  the  muscularis  mucosae. 

/'//('  muscularis  inncosac  and  the  diaphragm 

In  close  connection  with  the  inner  epithelium  of  both  the  anterior  and  posterior 
chamber  there  is  a  smooth  muscle  layer,  the  muscularis  mucosae.  This  is  ex- 
tremely thin  in  the  posterior  chamber  but  well  developed  in  the  anterior  chamber, 
especially  ventrally  in  connection  with  the  glandular  portion  of  the  epithelium. 
The  muscularis  mucosae  also  makes  a  large  contribution  to  the  diaphragm  where 
it  forms  a  sphincter  around  the  hole.  Tower  (1908)  observed  that  the  position 
of  the  diaphragm  varies  from  about  one-third  of  the  distance  from  the  posterior 
end  to  less  than  one-sixth  of  the  distance.  We  have  observed  the  same  variations. 
That  these  changes  of  the  position  of  the  diaphragm  are  due  to  reflex  movements 
of  the  muscularis  mucosae  is  shown  by  the  following  observations :  ( 1 )  In  a  speci- 
men in  which  gas  secretion  had  been  stimulated  by  emptying  the  bladder  three 
hours  earlier,  the  diaphragm  had  a  posterior  position,  by  which  consequence  the 
anterior  chamber  was  enlarged  and  the  posterior  chamber  diminished.  The  hole 
in  the  diaphragm  was  closed.  (2)  In  a  specimen  which  suffered  from  asphyxia- 
tion and  which  in  addition  had  received  an  injection  of  adrenaline  (0.1  ml., 
1:1000),  the  diaphragm  was  found  in  the  anterior  position  and  with  its  hole 
open.  (Asphyxia  and  adrenaline  each  stimulate  gas  resorption.)  (3)  In  indi- 
viduals, where  the  hole  in  the  diaphragm  was  initially  closed,  application  of  a  small 
drop  of  adrenaline  solution  to  the  margin  of  the  hole  caused  this  to  open  to  a  width 
of  2-3  mm.  It  is  evident  that  movements  of  the  muscularis  mucosae  are  among 
the  physiological  regulatory  mechanisms  which  control  reflexly  the  function  of  the 
secretory  chamber  (the  gas  gland)  and  the  resorbent  chamber  ("the  posterior 
vascular  organ"). 

The  blood  supply  of  the  swimbladder 

The  swimbladder  receives  its  blood  from  a  branch  of  the  coeliac  artery,  the 
swimbladder  artery  (Fig.  1).  The  individual  retia  of  the  anterior  chamber  are 
supplied  by  branches  from  the  swimbladder  artery.  Within  each  rete  the  arterial 
and  venous  capillaries  form  the  typical  counter-current  exchange  system  studied 
by  Woodland  (1911),  Haldane  (1922),  Krogh  (1929)  and  Scholander  (1954). 
All  the  blood  to  the  anterior  chamber  passes  through  the  retia.  The  entire  venous 
return  from  the  anterior  chamber  passes  back  through  the  retia  and  leaves  the 
swimbladder  by  the  swimbladder  vein,  which  joins  the  portal  vein  (Fig.  1,  ventral 
view). 

The  blood  supply  to  the  resorbent  capillary  network  (the  "posterior  vascular 
organ")  of  the  posterior  chamber  resembles  that  of  Fierasfcr  (Emery,  1880)  and 
the  eel  (Mott,  1950a,  1950b)  in  that  the  arterial  blood  is  supplied  from  the  swim- 
bladder  artery  instead  of  from  the  intercostal  arteries  as  in  most  physoclists. 
The  venous  return  is  to  the  cardinal  vein  system  (Fig.  1,  dorsal  view). 


SWIMBLADDER  OF  THE  TOADFISH 


177 


DISCUSSION 

The  swimbladder  of  the  toadfish  is  of  the  typical  euphysoclist  type  (Rauther, 
1922;  Fange,  1953).  It  shows  many  similarities,  both  physiologically  and  morpho- 
logically, with  that  of  the  eel. 

The  swimbladder  of  the  toadfish  is  apparently  specialized  for  the  production 
of  sounds  (Tower,  1908),  and  the  tunica  externa  forms  a  thick  capsule  enclosing 
both  the  anterior  and  posterior  chambers.  Removal  of  this  capsule  reveals  the 
homology  of  the  two  chambers  with  corresponding  parts  of  the  eel  swimbladder 
(Fig.  4).  (For  previous  descriptions  of  the  swimbladder  of  the  eel  see  Queckett 


1. 

Pneumatic 
duct 


oesophagus 


2. 


3. 


swim  bladder 
artery 


secretory  part 

resorbent  part 
(pneumatic  duct) 

resorbent  part 
(posterior  chamber) 


OPSANUS 


ANGUILLA 


Anterior     Posterior 
chamber     chamber 

EMBRYOLOGICAL 
DEVELOPMENT 

FIGURE  4.  The  swimbladder  of  the  toadfish  (Opsanus  tan)  and  the  eel  (Anguilla  anguilla) 
illustrating  the  similarity  in  general  structure.  The  embryological  stages  to  the  left  in  the 
figure  are  redrawn  from  Tracy  (1911).  Note  the  transformation  of  the  embryonic  pneumatic 
duct  into  the  posterior  chamber. 

(1844),  Woodland  (1911),  Rauther  (1922),  Fange  (1953).)  The  anterior 
chamber  of  the  toadfish  swimbladder  corresponds  to  the  swimbladder  per  sc  in  the 
eel  and  the  posterior  chamber  corresponds  to  the  pneumatic  duct  of  the  eel.  The 
homology  is  further  substantiated  by  the  embryonic  development  of  the  toadfish 
swimbladder  (Tracy,  1911)  during  which  the  posterior  chamber  develops  from 
the  embryonic  pneumatic  duct.  The  muscularis  mucosae  of  the  toadfish  and  the 
eel  respond  to  adrenaline  in  a  similar  manner ;  the  anterior  chamber  of  the  toadfish 
swimbladder  and  the  swimbladder  of  the  eel  both  are  contracted  by  adrenaline 
while  the  posterior  chamber  of  the  toadfish  swimbladder  and  the  pneumatic  duct 
of  the  eel  are  relaxed  (Fange,  1953). 

Woodland  (1911),  in  his  classic  description  of  the  gas  gland,  distinguishes  three 
major  types  of  gas  glands :  those  in  which  the  glandular  epithelium  is  composed 


178  RAGNAR  FANGE  AND  JONATHAN  B.  WITTENBERG 

of  a  single  layer  of  cells,  those  in  which  the  gland  is  massive,  and  those  in  which 
a  primitively  single  layer  of  cells  is  secondarily  folded  into  a  massive  structure. 
The  toadfish,  in  common  with  the  eel,  belongs  to  the  first  category  (Woodland, 
1911)  in  which  (p.  193)  "the  glandular  epithelium  is  composed  of  a  single  layer 
<of  cells  which  either  renains  unfolded  or  is  only  simply  folded.  .  .  ."  Micro- 
scopically, according  to  our  present  observations,  the  glandular  epithelia  of  the 
toadfish  and  the  eel  swimbladder  are  scarcely  distinguishable  in  appearance. 
Woodland  further  subdivides  his  first  class  of  gas  glands  on  the  basis  of  the  extent 
of  the  glandular  epithelium  and  the  degree  of  reunion  of  the  blood  vessels.  The 
toadfish  belongs  to  the  Syngnathus  subdivision,  type  Ib,  in  which  (p.  195)  "the 
glandular  epithelium  is  restricted  in  area,  not  lining  the  whole  of  the  bladder 
cavity,  and  the  rete  mirabile  is  contiguous  with  the  gas  gland,  although  a  small 
amount  of  reunion  of  the  capillaries  of  the  rete  may  occur  before  these  supply 
the  epithelium." 

The  gas  mixtures  secreted  into  the  swimbladder s  of  the  toadfish  and  the  eel 
are  markedly  similar  (Wittenberg,  unpublished  data;  Wittenberg,  1958).  The 
oxygen  content  is  very  high,  up  to  95  per  cent,  and  the  carbon  dioxide  content 
usually  is  low,  about  4  per  cent.  The  ratio  of  argon  to  nitrogen  is  very  high  and 
approaches  what  may  be  a  theoretical  maximal  value,  2.6  X  10~2  (Wittenberg, 
1958).  Both  the  toadfish  and  the  eel  possess  highly  efficient,  powerfully  developed, 
oxygen  transporting  mechanisms,  obviously  of  very  similar  nature. 

SUMMARY 

1.  The  anatomy  of  the  swimbladder  and  the  gas  gland  of  the  toadfish  (Opsanus 
tau  L.)    is  described.     The  swimbladder   is   of  the   euphysoclist   type.     The   gas 
gland  is  composed  of  a  single  cell  layer. 

2.  Both  physiologically  and  morphologically  the  swimbladder  of  the  toadfish 
shows  strong  resemblances  to  that  of  the  eel  (Anguilla) .     The  swimbladder  nor- 
mally has  a  high  proportion  of  oxygen,  an  unusual  feature  for  fishes   living  in 
shallow  water. 

LITERATURE  CITED 

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Fauna  c  Flora  Golfo  Napoli,  2 :  1-76. 
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Acta  Physiol.  Scand.,  30,  Snppl,  110:   1-133. 
GREENE,   C.   W.,   1924a.     Physiological   reactions  and  structure  of  the   vocal   apparatus   of   the 

California  Singing  Fish,  Porichthys  notatus.     Amcr.  J.  Physiol.,  70:  496-499. 
GREENE,  C.  W.,  1924b.     Analysis  of  the  gases  of  the  airbladder  of  the  California  Singing  Fish, 

Porichthys  notatus.     J.  Biol.  Chcm.,  59:  615-621. 

HALDANE,  J.  S.,  1922.     Respiration.     Yale  University  Press,  New  Haven,   Conn. 
KROGH,  A.,  1929.     The  Anatomy  and  Physiology  of  Capillaries.     Yale  University  Press,  New- 
Haven,  Conn. 
MOTT,  J.   C.,   1950a.     The  gross  anatomy  of  the  blood  vascular   system   in   Anguilla   anquUla. 

Proc.  Zool.  Soc.  Loud.,  120 :  503-518. 
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with   some  remarks  on  the  evidence  they  afford  of  the   true   function  of   that   organ. 

Trans.  Micr.  Soc.  London,  1:   98-108. 


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RAUTHER,   M.,   1922.     Zur  vergleichenden  Anatomic  der   Schwimmblase  der   Fische.     Ergebn. 

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somatischen  Muskeln  bei  den  Triglidae  und  anderen   Scleroparei.     Zool.  Jahrb.,  69 : 

159-250. 

ROMEIS,   B.,    1948.     Mikroskopische   Technik.     Leibniz   Verlag,    Miinchen. 
SCHOLANDER,   P.  F.,   1954.     Secretion  of  gases  against  high  pressures   in   the   swimbladder   of 

deep  sea  fishes.     II.  The  rete  mirabile.     Biol.  Bull.,  107 :  260-277. 
SCHOLANDER,   P.   F.,  L.  VAN   DAM,   C.   LLOYD   CLAFF  AND  J.   W.   KANWISHER,   1955.     Micro 

gasometric  determination  of  dissolved  oxygen  and  nitrogen.     Biol.  Bull.,  109 :  328-334. 
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toadfish.     Ann.  N.   Y.  Acad.   Sci.,   18:    149-180. 
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600-606;  638-649. 
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PhysioL,  41:   783-804. 
WOODLAND,  W.  N.  F.,  1911.     On  the  structure  and  function  of  the  gas  glands  and  retia  mira- 

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teleost  pancreas.     Proc.  Zool.  Soc.  London,  183-248. 


SALT  AND  WATER  ANATOMY,  CONSTANCY  AND  REGULATION 

IN   RELATED   CRABS   FROM    MARINE   AND 

TERRESTRIAL  HABITATS 

LAUNCE  J.  FLEMISTERi 

Edzvard  Martin  Biological  Laboratories,  Swarthmorc  College,  Swarthmore,  Pennsylvania, 
and  Bermuda  Biological  Station,-  St.  George's  West,  Bermuda 

Among  the  numerous  species  of  brachyuran  crabs  may  be  found  some  which 
are  terrestrial,  others  which  are  semi-terrestrial  and  many  which  are  marine. 
Within  this  definitive  group  of  animals  of  close  morphological  and  taxonomic 
similarities  there  is  a  spectrum  of  adaptation  and  the  implied  regulation  of  salt  and 
water.  This  adaptation  has  succeeded  across  the  marine-terrestrial  path  of 
emergence  which  has  proved  an  insurmountable  barrier  to  all  but  a  few  animals. 
Three  species  were  selected  to  represent  three  different  degrees  of  exposure  of  the 
animal  to  the  electrolyte  and  water  environment  of  the  sea.  The  relationship 
between  the  electrolyte  concentration  of  the  marine  environment  and  that  within 
these  animals  was  investigated  to  determine  the  degree  of  independence  and  the 
direction,  degree  and  pathways  of  electrolyte  and  water  regulation. 

The  common  land  crab,  Gecarcinus  latcralis  (Frem.),  was  selected  to  represent 
the  greatest  independence  from  the  marine  habitat.  It  is  found  in  burrows  suffi- 
ciently high  in  the  banks  of  beach  sand  on  Nonesuch  Island,  Bermuda,  that  these 
burrows  at  their  deepest  do  not  approach  within  a  meter  of  the  high  tide  level. 
Nocturnal  and  beach  scavenger  in  habit,  it  is  able  to  go  weeks,  or  even  months, 
without  entering  the  surf.  The  ghost  crab,  Ocypode  albicans  (Bosq),  selected 
to  represent  a  somewhat  closer  relationship  to  the  marine  habitat,  is  found  in 
burrows  near  and  above  high  tide  level  on  the  Delaware  ocean  beaches.  These 
burrows  approach  and  many  have  been  found  to  penetrate  high  tide  level  with 
consequent  flooding.  A  nocturnal  beach  scavenger,  it  goes  into  the  surf  briefly 
during  feeding.  The  mangrove  crab,  Goniopsis  cruentatus  (Latr.),  almost  con- 
tinuously in  water,  was  selected  to  represent  the  closest  relationship  to  the  marine 
habitat.  It  is  found  in  burrows  in  the  silt  and  coral  basins  of  mangrove  swamps 
on  St.  George's  Island,  Bermuda.  It  leaves  the  burrows  to  seek  food  at  night 
and  may  leave  the  water  for  brief  periods  by  climbing  out  on  mangrove  roots. 
Seldom  found  more  than  a  meter  above  the  water  and  seldom  more  than  two 
meters  from  a  usable  burrow,  this  sojourn  into  air  appears  superficially  to  be, 
timewise,  a  reciprocal  of  the  air-surf  relationship  shown  by  ghost  crabs. 

Gross  weight  changes  are  the  most  obvious  indicators  where  massive  inboard 
or  outboard  water  shifts  are  suspected,  but  in  box-like  animals  such  as  the 
brachyuran  crabs,  unilateral  water  shifts  and  resulting  weight  changes  may  be 

1  With  the  technical  assistance  of  Sarah  C.  Flemister. 

2  Contribution  No.  243  from  the  Bermuda  Biological  Station.     Assisted  by  a  Grant-in-Aid 
from  the  National  Science  Foundation  through  the  Bermuda  Biological  Station. 

180 


SALT  AND  WATER  REGULATION  IN  CRABS  181 

expected  to  be  of  small  magnitude.  A  second  indicator  might  be  blood  specific 
gravity  shifts  resulting  from  the  movement  of  water  into  or  out  of  the  circulating 
fluid  as  a  result  of  electrolyte  and  osmotic  imbalance,  this  indicated  by  a  greater 
or  smaller  fraction  of  the  blood  being  water. 

The  absence  of  gross  changes  in  fresh  weight  or  blood  specific  gravity  does  not 
preclude  the  possibility  of  electrolyte  and  water  movement,  but  instead  suggests 
that  this  movement  results  in  constant  water  volumes  and  electrolyte  concentra- 
tions. Although  chloride  ion  concentration  measurements  in  the  environment, 
in  the  blood  and  in  the  urine  give  clear  evidence  of  regulation  of  concentration  in 
the  ghost  crab  (Flemister  and  Flemister,  1951),  the  problem  of  rate  and  direction 
of  exchange  is  difficult,  if  not  impossible,  to  approach  from  the  chloride  ion  con- 
centration alone.  Rate  and  direction  of  exchange  may  be  determined  by  the  use 
of  an  ion  suitably  alike  to  chloride  in  its  distribution  and  biological  properties  in 
the  range  of  concentrations  required  for  analysis,  yet  subject  to  precise  measure- 
ment apart  from  chloride.  If  used  in  sufficiently  small  quantities,  the  resulting 
environmental,  blood  and  urine  concentrations  will  not  interfere  with  normal 
chloride  movements  which  would  be  occurring  at  the  same  time,  in  the  same 
direction,  and,  presumably,  at  about  the  same  rate.  It  is  assumed  on  the  basis 
of  an  extensive  mammalian  literature  that  thiocyanate  ions  may  be  used  to  measure 
the  space  into  which  chloride  ions  are  distributed,  this  space  being  profitably  termed 
"extracellular,"  although  some  cellular  absorption  and  concentration  of  both  ions 
are  known  to  occur  in  some  animals.  Such  concentrations  would  involve  only  a 
limited  portion  of  the  data  presented  here,  for  this  investigation  is  concerned  pri- 
marily with  the  rates  of  exchange  of  ions  and  water  between  blood  and  environ- 
mental fluids.  This  is,  in  effect,  a  matter  of  using  SCN  as  a  "tagged  chloride  ion." 
A  second  indicator  substance  is  necesary  for  such  a  study :  a  biologically  inert 
non-electrolyte  of  minimal  osmotic  effect  in  the  required  concentration  and  of 
known  pattern  of  movement  across  certain  membranes  relative  to  the  movement 
of  water.  Inulin  clearance  is  taken  to  indicate  the  rate  of  movement  of  fluid  across 
the  membrane  of  the  antennal  gland  in  a  manner  which  will  be  called  "filtration" 
for  the  sake  of  brevity.  The  simple  filtration  of  inulin  is  assumed  on  the  basis  of 
osmotic  and  hydrostatic  measurements  on  Care-inns  by  Picken  (1936),  inulin 
blood :  urine  ratios  of  unity  found  in  the  lobster,  Homarus,  by  Forster  and  Zia- 
Walrath  (1941)  and  by  Burger  (1955,  1957),  and  the  possibility  that  the  work 
of  Maluf  (1941),  originally  thought  to  indicate  inulin  secretion  in  the  crayfish, 
Cambarus,  may  be  interpreted  differently  as  pointed  out  by  Martin  (1957). 

The  measurement  of  chloride,  thiocyanate,  inulin  and  water  content  of  environ- 
ment, blood  and  urine  might  be  expected  to  illuminate  the  constancy  of  volumes 
and  of  electrolyte  and  water  proportions,  the  direction  and  rate  of  movement  of 
electrolytes  and  of  water  in  the  maintenance  of  the  constancy  against  variation  in 
the  proportion  of  electrolyte  to  water  in  the  environment. 

METHODS 

Ghost  crabs  (Ocypodc  albicans)  from  the  Delaware  beaches  and  land  crabs 
(Gecarcinus  lateralis)  from  Nonesuch  Island,  Bermuda,  were  kept  in  individual 
containers  in  which  there  was  enough  beach  sand  to  allow  burrow  digging.  Man- 
grove crabs  (Goniopsis  crucntatus)  from  mangrove  swamps  on  St.  George's 


182  LAUNCE  J.  FLEMISTER 

Island,  Bermuda,  were  kept  in  individual  containers  with  fresh  sea  water  about 
two  inches  deep  and  planks  on  which  they  could  get  out  of  the  water.  Ghost  crabs 
were  fed  all  the  fresh  fish  they  would  eat  each  night  and  allowed  to  swim  in  sea 
water  for  about  five  minutes.  The  once-a-day  feeding  and  bathing  routine  paral- 
leled natural  conditions  and  made  it  possible  to  keep  the  animals  in  good  condition 
for  ten  days  or  longer.  At  Bermuda,  where  fresh  land  crabs  and  mangrove  crabs 
could  be  obtained  more  easily  and  at  more  frequent  intervals,  no  effort  was  made 
to  sustain  a  large  number  of  crabs  in  the  laboratory.  Individuals  of  each  species 
were  in  good  condition  after  a  week  or  ten  days.  All  crabs  were  weighed  daily  and 
all  had  been  in  the  laboratory  at  least  twenty-four  hours  before  any  work  was 
done  with  them. 

The  total  water  content  of  the  animals  was  determined  as  the  difference  between 
fresh  weight  and  the  constant  weight  of  the  minced  carcass  after  drying  at  105°  C. 
and  cooling.  Blood  specific  gravity  of  ghost  crabs  was  determined  by  the  method 
of  Jacobsen  and  Linderstrom-Lang  (1940)  and  blood  total  water  was  determined 
as  the  difference  between  fresh  and  dried  weights  of  1-  to  2-cc.  blood  samples. 
None  of  the  crabs  used  for  these  determinations  were  used  in  any  other  procedure. 

Blood  concentrations  of  thiocyanate  and  inulin,  determined  at  30-minute  inter- 
vals on  animals  kept  in  dry  containers  during  the  three  hours  following  injection 
of  known  amounts  of  the  compounds,  were  used  as  the  basis  for  extrapolation  to 
the  concentrations  which  would  have  been  produced  by  complete  and  instantaneous 
distribution.  The  indicated  concentrations  were  used  to  calculate  the  volumes 
available  for  SCN  and  inulin  dilution.  The  variability  of  these  volumes  was  ap- 
praised in  relation  to  the  total  body  water  volume  in  8  to  12  animals  of  each 
species.  The  blood  and  urine  chloride  concentrations  and  their  variability  were 
determined  on  a  similar  number  of  animals  in  dry  containers.  This  quantitative 
characterization,  the  fluid  and  chloride  anatomy,  was  used  as  the  basis  for  the 
demonstration  of  regulation  of  electrolyte  and  water  proportions  in  animals  ex- 
posed to  environmental  salt  and  water  stress.  The  rates  of  SCN  and  inulin  loss 
and  of  SCN  absorption  were  used  to  determine  the  rate,  direction  and  pathway  of 
electrolyte  and  water  movement  in  these  stress  situations. 

One-tenth  of  a  cubic  centimeter  of  blood  was  drawn  from  the  sinus  within 
the  proximal  joint  of  one  of  the  legs,  using  a  No.  27  needle  fitted  to  a  clean,  dry 
one-quarter  cubic  centimeter  tuberculin  syringe  in  a  holder.  No  anticoagulant 
was  used.  It  was  found  that  quick,  smooth  handling  of  the  blood  could  effect 
the  measurement  and  transfer  of  aliquots  before  clotting  commenced.  Aliquots 
of  this  blood  sample  were  prepared  as  blanks  for  reference  setting  of  the  spectro- 
photometer  for  SCN  and  inulin  measurements  and  the  determination  of  control 
chloride  concentrations.  Injection  of  either  0.100  to  0.200  cc.  3%  NaSCN  (Merck 
Reagent),  0.100  to  0.200  cc.  5%  inulin  (Pfanstiehl  C.  P.,  re-crystallized),  or  0.100 
to  0.200  cc.  5%  inulin  in  3%  NaSCN  was  made  deep  into  the  same  sinus  from  a 
fixed-delivery  syringe  and  needle.  Immediately  the  same  volume  of  the  same 
solution  was  introduced  into  a  5-,  a  10-  and  a  25-cc.  volumetric  flask,  made  to 
volume  and  samples  taken  from  these  were  analyzed  along  with  the  blood  samples 
for  the  precise  determination  of  the  amounts  of  NaSCN  and  inulin  injected.  Slow, 
deep  introduction  of  injected  fluid  and  careful  sampling  from  deep  within  the 
sinus  usually  prevented  fluid  or  blood  loss  from  the  site  of  puncture.  In  the  few 
cases  where  fluid  loss  or  bleeding  did  occur,  the  animals  were  discarded. 


SALT  AND  WATER  REGULATION  IN  CRABS  183 

At  fixed  intervals  after  the  injection  of  SCN  and  inulin,  one-tenth  of  a  cubic 
centimeter  of  blood  was  drawn  from  the  sinus  of  the  proximal  joint  of  one  of  the 
legs  on  the  side  opposite  the  injection  site.  Two  0.500-cc.  samples  of  diluted  blood 
were  prepared  by  transferring  0.040  cc.  blood  to  3-cc.  test  tubes,  each  containing 
0.460  cc.  distilled  water.  The  transfers  were  made  by  separate,  clean,  dry  measur- 
ing micropipettes  of  the  Folin  type  which  were  flushed  into  the  0.460  cc.  distilled 
water  with  repeated  rinsing  of  the  pipette  lumen  with  the  resulting  0.500  cc.  of 
diluted  blood.  To  one  of  the  samples  of  diluted  blood  was  added  1.00  cc.  10% 
CClgCOOH,  the  mixture  shaken  thoroughly,  centrifuged,  and  1.00  cc.  of  the  super- 
natant fluid  transferred  to  a  Coleman  cuvette.  Two-tenths  of  a  cubic  centimeter 
10%  Fe(NO3)3-9  H2O  in  5%  HNO3  was  added  with  thorough  mixing,  and  the 
optical  density  of  the  resulting  Fe(SCN)3  was  read  immediately  at  490  m/A  and 
the  SCN  concentration  calculated.  This  procedure  is  a  modification  of  a  method 
introduced  by  Crandall  and  Anderson  (1934).  On  the  second  sample  of  diluted 
blood  a  Somogyi  (1930)  precipitation  of  protein  was  carried  out  by  adding  0.50  cc. 
10%  ZnSO4-7  H,O  with  thorough  mixing  and  then  adding  0.50  cc.  0.5  N  NaOH, 
the  mixture  mechanically  shaken  for  30  minutes,  centrifuged,  and  1.0  cc.  of  the 
supernatant  transferred  to  a  9-cc.  test  tube.  Following  in  principle  a  method 
introduced  by  Young  and  Raisz  (1952),  0.25  cc.  4  N  NaOH  was  added  with 
thorough  mixing,  the  tube  closed  by  a  glass  marble,  and  the  contents  heated  in 
a  boiling  water  bath  for  15  minutes.  The  contents  were  cooled,  and  6.25  cc. 
anthrone  reagent,  0.4%  anthrone  (Matheson,  Coleman  and  Bell)  in  75%  H2SO4, 
were  added  with  cooling.  The  tube  was  again  closed  by  a  glass  marble  and  the 
contents  heated  in  a  75°  C.  water  bath  for  5  minutes,  cooled,  allowed  to  stand  30 
minutes  at  room  temperature,  transferred  to  a  Coleman  cuvette,  the  optical  density 
read  at  630  m/*,  and  the  inulin  concentration  calculated. 

From  the  sample  remaining  in  the  syringe  and  needle  after  the  transfer  of  the 
two  0.040-cc.  portions,  blood  was  drawn  to  the  1  mark  in  a  Thoma  pipette  and 
diluted  to  the  11  mark  with  distilled  water.  The  contents  were  mixed  and  blown 
into  a  small  glass  cup  with  repeated  rinsing  of  the  pipette  lumen.  Duplicate  0.020- 
or  0.100-cc.  aliquots  of  the  diluted  blood,  depending  on  chloride  concentration,  were 
transferred  by  a  micro  blood  sugar,  Folin,  pipette  to  0.200  cc.  distilled  water  in 
each  of  several  depressions  in  a  Coors  porcelain  plate  with  rinsing  of  the  micro- 
pipette  lumen  with  the  now  doubly  diluted  blood.  Two-tenths  of  a  cubic  centi- 
meter of  1  N  H2SO.t  was  added.  With  mechanical  stirring,  0.010  N  AgNO3  was 
added  in  small  increments  from  a  Scholander  micrometer  burette  (Scholander 
ct  a/.,  1943)  and  the  potentiometric  titration  of  the  chloride  ion  was  accomplished 
by  the  method  of  Cunningham,  Kirk  and  Brooks  ( 1941 ) .  Appropriate  blanks  and 
the  determination  of  known  standards  accompanied  the  measurements  of  all 
unknowns. 

Urine,  collected  as  described  by  Flemister  and  Flemister  (1951),  was  diluted 
in  a  Thoma  pipette,  aliquots  taken  and  chloride,  SCN  and  inulin  concentrations 
determined  by  the  procedures  described  for  blood.  Appropriate  dilutions  were 
made  to  hold  concentrations  within  the  sensitive  range  of  the  methods. 

After  determination  of  sea  water  chloride  concentrations  by  the  method  de- 
scribed for  blood,  dilutions  were  made  with  distilled  water  and  concentrations  were 
accomplished  by  evaporation  at  room  temperature  to  prepare  environmental  fluids 
containing  120,  240.  360,  4SO,  600  and  720  mM.  Cl/L.  To  this  series,  which  was 


184  LAUNCE  J.  FLEMISTER 

checked  for  chloride  concentration  after  preparation,  was  added  distilled  water, 
presented  in  the  graphs  as  0  mM.  Cl/L.  Data  from  animals  exposed  to  air  rather 
than  environmental  fluids  are  presented  as  "dry."  Animals  were  exposed  indi- 
vidually to  the  various  environmental  fluids.  They  were  placed  in  glass  containers 
with  sufficient  volume,  200  to  300  cc.,  of  the  fluid  to  cover  their  bodies  in  the  resting 
position.  Fluids  were  renewed  every  twelve  hours.  Environmental  solutions 
were  prepared  for  SCN  uptake  by  adding  1.00  or  2.00  cc.  3%  NaSCN  to  each 
100  cc.  of  environmental  fluid.  The  amount  added  was  fixed  by  the  expected  rate 
of  absorption  in  order  to  keep  blood  concentrations  within  reasonable  physiological 
limits  and  within  the  sensitive  range  of  the  procedures  used  for  measurement. 

RESULTS 

Fresh  weights  of  land  crabs  (Gccarcinus  lateralis),  15  to  45  gm.,  ghost  crabs 
(Ocypodc  albicans),  20  to  50  gm.,  and  mangrove  crabs  (Goniopsis  cruentatus), 
20  to  50  gm.,  were  random  in  distribution  with  no  relation  to  sex  or  time  of  year. 
Gravid  females  were  not  collected.  All  animals  were  in  the  inter-molt  period 
during  the  time  they  were  in  the  laboratory.  There  was  no  significant  weight 
gain  or  loss  in  ghost  crabs  maintained  in  the  laboratory  for  as  long  as  three  weeks, 
nor  in  land  crabs  and  mangrove  crabs  kept  in  the  laboratory  for  a  week  or  ten 
days.  All  variations  in  individual  weights  during  captivity  were  less  than  2.8% 
of  the  first  weight  determined  soon  after  capture. 

There  was  no  appreciable,  consistent  change  in  fresh  weight  after  72  hours  in 
any  of  the  environmental  fluids  (120.  240,  360.  480,  600  and  720  mM.  Cl/L.) 
except  distilled  water  (0  mM.  Cl/L.).  In  land  crabs  and  ghost  crabs  exposed  to 
distilled  water  for  24  hours  and  in  mangrove  crabs  exposed  for  48  hours,  in- 
creases in  weight  never  exceeded  4.8%  of  the  fresh  weights  before  the  animals 
were  placed  in  the  environmental  fluid.  In  view  of  the  2.8%  variation  in  fresh 
weight  of  crabs  in  the  control  group  and  the  difficulty  of  removing  environmental 
fluid  from  the  gill  chambers  before  weighing  in  air.  these  weight  changes  are  not 
considered  significant. 

No  correlation  was  found  between  sex.  size,  or  time  of  capture  and  blood 
specific  gravity  in  152  recently  caught  ghost  crabs.After  the  initial  determinations, 
46  crabs  were  placed  on  sand  and  about  20  in  each  of  the  environmental  fluids. 
Among  the  46  crabs,  after  72  hours  on  sand,  the  mean  of  specific  gravity  was 
1.0442  with  a  standard  error  of  0.0009  and  the  mean  for  blood  total  water  was 
89.2%  with  a  standard  error  of  0.29.  Of  about  20  crabs  exposed  to  each  of  the 
environmental  fluids,  only  in  those  surviving  24  hours  in  distilled  water  was  there 
a  possibly  significant  change  in  blood  specific  gravity,  a  decrease,  with  a  "P" 
value  between  .01  and  .05. 

/.   Water  and  electrolyte  anatomy 

Reliable  data  on  volumes  of  fluid  and  electrolyte  concentrations  within  the 
animal  are  essential  to  an  attempt  to  determine  rates  of  exchange  of  water  and 
electrolytes.  Measurements  of  total  water  content  and  volume  of  fluid  available 
for  dilution  of  thiocyanate  and  inulin  on  8  to  12  crabs  of  each  of  the  three  species 
studied  are  presented  in  Table  I  in  terms  of  per  cent  of  fresh  weight.  The  second 


SALT  AND  WATER  REGULATION  IN  CRABS 


185 


TABLE  I 

Water  and  electrolyte  anatomy  of  land  crab  (Gecarcinus  lateralis),  ghost  crab 
(Ocypode  albicans)  and  mangrove  crab  (Goniopsis  cruentatus) 


Land  Crab 

Ghost  Crab 

Mangrove  Crab 

Total  water 

%  fresh  weight 

66.18 

69.93 

65.44 

S.  E. 

.52 

.70 

.63 

64.69-67.66 

67.49-72.37 

63.46-67.42 

SCN  space 

%  fresh  weight 

28.52 

34.18 

30.39 

S.  E. 

.26 

.58 

.56 

27.79-29.25 

32.18-36.18 

28.60-32.18 

%  total  water 

43.1 

48.9 

46.4 

Inulin  space 

%  fresh  weight 

18.80 

21.49 

19.92 

S.  E. 

.34 

.39 

.57 

17.84-19.76 

20.13-22.85 

18.67-21.17 

%  total  water 

28.4 

30.7 

30.4 

%  SCN  space 

65.9 

62.9 

65.5 

Blood  chloride 

mM.  Cl/L. 

385 

378 

422 

S.  E. 

9 

7 

3 

360-410 

354-402 

413-431 

Urine  chloride 

mM.  Cl/L. 

455 

602 

S.  E. 

13 

10 

409-501 

571-633 

Sea  water 

mM.  Cl/L. 

600 

480 

600 

item  under  each  tabulation  is  the  standard  error  of  the  mean.  The  third  item  in 
each  case  is  calculated  from  the  standard  deviation  and  is  the  range  within  which 
two-thirds  of  the  data  falls,  this  indicating  the  variability  of  the  data  making  up 
each  of  the  means.  Applying  the  "t"  test  to  the  data  and  taking  "P"  values  less 
than  .01  to  indicate  significance,  .01  to  .05  possible  significance,  and  values  greater 
than  .05  no  significance,  the  following  statements  can  be  made.  Although  there 
is  a  significant,  but  not  marked,  difference  between  the  SCN  spaces,  the  volumes 
of  the  three  compartments  in  terms  of  fresh  body  weight  are  much  alike  between 
land  crabs  and  mangrove  crabs.  The  volumes  of  the  compartments  of  ghost  crabs 
in  the  same  terms  are  significantly,  and  markedly,  larger  with  the  exception  of  the 
inulin  space  as  compared  with  that  in  mangrove  crabs.  Here  the  significance  of 
the  difference  is  questionable. 

Compared  as  fractions  of  total  water,  the  SCN  and  inulin  spaces,  presented  as 
the  fourth  item  in  the  tabulation  in  each  case  in  Table  I,  show  no  significant  differ- 
ences in  the  three  species  studied.  Therefore,  absolute  volumes,  though  showing 
the  differences  described  in  terms  of  per  cent  fresh  body  weight,  are  of  comparable 
size  relative  to  the  total  water  content  of  the  animal.  The  average  of  the  means 
of  SCN  space  is  46.2%  and  of  inulin  space  29.8%  that  of  the  total  water  content. 


186 


LAUNCE  J.  FLEMISTER 


The  average  of  the  means  of  inulin  space  is  64. 7 %  that  of  SCN  space,  fifth  item 
of  tabulation,  with  the  fractions  for  land  crabs  and  mangrove  crabs  being  close 
together  and  somewhat  larger  than  for  ghost  crabs. 

Blood  chloride  concentrations  of  mangrove  crabs  are  significantly  greater  than 
those  of  land  crabs  and  ghost  crabs,  in  which  the  concentrations  are  alike  (Table  I). 
A  significant  difference  was  found  between  urine  chloride  concentrations  of  ghost 
crabs  and  mangrove  crabs  immediately  after  capture.  The  greater  concentration 
in  mangrove  crabs  is  to  be  related  to  their  almost  continuous  exposure  to  sea  water 
of  high  chloride  content.  Urine  samples  could  not  be  obtained  from  land  crabs. 
Within  each  of  the  three  species,  no  correlation  was  found  between  sex,  size,  or 
time  of  capture  and  the  fluid  space  available  for  dilution  of  SCN  or  inulin,  the 
total  water  content,  or  the  concentration  of  chloride  ion  in  the  blood  or  urine. 

//.  Evidence  of  regulation 

The  effects  of  exposure  to  distilled  water  (0  mM.  Cl/L.),  120,  240,  360,  480, 
600  and  720  mM.  Cl/L.  sea  water  dilutions  and  concentrations  for  24,  48  and  72 
hours  on  blood  and  urine  chloride  concentrations  in  the  three  species  of  crabs 


2     20 

y 

S       .8 
J 

3 

S       .6 


< 

<r 


I" 

O         B 


OC       -6 

i  * 


ai 


LAND   CRAB 


GHOST   CRAB 


URINE 


BLOOD 


MANGROVE   CRAB 


A  URINE 


K./  / 


DRY      0         l?0     240     160    4«0     600      720 

ENVIRONMENTAL  CHLORIDE   uM/L 


DRY       0         I2O     24O     36O     460     600     720 


DRY      0         120    24O     36O     460     6OO     72D 


FIGURE  1.  Blood  and  urine  chloride  concentrations  of  land  crab  (Gecarcinus  latcralis), 
ghost  crab  (Ocypodc  albicans)  and  mangrove  crab  (Goniopsis  crucntatus)  related  to  environ- 
mental chloride  concentrations. 


studied  are  summarized  in  Figure  1.  The  normal  blood  chloride  for  each  indi- 
vidual animal  of  each  species  on  sand  or  in  dry  containers,  plotted  as  "dry"  and 
placed  opposite  "1.0"  on  the  ordinate,  serves  as  the  basis  for  representation  of  the 
concentrations  of  chloride  ion  after  exposure  to  experimental  environmental  fluids. 
The  mean  of  the  values  for  normal  urine  chloride  and  for  blood  and  urine  chloride 
ion  concentration  of  the  same  6  to  10  animals  after  exposure  to  experimental  fluids, 
expressed  separately  as  fractions  or  multiples  of  the  normal  blood  chloride  for  the 


SALT  AND  WATER  REGULATION  IN  CRABS  187 

same  animal,  is  plotted  for  each  experimental  condition  and  time  interval.  En- 
vironmental fluid  chloride  concentrations  are  represented  by  a  dotted  line,  blood 
chloride  by  a  solid  line  and  boxes  are  used  to  indicate  ranges  and  concentrations 
of  no  statistically  significant  change  during  exposure,  and  urine  chloride  by  a 
broken  line.  Additional  points  and  arrows  indicate  positions  and  directions  of 
shift  of  blood  and  urine  chloride  curves  during  exposure  from  24  to  48  hours  and 
from  48  to  72  hours.  Standard  deviations  of  these  individual  items  of  data  were 
relatively  small  and  are  not  indicated  to  avoid  congestion  of  the  graphs. 

Data  from  land  crabs  present  a  picture  of  effective  regulation  of  blood  chloride 
concentration  for  72  hours  in  mid-range  environmental  concentrations,  though 
elevated  by  10%  in  240  and  360  and  by  20%  in  480  mM.  Cl/L.  fluids.  A  break- 
down appears  before  24  hours  in  distilled  water,  soon  after  24  hours  in  720 
mM.  Cl/L.  with  no  survivors  at  48  hours  in  either  of  these  environments,  and 
between  48  and  72  hours  in  120  and  600  mM.  Cl/L.  fluids.  Blood  chloride  regu- 
lation is  somewhat  more  effective  in  hypotonic  (except  0  mM.  Cl/L.)  than  in 
hypertonic  ranges,  but  in  either,  once  the  breakdown  occurs,  blood  chloride  levels 
approach  environmental  fluid  concentrations.  Urine  samples  could  not  be  ob- 
tained from  land  crabs. 

In  ghost  crabs  blood  chloride  regulation  is  effective  up  to  72  hours  over  the 
range  from  120  to  600  mM.  Cl/L.  environmental  fluids,  maintaining  concentrations 
which  do  not  differ  significantly  from  those  found  in  animals  on  sand.  However, 
regulation  fails  during  the  first  24  hours  in  0  and  720  mM.  Cl/L.  fluids.  No  ghost 
crab  survived  to  48  hours  in  these  environmental  extremes.  Urine  chloride  con- 
centrations roughly  parallel,  but  are  much  higher  than,  environmental  levels  in  all 
except  the  high  concentrations  (600  and  720  mM.  Cl/L.).  The  antennal  gland 
clearly  wastes  chloride  in  isotonic  and  hypotonic  environmental  situations.  Urine 
chlorides  vary  little  for  48  hours  from  120  to  600  mM.  Cl/L.,  but  as  exposure  is 
prolonged  to  72  hours,  urine  chloride  concentrations  approach  blood  chloride  levels. 

The  blood  chloride  concentrations  of  mangrove  crabs  show  a  striking  con- 
stancy at  near  normal  levels  over  the  range  from  120  to  720  mM."  Cl/L.  environ- 
mental fluids  for  up  to  72  hours.  The  slight  elevations  at  the  extremes  fail  statis- 
tical tests  for  significance.  However,  in  animals  exposed  to  distilled  water,  blood 
chlorides  are  significantly  decreased  in  24  hours  and  markedly  so  in  48  hours  with 
no  survivors  at  72  hours.  Urine  chloride  concentrations  are  very  close  to  en- 
vironmental levels  in  600  and  720  mM.  Cl/L.  fluids,  significantly  above  environ- 
mental levels  in  360  and  120  mM.  Cl/L.  and  are  markedly  elevated  in  distilled 
water.  In  crabs  exposed  to  distilled  water,  urine  chloride  concentrations  decreased 
between  24  and  48  hours,  but  still  remained  high.  Urine  chloride  levels  of  ani- 
mals in  all  other  fluids  did  not  change  in  72  hours.  These  crabs  fall  short  of  the 
classical  picture  of  completely  effective  regulation  only  in  the  breakdown  in  distilled 
water  and  the  apparent  leakage  of  chloride  in  the  urine  when  exposed  to  120  and 
360  mM.  Cl/L.  environmental  fluids. 

///.  Evidence  of  regulatory  mechanisms 

Data  on  simultaneous  inulin  clearance  and  thiocyanate  loss  and  on  absorption  of 
thiocyanate  from  the  experimental  environmental  fluids  into  the  blood  of  crabs  of  the 
three  species  studied  are  summarized  in  Figure  2.  In  the  clearance  and  loss  curves, 


188 


LAUNCH  J.  FLEMISTER 


SCN  and  inulin  concentrations  of  blood  samples,  taken  at  1-  to  4-hour  intervals 
for  the  first  12  hours  of  exposure  to  environmental  fluids  (0  to  720  mM.  Cl/L.) 
and  then  every  8  to  12  hours,  are  presented  as  fractions  of  the  concentrations 
determined  immediately  prior  to  exposure  and  following  a  post-injection  equilibra- 
tion period  of  2  to  4  hours  in  dry  containers.  Thiocyanate  absorption  curves  are 
composed  of  blood  SCN  concentrations,  determined  at  similar  intervals  and  ex- 
pressed as  fractions  or  multiples  of  environmental  SCN  levels,  in  crabs  exposed 
to  environmental  fluids  (0  to  720  mM.  Cl/L.)  containing  small  amounts  (4  to  7 
mM./L.)  of  NaSCN,  after  a  2-  to  4-hour  period  in  dry  containers.  Corrections 


LAND  CRAB 


GHOST  CRAB 


MANGROVE   CRAB 


120 


120-720 


£00 


720^ 


360 


120 


DRY 


0-7*0 


12  24          36 


60  72 


12  24          36  44  60  72 


EXPOSURE    IN   HOURS 


FIGURE  2.  Inulin  and  thiocyanate  loss  and  thiocyanate  uptake  by  land  crab  (Gecarcinus 
lateralis) ,  ghost  crab  (Ocypode  albicans)  and  mangrove  crab  (Goniopsis  cruentatus)  exposed 
to  a  variety  of  environmental  chloride  concentrations. 

were  made  in  each  case  for  SCN  and  inulin,  or  SCN  alone,  removed  from  the 
animal  in  taking  samples.  Standard  deviations  are  not  represented.  They  were 
relatively  small  except  in  very  low  and  high  concentrations.  Sufficient  numbers 
of  animals,  4  to  18,  were  exposed  to  each  of  the  experimental  conditions  to  achieve 
statistically  significant  ("P"  value  of  less  than  .01)  separation  by  the  "t"  test. 
Determinations  were  made  on  fresh  animals  which  were  discarded  at  the  com- 
pletion of  the  72-hour  measurements. 

In  land  crabs  on  sand,  inulin  clearance  indicates  continuing  filtration  with  the 
rate  of  inulin  loss  slowly  and  steadily  decreasing  as  blood  level  falls.  Thiocyanate 
loss,  comparable  to  inulin  loss  at  first,  decreases  completely  as  blood  concentration 
levels  off,  suggesting  that  SCN  is  re-absorbed  from  the  fluid  which  continues  to 
be  filtered.  The  constancy  of  blood  chloride  concentration  and  of  blood  SCN 
levels  after  24  hours  indicates  re-absorption  of  filtered  water.  In  distilled  water, 
inulin  is  cleared  at  the  same  rate  as  on  sand,  but  SCN  blood  level  falls  more  rapidly 


SALT  AND  WATER  REGULATION  IN  CRABS  189 

and  to  about  half  the  beginning  value  in  24  hours.  This  is  comparable  to  chloride 
loss  in  distilled  water  and  indicates  a  breakdown  in  electrolyte  regulation  not 
dependent  on  filtration  through  the  antennal  gland.  Uninjected  animals  in 
0  mM.  Cl/L.  fluid  containing  4  to  7  mM.  SCN/L.  absorb  SCN  at  such  a  rate  that 
despite  concurrent  loss,  which  must  be  assumed,  blood  levels  equal  environmental 
concentrations  in  15  hours  and  are  twice  as  high  in  48  hours.  Though  striking, 
this  absorption  results  in  accumulation  of  only  about  7  mM.  SCN/L.  during  the 
24  hours  when  blood  chloride  levels  fall  162  mM.  Cl/L.  Land  crabs  in  120  and 
360  mM.  Cl/L.  fluids  clear  inulin  more  rapidly  than  those  on  sand  or  in  distilled 
water,  indicating  a  more  rapid  turnover,  absorption  and  excretion,  of  water.  In 
these  fluids,  SCN  loss,  more  rapid  and  more  complete  than  inulin  clearance,  sug- 
gests a  pathway  other  than  filtration.  Blood  SCN  concentrations  of  uninjected 
crabs  in  these  fluids,  to  which  small  amounts  of  SCN  were  added,  are  comparable 
and  become  steady  at  80%  of  environmental  SCN  concentration  in  48  hours.  In 
all  600  and  after  24  hours  in  720  mM.  Cl/L.  fluids,  inulin  clearances  and  SCN 
losses  are  more  rapid  and  more  complete  than  those  on  sand  or  in  distilled  water, 
and  less  rapid  and  less  complete  than  those  in  120  or  360  mM.  Cl/L.  environments. 
Again,  SCN  loss  rates,  greater  than  inulin  clearance  rates,  suggest  a  pathway  other 
than  filtration  for  electrolyte  loss.  Absorption  of  SCN  from  these  environmental 
fluids,  containing  small  amounts  of  NaSCN,  are  the  same  during  the  first  24  hours 
and  at  such  a  rate  that  blood  SCN  levels  off  at  45%  of  environmental  concentra- 
tion in  48  hours  in  animals  exposed  to  600  mM.  Cl/L.  fluids.  The  implication  is 
clearly  one  of  turnover  rates  reduced  from  those  in  hypotonic  and  near-isotonic 
fluids. 

There  appears  to  be  a  virtual  shutting  off  of  filtration  in  ghost  crabs  in  the 
dry  situation,  judging  from  the  fact  that  neither  inulin,  thiocyanate,  nor  chloride 
concentrations  fall  significantly  during  72  hours  on  sand.  It  was  increasingly 
difficult  to  get  urine  samples  as  exposure  to  a  dry  environment  was  prolonged. 
This  suggests  that  water  gained  during  brief  nightly  excursions  into  the  surf  is 
critical  for  adequate  urine  formation.  The  loss  of  SCN,  far  exceeding  that  of 
inulin  in  the  24  hours  these  crabs  survived  in  distilled  water,  is  parallel  to  the 
drop  in  blood  chloride  level,  and  is  much  greater  than  can  be  accounted  for  by  a 
failure  in  re-absorption  after  filtration.  Absorption  of  SCN  added  in  small  amount 
to  0  mM.  Cl/L.  fluids,  reaching  3.7  times  environmental  concentration  in  12  hours, 
6.2  in  24  and  8.0  in  48,  with  no  survivors  at  72  hours,  was  even  more  striking  than 
in  land  crabs,  and  indicated  that  25  mM.  SCN/L.  was  retained  in  the  blood  in  the 
24  hours  while  129  mM.  Cl/L.  was  being  lost.  In  120  mM.  Cl/L.  fluid,  inulin 
and  SCN  disappear  from  the  blood  at  about  the  same  rate  with  complete  removal 
in  30  hours.  In  this  time  sufficient  SCN  is  absorbed  from  SCN-containing  120 
mM.  Cl/L.  fluid  to  bring  the  blood  level  to  1.5  times  the  environmental  level. 
This  indicates  a  rapid  water  and  electrolyte  turnover  with  a  somewhat  excessive 
retention  of  ions  from  the  environment  which  may  be  compensatory  to  the  loss  of 
chloride  due  to  inadequate  re-absorption  by  the  antennal  gland.  Inulin  clearance 
and  SCN  loss  in  360  mM.  Cl/L.  environments  indicate  a  much  slower  filtration  and 
an  electrolyte  loss,  complete  in  30  hours,  by  a  route  other  than  filtration.  Blood 
SCN  levels  in  uninjected  animals  in  SCN-containing  360  mM.  Cl/L.  fluids  become 
constant  in  48  hours  at  about  90%  of  environmental  SCN  concentration  and  at  a 
time  when  urine  chloride  level  drops  from  a  high  toward  the  environmental,  and 


190  LAUNCE  J.  FLEMISTER 

blood,  concentration.  In  all  600  and  for  24  hours  in  720  mM.  Cl/L.  environ- 
ments, filtration  rate  is  intermediate  between  those  of  animals  in  120  and  360 
mM.  Cl/L.  fluids  and  is  inadequate  to  account  for  SCN  loss  which  is  complete 
in  30  hours.  Blood  SCN  levels,  resulting  from  absorption  of  SCN  added  in  small 
amount  to  these  fluids,  are  the  same  for  the  first  24  hours  and  reach  a  steady  level 
in  animals  exposed  to  600  mM.  Cl/L.  fluids  at  60%  of  the  environmental  con- 
centration in  48  hours. 

Inulin  clearance  rates  for  mangrove  crabs  are  the  same  for  environmental  fluids 
from  distilled  water  to  720  mM.  Cl/L.  This  indicates  a  remarkably  versatile 
adjustment  of  chloride  ion  concentration  in  urine,  if  the  regulation  of  the  internal 
environment  is  to  be  maintained,  as  it  apparently  is,  in  contrast  to  widely  different 
chloride  concentrations  in  the  environment.  During  48  hours  of  exposure,  SCN 
loss  in  distilled  water  is  much  less  rapid  than  in  the  other  environmental  fluids, 
less  rapid  even  than  inulin  clearance,  indicating  re-absorption  of  electrolyte  after 
filtration.  Thiocyanate  absorption  from  SCN-containing  0  mM.  Cl/L.  fluid,  much 
more  rapid  than  that  from  more  concentrated  environmental  fluids,  results  in  blood 
SCN  levels  2.2  times  the  environmental  level  in  24  hours,  2.8  in  48  and  3.0  in  72. 
The  9  mM.  SCN  retained  in  the  blood  at  24  hours  does  not  compare  favorably 
with  the  blood  chloride  loss,  72  mM./L.,  and  the  difference  becomes  greater  by 
48  hours.  Thiocyanate  loss  rates,  exceeding  inulin  clearances,  become  greater  as 
environmental  chloride  concentrations  increase  from  120  to  600  and  720  mM./L., 
indicating  electrolyte  loss  by  a  pathway  other  than  filtration.  Blood  concentration 
of  SCN  absorbed  from  120  to  600  and  720  mM.  Cl/L.  fluids  containing  small 
amounts  of  NaSCN  has  not  reached  a  plateau  after  72  hours  in  120,  has  become 
steady  at  1.2  times  environmental  level  in  360,  and  at  80%  of  environmental  con- 
centration in  600  and  720  in  36  hours. 

DISCUSSION 

The  absence  of  significant  changes  in  the  fresh  weight  of  crabs  exposed  to 
environmental  fluids  of  a  wide  range  of  electrolyte  concentration  makes  it  apparent 
that  the  volume  of  fluid  within  the  animals  is  held  constant  although  exchanges 
occur.  The  persistence  of  normal  blood  specific  gravity  in  the  ghost  crab  under 
such  experimental  conditions  further  indicates  that  there  is  no  appreciable  change 
in  water  or  salt  content  of  blood.  Only  after  24  hours  in  distilled  water  did  this 
constancy  of  body  weight  and  blood  specific  gravity  show  any  signs  of  weakening. 
The  significance  of  even  these  changes  was  questionable.  The  presence  of  regu- 
lation, therefore,  is  obvious. 

Although  there  are  statistically  significant  differences  between  the  volumes  of 
thiocyanate  space,  inulin  space  and  total  body  water  in  the  three  species  studied, 
there  is  no  apparent  correlation  with  dry  or  wet  habitats.  A  lack  of  fundamental 
differences  in  the  partitioning  of  water,  SCN  and  inulin  spaces  and  the  implied 
cellular  space,  becomes  apparent  when  these  volumes  are  related  to  the  volume 
of  total  body  water. 

The  blood  chloride  concentration  in  the  mangrove  crab  (Goniopsis  cruentatus), 
significantly  higher  than  those  in  the  land  crab  (Gecardnus  lateralis}  and  the 
ghost  crab  (Ocyf>odc  albicans},  bears  a  correlation  to  this  crab's  almost  continuous 
exposure  to  sea  water  of  high  salinity.  This  correlation  is  also  found  by  compar- 


SALT  AND  WATER  REGULATION  IN  CRABS  191 

ing  mangrove  crab  and  ghost  crab  urine  chloride  levels.  It  is  interesting  that 
urine  taken  from  ghost  crabs  soon  after  capture  on  Nonesuch  Island,  Bermuda, 
did  not  differ  appreciably  in  chloride  content  from  that  taken  from  the  ones  cap- 
tured on  the  Delaware  beaches.  The  difference  in  salinity  of  the  sea  water  avail- 
able to  the  two  habitats  does  not  impose  a  difference  in  urine  chloride  clearance. 
This  might  be  expected  in  view  of  the  brief  nightly  exposure  to  the  surf  during 
feeding.  However,  mangrove  crabs,  constantly  exposed  to  600  mM.  Cl/L.  sea 
water,  did  show  the  effect  of  high  environmental  salinity. 

During  the  first  two  hours  after  injection,  inulin  became  diluted  in  a  volume 
of  fluid  about  two-thirds  the  indicated  thiocyanate  space.  This  suggests  that 
either  (1)  the  blood  SCN  after  injection  is  less  concentrated,  indicating  a  larger 
dilution  volume,  due  to  absorption  of  SCN  by  cells,  or  (2)  inulin  more  slowly 
penetrates  the  remote  spaces  invaded  more  rapidly  by  SCN.  The  similarity  of  the 
slope  of  the  dilution  curves  for  massive  and  light  SCN  injections  and  the  similarity 
between  simultaneous  SCN  and  inulin  curves  suggest  that  only  the  mechanical 
factors  of  spreading  are  involved.  Recovery  determinations  on  ghost  crabs,  ac- 
counting for  87  to  97%  of  injected  SCN  under  a  variety  of  environmental  condi- 
tions, indicate  that  little,  if  any,  SCN  is  bound  by  cells.  Whether  or  not  inulin 
eventually  invades  all  of  the  SCN  volume  can  only  be  suggested  on  the  basis  of 
data  presented  here.  The  apparent  cessation  of  antennal  gland  activity  in  ghost 
crabs  on  sand  appears  to  offer  some  opportunity  for  an  answer.  So  far,  it  appears 
that  in  the  70  hours  following  the  first  two,  inulin  still  occupies  only  two-thirds  of 
the  SCN  space.  The  suggestion  of  a  functionally  closed  circulation,  inulin  space, 
within  the  larger  extracellular  compartment,  SCN  space,  is  an  interesting  one  for 
which  the  mechanical  factors  of  lumen  flow  and  stream  boundary  diffusion  seem 
reasonable. 

The  breadth  of  the  range  within  which  two-thirds  of  the  data  are  estimated  to 
fall,  presented  in  Table  I,  is  taken  to  be  a  reliable  indication  of  the  effectiveness  of 
regulation.  Comparison  of  these  ranges  reveals  that  the  three  fluid  compartments, 
total  water,  SCN  space  and  inulin  space,  are  more  closely  regulated  in  land  crabs 
than  in  ghost  crabs  and  mangrove  crabs.  A  greater  difference  in  the  regulation 
of  these  volumes  might  be  expected  between  ghost  crabs  and  mangrove  crabs  in 
view  of  the  difference  in  the  stress  imposed  by  their  normal  habitats.  Chloride 
concentrations  in  the  blood  and  urine  of  mangrove  crabs  are  much  more  closely 
regulated  than  in  land  crabs  and  ghost  crabs.  This  indicates  that  the  land  crab 
is  farther  along  in  the  evolution  of  volume  regulation  and  that  the  mangrove  crab 
has  a  more  definitive  control  of  chloride  concentration. 

Comparison  of  chloride  and  SCN  loss  from  the  blood  of  crabs  exposed  to  the 
various  environmental  fluids  shows  that  these  ions  move  at  about  the  same  rate 
in  each  species  and  in  each  situation.  Urine  SCN  concentrations  stood  in  the  same 
ratio  and  range  to  blood  SCN  levels  as  did  these  respective  concentrations  of 
chloride.  The  graphs  summarizing  data  are  not  further  complicated  by  adding 
these  items,  inasmuch  as  they  duplicate  the  chloride  data.  These  observations 
indicate  that  it  is  valid  to  use  SCN  as  "tagged  chloride"  in  an  effort  to  determine 
the  movement  of  chloride  ions  under  conditions  of  electrolyte  and  water  stress. 
The  presence  of  inulin  or  SCN  in  the  blood  did  not  affect  the  clearance,  rate  or 
degree,  of  the  other.  Inulin  was  not  absorbed  from  the  environmental  fluids. 
Its  presence  in  the  environment  did  not  affect  the  rate  or  degree  of  absorption  of 


192  LAUNCH  J.  FLEMISTER 

SCN  from  the  environmental  fluids,  or  the  rate  or  degree  of  inulin  or  SCN  clear- 
ance from  injected  animals.  This  was  true  even  when  sufficient  inulin  was  added 
to  the  environmental  fluids  to  make  them  equal  in  concentration  to  the  blood  of 
animals  injected  for  the  determination  of  inulin  clearance.  Therefore,  inulin  was 
judged  to  exert  no  appreciable  effect  on  the  direction,  rate  or  degree  of  electrolyte 
and  water  shifts  in  the  concentrations  used.  The  presence  of  SCN  in  the  environ- 
ment in  concentrations  used  did  not  affect  the  rate  or  degree  of  inulin  clearance, 
but  it  did  affect  the  rate  of  fall  of  SCN  levels  in  injected  animals.  In  injected 
animals  placed  in  fluids  to  which  SCN  had  been  added,  blood  concentrations  of 
SCN  fell  more  slowly  and  only  to  a  point  well  above  equilibrium  with  environ- 
mental SCN  in  120  mM.  Cl/L.,  about  equal  in  360  and  well  below  in  600,  but 
not  cleared. 

Of  the  three  species,  only  the  land  crab  and  the  ghost  crab  survived  24  hours 
out  of  water.  This  was  to  be  expected  from  the  differences  in  habitat  and  was 
one  basis  on  which  the  three  species  were  selected.  The  inulin  and  SCN  clear- 
ance in  land  crabs  and  ghost  crabs  on  sand  for  72  hours,  during  which  blood  chlo- 
rides remained  constant,  indicate  a  difference  in  antennal  gland  activity.  The 
indicated  re-absorption  of  filtered  water  in  land  crabs  could  account  for  the  lack 
of  obtainable  urine.  If  the  re-absorption  of  electrolytes  is  obligatory,  it  could  be 
a  cause  of  the  elevated  chloride  levels  found  in  land  crabs  exposed  to  hypertonic 
environments.  In  ghost  crabs,  such  a  continuing  nitration  and  re-absorption  does 
not  appear  to  exist.  The  dependence  on  contact  with  the  sea  for  filtration  and 
resulting  urine  formation  is  in  agreement  with  the  observations  by  Burger  (1957) 
that  haemoconcentration,  from  keeping  lobsters  in  air,  suppresses  urine  formation. 
His  interpretation  is  that  non-diffusible  molecules  in  the  blood  draw  in  water 
principally  through  the  gills,  and  that  this  water  is  bailed  out  as  urine. 

The  similarity  between  inulin  clearance  rates  in  land  crabs  on  sand  and  for 
24  hours  in  distilled  water  is  interesting.  The  same  is  true  of  ghost  crabs.  The 
persistent  high  inulin  concentrations  in  these  latter  animals  suggest  very  little 
filtration  in  distilled  water.  The  possibility  is  immediately  obvious  that  cellular 
osmotic  swelling  in  gill  membranes  and  branchial  epithelium  may  cause  mechanical, 
if  not  metabolic,  interference  with  absorption  of  water  by  crabs  in  such  environ- 
ments. If  this  should  be  the  case,  why  are  mangrove  crabs  different? 

Chloride  and  SCN  loss  in  0  mM.  Cl/L.  fluid,  most  rapid  in  land  crabs  and 
least  so  in  mangrove  crabs,  appears  to  be  compensated  for  by  the  absorption  of 
available  ion,  SCN,  from  the  environmental  fluids.  The  rate  and  degree  of  net 
gain,  blood  concentration,  of  the  absorbed  ion  is  not  proportional  to  the  rate  or 
degree  of  blood  chloride,  or  SCN,  loss.  The  three  species  clearly  differ  in  their 
ability  to  retain  normally  present  chloride  ions  and  to  absorb  and  hold  SCN  ions. 
The  blood  chloride  level  in  land  crabs  seems  to  be  least  well  held  and  the  least  well 
protected  by  absorption  rates.  The  blood  chloride  of  ghost  crabs  is  somewhat 
better  held  and  is  better  protected  by  a  remarkably  rapid  absorption  rate.  Reten- 
tion of  blood  chloride  in  mangrove  crabs,  best  of  the  three,  is  supported  by  an 
intermediate  absorption  rate.  The  apparent  superiority  of  the  holding  and  com- 
pensatory mechanisms  in  mangrove  crabs  is  reflected  by  their  longer  survival, 
past  48  hours.  It  should  be  pointed  out,  however,  that  in  all  three  of  these  species, 
the  net  absorptions  are  inadequate  to  compensate  for  a  falling  blood  chloride. 
The  significance  of  some  ion,  however  dilute,  to  the  survival  of  crabs  in  0  mM.  Cl/L. 


SALT  AND  WATER  REGULATION  IN  CRABS  193 

fluids  is  shown  by  the  doubling  of  survival  time  by  the  retention  in  the  blood  of 
7  mM.  SCN/L.  for  162  mM.  Cl/L.  lost  in  land  crabs,  25  for  129  in  ghost 
crabs  and  9  for  72  in  mangrove  crabs  during  the  first  24  hours  of  exposure. 
There  was  no  such  increase  in  the  survival  time  of  crabs  in  720  mM.  Cl/L.  fluids 
to  which  similar  amounts  of  Na  SCN  had  been  added.  None  of  the  animals 
showed  any  signs  of  depression. 

Urine  chloride  of  ghost  crabs  exposed  to  distilled  water  for  24  hours  was 
48%  of  blood  concentration,  indicating  that  some,  though  obviously  not  all, 
filtered  chloride  is  re-absorbed.  This  is  also  indicated  by  comparable  SCN  data. 
The  high  urine  chloride  is  not  high  enough  to  suggest  chloride  secretion  by  the 
antennal  gland.  The  impression  that  these  animals  formed  very  little  urine  is 
supported  by  the  fact  that  only  10%  of  the  injected  inulin  is  cleared  during  this 
24-hour  period.  The  less-than-blood  concentration  and  the  small  volume  of  urine 
and  the  loss  of  one-third  of  blood  chloride  suggest  a  removal  of  chloride,  and 
SCN,  from  the  blood  by  a  pathway  other  than  the  antennal  gland.  The  urine 
chloride  concentration  in  mangrove  crabs,  62%  of  blood  level,  after  a  similar 
exposure  indicates  partial  chloride  recovery  by  the  antennal  gland.  This  re- 
absorption  continues  through  48  hours,  but  fails  to  repair  a  falling  blood  chloride 
concentration. 

Comparison  between  a  35-gram  ghost  crab  and  a  similar  mangrove  crab,  cal- 
culated from  data  presented  in  Table  I,  serves  to  demonstrate  the  possible  differ- 
ence in  pathways  of  chloride  loss  in  animals  exposed  to  distilled  water.  From 
Figures  1  and  2,  it  may  be  seen  that  88  mgm.  NaCl  were  lost  from  the  blood  of 
such  a  ghost  crab  during  24  hours  in  distilled  water  and  that  the  concurrently 
formed  urine  contained  11  mgm.  NaCl  per  cubic  centimeter.  For  the  chloride 
lost  from  the  blood  to  have  been  cleared  by  only  the  antennal  gland,  8.0  cc.  of  urine 
would  have  had  to  be  formed.  As  calculated  by  inulin  clearance,  only  0.8  cc.  of 
fluid  was  filtered  during  this  period.  Urine  inulin  concentrations  were  roughly 
equal  to  blood  levels,  indicating  little  or  no  water  re-absorption  or  secretion  after 
filtration.  Ninety  per  cent  of  the  chloride  loss  must  have  been  by  another  route 
in  the  ghost  crab.  From  Figures  1  and  2  it  appears  that  45  mgm.  NaCl  were 
lost  from  a  comparable  mangrove  crab  during  a  similar  exposure  and  that  the 
urine  formed  contained  15  mgm.  NaCl  per  cubic  centimeter.  The  filtration  and 
excretion  of  3.0  cc.  of  this  urine  would  account  for  the  blood  chloride  loss.  Accord- 
ing to  inulin  clearance,  3.5  cc.  fluid  were  filtered,  and  according  to  urine  inulin 
concentrations  there  was  no  appreciable  re-absorption  of  water.  In  spite  of  the 
clearance  of  proportionately  less  chloride  than  water  by  the  antennal  gland,  sug- 
gesting re-absorption  of  chloride,  this  is  the  only  pathway  necessary  to  account 
for  the  observed  failure  in  chloride  ion  regulation  in  mangrove  crabs  exposed  to 
distilled  water.  Re-absorption  of  chloride  occurred  in  both  species  in  distilled 
water.  Since  0.8  cc.  blood  was  filtered  in  the  ghost  crab,  18.0  mgm.  NaCl  crossed 
over  into  the  lumen  of  the  antennal  gland.  Since  urine  contained  48%  blood 
chloride  concentration,  8.6  mgm.  NaCl  were  lost,  and  the  remaining  9.4  mgm. 
must  have  been  re-absorbed.  The  3.5  cc.  blood  filtered  in  the  mangrove  crab 
carried  86.4  mgm.  NaCl  into  the  antennal  gland.  The  urine,  containing  62% 
blood  chloride  concentration,  removed  53.6  mgm.  NaCl,  leaving  32.8  mgm.  to  be 
re-absorbed.  This  is  in  agreement  with  the  observed  chloride  loss.  The  removal 
of  more  water  than  electrolyte  from  the  blood  of  ghost  crabs  and  mangrove  crabs, 


194  LAUNCH  J.  FLEMISTER 

and  the  decrease  in  blood  chloride  concentrations  of  all  three  species  exposed  to 
distilled  water  for  24  hours,  make  it  apparent  that  the  water  entering  the  animals 
is  flushing  chloride  out  through  the  antennal  gland.  Moreover,  loss  of  chloride 
through  another  pathway  is  indicated  in  land  crabs  and  ghost  crabs,  but  not 
necessarily  in  mangrove  crabs. 

Although  complete  extraction  of  electrolytes  in  one  passage  through  the  gill 
chamber  can  not  be  assumed,  comparisons  of  absorption  rates  can  be  made.  The 
absorption  of  SCN  added  to  0  mM.  Cl/L.  environmental  fluids  indicates  a  with- 
drawal from  a  volume  of  environmental  fluid  equal  to  the  SCN  space,  about  11  cc. 
for  a  35-gram  animal,  in  2  hours  for  ghost  crabs,  6  hours  for  mangrove  crabs  and 
15  hours  for  land  crabs.  Leveling  off  of  the  concentration  curves  in  time  suggests 
that  if  absorption  rates  hold,  the  rate  of  diffusion  outward  increases  with  increasing 
concentration.  This  suggests  a  far  more  rapid  turnover  at  the  gill  membrane  and, 
perhaps,  branchial  epithelium  than  clearance  rates  in  the  antennal  gland  would 
indicate.  The  gill  and,  perhaps,  branchial  epithelium  appear  to  be  the  site  of  this 
absorption  activity  since  animals  whose  digestive  tracts  were  closed  at  both  ends 
with  grafting  wax  did  not  differ  in  absorption  rates  from  those  animals  not 
blocked.  Similar  blocking  prior  to  electrolyte  and  inulin  loss  determinations  indi- 
cated that  the  digestive  tract  has  no  significant  role  in  the  clearances  observed. 

The  appreciable,  W%  rise  in  blood  chloride  concentrations  in  land  crabs  ex- 
posed to  120,  240,  and  360  mM.  Cl/L.  fluids  indicates  that  the  rate  of  absorption 
of  chloride  from  these  environments  exceeds  the  rate  of  loss  until  a  new  steady-state 
is  reached.  The  steadily  maintained  higher  blood  level,  failing  only  in  120  mM. 
Cl/L.  at  72  hours,  shows  that  the  regulation  is  effective,  if  not  compensating. 
The  much  more  elevated,  20%  higher,  yet  steady  concentrations  found  in  animals 
exposed  to  480  mM.  Cl/L.  for  72  hours,  600  for  48  and  720  for  24,  indicate  that 
this  regulation  persists  and  has  some  flexibility  and  upper  limits  in  situations  hyper- 
tonic  to  the  blood.  Absorption  rates  are  greater  than  indicated  by  the  concen- 
tration curves,  for  it  must  be  assumed  that  during  absorption  the  ions  are  being 
lost  at  rates  suggested  by  the  SCN  loss  curves.  The  slower  rate  of  SCN  loss 
from  injected  animals,  the  slower  rate  of  SCN  absorption  by  uninjected  ones  and 
the  slower  rate  of  filtration  in  600  and  720  mM.  Cl/L.  fluids  indicate  that  there  is 
reduced  exchange  with  the  environmental  fluids  perhaps  due  to  reduced  exposure 
which  in  turn  may  be  due  to  a  partial  restriction  of  gill  chamber  volume  or  flow 
as  suggested  by  the  work  of  Gross  (1957)  on  the  brachyuran  shore  crab  (Pachy- 
gmpsus  crassipes}  exposed  to  hypertonic  fluids.  The  loss  of  ions  across  the  gill 
membrane  and,  possibly,  the  branchial  epithelium  and  the  persisting,  though  re- 
duced, filtration  through  the  antennal  gland  are  not  sufficiently  rapid  to  prevent 
an  accumulation  of  ions  from  the  environment  resulting  in  the  elevated  blood 
chloride  level  observed.  Although  the  lack  of  urine  data  precludes  further  analy- 
sis and  appraisal  of  this  regulation,  it  appears  that  there  is  a  correlation  between 
the  dry  habitat  of  land  crabs  and  their  relatively  slow  electrolyte  clearance  resulting 
in  elevated  blood  chloride  levels  even  in  hypotonic  environmental  fluids. 

The  regulation  of  blood  chloride  concentration  in  ghost  crabs  is  more  rigid 
from  120  to  600  mM.  Cl/L.  than  in  land  crabs.  The  similarity  of  the  SCN  loss 
curves  for  360  to  720  mM.  Cl/L.  fluids,  faster  than  inulin  clearance,  indicates  that 
the  antennal  gland  is  of  only  secondary  importance  in  electrolyte  loss  in  near-isotonic 
and  hypertonic  environments.  Since  in  all  fluids  the  injected  SCN  is  cleared  in 


SALT  AND  WATER  REGULATION  IN  CRABS  195 

about  24  hours,  the  constancy  of  the  blood  chloride  concentration  would  appear  to 
depend  on  the  net  absorption,  or  retention  of  the  same  quantity  of  electrolyte 
irrespective  of  environmental  concentration.  This  can  be  concluded  to  happen 
from  the  net  absorption  curves.  Proportional  to  chloride  ions  present  in  environ- 
mental fluids,  there  is  about  three  times  as  much  SCN  in  120  mM.  Cl/L.  fluids  as 
in  360  and  almost  twice  as  much  in  360  as  in  600.  This  is  approximately  the  ratio 
of  net  absorption  concentration  of  SCN  accumulating  in  the  blood  during  exposure 
to  the  various  environments.  The  greater  volume  of  environmental  water  involved 
in  this  extraction  process  in  120  mM.  Cl/L.  fluids  is  reflected  in  the  more  rapid 
filtration  through  the  antennal  gland. 

Blood  chloride  is  held  constant  over  a  wider  range,  120  to  720  mM.  Cl/L.,  for 
72  hours  in  mangrove  crabs  than  in  either  of  the  other  two  species.  The  close 
approximation  of  urine  chloride  concentrations  to  those  of  environmental  fluids 
suggests  that  the  regulation  is  closely  held  and  yet  flexible  in  that  absorbed  ions 
are  apparently  retained  in  hypotonic  situations  and  cleared  in  hypertonic  ones. 
The  fact  that  filtration  continues  at  the  same  rate  for  all  environmental  fluids, 
even  distilled  water,  shows  that  constant  blood  chloride  levels  must  be  maintained 
by  prompt  re-absorption  of  ions  and  water  by  the  antennal  gland  and  by  absorption 
and  loss  by  any  other  route  of  exchange  involved.  The  more  rapid  loss  of  SCN 
in  hypertonic  environments  than  in  near-isotonic  ones,  and  these  more  rapid  than 
in  hypotonic  ones,  at  a  time  when  blood  chlorides  are  constant,  shows  that  the 
turnover,  absorption  and  loss,  of  electrolytes  is  more  rapid  in  the  more  concen- 
trated environments.  This  may  account  for  the  fact  that  the  accumulated  SCN 
curves  fail  to  level  off  at  points  which  suggest  the  ratios  of  proportionate  SCN  and 
chloride  concentrations,  as  was  found  in  ghost  crabs.  The  outbound  passage  of 
the  same  amount  of  water  through  the  antennal  gland  in  all  environmental  con- 
centrations, indicated  by  inulin  clearance,  fails  to  account  for  electrolyte  clearance, 
except  from  crabs  in  distilled  water. 

The  fact  that  urine  chloride  concentrations  approach  environmental  fluid  levels 
and  not  blood  levels  during  exposure  to  120  to  720  mM.  Cl/L.  environments  for 
up  to  48  hours  in  ghost  crabs  and  72  hours  in  mangrove  crabs  suggests  that  the 
antennal  gland  re-absorbs  some  chloride  in  hypotonic  and  some  water  in  hypertonic 
situations  after  filtration.  It  is  apparent  that  the  reabsorption  of  chloride  is  not 
completely  adequate  in  hypotonic  environments  in  either  species  and  begins  to  fail 
earlier  in  ghost  crabs  than  in  mangrove  crabs.  The  re-absorption  of  water  in 
hypertonic  environments  is  more  effective  in  both  species.  Urine  electrolyte  and 
inulin  concentrations  indicate  that  the  high  urine  chloride  in  hypertonic  and  near- 
isotonic  environments  is  due  to  re-absorption  of  water.  Inasmuch  as  blood  chlo- 
ride levels  continue  to  be  maintained,  and  inulin  data  indicate  only  a  moderate 
increase  in  filtration  and  only  a  moderate  decrease  in  water  re-absorption  by  the 
antennal  gland,  the  markedly  reduced  level  of  urine  chloride  in  hypertonic  environ- 
ments at  72  hours  implies  a  closing  of  a  portal  of  entry  of  chloride  in  the  ghost 
crab.  This  might  be  due  in  part  to  restricted  gill  chamber  exposure  suggested  in 
the  shore  crab  in  hypertonic  fluids  by  Gross  (1957).  The  marked  increase  in 
urine  chloride  concentration  in  the  ghost  crab  in  120  mM.  Cl/L.  fluid  in  72  hours, 
when  blood  chloride  level  remains  constant  and  urine  inulin  concentrations  indi- 
cate no  re-absorption  of  water,  suggests  accelerated  chloride  absorption  from  the 
environment.  It  is  interesting  that  urine  chloride  concentrations  in  the  two  species 


196  LAUNCH  J.  FLEMISTER 

are  near  normal  when  the  mangrove  crab  is  in  600  mM.  Cl/L.  fluid,  its  normal 
habitat,  and  when  the  ghost  crab  is  in  360  mM.  Cl/L.  fluid,  near  isotonicity  with 
its  blood.  It  is  also  interesting  that  urine  and  blood  chloride  concentrations  are 
equal  when  the  animals  are  exposed  to  environmental  chlorides  100  mM.  Cl/L. 
less  concentrated  than  the  blood.  This  gives  a  rough  estimate  of  the  re-absorption 
gradient  in  the  antennal  gland  and  indicates  that  similar  mechanisms  and  thresholds 
are  involved  in  the  two  species. 

When  animals  of  these  three  species  are  exposed  to  environmental  fluids  rang- 
ing from  120  to  600  mM.  Cl/L.,  the  rate  of  turnover,  absorption  and  loss,  of  elec- 
trolytes and  the  rate  of  filtration  are  less  in  the  land  crabs  than  in  the  others.  The 
difficulty  in  getting  urine  samples  suggests  re-absorption  of  most  of  the  filtered 
water,  which  might  be  expected  in  view  of  this  crab's  adaptation  to  a  dry  habitat. 
The  electrolyte  turnover  and  filtration  rates  are  most  rapid  in  the  ghost  crab  in 
hypotonic  and  in  the  mangrove  crab  in  hypertonic  environmental  fluids.  There  is 
an  apparent  correlation  between  the  almost  constant  exposure  of  the  mangrove 
crab  to  sea  water  hypertonic  to  its  own  blood  and  a  rapid  turnover  and  clearance 
rate.  It  appears  that  the  defense  in  the  ghost  crab  is  against  the  inbound  move- 
ment of  hypotonic  fluids  and  that  this  is  a  poor  defense  at  best  in  view  of  the  in- 
efficient re-absorption  of  chloride  by  its  antennal  gland.  It  is  interesting  that  when 
animals  of  these  species  are  exposed  to  environmental  fluids  which  are  near  iso- 
tonic  to  their  own  blood  concentrations,  the  filtration  rates  through  the  antennal 
glands  are  similar.  This  indicates  that  the  hydrostatic  and  osmotic  factors  in  filtra- 
tion are  similar  in  all  three  of  the  species.  This  augments  the  interpretation  based 
on  the  uniformity  of  re-absorption  gradients  that  similar  mechanisms  and  thresholds 
are  involved  in  antennal  gland  function  in  the  three  species. 

In  the  early  intervals  of  SCN  absorption  and  loss  determinations,  before  blood 
levels  are  much  altered,  absorption  rates  exceed  loss  rates  in  ghost  crabs  and  man- 
grove crabs  and  are  about  equal  in  land  crabs  in  hypotonic,  120  mM.  Cl/L.,  fluids. 
Land  crabs  and  ghost  crabs  hold  about  equal  in  near-isotonic,  360  mM.  Cl/L., 
fluids,  but  mangrove  crabs  show  an  absorption  advantage  in  the  same  fluid,  which 
is  hypotonic  to  their  blood.  Early  loss  rates  exceed  early  absorption  rates  in  all 
three  species  in  hypertonic,  600  mM.  Cl/L.,  sea  water.  These  absorption  and 
loss  rate  differences  are  parallel  to  the  leveling-off  points  in  the  SCN  accumulation 
curves,  \vhich  are  interpreted  to  arise  from  the  equating  of  outbound  and  inbound 
passage  of  ions  across  gills  and,  perhaps,  branchial  epithelium  as  blood  concentra- 
tions are  increased  as  a  result  of  absorption  exceeding  loss  earlier.  Comparison 
of  these  leveling-off  concentrations  of  net  absorbed,  accumulated,  SCN  and  the 
ratio  of  the  concentration  of  chloride  maintained  in  the  blood  and  that  imposed  by 
the  environmental  fluid  shows  close  agreement  for  near-isotonic  and  for  hypertonic 
situations.  In  both  the  ghost  crab  and  the  mangrove  crab,  the  plateau  has  not 
been  reached  in  hypotonic  fluid,  but  in  the  land  crab  there  is  evidence  of  both  a 
leveling-off  and  a  breakdown  in  blood  chloride  regulation  at  72  hours.  In  crabs 
of  all  three  species  exposed  to  distilled  water,  the  plateau  is  so  remote  and  the 
breakdown  so  severe  that  no  conclusions  can  be  drawn.  Leveling-off  of  loss 
curves  in  time  is  interpreted  to  reflect  rates  markedly  reduced  by  the  falling  blood 
concentration.  After  ten  days  no  SCN  or  inulin  could  be  found  in  injected  ani- 
mals kept  in  the  laboratory  under  normal  conditions.  The  evidence  from  the  net 
absorption  curves  is  that  electrolyte  movement  is  rapid  and  precise.  In  hypotonic 


SALT  AND  WATER  REGULATION  IN  CRABS  197 

environments  an  appreciably  longer  time  is  required  to  reach  a  plateau  than  is 
required  to  clear  the  ion  once  it  is  injected,  which  suggests  that  there  is  a  choke  on 
the  rate  of  absorption  of  ions  from  hypotonic  fluids.  This  is  altogether  reasonable 
when  the  handling  of  the  required  amount  of  fluid  is  considered. 

The  loss  of  injected  SCN  to  a  level  well  above  a  concentration  in  equilibrium 
with  SCN  added  to  120  mM.  Cl/L.  environmental  fluid  coincides  with  and  sup- 
ports the  evidence  from  urine  chlorides  of  animals  in  such  environments  that 
electrolyte  loss  continues  even  in  situations  where  ions  must  be  acquired  to  main- 
tain constancy.  The  fact  that  injected  SCN  falls  to  approximate  equilibrium  with 
environmental  SCN  in  animals  exposed  to  near-isotonic  fluids  also  supports  this 
evidence.  The  loss  of  injected  SCN  to  a  concentration  less  than  environmental 
in  animals  in  600  mM.  Cl/L.  fluids,  in  which  absorbed  SCN  is  held  to  less  than 
equilibrium  concentration,  shows  that  the  capacity  to  lose  electrolyte  is  not  satu- 
rated by  this  degree  of  hypertonicity.  It  is  clear  that  these  loss  rates  are  much 
greater  than  can  be  accounted  for  by  passage  through  the  antennal  gland. 

On  the  basis  of  early  clearance  rates,  before  blood  concentrations  are  greatly 
decreased,  fluid  equal  to  the  inulin  space  volume  is  filtered  by  the  antennal  gland 
of  land  crabs  in  their  normal  habitat  on  sand  in  about  60  hours,  and  in  mangrove 
crabs  in  600  mM.  Cl/L.  sea  water,  their  normal  habitat,  in  24  hours.  In  ghost 
crabs  on  sand  no  appreciable  filtration  was  found.  However,  in  sea  water  between 
360  and  600  mM.  Cl/L.,  to  which  ghost  crabs  normally  have  access,  the  filtration 
rates  are  similar  to  those  of  mangrove  crabs.  This  indicates  the  importance  of 
the  ghost  crab's  brief  nightly  exposure  to  the  surf.  It  is  assumed  that  in  their 
normal  habitat,  ghost  crabs  filter  somewhat  slower  than  do  mangrove  crabs,  but 
that  they  do  filter  is  apparent  from  the  fact  that  fresh-caught  crabs  have  urine. 
Therefore,  an  obvious  correlation  exists  between  filtration  rate  and  type  of  habitat 
in  these  three  species. 

The  turnover  rates  indicate  the  activity  in  electrolyte  and  water  movement 
which  goes  on  during  the  maintenance  of  constancy  of  volumes  and  concentrations 
in  the  water  compartments  measured.  The  persistence  of  normal  values  for  these 
quantities  in  the  variety  of  devised  and  imposed  environmental  stress  situations  is 
as  remarkable  as  the  rate  of  continuous  change  which  underlies  it.  It  must  be 
concluded  that  in  submerged  crabs  of  these  three  species,  the  gills  and,  possibly, 
the  branchial  epithelium  provide  the  principal  pathway  for  this  rapid  and  precise 
absorption  and  loss  of  electrolytes  and  water,  and  that  the  antennal  gland  plays 
only  a  limited  role  in  this  turnover.  However,  the  urine  chloride,  thiocyanate  and 
inulin  concentrations  indicate  that  clearance  through  the  antennal  gland  may  provide 
the  all-important  fine  adjustment  in  blood  concentration  of  electrolytes  and  water. 

Among  these  three  species,  found  in  different  degrees  of  exposure  to  seas  of 
different  salinity,  the  mangrove  crab,  most  constantly  and  continuously  exposed 
intimately  to  the  stable  environment  of  the  sea,  is  the  one  showing  the  greatest 
capacity  to  regulate  the  concentration  of  blood  chloride  when  subjected  to  environ- 
ments of  widely  differing  salinities.  The  crab  most  independent  of  the  sea,  the 
land  crab,  has  the  most  definitively  regulated  volume  of  total,  inulin  space  and 
SCN  space  water,  and  an  adequate  electrolyte  regulation  when  exposed  to  a 
limited  hypotonic  range  or  to  food  containing  proportionately  more  water  than  salt, 
but  little  or  none  when  environmental  chloride  exceeds  that  of  blood  as  does  the 
sea  water  accessible  in  its  habitat.  In  the  ghost  crab,  intermediate  between  them. 


198  LAUNCE  J.  FLEMISTER 

there  appear  to  be  the  mechanisms  for  effective  regulation  with  diminished  chloride 
absorption  in  hypertonic  fluids  and  increased  absorption  in  hypotonic  ones,  but 
with  the  threat  of  an  extravagantly  wasteful  chloride  loss  through  the  antennal 
gland.  The  independence  of  the  land  crab  from  the  sea  depends  on  the  mainte- 
nance of  a  gradient  and  not  on  effective  regulation.  The  land  habitat  of  the  ghost 
crab  is  critically  dependent  on  access  to  the  surf,  albeit  for  a  short  nightly  exposure. 
The  sea  habitat  of  the  mangrove  crab  is  a  complete  commitment  despite  a  wide 
range  of  effective  regulation.  There  emerges  a  picture  of  independence  which 
depends  on  the  constancy  of  a  normal  gradient,  and  the  capacity  to  tolerate  a 
changing  gradient  which  depends  on  effective  regulation.  They  afford  the  mecha- 
nisms of  adaptation  to  totally  different  habitats. 

SUMMARY 

1.  Exposure  to  environmental  salinities  ranging  from  120  to  720  mM.  Cl/L. 
for  72  hours  did  not  produce  changes  in  fresh  weights  of  the  land  crab  (Gccarcinus 
lateralis),  the  ghost  crab    (Ocypode  albicans)   or  the  mangrove  crab    (Goniopsis 
cruentatus).     There  was  an  increase  in  weight  of  questionable  significance  after 
24  hours  in  crabs  exposed  to  distilled  water.     Only  in  distilled  water  was  there  any 
change  in  the  blood  specific  gravity  of  ghost  crabs.     Even  this  change   was  of 
questionable  significance. 

2.  The  total  body  water  content  of  ghost  crabs  is  significantly  larger  than  those 
of  land  crabs  and  mangrove  crabs,  which  are  similar.     The  fractions  of  total  water 
content  which  are  available  for  the  dilution  of  thiocyanate  and  inulin  are  similar 
in  the  three  species.     The  volumes  available  for  the  dilution  of  inulin  are  about 
two-third  the  volumes  in  which  SCN  appears  to  be  diluted.     This  suggests  the 
interesting  possibility  of  a  functionally  closed,  lumen  flow,  circulation. 

3.  The   blood  chloride  concentration   of   mangrove   crabs,   although   less   than 
that  of  their  environment,  is  significantly  greater  than  those  of  the  more  terrestrial 
ghost  crabs  and  land  crabs,  which  are  similar.     The  urine  chloride  concentration 
of  mangrove  crabs  is  identical  to  that  of  its  environment  and  is  more  concentrated 
than  that  of  ghost  crabs. 

4.  Exposed  to  environmental  fluids  of  120  to  600  mM.  Cl/L.  sea  water  for 
72  hours,   land  crabs  show  adequate  regulation   of  blood   chloride   concentration 
over  a  limited  hypotonic  range,  but  little  or  no  regulation  in  fluids  hypertonic  to  its 
blood  chloride.     Blood  chloride  regulation  in  ghost  crabs  is  adequate  over  this 
range,  but  with  the  production  of  a  urine  which  wastes  chloride  in  hypotonic  fluids. 
Mangrove  crabs  show  an  adequate  and  closely  held  regulation  of  blood  chloride 
concentration  in  this  range  and  the  production  of  a  urine   with  chloride   levels 
similar  to  those  of  the  environment,  but  with  some  chloride  leakage  in  hypotonic 
fluids.     Blood  chloride  regulation  failed  in  all  three  species  wrhen  exposed  to  dis- 
tilled water  for  24  hours,  and  in  land  crabs  and  ghost  crabs  exposed  to  720  mM. 
Cl/L.  for  about  24  hours.     Mangrove  crabs  survived  72  hours  in  720  mM.  Cl/L. 
fluid  with  regulation  intact,  but  could  not  survive  24  hours  in  air. 

5.  On  dry  sand,  land  crabs  filter  across  the  antennal  gland  a  volume  equal  to 
their  inulin  space  in  60  hours.     It  also  re-absorbs  most  of  the  water  of  the  urine 
thus  formed.     This  is  not  true  of  ghost  crabs  in  which  the  formation  of  urine  ap- 
pears to  depend  on  water  gained  during  brief  nightly  exposures  to  the  surf.     When 


SALT  AND  WATER  REGULATION  IN  CRABS  199 

0 

exposed  to  600  mM.  Cl/L.  sea  water,  their  normal  habitat,  mangrove  crabs  filter 
their  inulin  volume  in  24  hours.  There  is  an  apparent  correlation  between  these 
filtration  rates  and  the  availability  of  water  in  the  habitat. 

6.  Antennal  gland  filtration  and  re-absorption  rates  are  adequate  to  account 
for  the  rate  of  chloride  loss  in  mangrove  crabs  in  distilled  water.     This  is  not  true 
for  ghost  crabs  and  land  crabs  in  which  filtration  rates  are  not  much  faster  than 
those    on   sand.     Electrolytes    are    escaping   across    some   other   membrane,    sup- 
posedly gills  and,  perhaps,  branchial  epithelium.     The  loss  of  electrolyte  by  a  route 
other  than  the  antennal  gland  is  also  apparent  in  animals  of  all  three  species  ex- 
posed to  environmental  fluids  from  120  to  720  mM.  Cl/L. 

7.  Re-absorption  of  chloride  by  the  antennal  gland  of  ghost  crabs  and  man- 
grove crabs  exposed  to  hypotonic  fluids  and  of  water  in  animals  exposed  to  hyper- 
tonic  fluids  is  apparent  from  the  similarity  between  urine  and  environmental  chlo- 
ride concentrations.     Similar  re-absorptions  can  be  inferred  from  data  presented 
on  land  crabs. 

8.  The  similarity  of  the  mechanisms  and  thresholds  involved  in  antennal  gland 
function  is  indicated  by   (1)  the  approach  of  urine  chloride  concentrations  to  the 
blood  chloride  levels  when  ghost  crabs  and  mangrove  crabs  are  exposed  to  environ- 
mental fluid  chloride  levels  100  mM.  Cl/L.  less  concentrated  than  the  blood,  and 
(2)  the  similarity  in  filtration  rates  in  all  three  species  when  animals  are  exposed 
to    environmental    fluids    which   are    near    isotonic    to    their    own    blood    chloride 
concentrations. 

9.  The  blood  concentrations  of  SCN  absorbed  from   120  to  720  mM.   Cl/L. 
environmental  fluids  tend  to  plateau,  due  to  equating  of  inbound  and  outbound 
ion  passage,  at  a  point  roughly  equal  to  the   ratio  between  blood  chloride  and 
environmental  chloride  levels.    The  point  of  plateau  is  reached  more  slowly  in  hypo- 
tonic  situations  indicating  the  difficulty  of  handling  the  required  volume  of  environ- 
mental fluid.     The  persistence  of  electrolyte  loss,  even  in  situations  where  ions 
must  be  rapidly  absorbed  to  maintain  constancy,  is  indicated  by  the  SCN  loss  rate 
curves  for  the  various  environments. 

10.  The  rates  of  turnover  of  water  and  electrolyte  are  as  remarkable  as  the  con- 
stancy of  the  regulation  from  which  they  result  and  for  which  they  are  responsible. 
The  effectiveness  of  this  regulation  in  mangrove  crabs  and  the  maintenance  of  a 
concentration  gradient  in  land  crabs  can  be  related  to  the  successful  adaptation  of 
these  two  species  to  totally  different  habitats. 

LITERATURE  CITED 

BURGER,  J.  W.,  1955.     Excretion  in  the  lobster,  Homanis.     Anat.  Rec.,  122:  460-461. 
BURGER,  J.   W.,   1957.     The  general  form  of  excretion   in   the   lobster,   Homarus.     Biol.   Bull., 

113:  207-223. 
CRANDALL,  L.  A.,  AND  M.  X.  ANDERSON,  1934.     Estimate  of  the  state  of  hydration  of  the  body 

by  the  amount  of  water  available  for  the  solution  of  sodium   thiocyanate.     Amer.  J. 

Digest.  Dis.  and  Nutr.,  1 :  126-131. 
CUNNINGHAM,  B.,  P.  L.  KIRK  AND  S.  C.  BROOKS,  1941.     Quantitative  drop  analysis:  XIV.  Po- 

tentiometric  determination  of  chloride.     /.  Biol.  Chem.,  139:   11-19. 
FLEMISTER,  L.  J.,  AND  S.  C.  FLEMISTER,  1951.     Chloride  ion  regulation  and  oxygen  consumption 

in  the  crab  Ocypodc  albicans  (Bosq).     Biol.  Bull.,  101:  259-273. 
FORSTER,  R.  P.,  AND  P.  ZiA-\VALRATH,  1941.     The  absence  of  active  secretion  as  a  factor  in 

the   elimination    of   inulin    and   other    substances    by    the   green    gland   of   the    lobster, 

Homanis  americanus.     Anat.  Rcc.,  81  :  siippl.   128. 


200  LAUNCE  J.  FLEMISTER 

GROSS,  W.  J.,  1957.     An  analysis  of  response  to  osmotic  stress  in  selected  decapod  Crustacea. 

Biol.  Bull.,   112:  43-62. 
JACOBSEN,  C.  F.,  AND  K.  LINDERSTROM-LANG,  1940.     Method  for  rapid  determination  of  specific 

gravity.     Acta.  Physiol.  Scand.,  1  :_  149-152. 
MALUF,  N.  S.  R.,  1941.     Secretion  of  inulin,  xylose  and  dyes  and  its  bearing  on  the  manner  of 

urine  formation  by  the  kidney  of  the  crayfish.     Biol.  Bull,  81 :  235-260. 
MARTIN,  A.  W.,  1957.     Recent  advances  in  knowledge  of  invertebrate  renal  function.     Recent 

Advances  in  Invertebrate  Physiology.     Univ.  of  Oregon  Publications. 
PICKEN,  L.  E.  R.,  1936.     The  mechanism  of  urine  formation  in  invertebrates.     I.  The  excretion 

mechanism  in  certain  Arthropoda.     /.  Exp.  Biol.,  13 :  309-328. 
SCHOLANDER,    P.    F.,   G.    A.    EDWARDS   AND    L.    IRVING,    1943.     Improved   micrometer    burette. 

/.  Biol.  Chan.,  148 :  495-500. 
SOMOGYI,  M.,   1930.     A  method  for  the  preparation  of  blood  filtrates  for  the  determination  of 

sugar.     J.  Biol.  Chcm.,  86:  655-663. 
YOUNG,  M.  K.,  AND  L.  G.  RAISZ,  1952.     An  anthrone  procedure  for  determination  of  inulin  in 

biological  fluids.     Proc.  Soc.  Exp.  Biol.  Mcd.,  80:  771-774. 


REGIONAL  LOCALIZATION  OF  NEURAL  AND  LENS  ANTIGENS 
IN  THE  FROG  EMBRYO  IN  RELATION  TO  INDUCTION 

REED  A.  FLICKINGER 

Department  of  Zoology,  State  University  of  loiva,  lo-iva  City,  Iowa 

A  number  of  embryologists  recently  have  attempted  to  characterize  embryonic 
cells  by  their  constituent  proteins.  This  approach  is  of  particular  interest  when 
this  characterization  is  attempted  before,  or  at  the  time  of,  embryonic  determination 
since  it  might  be  expected  that  a  protein,  or  proteins,  usually  associated  with  a 
given  tissue  would  increase  in  amount  once  the  differentiation  and  growth  of  that 
tissue  has  already  begun.  The  serological  experiments  of  Ebert  et  al.  (1955)  in 
localizing  cardiac  myosin  and  actin  in  the  early  chick  blastoderm,  and  those  of 
Ten  Cate  and  Van  Doorenmaalen  (1950)  in  determining  the  time  of  appearance 
and  location  of  the  lens  antigen  in  frog  and  chick  embryos  are  examples  of  this 
approach. 

In  relation  to  embryonic  induction  it  would  appear  to  be  of  some  theoretical 
significance  to  be  able  to  map  or  localize  the  protein  that  may  characterize  the  re- 
acting tissue  in  an  induction  system.  In  the  induction  of  the  medullary  plate  by 
the  underlying  chorda  mesoderm,  where  is  the  greater  amount  of  neural  antigen 
localized  just  before  this  induction  occurs?  Is  there  more  specific  neural  protein 
in  the  inductor  (chorda  mesoderm)  or  in  the  reacting  tissue  (gastrula  ectoderm)  ? 
If  the  inductor  has  more  neural  protein,  then  this  may  imply  the  passage  of  spe- 
cific protein  from  the  inducing  to  the  reacting  tissue  and  subsequent  synthesis  of 
this  protein  in  the  reacting  tissue.  If,  on  the  other  hand,  more  of  the  neural 
antigen  is  present  in  the  gastrula  ectoderm,  this  implies  that  the  induction  stimulus 
is  of  a  less  specific  nature  and  may  merely  be  an  activator  for  the  synthesis  of  more 
neural  protein  in  the  reacting  tissue. 

MATERIALS  AND  METHODS 

In  order  to  examine  this  question,  antisera  were  developed  against  adult  male 
frog  brains  (Rana  pipiens)  and  antisera  developed  against  adult  frog  lenses  and 
cattle  lenses  for  a  previous  investigation  (Flickinger  et  al.,  1955)  were  also  utilized. 
The  method  of  preparing  the  anti-lens  sera  was  presented  in  the  above  paper. 
Two  antisera  against  adult  male  frog  brains  were  prepared  by  cutting  open  the 
brains  and  freeing  them  of  all  visible  blood,  washing  them  several  times  in  cold 
0.65%  saline,  homogenizing  them  in  a  glass  tissue  grinder  in  the  cold,  and  injection 
of  the  supernate  obtained  from  centrifugation  at  3000  g.  This  supernate  was 
about  1%  protein  as  shown  by  nitrogen  determinations.  Intravenous  injections 
of  0.5,  1.0  and  2.0  ml.  and  an  intraperitoneal  injection  of  4.0  ml.  were  given  on 
alternate  days  and  constituted  an  injection  series.  Three  such  series  of  injections 
were  administered  a  week  apart  with  the  modification  that  the  whole  uncentrifuged 
homogenate  was  injected  intraperitoneally  in  the  third  series  of  injections.  The 
rabbits  were  bled  7  days  after  completion  of  injections.  One  of  the  antisera 

201 


202 


REED  A.  FLICKINGER 


reacted  with  a  0.1  %  protein  supernate  from  adults'  brains  at  an  antiserum  dilution 
of  1/128.     Obviously  extracts  of  adult  frog  brains  are  not  highly  antigenic. 

In  the  preparation  of  test  antigens,  early  gastrulae  (Shumway  stage  10)  were 
operated  upon  under  sterile  conditions  and  cut  in  four  regions ;  ectoderm,  dorsal 
mesoderm,  ventral  mesoderm  and  endoderm,  as  seen  in  Figure  1.  Most  of  the 
large  white  yolky  cells  were  removed  from  the  ventral  and  dorsal  mesoderm 
tissues.  Recently  hatched  larvae  (Shumway  stage  19-20)  were  cut  into  three 
parts;  head,  trunk  and  gut  (Fig.  1).  The  heads  were  removed  by  cutting  verti- 


ECTODERM 


HEAD 


TRUNK 


VENTRAL 
MESODERM 


DORSAL 
MESODERM 


ENDODERM 


GUT 


FIGURE  1.  See  text  for  explanation. 


cally  just  posterior  to  the  gill  plate;  the  trunks  were  separated  from  the  guts  by 
cutting  horizontally  just  ventral  to  the  somites.  No  attempt  was  made  to  count 
the  number  of  gastrulae  and  larvae  that  were  operated  upon,  but  for  each  prepara- 
tion of  test  antigen  from  an  embryo  fraction,  several  hundred  of  the  appropriate 
tissues  were  homogenized  with  an  equal  volume  of  0.9%  NaCl  in  micro-tissue 
grinders  and  then  centrifuged  in  micro-centrifuge  tubes  (3  mm.  inside  diameter 
(  55  mm.  length)  in  adaptors  designed  for  the  7-ml.  high  speed  head  of  the  Inter- 
national centrifuge.  After  removal  of  the  pigment,  yolk,  and  lipid  cap  by  re- 
peated centrifugation  at  15,000  g,  some  0.2-0.3  ml.  of  a  centrifugal  supernate 
is  obtained  which  can  be  used  as  the  test  antigen.  The  precipitin  reactions  were 
carried  out  in  the  same  type  of  small  tubes  used  for  the  centrifugations  so  as  to 
conserve  the  antigen  preparations.  In  some  cases  more  concentrated  embryo  anti- 
gen preparations  wrere  obtained  by  homogenizing  the  embryo  parts  with  an  equal 
volume  of  supernate  from  a  previous  fractionation  of  the  same  tissues.  A  number 
of  nitrogen  determinations  were  made  upon  the  test  antigen  preparations  by  the 
nesslerization  method.  The  protein  concentrations  ranged  from  .35-1.5%  pro- 
tein, but  for  any  given  fractionation  the  protein  concentrations  were  usually  fairly 
similar  for  the  four  parts  of  the  gastrula  or  the  three  regions  of  the  larva.  The 
test  antigen  preparations  were  adjusted  to  the  same  protein  concentration  before 
any  given  set  of  serological  tests. 

RESULTS 

The  anti-brain  serum  was  not  organ-specific  and  cross-reacted  with  frog  serum, 
muscle,  heart  and  kidney.  It  was  found  that  serial  absorption  of  1.0  ml.  of  anti- 
brain  serum  with  a  total  of  1.15  ml.  of  female  frog  serum,  1.0  ml.  of  a  centrifugal 
supernate  from  a  frog  heart  homogenate  and  0.95  ml.  of  a  frog  kidney  supernate 


ANTIGEN  LOCALIZATION  AND  INDUCTION  203 

was  sufficient  to  render  the  anti-brain  serum  specific.  If  the  frog  serum  was  not 
used  in  the  absorptions  then  it  required  2.4  ml.  each  of  the  heart  supernate  and  the 
kidney  supernate  to  render  1.0  ml.  of  the  anti-brain  serum  organ  specific.  After 
absorption  the  antiserum  did  not  react  with  centrifugal  supernates  obtained  from 
adult  frog  heart,  kidney,  muscle,  liver,  spleen,  ovary  and  frog  serum. 

In  four  separate  experiments  where  the  anti-adult  male  frog  brain  serum  had 
been  absorbed  to  completion  with  male  frog  serum,  the  antiserum  showed  a  pre- 
cipitin  reaction  with  female  frog  serum.  This  was  also  found  to  hold  true  for 
similar  absorption  of  an  anti-adult  male  muscle  serum.  Upon  further  dilution  with 
male  frog  serum  the  reactivity  towards  female  frog  serum  disappeared.  If  the 
anti-brain  serum  was  diluted  1/4  with  normal  rabbit  serum  and  reacted  against 
male  and  female  frog  sera,  the  latter  reaction  occurred  at  once  while  a  faint  pre- 
cipitate did  not  appear  in  the  male  serum  reaction  for  a  period  of  twenty  minutes. 
At  the  end  of  an  hour  the  female  serum-antiserum  precipitate  was  much  heavier 
than  the  male  serum-antiserum  reaction.  It  is  known  from  agar-plate  serology 
experiments  of  Flickinger  and  Rounds  (1956)  that  an  anti-embryonic  yolk  frac- 
tion serum  gives  5  precipitate  bands  with  female  frog  serum,  and  only  two  with 
male  frog  serum,  so  that  the  female  frog  serum  apparently  contains  some  proteins 
in  higher  concentration,  or  with  different  determinate  groups,  than  those  found  in 
serum  of  the  male  frog.  It  is  more  difficult  to  understand  the  situation  in  this 
work  where  the  antibodies  have  been  formed  in  response  to  injections  of  an  adult 
organ  from  a  male  frog.  It  might  be  expected  that  absorption  to  completion  with 
male  frog  serum  would  not  only  remove  activity  to  the  male  serum  but  also  to 
female  serum,  especially  since  the  serum  contaminating  the  injection  antigen  was 
male  serum.  The  most  likely  explanation  is  that  the  common  frog  serum  proteins 
in  male  and  female  are  in  higher  concentration  in  the  female  serum  and  therefore 
the  female  serum  is  a  better  antigen  and  can  still  react  with  anti-adult  male  organ 
sera  absorbed  to  completion  with  male  frog  serum.  This  explanation  seems  more 
plausible  than  trying  to  invoke  any  type  of  pangenesis  for  adult  organ  antigens. 

Anti-brain  serum,  rendered  specific  by  absorption,  reacted  positively  with  all 
test  antigen  preparations  from  the  four  parts  of  the  gastrula  and  the  three  regions 
of  the  larva  (Fig.  1).  The  precipitates  in  every  case  were  distinct  after  twenty 
minutes  at  room  temperature;  after  twenty  minutes  at  37°  C.  and  after  twenty 
minutes  at  6°  C.  the  precipitates  were  still  of  equal  intensity.  Normal  rabbit 
serum-antigen  controls  were  negative.  No  attempt  was  made  to  titer  the  anti- 
serum  against  the  various  test  antigen  fractions  since  it  was  apparent  that  the 
antigens  reacting  with  the  brain-specific  antiserum  were  to  be  found  in  all  regions 
of  the  gastrula  and  larva  in  approximately  equal  amounts.  Absorption  of  pre- 
viously unabsorbed  anti-brain  serum  with  the  antigen  fraction  from  the  larval  gut 
(  where  no  elements  of  the  nervous  system  are  present)  also  removed  activity 
against  all  other  antigen  fractions,  thus  indicating  in  another  way  that  proteins 
1  tearing  the  determinate  groups  that  react  with  the  anti-adult  brain  serum  are 
located  in  all  regions  of  the  frog  gastrula  and  larva. 

In  order  to  see  if  different  proteins  may  be  given  off  by  different  tissues,  as 
some  of  the  work  of  Niu  (1956)  suggests,  a  series  of  forty  explantation  experiments 
was  carried  out  in  which  20-30  pieces  of  larval  brain  (stage  19)  or  larval  trunk 
(Fig.  1)  were  stripped  of  their  epidermis  and  cultured  in  the  bottoms  of  deep  well 
depression  slides  in  Niu-Twitty  solution  (1953).  After  a  week  of  explantation 


204  REED  A.  FLICKINGER 

the  culture  fluids  were  collected  from  above  the  tissues,  centrifuged,  and  the  super- 
nates  from  the  various  cultures  of  denuded  heads  were  pooled,  as  were  the  trunk 
culture  supernates.  There  was  some  slight  cytolysis  in  these  cultures  which  would 
account  for  the  release  of  some  soluble  protein  into  the  medium,  but  it  is  felt  that 
some  of  the  protein  probably  was  released  into  the  medium  by  "natural  means" 
other  than  cytolysis.  These  solutions  were  dialyzed  against  distilled  water  and 
then  evaporated  in  dialysis  bags  suspended  in  front  of  a  fan  in  a  cold  room 
(2-4°  C.).  An  individual  dialysis  bag  usually  contained  about  8  ml.  of  the  culture 
supernate  and  this  volume  was  reduced  to  about  0.2  ml.  Nitrogen  determinations 
in  one  case  revealed  the  protein  level  of  the  concentrated  supernate  to  be  0.06% 
protein.  The  absorbed  brain-specific  antiserum  gave  positive  ring  tests  of  equal 
intensity  with  both  head  and  trunk  supernates  twenty  minutes  after  the  test  anti- 
gens were  layered  over  the  antiserum.  It  would  appear  that  the  proteins  given 
off  from  the  denuded  heads  and  trunks  bear  similar  determinate  groups  that  react 
with  the  brain-specific  antibodies.  These  results  are  certainly  not  definite  enough 
to  state  that  proteins  or  nucleoproteins  (inducing  substances?)  given  off  by  differ- 
ent inductors  are  similar,  even  though  much  of  the  protein  from  the  trunk  cultures 
would  be  derived  from  the  exposed  myotomes  and  that  from  the  head  cultures  would 
come  from  the  brain,  since  the  antiserum  lacks  the  desired  degree  of  specificity. 
However,  the  data  tend  to  support  the  many  biological  examples  of  non-specificity 
of  the  inducing  agent  (Holtfreter,  1951). 

The  obvious  disadvantage  to  the  use  of  anti-brain  serum  is  its  lack  of  speci- 
ficity. Hence  it  was  decided  to  use  anti-lens  sera  where  it  is  known  that  this 
organ  has  a  greater  degree  of  organ  specificity.  This  organ  is  of  course  also  an 
induced  organ  and  the  localization  of  lens  antigen  in  the  embryo  would  be  of  value 
in  relation  to  the  induction  problem.  In  a  previous  investigation  (Flickinger  ct 
al.,  1955)  anti-frog  lens  serum  was  used  to  demonstrate  the  presence  of  lens  anti- 
gen in  the  anterior  half,  but  not  in  the  posterior  half,  of  feeding  frog  larvae.  How- 
ever, the  negative  results  do  not  necessarily  mean  an  absence  of  the  lens  antigen 
but  might  imply  only  a  reduced  amount  of  the  antigen  in  the  posterior  region 
of  the  embryo. 

As  a  first  attempt  to  improve  the  means  of  localizing  lens  antigen,  flank  ecto- 
derm, which  is  known  to  possess  the  ability  to  respond  to  an  induction  stimulus 
and  form  lens,  was  stripped  from  several  hundred  hatched  larvae  (Shumway  stage 
19),  homogenized  in  0.9%  NaCl,  and  a  supernate  fraction  obtained  by  centrifuga- 
tion.  This  extract  did  not  react  in  the  ring  tests  with  an  anti-cattle  lens  serum, 
or  an  antiserum  to  cattle  lens  a-  crystalline  (previously  provided  to  me  by  Dr.  Ten 
Gate  of  the  University  of  Amsterdam).  The  anti-a-crystalline  was  also  negative 
against  the  gastrula  ectoderm  and  chorda  mesoderm  test  antigens.  It  is  believed 
that  the  epidermis  alone  did  not  provide  sufficient  soluble  protein  to  give  a  pre- 
cipitin  reaction.  Therefore,  in  order  to  concentrate  the  test  antigen  preparations, 
it  was  decided  to  use  test  antigens  from  the  head  and  trunk  regions  of  hatched 
larvae  in  which  the  tissues  were  homogenized  with  an  equal  volume  of  supernate 
of  heads  or  trunks  from  a  previous  fractionation.  This  has  an  advantage  over  the 
use  of  posterior  halves  of  larvae  (Flickinger  et  al.,  1955)  in  that  the  less  metaboli- 
cally  active  gut  region,  containing  more  storage  protein,  is  not  included  with  the 
trunk  tissues. 

The  anti-frog  lens  serum  gave  immediate  strong  ring  tests  with  both  the  head 


ANTIGEN  LOCALIZATION  AND  INDUCTION  205 

and  trunk  antigen  preparations,  but  this  antiserum  also  reacted  with  adult  frog 
serum  and  therefore  was  not  organ-specific.  After  absorption  of  1  volume  of  anti- 
frog  lens  serum  with  l/2  volume  of  female  frog  serum  the  antiserum  was  negative 
to  serum,  and  still  gave  definite  ring  tests  after  20  minutes  with  head  and  trunk 
supernates.  This  antiserum  was  negative  against  test  antigen  preparations  from 
adult  muscle,  heart,  kidney  and  ovarian  supernate,  but  it  did  give  a  weak  positive  re- 
action with  an  adult  brain  supernate  after  one  hour.  Therefore  three  volumes  of  the 
anti-frog  lens  serum  were  absorbed  with  two  volumes  of  a  centrifugal  supernate 
obtained  from  homogenizing  adult  frog  brains  with  an  equal  volume  of  frog  serum. 
This  absorption  left  the  anti-frog  lens  serum  negative  to  serum  and  brain  and  the 
antiserum  gave  a  reaction  with  the  test  antigen  preparation  from  larval  trunks. 

The  use  of  antiserum  against  adult  frog  lens  in  localizing  the  presence  of  lens 
protein,  or  a  protein  bearing  lens  determinate  groups,  has  the  disadvantage  that 
the  antibodies  are  directed  against  determinate  groups  characteristic  of  frog  pro- 
teins as  well  as  those  characteristic  of  lens  protein.  It  would  be  preferable  to  use 
an  organ-specific  anti-cattle  lens  serum  where,  if  the  antibodies  did  react  with 
frog  embryo  test  antigens,  the  reaction  would  most  likely  be  due  to  the  lens  deter- 
minate groups  of  the  frog  antigens  reacting  with  the  antibodies  directed  against 
cattle  lens  proteins. 

The  anti-cattle  lens  serum  was  tested  against  frog  serum,  brain  and  kidney  and 
found  to  give  no  reaction.  However,  this  antiserum  gave  immediate  strong  posi- 
tive reactions  with  the  concentrated  head  and  trunk  antigens  from  the  hatched 
larva.  The  appropriate  normal  rabbit  serum-test  antigen  controls  were  negative. 
The  anti-cattle  lens  serum  did  not  react  with  test  antigen  preparations  from  the 
four  parts  of  the  early  gastrula.  This  negative  result  might  be  explained  by  the 
fact  the  gastrula  supernates  were  not  prepared  as  concentrated  antigens,  as  in 
the  case  of  the  larval  heads  and  trunks,  but  it  is  also  likely  that  the  lens  antigen 
is  present  in  lower  concentration  at  earlier  stages. 

In  looking  back  at  the  previous  work  with  this  antiserum  (Flickinger  ct  a!., 
1955)  it  was  noted  that  the  antiserum  reacted  with  supernates  from  both  the 
anterior  and  posterior  halves  of  69-hour  chick  embryos  as  well  as  with  the  ovarian 
supernate  and  hatching  larva  supernate  from  frog  embryos. 

DISCUSSION 

If  antigens  with  neural  determinate  groups  are  localized  in  all  parts  of  the 
gastrula  and  tailbud  larva,  and  particularly  in  the  more  critical  case  where  antigens 
with  lens  determinate  groups  are  situated  in  the  trunk  region  (somites,  neural 
tube,  notochord,  and  dorsal  epidermis)  of  the  early  larva,  this  indicates  that  organ 
antigens  may  be  more  disperse  for  a  certain  time  than  the  organ-forming  areas  in 
the  embryos.  Ebert  et  al.,  (1955)  have  shown  this  to  be  the  case  for  cardiac 
myosin  in  the  chick  blastoderm,  although  cardiac  myosin  did  become  localized  in 
the  heart-forming  areas  after  a  period  of  time.  Cardiac  actin  was  always  confined 
to  these  heart-forming  regions. 

It  seems  that  the  presence  of  organ  antigens  outside  their  organ-forming  dis- 
tricts does  not  invalidate  the  idea  that  one  may  in  part  characterize  a  cell,  tissue 
or  organ  by  the  structural  and  functional  (enzymes)  proteins  they  contain.  The 
wide  distribution  of  organ-specific  proteins  is  an  indication  of  the  totipotency  of 


206  REED  A.  FLICKINGER 

various  regions  of  the  embryo  which  has  been  demonstrated  by  numerous  trans- 
plantation experiments.  It  would  also  tend  to  support  the  idea  that  embryonic 
induction  could  be  any  one  of  a  number  of  stimuli  which  might  evoke  protein 
synthesis  at  a  particular  region  in  the  embryo. 

For  example :  any  specific  protein,  which  the  genetic  machinery  of  the  cells 
would  allow  to  be  synthesized,  might  be  stimulated  (induced)  to  this  synthesis  by 
a  number  of  factors.  Ribonucleic  acid  or  ribonucleo-protein  is  a  critical  component 
for  protein  synthesis  (Brachet,  1950;  Gale  and  Folkes,  1954)  and  for  embryonic 
induction  (Niu  and  Twitty,  1953).  It  is  known  that  protein  synthesis  is  an 
•endergonic  process  demanding  energy  (Fruton  and  Simmonds,  1953)  and  Miller 
(1939)  has  demonstrated  the  reversal  of  the  anterior-posterior  polarity  of  a  section 
•of  stem  of  a  regenerating  hydroid  by  raising  the  temperature  (and  therefore  the 
level  of  energy-yielding  reactions)  at  the  posterior  end  of  the  stem.  The  presence 
of  free  amino  acids  is  known  to  be  necessary  for  adaptive  enzyme  formation 
(Halvorson  and  Spiegelman,  1953)  and  it  is  known  from  the  work  of  Earth 
(1941),  Holtfreter  (1945),  Yamada  (1950)  and  Flickinger  (1958)  that  competent 
tissues  can  be  stimulated  to  differentiate  independently  of  an  induction  stimulus 
from  another  tissue  by  pH  shock  treatments  wdiich  can  dissolve  yolk  (Holtfreter, 
1946).  Flickinger  (1957)  has  emphasized  that  the  solubilization  of  yolk  protein, 
which  provides  the  material  from  which  cytoplasmic  proteins  are  synthesized,  can 
be  a  causal  step  in  embryonic  induction.  Even  after  specific  cytoplasmic  proteins 
have  been  synthesized  the  provision  of  an  enzymatic  substrate,  as  in  Wilde's  (1955) 
conversion  of  gastrula  ectoderm  cells  into  melanophores  by  giving  them  phenylala- 
nine,  could  be  considered  an  embryonic  induction.  Activators  or  inhibitors  of  an 
enzymatic  reaction  might  then  also  act  in  an  induction  system.  Viewed  in  this 
manner  there  may  be  multiple  aspects  of  embryonic  induction  with  any  factor,  or 
combination  of  factors,  that  would  facilitate  the  synthesis,  or  activity,  of  specific 
proteins  being  considered  an  inductor.  This  is  somewhat  similar  to  the  case  shown 
by  Spiegelman  and  Reiner  (1947)  where,  under  conditions  optimal  for  growth 
and  protein  synthesis,  adaptive  enzymes  may  be  formed  without  the  substrate  or 
inducer  being  present. 

If  a  sub-differentiation  threshold  level  of  any  given  specific  protein,  or  proteins, 
exists  throughout  the  embryo,  it  may  be  that  a  preferential  hierarchy  of  cell  and 
tissue  specialization  exists  such  that  when  conditions  become  optimal  for  protein 
synthesis  in  a  given  region  of  the  embryo  then  a  specific  cell  type  or  tissue  will  be 
formed.  That  there  is  some  kind  of  preferential  hierarchy  can  be  seen  from  the 
tendency  for  gastrula  ectoderm  activated  by  sub-lethal  cytolysis  to  form  forebrain 
structures  (Holtfreter,  1944).  This  tendency  to  form  head  structures  first  can 
be  seen  in  Sabella  regeneration  (Berrill,  1931)  where  the  most  anterior  part  forms 
first  and  then  fills  in  the  missing  parts.  From  studies  of  regeneration  and  embry- 
onic development  it  is  evident  that  differentiation  occurs  time-wise  along  anterior- 
posterior  and  dorsal-ventral  axes  with  the  anterior  and  dorsal  differentiations 
usually  preceding  the  posterior  and  ventral  ones.  Possibly  these  anterior-posterior 
and  dorsal-ventral  patterns  of  specialization  are  due  to  gradients  of  factors  which 
favor  protein  synthesis,  and  that  certain  types  of  cell  or  tissue  specialization  are 
favored  when  conditions  for  protein  synthesis  become  optimal.  For  example, 
Flickinger  (1957)  has  hypothesized  that  the  primary  organizer  area  forms  where 
the  first  and  most  active  conversion  of  yolk  to  cytoplasm  occurs.  The  biological 


ANTIGEN  LOCALIZATION  AND  INDUCTION  207 

totipotency  of  most  parts  of  the  embryo,  and  the  serological  evidence  tend  to  indi- 
cate that  some  organ-specific  proteins  may  be  more  widely  distributed  than  the 
corresponding  specific  organ-forming  areas.  Possibly  the  postulated  sequential 
protein  synthesis,  and  cell  specializations  and  growth  that  may  depend  upon  these 
syntheses,  are  of  a  self-limiting  type  as  postulated  by  Rose  (1952,  1957)  and 
Weiss  (1952).  The  question  why  the  synthesis  of  a  given  type  of  protein  may  be 
favored  when  conditions  become  optimal  for  protein  synthesis  is  indeed  puzzling. 
It  is  apparently  not  due  to  a  purely  qualitative  distribution  of  protein,  or  to  unequal 
nuclei  (Briggs  and  King,  1952).  It  might  be  ascribed  to  a  quantitative  distribu- 
tion of  various  organ-specific  proteins,  or  nucleoproteins,  but  although  there  are 
gradient-wise  distributions  of  soluble  proteins  and  ribonucleoproteins,  there  is  as 
yet  no  evidence  concerning  the  specificity  of  these  compounds.  Another  possi- 
bility might  be  a  preferential  sequence  of  activity  of  different  specific  genes. 

It  may  be  well  at  this  time  to  review  the  idea  of  Driesch  that  the  fate  of  a  cell  or 
tissue  is  a  function  of  its  position.  It  is  well  known  that  undetermined  embryonic 
cells  and  tissues  tend  to  "fit  in"  to  the  particular  locale  in  which  they  find  them- 
selves. The  fact  that  the  nuclei  of  the  cells  of  the  determined  neural  plate  are 
apparently  undifferentiated  and  still  able  to  promote  complete  development  when 
injected  into  the  enucleated  egg  (King  and  Briggs,  1954)  and  the  apparent  deter- 
mination of  the  whole  mouse  embryonic  shield  before  the  determination  of  its 
individual  constituent  cells  (Grobstein,  1952)  argues  for  some  sort  of  "supra- 
cellular  patterning,"  perhaps  of  a  polar  or  axial  type  (Child,  1941;  Rose,  1957), 
which  precedes  cell  specialization.  This  is  a  question  which  deserves  a  good  deal 
of  attention  from  embryologists. 

SUMMARY 

1.  Anti-adult  male  frog  brain  and  muscle  sera  absorbed  to  completion  with  male 
frog  serum  still  react  with  female  frog  serum.     It  is  believed  that  serum  proteins 
common  to  the  male  and  female  may  be  in  higher  concentration  in  the  female  serum 
and  hence  account  for  this  reaction. 

2.  Anti-brain  serum,  rendered  organ-specific  by  absorption,  reacted  positively 
with  test  antigen  preparations  from  four  regions  of  the  early  frog  gastrula  (ecto- 
derm, dorsal  mesoderm,  ventral  mesoderm,  and  endoderm)   and  three  regions  of 
the  hatched  frog  larva  (head,  trunk  and  gut).     The  proteins  bearing  brain  deter- 
minate groups  are  apparently  situated  throughout  the  embryo  at  these  stages. 

3.  The  organ-specific  anti-brain  serum  gave  positive  precipitin  reactions  with 
culture  supernates  from  both  larval  heads  and  trunks  which  had  been  denuded  of 
their  epidermis  and  explanted  for  a  period  of  a  week.     The  proteins  given  off  by 
these  cultured  heads  and  trunks  bear  similar  determinate  groups  that  react  with 
the  brain  antibodies. 

4.  An    absorbed    organ-specific    anti-frog    lens    serum,    and    an    organ-specific 
anti-cattle  lens  serum,   reacted  with  concentrated  test  antigen  preparations  from 
both  the  heads  and  trunks  of  hatched  frog  larvae.     It  seems  that  lens  antigen,  or 
protein  bearing  lens  determinate  groups,  is  localized  in  areas  other  than  the  lens- 
forming  region. 

5.  The  significance  of  these  results  is  discussed. 


208  REED  A.  FLICKINGER 

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STUDIES  ON  NEUROMUSCULAR  TRANSMISSION  IN  LIMULUS 

GRAHAM  HOYLE * 
Marine  Biological  Laboratory,  Woods  Hole,  Mass.2 

Among  invertebrates  only  certain  crustaceans  and  insects  have  been  the  sub- 
jects of  detailed  study  in  regard  to  neuromuscular  mechanisms.  There  have 
proved  to  be  very  considerable  differences  between  the  various  arthropod  mecha- 
nisms encountered  on  the  one  hand  (Wiersma,  1957 — Crustacea;  Hoyle,  1957 — 
insects)  and  those  of  vertebrates  on  the  other  (Fatt,  1954).  The  differences  con- 
tribute to  the  difficulty  in  arriving  at  a  general  concept  of  the  way  in  which 
coupling  between  excitation  of  the  surface  membrane  of  the  muscle  fiber,  which 
is  achieved  by  nervous  action,  and  shortening  of  the  contractile  material,  is  brought 
about.  But  they  also  show  that  certain  favored  hypotheses  in  regard  to  vertebrate 
muscle  are  either  of  only  limited  applicability  for  muscle  as  a  whole,  or  are  wide 
of  the  mark.  There  is  a  strong  difference  of  opinion  regarding  the  relevance  of 
the  electrical  activity  of  the  muscle  fiber  membrane  in  the  process.  Most  recent 
authors  (cf.  Sten-Knudsen,  1954;  Huxley,  1956)  have  regarded  the  contractile 
machinery  as  being  in  some  way  connected  with  the  membrane  potential.  Some 
(e.g.,  Csapo  and  Suzuki,  1957)  believe  that  contraction  is  initiated  by  current  flow 
resulting  from  membrane  action  potentials.  For  the  Crustacea,  it  has  been  found 
necessary  to  postulate  a  separate  coupling  mechanism  within  the  muscle  fiber 
which  may  be  activated  differently  by  neuromuscular  transmitter  action  in  different 
cases.  In  some  (Hoyle  and  Wiersma,  1958b)  there  may  be  a  direct  action  by  the 
transmitter  substance  on  the  coupling  mechanism,  the  electrical  intermediate  (or 
propagation)  stage  having  been  by-passed.  In  others,  electrical  changes,  or  the 
ionic  fluxes  associated  with  them,  affect  the  coupling  mechanism. 

From  this  it  seems  likely  that  in  the  elucidation  of  the  general  problems  of 
excitation-contraction  coupling,  the  arthropods  will  provide  favorable  material. 
In  them  single  muscle  fibers  are  innervated  by  more  than  one  motor  axon,  each 
having  different  motor  effects,  and  in  the  Crustacea  there  are  also  inhibitory  axons 
which  uncouple  the  excitatory  action.  In  many  arthropod  systems  the  unit  of 
contraction  is  not  an  all-or-nothing  twitch,  and  contractions  are  minutely  graded. 
This  difference  between  arthropod  muscle  and  ordinary  skeletal  muscle  of  verte- 
brates is  probably  attributable  to  the  absence  of  propagated  muscle  action  poten- 
tials in  the  former.  In  spite  of  their  potential  interest,  and  the  variety  of  their 
mechanisms,  several  major  subdivisions  of  the  phylum  remain  unexplored,  no 
arachnid,  for  example,  having  been  examined  in  regard  to  its  detailed  neuro- 
muscular mechanisms.  It  seems  desirable,  therefore,  to  have  information  regard- 
ing the  motor  mechanisms  of  the  particularly  interesting  primitive  arachnids,  the 
Xiphosura.  Accordingly  a  preliminary  study  has  been  made  on  Limuhis  polyphe- 
mus  Latr.  and  has  revealed  several  interesting  features  which  are  reported  here. 

1  Fellow  of  the  Rockefeller  Foundation. 

-  Permanent  address :  Department  of  Zoology,  University  of  Glasgow,  Scotland. 

209 


210 


GRAHAM  HOYLE 


METHODS 

The  walking  legs,  except  the  specialized  fifth  pair,  have  been  examined  from 
specimens  16"-22"  long,  obtained  at  Woods  Hole,  with  a  view  to  finding  suitable 
nerve-muscle  preparations.  The  legs  were  severed  by  a  quick  snip  of  the  coxo- 
trochanteral  joint.  A  few  of  the  leg  muscles  can  be  used,  in  particular  the  closer 
of  the  claw  (adductor  or  depressor  of  the  tarsus)  and  the  flexor  (levator)  tibiae 
(situated  in  the  patella).  The  present  studies  were  carried  out  entirely  on  the 
claw  closer.  This  muscle  exhibits  in  the  freshly-excised  leg  a  remarkable  pseudo- 
reflex.  If  the  inside  of  the  pollex  (fixed  extension  of  the  tibia)  is  gently  stroked, 
the  clawr  closes  sharply.  This  reflex  can  be  obtained  repeatedly  for  up  to  15 
minutes  or  so  after  removal  of  the  leg.  It  seems  highly  improbable  that  any 
nervous  machinery  of  true  synaptic  type  can  be  present  in  the  isolated  leg  to 
account  for  this  curious  phenomenon.  Similar  phenomena  have  been  described 


Electrodes 
for  stimulating 
axon   bundle 


FIGURE  1.  Drawing  of  the  preparation  seen  from  above.     The   leg  is   placed  in  a   trough   cut 
in  a  wax  block.     The  opener  of  the  claw  has  been  removed,  exposing  the  closer  muscle. 

in  excised  crustacean  legs  in  which  stretching  of  the  chela,  for  example,  can  lead 
to  its  opening.  Wiersma  (unpublished)  has  suggested  as  an  explanation  of  the 
crustacean  responses  that  following  excision  the  excitability  of  the  cut  ends  of  the 
motor  nerves  is  raised  to  such  an  extent  that  an  ephaptic  transmission  occurs  from 
adjacent  sensory  axons. 

To  make  a  preparation,  the  excised  leg  was  laid  in  a  sculptured  trough  of  wax 
and  stapled  into  position  with  the  tarsus  uppermost.  The  main  leg  nerve  can 
then  be  easily  exposed  in  the  femur  by  cutting  away  the  shell  and  removing  part 
of  the  extensor  patella  muscle.  The  nerve  has  no  surrounding  sheath  and  very 
little  connective  tissue  so  that  it  can  easily  be  split  into  bundles.  These  may  be 
stimulated  in  turn  and  any  having  an  effect  on  the  tarsus  retained,  the  rest  being 
cut  away.  The  retained  bundles  can  then  be  split  again  until  either  very  small 
bundles,  or  eventually  single  axons,  remain. 

In  this  way  it  was  ascertained  that  the  closers  of  the  claws  of  legs  I-IV  are 
innervated  by  two  motor  axons.  No  inhibitory  axons  were  found.  In  this  re- 
spect Linuihis  resembles  the  insects  rather  than  the  crustaceans. 

There  is  no  tested  physiological  saline  for  Linnthts  so  filtered  sea  water  was 


LIMULUS  NEUROMUSCULAR  TRANSMISSION 


211 


used  to  bathe  the  preparation.  Cole  (1940)  has  analyzed  the  haemolymph  and 
found  that  the  mineral  composition  approximates  very  closely  indeed  that  of  the 
local  sea  water  in  two  different  localities,  one  of  which  was  Woods  Hole.  Since 
the  present  work  was  done,  a  physiological  saline  has  been  developed  for  the 
Japanese  horseshoe  crab,  Tachypleus  tridentatiis  (Kikuchi  and  Tanaka,  1957). 

At  this  stage  a  strip  of  shell  was  carefully  snipped  away  from  the  margin  of 
the  tibia  in  order  to  expose  the  outer  edge  of  the  opener  muscle  (abductor  tarsi). 
The  opener  apodeme  was  then  cut  close  to  the  tarsus,  grasped  with  forceps,  lifted 
and  stretched  until  the  whole  muscle  came  away.  This  leaves  the  V-shaped  closer 
muscle  exposed,  with  its  innervation  intact. 

The  pollex  was  fixed  in  a  hole  in  the  wax  block  and  the  tip  of  the  tarsus  was 
attached  by  a  thread  to  an  electromechanical  transducer.  Pairs  of  fine  silver  wires 
were  micromanipulated  onto  the  exposed  nerve  bundles.  A  drawing  of  the  prepa- 
ration, seen  from  above,  is  presented  in  Figure  1. 

The  muscle  fibers  are  of  fairly  uniform  diameter  but  only  25-40  ^  thick,  i.e., 
they  are  appreciably  thinner  than  many  insect  muscle  fibers  and  very  much  thinner 
than  those  of  the  larger  decapod  crustaceans.  Glass  capillary  micro-electrodes, 
filled  with  3  M  KC1,  were  used  to  record  trans-membrane  potentials  from  muscle 
fibers  of  the  claw  closer.  The  nerve  bundles  were  stimulated  with  brief  rectangu- 
lar pulses  isolated  by  radiofrequency  coupling  units.  Display  was  conventional. 

RESULTS 

In  the  more  vigorous  preparations  a  single  stimulus  applied  to  either  of  the 
two  nerve  fibers  evokes  in  each  case  a  small  twitch.  Repetitive  stimuli  lead  to 
partial  and  complete  tetani.  The  mechanical  response  to  one  of  the  two  axons  is, 
however,  always  larger  than  the  other,  and  at  a  given  frequency  of  stimulation 
also  appears  slower.  Hence  the  two  axons  may  be  referred  to  as  "fast"  and 
"slow"  according  to  the  nature  of  the  contraction  evoked,  as  is  customary  in  deal- 


lOOrnsec 


J^     i*       i*     r^         r 


FIGURE  2.  "Spontaneous"  potentials.  Four  records  from  claw-closer  muscle  fibers  of  a 
fresh-excised  Limidus  leg,  showing  recurring  potentials.  The  deflections  marked  +  are  attrib- 
uted to  discharges  in  the  fast  axon ;  those  marked  •  to  the  slow  axon.  The  single  spike 
response  in  b  was  associated  with  a  twitch  which  must  have  caused  the  electrode  to  be  jerked 
out  of  the  fiber.  The  upper  trace  in  each  record  marks  the  zero  baseline  and  the  lower  one 
the  internal  potential  recorded  with  a  3  M  KCl-filled  glass  capillary  micro-electrode. 


212  GRAHAM  HOYLE 

ing  with  crustacean  motor  nerve  fibers  (Wiersma,  1941).  The  corresponding 
responses  are  then  called  fast  and  slow,  respectively. 

"Spontaneous"  responses.  When  the  preparation  is  very  fresh,  discharges 
originating  in  the  hypersensitive  cut  ends  of  the  axons  lead  to  spontaneous  "tone"  in 
the  closer  muscle  and  contractions  which  cause  small  movements  of  the  tarsus. 
If  a  micro-electrode  is  inserted  at  random  into  a  muscle  fiber  of  the  closer  at  this 
time,  recurring  electrical  potentials  of  small  size  are  seen  (Fig.  2).  The  resting 
potentials  of  the  muscle  fibers  are  of  small  magnitude,  ranging  from  35-55  mV. 
The  peak  amplitudes  of  the  recurring  potentials  are  from  0.5  mV  to  a  maximum 
of  25  mV  in  different  fibers.  In  any  one  fiber  they  are  clearly  of  two  distinct  sizes, 
the  smaller  being  due  to  the  slow  axon  and  the  larger  to  the  fast.  Single  small 
potentials  are  not  usually  associated  with  visible  twitches  although  in  the  more 
vigorous  preparations,  when  they  occurred  singly,  this  was  the  case,  and  twitches 
were  seen.  A  small  proportion  of  muscle  fibers  gave  "fast"  potentials  which  were 
compound,  i.e.,  they  had  an  initial  component  resembling  an  ordinary  end-plate- 
potential  (e.p.p.)  giving  rise  to  a  small  spike  response  (Fig.  2b). 

The  slozv  responses.  Responses  attributable  to  the  "slow"  axon  could  be 
observed  in  about  60%  of  those  muscle  fibers  in  which  any  appreciable  electrical 


o  • 

mV 


SLOW 
40  |        i        *"        I          i 


FAST 


lOOmsec 


lOO-xsec 


FIGURE  3.  Potentials  and  tension  due  to  single  excitations  applied  to :  a,  the  slow  axon 
and  b,  the  fast  axon.  Left  hand  side :  electrical  responses  from  the  same  single  muscle  fiber. 
Right  hand  side :  mechanical  responses  of  whole  muscle  recorded  at  tarsal  tip. 

change  could  be  obtained  during  stimulation  of  both  fast  and  slow  axons  (usually 
the  bundles  containing  them)  together.  The  single  electrical  response  was  always 
a  very  small  one  resembling  a  small  e.p.p.  It  will  be  referred  to  as  a  junctional 
potential  (j.p.)  rather  than  an  e.p.p.  since  nothing  is  known  of  the  nature  of  the 
nerve  terminals  in  Limulus  muscle.  To  distinguish  it  from  the  corresponding 
response  to  the  "fast"  axon  it  will  be  called  a  slow  junctional  potential  (s.j.p.) 
The  long  latency  following  the  stimulus  artifact,  which  is  apparent  in  the  records, 
is  clue  largely  to  the  conduction  time  of  the  nerve  impulse  along  the  nerve  in  the 
femur  and  patella  into  the  tibia. 

The  s.j.p.'s  rise  to  a  peak  in  12-18  msec,  and  decay  in  about  60  msec.  The 
largest  one  found  had  a  peak  amplitude  of  5  mV.  Although  no  tension  was 
usually  recorded  at  the  tip  of  the  tarsus  during  stimulation  of  the  slow  axon  with 
a  single  shock,  some  preparations  did  show  a  small  twitch,  giving  not  more  than 


LIMULUS  NEUROMUSCULAR  TRANSMISSION 


213 


o- 


4O 


lOOmsec 


II 

III 

FIGURE  4.  The  responses  to  short  trains  of  stimuli  in  three  different  muscle  fibers  (i-iii). 
(i)  Slow  axon.  Three  responses  from  the  same  fiber  showing  the  summation  of  s.j.p.'s  and 
the  small  degree  of  facilitation.  A  small  spike  arises  in  c  from  the  plateau  of  depolarization, 
(ii  and  iii)  Fast  axon.  a,  low  frequency;  b-d,  higher  frequency.  Successive  steps  (f.j.p.'s) 
are  progressively  larger  (facilitation).  Summation  is  evident;  occasional  spikes  arise  from 
the  depolarization  plateau. 

0.5  gm.  tension  at  the  tip  of  the  tibia  (Fig.  3).  On  repetitive  stimulation  appre- 
ciable tension  developed,  increasing  with  increasing  frequency  of  stimulation  up 
to  a  maximum  of  just  over  50  gm.  at  200  per  second.  Thus  the  tetanus/twitch 
ratio  was  more  than  100:  1.  The  s.j.p.'s  initially  increased  in  magnitude  by  two 
or  three  times  during  a  train  of  stimulation,  a  phenomenon  usually  referred  to  as 
facilitation,  but  later  diminished  as  they  also  summated  to  give  a  plateau  of  de- 
polarization. From  the  plateau  occasionally  a  small  spike  arises  (Fig.  4i,  c). 

The  fast  response.  The  fast  axon  evoked  electrical  responses  in  most  of  the 
muscle  fibers  penetrated.  They  were  often  very  small,  but  they  were  always  larger 


OT 


mV 


40-L 


lOOmsec 


FIGURE  5.  Electrical  response  of  one  fiber  and  total  mechanical  response  at  tip  of  tarsus 
to:  single  (a)  and  paired  (b)  stimulation  of  the  fast  axon.  Note  that  the  f.j.p.  is  followed 
by  a  small  spike  in  each  case. 


214 


GRAHAM  HOYLE 


than  the  corresponding  slow  responses,  when  these  were  seen,  in  the  same  fibers. 
There  was  no  overlap  of  s.j.p.  and  f.j.p.  magnitudes  in  individual  libers,  such  as 
was  found  in  several  muscles  of  decapod  crustaceans  (Hoyle  and  Wiersma,  1958a). 
The  typical  response  to  a  single  shock  is  shown  in  Figure  2.  The  response,  like 
the  s.j.p.,  is  of  end-plate-potential  type  and  will  be  referred  to  as  the  fast  junctional 
potential  (f.j.p.).  The  rise-time  of  the  f.j.p.'s  was  usually  about  the  same  as  that 
of  the  s.j.p.'s,  i.e.,  12-18  msec,  and  the  decay  likewise  about  60  msec.  But  occa- 
sionally an  f.j.p.  had  a  faster  rise-time  of  only  5-6  msec,  and/or  a  faster  decay  of 
about  40  msec.  In  some  fibers  the  f.j.p.  leads  to  a  small  spike  of  10-15  mV. 
The  larger  f.j.p.'s  reached  a  peak  amplitude  of  11  mV. 


so 


OT 


100 


\OO<jm 

FIGURE  6.  Tension  recorded  during  stimulation  of  the  fast  axon  to  show  the  development 
of  tetanus  at  the  various  frequencies  indicated. 

When  pairs  of  shocks  are  applied  to  the  axon  the  mechanical  responses  sum- 
mate  and  also  show  facilitation  (Fig.  5)  as  the  interval  between  the  shocks  is 
reduced.  If  the  first  f.j.p.  evokes  a  spike  then  the  second  one,  at  intervals  up  to 
200  msec.,  seldom  does  so  or  gives  a  much  smaller  one,  i.e.,  there  is  a  long  rela- 
tively refractory  period  for  the  spike.  If  the  first  f.j.p.  is  of  relatively  large  size 
but  does  not  give  a  spike  then  the  second  usually  evokes  one.  When  the  paired 
shocks  are  applied  regularly,  at  a  low  repetition  rate,  the  character  of  the  response 
is  seen  to  change  from  time  to  time.  Thus  the  first  f.j.p.  will  soon  fail  to  evoke 


LIMULUS  NEUROMUSCULAR  TRANSMISSION 


215 


a  spike  but  the  second  will  lead  to  one  and  vice  versa,  the  process  being  reversed 
again  after  a  while.  The  spike  mechanism  either  fatigues  very  easily  or  it  is  a 
very  labile  response. 

With  prolonged  repetitive  stimulation,  whether  there  is  spiking  or  not,  a 
plateau  of  depolarization  builds  up  and  is  maintained.  Brief  bursts  of  stimulation 
illustrate  the  way  in  which  the  plateau  builds  up  (Fig.  4ii,  b-d).  At  the  higher 
frequencies  spikes,  taking  off  from  the  depolarization  plateau,  may  just  reach  and 
occasionally  overshoot  the  zero  baseline  (Fig.  4i,  c).  The  total  tetanus  tension  and 
also  the  rate  of  rise  of  tension,  continue  to  increase  with  increasing  frequency  of 
stimulation  up  to  a  maximum  at  about  200  per  second  (Fig.  6).  The  tetanus 
tension  measured  at  the  tip  of  the  tarsus  then  exceeds  100  gm.  The  tetanus/twitch 
ratio  is  ordinarily  about  30 :  1  but  it  increases  as  the  preparation  ages,  eventually 
becoming  infinite  as  the  twitch  response  just  fails. 


-O 


normal 
&    6O 


sec 


30 
20 

IO&  15 
5 


lOOmsec 

FIGURE  7.  Records  of  tension  developed  in  response  to  a  single  shock  applied  at  various 
intervals   (as  indicated  in  seconds)   after  a  brief  tetanus   (100  shocks  at  100/sec.)- 

The  electromechanical  transducer  used  was  a  fluid  potentiometer.  When  lightly- 
loaded  it  recorded  the  twitch  tensions  associated  with  the  slow  axon  of  0.5  gm. 
and  less.  Under  these  loading  conditions  the  tension  records  did  not  have  the 
usual  shape  for  a  twitch  but  instead  showed  a  plateau  of  tension.  This  may  have 
been  caused  in  part  by  sluggishness  of  the  potentiometer,  but  the  plateau  was  too 
long  to  be  due  entirely  to  this.  Thus,  in  the  absence  of  a  large  restoring  force 
(the  twitch  is  normal  in  appearance  with  a  spring  attached  to  the  load),  tension 
is  maintained  for  about  half  a  second,  after  which  relaxation  occurs. 

Post-tetanic  potentiation.  Following  a  very  brief  tetanus  there  is  an  enormous 
potentiation  of  the  twitch  response  which  is  regularly  5  times,  and  may  be  as  much 
as  7  times,  greater  than  the  normal  twitch  tension.  The  effect  subsides  gradually 
over  a  period  of  45-60  seconds  (Fig.  7).  The  intracellular  leads  showed  no 
electrical  concomitant  of  this  enhancement  in  the  individual  muscle  fibers  examined. 
It  would  of  course  be  necessary  to  examine  a  large  number,  particularly  in  respect 
to  increased  tendency  to  give  spikes,  in  order  to  be  sure  that  there  was  no  signifi- 
cant electrical  effect  and  this  has  not  been  attempted.  In  the  fibers  examined  the 
f.j.p.'s  were  facilitated  following  the  tetanus  but  only  for  a  few  seconds,  a  fraction 
of  the  time  during  which  the  tension  is  potentiated. 


216  GRAHAM  HOYLE 

DISCUSSION 

From  the  electrical  activity  recorded  in  various  muscle  fibers  of  the  closer  of 
the  claw  of  the  walking  leg  of  Limit! us  it  may  be  inferred  that  the  pattern  of 
innervation  is  substantially  similar  to  that  found  in  doubly-innervated  crustacean 
muscles  and  non-specialized  insect  muscles.  That  is,  most  of  the  muscle  fibers 
are  themselves  innervated  by  both  slow  and  fast  motor  axons  (polyneural  innerva- 
tion). It  has  not  been  established  in  this  investigation  that  the  innervation  is  also 
multi-terminal,  as  it  is  in  those  insects  and  crustaceans  which  have  been  examined 
closely,  i.e.,  that  the  axons  make  synapse  with  the  muscle  fibers  at  several  points 
along  their  length. 

The  rather  low  resting  potentials  and  small  size  of  the  electrical  responses  might 
suggest  that  the  muscles  deteriorate  following  excision  of  the  limb.  But  for  periods 
up  to  two  hours  in  which  the  preparation  was  used  there  usually  was  no  (further?) 
decline  in  their  value.  Thereafter,  decline  was  fairly  rapid.  Tetanus  tension 
measured  at  the  tarsal  tip  in  the  preparation  is  at  least  as  great  as  that  which  can 
be  obtained  by  evoking  reflex  closure  of  the  claw  in  the  intact  animal. 

Both  the  s.j.p.'s  and  the  f.j.p.'s  differ  in  peak  amplitude  in  different  fibers 
although  the  fast  is  always  larger  than  the  slow.  Their  rise  and  decay  times  have 
not  been  determined  critically  in  the  present  experiments,  partly  because  they  were 
somewhat  variable.  In  some  fibers  the  fast  response  had  both  a  faster  rise  time 
and  a  faster  decay  time  than  the  slow,  but  this  was  not  often  encountered  and  in 
most  cases  they  had  similar  values.  There  was  no  evidence  of  a  "paradox"  situa- 
tion similar  to  that  found  in  certain  Crustacea  (Hoyle  and  Wiersma,  1958b)  ; 
i.e.,  the  slow  axon  did  not  give  tension  at  lower  frequencies  of  excitation  than  those 
which  just  failed  for  the  fast.  There  was,  in  fact,  unlike  the  situation  in  many 
crustaceans  (Hoyle  and  Wiersma,  1958a)  nothing  to  indicate  that  the  slow  and 
fast  transmitter  substances  need  be  regarded  as  qualitatively  different  chemically. 
The  preliminary  results  could  be  interpreted  on  the  basis  of  quantitatively  different 
extents  of  release  of  one  transmitter  substance  from  the  terminals  of  the  fast  and 
slow  axons. 

Each  junctional  potential  attains  a  constant  height  over  long  periods  of  inter- 
mittent stimulation,  but  the  secondary,  small  spike  responses  are  extremely  un- 
predictable in  their  appearance  and  magnitude.  They  arise  only  from  the  larger 
f.j.p.'s  or  from  the  plateaux  of  depolarization  in  tetanus.  But  they  cannot  be  said 
to  appear  at  a  particular  level  of  membrane  potential.  They  may  be  present  on 
one  occasion  and  absent  on  the  next  although  the  same  j.p.  deflection  is  reached 
in  both.  Also,  they  occur  randomly,  not  synchronously,  in  the  population  of  fibers 
so  that  it  cannot  be  determined  whether  or  not  their  appearance  leads  to  extra 
tension. 

Facilitation  of  junctional  potentials  is  present  in  both  fast  and  slow  systems, 
quite  markedly  in  some  fibers,  hardly  at  all  in  others.  It  is  more  marked  than  it 
appears  in  the  records.  The  long  time-course  ensures  that  there  is  summation 
even  at  low  frequencies  of  stimulation.  Hence  the  later  j.p.'s  in  a  train  appear 
at  lower  and  lower  levels  of  membrane  potential.  Since  the  magnitude  of  a  j.p. 
is  proportional  to  the  magnitude  of  the  membrane  potential,  they  thus  appear 
quite  a  bit  smaller  than  they  would  if  the  same  amount  of  transmitter  action  occurred 
at  the  normal  resting  potential  level. 


LIMULUS  NEUROMUSCULAR  TRANSMISSION  217 

The  total  tension  is  related  to  the  extent  of  maintained  depolarization,  in  the 
randomly-selected  muscle  fibers  studied,  during  tetanus  at  different  frequencies. 
But  the  enhanced  tension  which  occurs  in  the  period  following  shortly  after  a 
tetanus  is  not  reflected  in  increased  depolarization.  This  argues  against  there 
being  a  simple  causal  connection  between  membrane  potential  and  tension.  There 
is  probably  a  connection  between  the  strong  post-tetanic  potentiation  and  the  fact 
that  there  is  a  high  tetanus/twitch  ratio,  but  both  must  be  attributed  to  intra- 
muscle-fiber  events  rather  than  to  neuromuscular  junctional  ones.  All  these  facts 
make  it  seem  probable  that  further  and  more  detailed  investigations  of  neuro- 
muscular transmission  in  Liniuhis  will  make  valuable  contributions  to  our  under- 
standing of  excitation-contraction  coupling  in  muscle. 

I  wish  to  thank  Professor  H.  Grundfest  for  the  generous  facilities  which  he 
placed  at  my  disposal  in  his  laboratory  at  Woods  Hole. 

SUMMARY 

1.  The    electrical   responses    occurring   in    single   muscle    fibers    of   the    closer 
muscles  of  the  chelae  of  the  walking  legs  of  Litnuliis  have  been  studied  with  the 
aid  of  intracellular  electrodes  and  electrical  stimulation  of  the  motor  axons.     At 
the  same  time  the  total  tension  of  the  muscle  was  recorded  at  the  tarsal  tip. 

2.  The  muscle  is  supplied  by  only  two  motor  nerve  fibers,  one  of  which   (the 
"fast"  axon)  evokes  larger  mechanical  and  electrical  responses  than  does  the  other 
(the  "slow"  axon). 

3.  No  inhibitory  nerve  fiber  was  found. 

4.  The  electrical  responses  consist  typically  of  junctional  potentials  resembling 
small  end-plate  potentials.     The  fast  junctional  potentials  may  give  rise  to  small 
spike  potentials. 

5.  On  repetitive  stimulation  both  axons  give  rise  to  plateaux  of  depolarization, 
from  which  small  spikes  may  arise. 

6.  The  mechanical  responses  consist  of  very  small  twitches  to   single  shocks 
and  tetani  to  repetitive  excitation.     The  tetanus/twitch  ratio  is  more  than  30 :  1  for 
the  fast  axon,  more  than  100:  1  for  the  slow  axon. 

7 .  There  is  post-tetanic  potentiation  of  the  twitch  response  of  up  to  5  times 
in  the  mechanical  response  to  a  single  shock  applied  to  the  fast  axon.     This  decays 
slowly  over  a  period  of  about  a  minute. 

LITERATURE  CITED 

COLE,  W.  H.,  1940.  The  composition  of  fluids  and  sera  of  some  marine  animals  and  of  the 
sea  water  in  which  they  live.  /.  Gen.  Physiol..  23  :  575-584. 

CSAPO,  A.,  AND  T.  SUZUKI,  1957.  A  preliminary  note  on  excitation  contraction  coupling. 
Proc.  Nat.  Acad.  Sci.,  43:  278-281. 

FATT,  P.,  1954.     Biophysics  of  junctional  transmission.     Physiol.  Rev.,  34 :   674-710. 

HOYLE,  G.,  1957.  Nervous  control  of  insect  muscles.  Recent  Advances  in  Invertebrate  Physi- 
ology. University  of  Oregon  Publications ;  pp.  304. 

HOYLE,    G.,    AND    C.    A.    G.    WIERSMA,    1958a.     Neuromuscular    transmission    in    Crustacea. 

I.  Excitation.     /.  Physiol.,  in  press. 

HOYLE,    G.,    AND    C.    A.    G.    WIERSMA,    1958b.     Neuromuscular    transmission    in    Crustacea. 

II.  Coupling  of  membrane  potential  to  contraction.     /.  Physiol.,  in  press. 


218  GRAHAM  HOYLE 

HUXLEY,  A.  F.,  1956.  Interpretation  of  muscle  striation :  evidence  from  visible  light  micros- 
copy. Brit.  Mcd.  Bull.,  12:  167-170. 

KIKUCHI,  R.,  AND  I.  TANAKA,  1957.  Physiological  saline  solution  for  Japanese  horseshoe  crab, 
Tachypleus  tridcntatns.  Annot.  Zool  Jap.,  30:  177-180. 

STEN-KNUDSEN,  O.,  1954.  The  ineffectiveness  of  the  "window-field"  in  the  initiation  of  muscle 
contraction.  /.  Pliysiol..  125:  396-404. 

WIERSMA,  C.  A.  G.,  1941.     The  efferent  innervation  of  muscle.     Biol  Syinp.,  3  :  259-289. 

'\YIERSMA,  C.  A.  G.,  1957.  Neuromuscular  mechanisms.  Recent  Advances  in  Invertebrate 
Physiology.  University  of  Oregon  Publications ;  pp.  304. 


THE  TOXICITY  OF  PHYSALIA  NEMATOCYSTS  1 

CHARLES  E.  LANE  AND  ELEANOR  DODGE 

The  Marine  Laboratory,  University  of  Miami,  Miami  49,  Florida 

The  siphonophore  Physalia  physalis  (Portuguese-man-of-war)  possesses  a  well- 
merited  evil  reputation  throughout  its  geographical  range.  Contact  with  the  tenta- 
cles of  this  animal  is  always  painful  to  man  and  may  result  in  vasomotor  dysfunction 
and  collapse.  Although  toxic  substances  have  been  previously  isolated  from 
Physalia  tentacles  (Richet  and  Portier,  1936),  there  appears  to  have  been  no 
examination  of  the  toxic  compounds  which  originate  specifically  within  the 
nematocyst. 

Phillips  (1956)  described  a  modification  of  the  method  of  Glaser  and  Sparrow 
(1909)  by  which  the  nematocysts  of  Metridiuin  could  be  isolated,  washed  and  dis- 
charged into  distilled  water.  The  methods  presented  here  are  similar  to  his.  It  is 
our  object  to  present  details  of  the  separation  of  nematocysts  from  Physalia  and 
preliminary  data  on  the  composition  and  toxicity  of  the  separated  components. 
Although  Physalia  is  a  colonial  form,  for  simplicity,  members  of  the  colony  will  be 
referred  to  as  if  they  were  anatomical  parts  and  the  whole  colony  as  a  single  entity. 

MATERIALS 

Physalia  appears  on  southeast  Florida  beaches  during  periods  of  prolonged 
on-shore  winds  of  greater  than  usual  intensity.  Locally  these  wrinds  may  be 
expected  seasonally,  from  October  through  March.  Small  animals  generally  appear 
early  in  the  season.  Specimens  of  Uca  pugilator  were  purchased  from  a  commer- 
cial distributor  in  the  vicinity  of  Englewood,  Florida. 

METHODS 

Specimens  were  collected  as  they  stranded  and  were  placed  in  clean  sea  water 
to  remove  sand.  The  fishing  tentacles  were  removed,  combined  with  the  tentacles 
from  other  animals,  and  allowed  to  autolyze  at  4°  C.  for  24  to  48  hours.  Then 
the  mixture  was  diluted  with  one  or  more  volumes  of  sea  water  and  put  through 
graded  screens  of  24  and  115  meshes  per  inch.  This  removed  most  of  the  muscle 
and  connective  tissue  of  the  tentacle  and  permitted  passage  of  undischarged  nemato- 
cysts. The  screened  suspension  was  allowed  to  settle  overnight  in  the  cold.  The 
supernatant  solution  was  decanted  and  discarded.  The  residue,  which  was  com- 
posed chiefly  of  nematocysts,  was  centrifuged  at  300-400  g  for  15  to  30  minutes. 
The  supernatant  solution  was  again  discarded  and  the  residue  re-suspended  in  sea 
water.  These  processes  were  continued  until  injection  of  0.1  ml.  of  the  super- 
natant solution  into  the  hemocoele  of  the  fiddler  crab,  Uca,  was  without  apparent 

1  Contribution  number  210  from  the  Marine  Laboratory,  University  of  Miami.  These 
studies  were  supported  by  U.S.P.H.S.  Grant  RG-5272. 

219 


220 


CHARLES  E.  LANE  AND  ELEANOR  DODGE 


effect.  The  nematocyst  suspension  at  this  time  (Fig.  1)  was  almost  completely 
free  of  tentacular  tissue  fragments  and  contained  approximately  55  million  nemato- 
cysts  per  wet  gram,  very  few  of  them  discharged.  Nematocysts  ranged  in  size 
from  8.8  to  42.3  micra.  They  fell  into  two  size  groups :  one  with  a  mean  diameter 
of  11.3  micra  made  up  23%  of  the  total  sample.  The  remainder  varied  about  a 
mean  diameter  of  26.8  micra.  The  packed  nematocysts  were  frozen  and  stored 
at  --  5°  C.  Nematocysts  were  still  reactive  after  20  weeks  of  frozen  storage.  An 
initial  sample  of  3.4  liters  of  isolated  fishing  tentacles  yielded  60  grams  of  packed 
wet  "purified"  nematocysts. 


FIGURE  1.  Photomicrograph  of  isolated,   purified,   still-reactive 
nematocysts  of  Physalia.     X  1400. 

The  contents  of  the  isolated  nematocysts  were  liberated  by  homogenization  in  a 
chilled  Potter-Elvejhem  homogenizer.  Amphibian  Ringer's,  sea  water  or  distilled 
water  may  be  used  as  the  diluent.  The  sample  was  examined  microscopically  at 
intervals  and  homogenization  was  continued  until  about  90%  of  the  capsules  were 
fragmented  (Fig.  2).  The  homogenate  wras  centrifuged  at  600  g  for  ten  minutes 
to  separate  capsules  and  capsular  fragments  from  the  diluted  capsular  contents. 
The  supernatant  solution  was  cloudy,  yellowish-white  in  color,  and  extremely 
toxic  to  crabs,  fish  and  small  mammals.  Precautions  must  be  taken  to  avoid 
exposure  of  skin  to  contamination  by  any  mixture,  wet  or  dry,  which  contains 
undischarged  nematocysts.  Nematocysts  on  laboratory  surfaces  or  clothing  retain 
their  reactivity  for  at  least  two  weeks  as  unpleasant  reminders  of  previous  careless- 


TOXICITY  OF  PHYSALIA  NEMATOCYSTS  221 

ness.  Nematocysts  on  the  tentacles  of  large  living  Physalia  may  occasionally 
penetrate  heavy-gauge  surgical  gloves.  Surfaces,  clothing  and  skin  can  be  de- 
contaminated by  the  application  of  95%  ethanol.  Although  this  treatment  does 
not  reduce  the  pain  of  stings  already  received,  it  appears  to  prevent  the  discharge 
of  additional  nematocysts. 

Fiddler  crabs  (Uca  pngilator)  have  been  employed  for  initial  screening  of 
toxic  extracts.  Doses  of  0.1  ml.  of  material  to  be  assayed  were  injected  into  the 
hemocoele  through  the  articular  membrane  of  the  third  walking  leg.  When  sea 


FIGURE  2.  Photomicrograph  of  homogenized  nematocysts,  x  250.  Fragments  of  capsules, 
everted  tubules  and  tubule  fragments  constitute  most  of  the  visible  formed  elements.  At  least 
90%  of  the  capsules  have  been  discharged. 

water  alone  was  administered  by  this  route  no  effect  was  produced,  but  when 
capsular  contents  were  present  in  the  sea  water,  paralysis  and  death  ensued.  Ten 
animals  were  routinely  injected  with  each  extract  to  be  assayed.  Other  animals 
employed  in  toxicity  determination  and  evaluation  include  several  species  of  fish, 
the  frog,  and  the  heart  of  the  clam  Merccnaria  campechiensis  (frequently  designated 
Venus  mercenaria} .  Acute  toxicity  studies  were  done  on  30-gram,  male  Swiss 
mice,  according  to  the  method  of  Deichmann  and  LeBlanc  (1943). 

Total  nitrogen  was  determined  by  the  micro-Kjeldahl  method;  moisture  by 
co-distillation  with  toluene  ;  and  ash  by  incineration.  Dry  samples  of  known  weight 
were  hydrolyzed  in  6  N  HC1  in  a  sealed  capsule  at  100°  C.  for  24  to  36  hours.  The 


222 


CHARLES  E.  LANE  AND  ELEANOR  DODGE 


ammo  acid  content  of  the  neutralized  hydrolysate  was  determined  by  two-dimen- 
sional chromatography  on  Whatman  Xo.  1  paper.  Two-dimensional  chromato- 
grams  employed  n-butanol,  acetic  acid  and  water  (4:1:5)  as  the  first  solvent  and 
water-saturated  phenol  as  the  second  solvent.  Chromatograms  were  developed  in 
0.2%  ninhydrin  in  acetone.  The  approximate  concentrations  of  amino  acids  in  va- 
rious samples  were  estimated  by  size  and  density  of  the  amino  acid  spots  on  the 
finished  chromatograms. 

RESULTS 

The  distribution  of  amino  acids  in  Pliysalia,  and  their  approximate  concentra- 
tions  are   shown   in   Table    II.     The   predominant   amino   acids    in   undischarged 

TABLE  I 

Composition  of  entire  colonies  and  of  component  parts  of  Physalia 


Material 

Moisture 

Fat 

Solids 

Ash 

%  Total  N 

Entire,  living  Phystilia 

82.2% 

0.23% 

17.5% 

Floats,  only 

88.5 

11.48 

10.94%* 

Fishing  tentacles 

88.07 

11.93 

Gonozooids 

88.78 

11.22 

Undischarged  nematocysts** 

77.8 

22.2 

3.35 

2.58 

Capsule  contents 

97.54 

1.46 

0.598 

Discharged  capsules  (residue) 

88.0 

12.0 

1.48 

First  wash  of  discharged  capsules 

0.12 

*  This  determination  refers  to  dry  material;  all  other  X  determinations  are  wet-weight  basis. 
**  This  was  a  standard  preparation  numbering  55.2  millions  of  capsules  per  milliliter. 


TABLE  II 
Amino  acids  of  Physalia 


Amino  acid* 

Entire 
Physalia 

Fishing 
tentacle 

Undischarged 
nematocysts 

Nematocyst 
contents 

Discharged 

capsules 

Cystine 

X 

X 

X 

X 

X 

Cysteine 

X 

X 

X 

X 

X 

Glutamic  acid 

XXX 

XXX 

xxx 

xxxx 

X 

Glycine 

X 

XX 

XX 

X 

XX 

Alanine 

X 

X 

XX 

X 

xxx 

Tyrosine 

0 

0 

0 

0 

0 

Proline 

X 

X 

XX 

0 

xxx 

Hydroxyproline 

X 

X 

X 

X 

X 

Leucine 

X 

X 

X 

X 

X 

Isoleucine 

X 

X 

X 

X 

X 

Methionine 

X 

X 

X 

0 

X 

Lysine 

X 

X 

X 

X 

0 

Threonine 

X 

0 

0 

0 

0 

Aspartic  acid 

X 

X 

0 

0 

0 

Histidine 

X 

XX 

0 

0 

0 

Serine 

X 

X 

0 

0 

0 

*  X  means  not  more  than  2.5  micrograms  of  amino  acid  in  50  micrograms  dry  hydrolysate. 
no  spot. 


TOXICITY  OF  PHYSALIA  NEMATOCYSTS  223 

nematocyst  capsules  appear  to  lie  glutamic  acid,  glycine,  alanine  and  proline.  Of 
these,  glutamic  acid  is  chiefly  a  constituent  of  the  fluid  contents  of  the  capsule, 
and  the  others  occur  chiefly  in  the  solid  components  of  the  capsule  wall.  Lysine, 
present  in  the  nematocyst  complex  in  small  quantity,  apparently  also  is  concerned 
only  with  the  fluid  contents.  Aspartic  acid,  histidine,  threonine  and  serine  are 
present  in  the  intact  animal,  but  are  apparently  not  present  in  the  capsular  complex. 

When  an  active  extract  in  sea  water  was  administered  to  the  fiddler  crab,  the 
response  was  immediate  and  predictable.  When  returned  to  the  container,  the 
injected  crab  made  a  short,  abrupt  run,  stopped  precipitately,  contracted  the  exten- 
sors of  the  walking  legs  vigorously.  This  made  the  animal  appear  to  rise  on 
tiptoes.  It  remained  motionless  during  the  imperceptible  relaxation  which  cul- 
minated in  death.  If  the  crabs  were  handled  after  relaxation  began,  responses  were 
limited  to  the  eyestalks  and  to  very  slow  movements  of  the  walking  legs.  The 
animals  appeared  to  be  paralyzed.  If  legs  of  "paralyzed"  crabs  were  crushed  with 
a  hemostat,  the  number  of  legs  autotomized  was  only  one-third  that  observed  in 
uninjected  crabs  similarly  treated. 

Activity  of  the  fluid  contents  of  the  capsule  was  markedly  decreased  by  heating 
to  60°  C.  for  15  minutes,  by  precipitation  with  acetone  or  by  extraction  with  ether. 
The  toxin  was  non-dialyzable.  It  was  positive  to  ninhydrin  and  negative  to  Bene- 
dict's reagent,  both  before  and  after  acid  hydrolysis.  Activity  persisted  without 
significant  quantitative  change  for  at  least  two  months  when  stored  at  -  5°  C. 
When  the  capsule  contents  were  precipitated  by  alcohol  and  then  assayed  on  crabs, 
a  qualitative  fractionation  of  the  total  activity  was  observed.  Before  treatment 
with  alcohol  the  extract  produced  immediate  death  of  test  crabs  throughout  the 
effective  concentration  range.  After  precipitation  in  alcohol  and  re-solution  of  the 
precipitate  in  sea  water,  or  variation  of  the  pH,  the  lethal  response  was  delayed 
as  much  as  24  hours,  but  the  extract  produced  immediate  autotomy  of  the  walking 
legs.  Similarly,  adsorption  of  the  toxin  on  paper  and  subsequent  elution  released 
only  the  autotomy-producing  activity.  The  residue  on  the  paper,  as  well  as  the 
eluate,  remained  ninhydrin-positive. 

The  approximate  lethal  dose  for  mice  of  a  toxin  sample  which  contained  0.201% 
total  N  was  2.1  ml. /kilo,  when  the  material  was  injected  subcutaneously  (12  mice) 
and  0.037  ml./kilo.  when  it  was  injected  intraperitoneally  (23  mice).  Sub- 
cutaneous injection  caused  depression  after  about  two  hours,  and  death,  apparently 
due  to  respiratory  failure,  occurred  12  to  18  hours  after  injection.  Post-mortem 
examination  of  a  single  mouse  immediately  after  it  had  stopped  breathing  showed 
the  heart  to  be  still  beating,  indicating  that  death  was  due  primarily  to  respiratory 
failure.  After  intraperitoneal  injection  there  was  an  immediate  onset  of  intoxi- 
cation— reminiscent  of  the  almost  instantaneous  response  of  the  fiddler  crab. 
Symptoms  included  increased  activity  and  tremors  probably  due  to  local  irritation. 
After  10  minutes  there  were  ataxia,  decreased  muscle  tone,  flaccid  paralysis,  slowed 
and  labored  breathing,  defecation,  aphrodisia,  marked  myosis,  cyanosis,  anoxic 
convulsions  and  death.  Survival  time  was  1  to  48  hours,  depending  on  the  dose 
administered.  Post-mortem  examination  showed  the  following  gross  pathology : 
lungs,  blanched ;  heart,  contracted,  especially  the  left  ventricle ;  hemorrhagic  edema 
in  the  peritoneal  cavity ;  skin  of  nose  and  ears  very  white ;  cornea,  cloudy ;  colon, 
no  formed  stools ;  urinary  bladder,  empty. 


224  CHARLES  E.  LANE  AND  ELEANOR  DODGE 

A  dose  of  0.5  ml.  of  crude  toxin  containing  2.43  p.  gm.  N  per  ml.  was  uniformly 
lethal  when  injected  into  the  left  ventral  lymph  sac  of  each  of  eight  frogs  (Rana 
pipiens).  Within  five  minutes  the  white  ventral  surface  of  the  frog  developed 
irregular  red  patches  which  suggested  a  localized  hemodynamic  response  if  not 
actual  escape  of  blood  cells  from  the  capillaries.  Breathing  became  rapid  and 
shallow.  Righting  and  postural  reflexes  deteriorated  progressively  during  the 
first  hour.  At  the  time  the  animal  first  failed  to  respond  to  visual  stimulation 
(about  30  minutes),  he  could  be  turned  over  if  stimulation  of  peripheral  end 
organs  were  minimized.  At  this  time,  spinal  reflexes  appeared  to  be  normal. 
After  75  minutes,  breathing  movements  ceased  and  spinal  reflexes  disappeared. 
Electrical  stimulation  of  the  sciatic  nerve  elicited  no  response  at  this  time,  but 
direct  stimulation  of  the  gastrocnemius  muscle  showed  it  to  be  normally  reactive. 
The  heart  continued  to  beat  for  12  to  24  hours.  Large  amounts  of  lymph  accumu- 
lated subcutaneously  in  the  abdominal  area.  Viscera  were  hyperaemic,  bladder 
and  intestine,  empty  and  in  several  instances  large  amounts  of  bloody  intraperi- 
toneal  fluid  were  observed. 

Fish  responded  immediately  to  intramuscular  injection  of  lethal  doses  of  crude 
toxin  by  hyperventilation  and  rapid  swimming.  Petechiae  often  appeared  at  the 
sclero-corneal  junction.  After  five  minutes  to  several  hours,  depending  on  dosage, 
the  fish  became  disoriented,  sank  to  the  bottom  of  the  tank,  and  died  after  a  period 
of  one  to  four  hours.  This  response  is  typical  of  the  pilchard  Harcngnla  hitiucr- 
alis,  silversides  Hepsitia  stipes  Miiller  and  Troschel,  and  Fitnditlits  lictcro- 
clitus.  These  species  exhibited  chromatophoric  responses  to  injection,  usually 
blanching  at  the  immediate  site  of  the  injection  and  darkening  over  the  general 
body  surface.  Examination  of  two  pilchard  which  had  fallen  to  the  bottom  of  the 
tank  immobilized,  showed  the  heart  of  each  to  be  beating  normally.  No  abnormal 
effects  were  observed  after  intraperitoneal  injection  of  a  lethal  dose  of  toxin. 
Washed  erythrocytes  of  the  mullet  (Mugil  ccphalus}  did  not  hemolyze  when 
incubated  at  37°  C.  with  several  dilutions  of  crude  toxin. 

The  heart  of  the  clam  Mcrccnaria  ccnnpcchicnsis,  isolated  by  the  method  of 
Welsh  and  Taub  (1948)  responded  to  administration  of  crude  toxin  at  a  concen- 
tration of  5.2  /A  gm.  N/ml.  of  bath.  The  pattern  of  response  was  similar  to  that 
obtained  with  acetylcholine,  i.e.,  cessation  of  beat  in  diastole.  The  crude  toxin 
appeared  to  produce  irreversible  changes  which  prevented  the  heart  from  giving 
an  equivalent  response  to  a  similar  dosage  later.  Administration  of  the  crude 
toxin  did  not  modify  the  response  of  the  heart  to  acetylcholine. 

DISCUSSION 

The  crude  toxin  of  the  nematocyst  is  apparently  a  protein  complex  or  is  asso- 
ciated with  a  protein.  The  lethal  components  obscure  secondary  or  side  reactions. 
After  warming  to  60°  C.,  adsorption  on  paper  and  subsequent  elution,  precipita- 
tion with  ethanol  and  subsequent  re-solution,  manipulation  of  pH  or  other  mild 
treatments,  at  least  two  fractions  of  the  total  activity  were  resolved  and  then  exerted 
separate  effects  on  test  animals.  Welsh  (1956)  has  shown  that  many  substances, 
including  extracts  of  PJiysalia  and  various  other  coelenterates,  modify  the  autotomy 
reflex  in  crustaceans.  It  is  therefore  of  considerable  interest  that  a  compound 


TOXICITY  OF  PHYSALIA  NEMATOCYSTS  225 

\vhich  caused  autotomy  appeared  in  the  capsular  contents  only  after  this  material 
had  been  subjected  to  drying,  heating,  or  other  procedures  which  cause  denatura- 
tion.  This  effect  has  not  been  produced  by  unmodified  extracts. 

Lenhoff,  Kline  and  Hurley  (1957)  have  described  a  characteristic  chemical 
composition  of  nematocyst  capsules  of  other  coelenterates.  They  have  suggested, 
together  with  Phillips  (1956),  that  the  capsule  is  similar  in  chemical  composition 
to  the  collagenous  group  of  proteins  of  higher  animals.  In  homogenized  prepara- 
tions of  Physalia  nematocysts,  the  capsules  tend  to  retain  their  general  shape  though 
they  be  ruptured  or  even  broken  completely  in  two.  This  observation  provides  a 
certain  amount  of  support  for  the  concept  that  the  capsule  wall  is  semi-rigid. 

Our  data  suggest  that  Physalia  differs  from  Metridiiini  in  that  the  amino  acid 
spectrum  of  the  capsule  contents  differs  both  qualitatively  and  quantitatively  from 
that  of  the  capsule  wall.  Apparently  no  hexose  constituents  are  present  in  Phy- 
salia although  Phillips  describes  hexoseamines  from  Mctridiiiui. 

Our  methods  of  isolation  of  nematocysts  of  Physalia  require  no  other  diluent 
than  sea  water,  with  which  the  nematocysts  are  presumably  normally  in  contact. 
This  avoids  the  introduction  of  extraneous  salts  and  may  contribute  to  the  long 
persistence  of  reactivity  we  have  observed.  We  have  elected  to  liberate  the  cap- 
sule contents  by  homogenization  rather  than  to  await  the  considerable  time  that 
may  be  required  for  normal  discharge.  The  lability  of  Physalia  toxin  necessitates 
a  minimum  of  delay  in  processing. 

Injection  of  crude  toxin  apparently  produces  a  general  paralysis.  It  appears 
to  affect  the  nervous  system,  especially  respiratory  centers,  before  the  muscular 
system.  In  the  frog,  the  central  nervous  system  is  apparently  affected  before  the 
peripheral  nervous  system.  Crude  toxin  seems  to  alter  the  permeability  of  capillary 
walls  in  mice,  fish  and  frogs.  Hemolysis  was  not  observed. 

Since  the  toxicity  of  the  capsule  contents  of  Physalia  is  reduced  by  some  organic 
solvents,  and  since  these  solvents  also  inactivate  adherent  nematocvsts,  the  local 

j 

application  of  alcohol  to  the  skin  of  a  swimmer  stung  by  Physalia  is  an  effective 
palliative  measure. 

SUMMARY  AND  CONCLUSIONS 

The  general  composition  and  conditions  of  reactivity  of  the  nematocysts  and 
nematocyst  contents  of  Physalia  are  described.  A  method  is  presented  for  isolation 
of  nematocysts  without  contamination  by  other  tentacular  material.  The  nemato- 
cyst content  appears  to  be  a  highly  labile  protein  complex.  The  toxicity  of  the 
capsule  contents  is  destroyed  or  denatured  by  heating  to  60°  C.,  by  drying,  by 
treatment  with  ethyl  ether,  acetone,  or  ethanol.  Activity  may  be  preserved  for 
two  months  when  the  material  is  stored  at  -  -  5°  C.  The  approximate  lethal  dose 
for  mice,  when  the  toxin  was  injected  intraperitoneally,  was  0.037  ml. /kilo,  of  a 
preparation  which  contained  0.201^  total  N.  The  toxin  was  shown  to  be  devoid 
of  hemolytic  activity  for  fish  erythrocytes.  When  tested  in  fish,  frogs  or  mice  it 
appeared  to  affect  the  nervous  system,  particularly  the  respiratory  centers,  before 
voluntary  muscles.  Localized  changes  in  cardiovascular  tone  have  been  observed 
in  some  test  animals.  Physalia  toxin  elicited  responses  in  the  isolated  heart  of  the 
clam  which  were  similar  to  those  caused  by  acetylcholine. 


226  CHARLES  E.  LANE  AND  ELEANOR  DODGE 

LITERATURE  CITED 

DEICHMANN,  WM.  B.,  AND  T.  J.  LEBLANC,   1943.     Determination  of  the  approximate   lethal 

dose  with  about  six  animals.     /.  Indust.  Hy;/.,  25:  415—417. 
GLASER,  O.  C.,  AND  C.  M.   SPARROW,  1909.     The  physiology  of  nematocysts.     /.   Exp.   Zool., 

6:  361-382. 
LENHOFF,  H.  M.,  E.   S.  KLTNE  AND  R.   HURLEY,   1957.     A  hydroxyproline-rich,   intracellular, 

collagen-like  protein  of  Hydra  nematocysts.     Biochim.  Biophys.  Acta,  26 :  204-205. 
PHILLIPS,    JOHN    H.,    1956.     Isolation    of   active   nematocysts    of    Mctridlum    senile    and    their 

chemical  composition.     Nature,  178:  932. 
RICHET,   CHARLES,  AND  D.    PORTIER,    1936.     Recherches   sur   la   toxine   des   coelenteres   et   les 

phenomenes  d'anaphylaxie.     Result.   Camp.  Sci.  Monac.,  95 :   3-24. 
WELSH,  JOHN  H.,  1956.     /;;:   Papers  in  Marine  Biology  and  Oceanography.     Pergamon  Press 

Ltd.,  London.     Pp.  287-297. 
WELSH,  JOHN  H.,  AND  R.  TAUB,  1948.     The  action  of  choline  and  related  compounds  on  the 

heart  of  Venus  mcrccnaria.     Biol.  Bull.,  95 :  346-350. 


ON    THE    EVOLUTION    OF    HEMOGLOBIN.     RESPIRATORY 
PROPERTIES  OF  THE  HEMOGLOBIN  OF  THE  CALIFORNIA 
HAGFISH,   POLISTOTREMA   STOUTI  l 

CLYDE  MANWELL 
Scripps  Institution  of  Oceanography  of  the  University  of  California,  La  Jolla,  California 

The  Cyclostomata,  which  is  composed  of  the  hagfishes  (Myxinoidia)  and  the 
lampreys  (Petromyzontia),  is  considered  on  morphological  (Young,  1950)  and 
biochemical  (Florkin,  1949;  Wald,  1952)  evidence  to  be  the  most  primitive  group 
of  living  craniate  vertebrates.  Whereas  the  molecular  weight  of  vascular  hemo- 
globin of  all  known  non-cyclostome  vertebrates  corresponds  to  four  oxygen- 
combining  units  (hemes)  per  molecule,  the  hemoglobin  of  both  hagfishes  and 
lampreys  consists  of  but  a  single  heme  per  molecule  (Svedberg,  1933;  Lenhert, 
Lowe  and  Carlson,  1956).  With  regard  to  amino  acid  composition,  cyclostome 
hemoglobin  appears  to  be  intermediate  between  vertebrate  and  invertebrate  hemo- 
globins (Florkin,  1949). 

The  oxygen  equilibrium  of  hemoglobin  solutions  prepared  from  the  blood  of 
the  sea  lamprey,  Petromyzon  marinus,  has  been  recently  studied  (Wald  and  Riggs. 
1951).  This  hemoglobin  possesses  a  hyperbolic  oxygen  dissociation  curve  (as 
would  be  expected  on  the  basis  of  the  above-mentioned  molecular  weight),  a  low 
oxygen  affinity,  and  an  extremely  large  Bohr  effect.  Wald  (1952)  has  claimed 
that  the  evolution  of  hemoglobin  has  proceeded  in  three  stages  (p.  366)  :  "(I)  the 
heme  enzymes  of  cellular  respiration  [cytochrome  oxidase  being  considered  as  the 
phylogenetic  precursor  of  hemoglobin  (Wald  and  Allen,  1957)];  (2)  cell  and 
tissue  hemoglobins  concerned  primarily  with  oxygen  storage;  and,  (3)  circulatory 
hemoglobins,  concerned  with  the  transport  of  oxygen  from  the  lungs,  gills,  and 
skin  to  the  internal  tissues."  Wald  emphasizes  that  in  this  progression  the  three 
main  biochemical  aspects  of  the  combination  of  hemoglobin  with  oxygen  are  altered : 
(1)  the  oxygen  dissociation  curve  changes  from  hyperbolic  to  sigmoid — i.e.,  heme- 
heme  interaction  develops;  (2)  the  affinity  for  oxygen  decreases — i.e.,  the  oxygen 
molecule  is  held  less  tightly  to  the  heme;  and  (3)  the  oxygen  affinity  becomes  a 
function  of  pH— i.e.,  a  Bohr  effect  is  developed. 

In  view  of  these  facts  concerning  the  cyclostomes,  and  Wald's  (1952)  theory 
on  the  evolution  of  hemoglobin,  it  is  of  interest  to  evaluate  the  oxygen  equilibrium 
of  the  hemoglobin  of  the  California  hagfish,  which  is  perhaps  an  even  more  primi- 
tive vertebrate  than  the  lamprey. 

The  author  wishes  to  thank  Denis  L.  Fox  and  the  other  members  of  the 
Division  of  Marine  Biology  for  their  assistance  in  and  discussion  of  these  studies, 

1  Contribution  from  the   Scripps   Institution  of   Oceanography.     New    Series. 

227 


CLYDE  MANWELL 

and   David   Jensen   for   provision   of   hagfish   and   for   assistance    in    bleeding    of 
specimens. 

MATERIALS  AND  METHODS 

Blood  was  obtained  from  30  specimens  of  the  California  hagfish,  Polistotrema 
stonti  ( Lockington ) ,  formerly  called  Bdcllostoma  or  Eptatrctus  stouti.  The  blood 
was  collected  by  placing  capillary  tubes  adjacent  to  the  severed  ends  of  blood  ves- 
sels ;  no  anticoagulant  was  necessary,  for  the  blood  of  this  animal  has  little  clotting 
ability.  The  blood  was  occasionally  contaminated  with  a  trace  of  the  ubiquitous 
slime ;  this  was  easily  removed  by  diluting  the  blood  with  isotonic  phosphate- 
buffered  saline  and  filtering  through  glass  wool.  In  several  cases  blood  from  a 
single  animal  provided  enough  hemoglobin  for  a  single  oxygen  equilibrium  deter- 
mination. However,  the  hemoglobin  concentration  is  low  (3-4%),  and  usually 
less  than  1  cc.  of  blood  is  available  from  each  animal ;  therefore,  blood  from  several 
animals  was  often  pooled.  Erythrocytes  were  either  (1)  washed  once  in  15  cc. 
of  isotonic  saline  and  used  immediately  for  determination  of  the  oxygen  equilibrium 
of  dilute  erythrocyte  suspensions  (equivalent  to  whole  blood),  or  (2)  washed  two 
more  times  and  then  hemolyzed.  Distilled  water  hemolysis  did  not  give  satis- 
factory results ;  up  to  80%  of  the  hemoglobin  remained  inside  the  cell.  Therefore, 
a  trace  of  powdered  saponin  was  added  to  a  suspension  of  one  volume  of  cells  to 
two  volumes  of  distilled  water.  Several  hours  later  the  hemoglobin  solution  was 
separated  as  a  supernatant  by  centrifugation,  diluted  with  an  equal  volume  of 
potassium  phosphate  buffer  (F/2  ==  0.4)  of  the  desired  pH,  and  then  filtered 
through  Whatman  No.  5  paper.  Such  a  hemoglobin  solution  is  stable  for  days, 
although  (except  where  specifically  indicated)  it  was  used  immediately  for  oxygen 
equilibrium  measurements.  Preparation  of  hemoglobin  was  at  0-1°  C.,  except 
for  centrifugation  at  8-12°  C. 

Oxygen  equilibria  were  evaluated  as  in  previous  studies  (Manwell,  1958a, 
1958b).  Erythrocytes  were  suspended  in  9  parts  isotonic  sodium  chloride  (0.54 
M)  to  1  part  potassium  phosphate  buffer  of  desired  pH.  To  eliminate  rapid 
settling  of  cells  during  spectrophotometric  determination  of  oxy hemoglobin,  and 
to  reduce  light-scattering  effects,  many  erythrocyte  suspensions  were  diluted  3  :  1 
with  Karo  (a  mixture  of  sugars,  dextrins,  and  soluble  starch,  which  has  a  high 
refractive  index  and  thus  effects  a  partial  clarification  of  the  cell  suspension). 
Erythrocytes  could  be  stored  for  a  week  in  such  a  medium  without  hemolysis, 
although  this  undesirable  effect  took  place  to  a  slight  extent  in  a  few  experiments 
involving  prolonged  equilibration.  Therefore,  some  experiments  \vere  performed 
on  cells  simply  suspended  in  buffered  saline  to  which  a  trace  of  powdered  bovine 
serum  albumin  was  added  to  increase  cell  stability ;  in  these  instances  absolutely  no 
hemolysis  was  observed  during  or  for  a  day  after  equilibrium  measurements, 
although  there  was  greater  fluctuation  in  spectrophotometric  readings  due  to  settling 
of  cells  and  rouleaux. 

Most  experiments  were  performed  at  18°  C.,  slightly  above  the  upper  limit 
of  the  physiological  temperature  range  of  the  hagfish.  However,  in  connection 
with  a  determination  of  the  heat  of  oxygenation  of  this  hemoglobin  some  studies 
were  made  at  11°  C.,  well  within  the  normal  temperature  range,  and  at  29-30°  C. 


HEMOGLOBIN  EVOLUTION 


229 


RESULTS 

Instead  of  presenting  all  data  in  the  form  of  the  usual  "oxygen  dissociation 
curve,"  the  linear  transformation  based  on  the  Hill  approximation, 


y  =  100 


+ 


is  used  in  Figures  1  and  3   (Lemberg  and  Legge,  1949).     The  variables  y  and  p 
are  the  per  cent  oxyhemoglobin  and  the  partial  pressure  of  oxygen,  respectively. 


100 -y 


0.1 


Hemoglobin     Solutions 


• 
o 
o 
« 
e 
o  7.32 

•  6.89  (  »  2thr«.  later) 
«  6.80 

*  5.80 
»                6.50 


Erythrocyte     Suspensions 


pH 

7.8* 
7.22 
7.20 

5.30 
6.28 


0.1 


10 


100 


FIGURE  1.  Oxygen  equilibrium  of  hemoglobin  of  the  California  hagfish,  Polistotrcma  stouti. 
Three  to  four  per  cent  hemoglobin  solutions  in  potassium  phosphate  buffer ;  final  ionic 
strength  =  0.2.  Erythrocyte  suspensions  in  isotonic  phosphate-buffered  saline  with  Karo  added 
as  explained  in  text.  Temperature  =  18°  C.  The  solid  lines  are  drawn  arbitrarily  with  a 
slope  (n)  =  1.00  and  a  />.,„  corresponding  to  approximately  physiological  pH's. 

That  value  of  p  for  which  y  equals  50%  is  the  />.|0.  The  "sigmoid  coefficient,"  n, 
is  a  measure  of  the  heme-heme  interactions.  Hence,  />-„  is  an  inverse  measure  of 
the  oxygen  affinity,  and  n  determines  the  shape  of  the  oxygen  dissociation  curve. 
If  the  slope  of  the  transformation,  log  [v/(100  --  y)]  as  a  function  of  log  p,  is  one, 
then  the  hemes  are  totally  independent — i.e.,  there  is  no  heme-heme  interaction. 
As  can  be  seen  from  Figures  1  and  3,  where  the  solid  lines  are  drawn  with  a  slope 
of  1.00,  this  is  true  of  hagfish  hemoglobin  inside  and  outside  the  erythrocyte,  and 
at  high  and  low  temperatures. 


230 


CLYDE  MANWELL 


Between  pH  6.7  and  9.0  hagfish  hemoglobin  in  solution  shows  no  detectable 
Bohr  effect.  Outside  that  pH  range  a  significant  decrease  in  oxygen  affinity 
occurs ;  however,  this  effect  appears  to  be  a  prelude  to  more  drastic  changes 
(methemoglobin  formation  and  decrease  in  solubility),  which  become  apparent 
several  hours  after  equilibrium  measurements.  This  is  in  contrast  to  the  solutions 
at  intermediate  pH  which  are  stable  for  days  and  display  identical  oxygen  equi- 
libria when  re-analyzed  one  or  two  days  after  the  original  measurements  (see 
Figure  1 ) .  No  Bohr  effect  was  observed  for  erythrocyte  suspensions  at  pH's 
above  neutrality ;  however,  paralleling  the  behavior  of  hemoglobin  in  solution,  a 
slight  oxygen  affinity  decrease  occurs  at  acid  pH's.  The  effect  was  shown  not  only 


100 


0 


50 


X 

o 


Polistotrenta.       stoi/ti 


PH 

7.21 
6.17 


0 


10 


20 


Partial      Pressure       of 


30 

I 


(mm. 


FIGURE  2.  Oxygen  dissociation  curves  of  erythrocyte  suspensions  of  the  California  hagfish, 
Polistotrema  stouti,  at  two  different  pH's,  showing  the  possible  very  slight  Bohr  effect. 
Erythrocytes  in  phosphate-buffered  saline;  no  Karo  present.  Temperature  =  20-21°  C. 


by  the  partially  clarified  suspensions  (Fig.  1),  but  also  when  no  Karo  was  present 
(Fig.  2).  In  contrast  to  hemoglobin  solutions  such  acidic  erythrocyte  suspensions 
were  stable,  possibly  because  of  the  presence  of  cellular  reducing  systems  able  to 
reduce  any  methemoglobin.  The  observed  decrease  in  oxygen  affinity  could  rep- 
resent a  very  small  Bohr  effect ;  however,  until  it  is  shown  that  the  decrease  in 
oxygen  affinity  is  rapid  and  entirely  reversible,  the  possibility  of  slight  denaturative 
changes  in  the  protein  cannot  be  overlooked,  especially  in  view  of  the  results 
obtained  for  hemoglobin  solutions. 

The  presence  of  CCX  specifically  decreases  the  oxygen  affinity,  in  addition  to 
its  effect  resulting  from  the  increase  in  acidity,  for  hemoglobin  of  the  horse  (Mar- 
garia  and  Milla,  1955)  and  the  teleost  Sebastodcs  ruberrinms  (Manwell,  unpub- 


HEMOGLOBIN  EVOLUTION 


231 


lished  data).  That  CO2  does  not  cause  any  special  Bohr  effect  for  hagfish  hemo- 
globin is  shown  in  Figure  3. 

Because  the  possible  Bohr  effect  of  hagfish  hemoglobin  is  so  small  and  occurs 
at  almost  one  pH  unit  below  the  normal  pH  of  hagfish  blood  (7.5-7.7;  Prosser  ct 
ol,  1950;  David  Jensen,  personal  communication),  it  is  reasonable  to  assume  that 
it  is  of  no  physiological  significance,  especially  as  CO2  does  not  have  any  specific 
effect. 

Knowledge  of  the  heat  of  oxygenation  (AH°)  of  hagfish  hemoglobin  enables 
one  to  predict  the  position  of  the  oxygen  equilibrium  at  any  particular  physiological 


10 


-y 


PoliS  to  tr  etna       sfov  ft 


«/»  II 

V*  prj 

•        11.5  7.50 

o       29.5  7.3* 

0       Ig.O  7.30 


Pco2 

0 
0 


10 


p 


FIGURE  3.  Oxygen  equilibria  under  a  variety  of  conditions  of  hemoglobin  solutions  prepared 
from  the  blood  of  the  California  hagfish,  Polistotrema  stouti.  Dashed  lines  are  drawn  to  ap- 
proximate the  oxygen  equilibrium  at  11.5  and  29.5°  C.  The  solid  line  is  drawn  on  the  basis  of 
data  presented  in  Figure  1  in  order  to  facilitate  the  comparison  of  hemoglobin  solutions  in  the 
presence  and  in  the  virtual  absence  of  CO-,. 

temperature.  In  addition,  absence  of  a  Bohr  effect  facilitates  the  evaluation  of 
AH°,  for  corrections  representing  the  effects  of  ionizations  of  heme-linked  groups 
do  not  need  to  be  applied.  Using  data  presented  in  Figure  3,  a  value  of  AH° 
-9.3  kcal.  (one  atmosphere  of  dissolved  oxygen  gas  at  the  standard  state)  was 
obtained  as  in  a  previous  study  (Manwell,  1958a).  This  value  of  AH°  is  similar 
to  those  observed  for  hemoglobin  of  sheep  (—  8.2  kcal. ;  Paul  and  Roughton,  1951), 
the  holothurian  Cucumaria  miniata  (—8.4  kcal.;  Manwell,  1958f),  and  adult  and 
fetal  spiny  dogfish,  Sqnalns  siicklcyi  (-8.7  to  -9.5  kcal.;  Manwell,  1958d). 
This  group  of  values  for  the  heat  of  oxygenation  of  various  hemoglobins  is  not 


CLYDE  MANWELL 

characterized  by  the  extensive  variation  seen  in  older  data  (reviewed  by  Paul  and 
Roughton,  1951)  ;  theoretical  considerations  imply  that  there  be  relatively  little 
variation  in  these  values  for  a  particular  respiratory  pigment,  although  significant 
differences  occur  between  hemoglobin,  hemocyanin,  and  hemerythrin  (Klotz  and 
Klotz,  1955;  see,  also,  Manwell,  1958a). 

DISCUSSION 
Biochemical  and  Physiological 

Hagfish  hemoglobin  has  a  hyperbolic  oxygen  dissociation  curve.  Beyond  that 
point,  however,  resemblance  to  sea  lamprey  hemoglobin  (Wald  and  Riggs,  1951  ) 
ceases.  Hagfish  hemoglobin  lacks  a  Bohr  effect  over  a  pH  range  well  in  excess 
of  pH's  to  be  expected  in  living  hagfish.  The  hagfish  is,  accordingly,  the  first 
adult  vertebrate  whose  blood  is  known  to  lack  a  Bohr  effect.  In  addition,  the 
oxygen  affinity  of  hagfish  blood  is  very  high — at  physiological  conditions  as  high, 
if  not  higher  than  that  of  any  known  vertebrate  blood ;  the  />-„  is  2-4  mm.  Hg  -  over 
the  temperature  range  of  5-15°  C.  By  way  of  comparison,  />-0  for  human  blood 
is  26  mm.  Hg  at  pH  ==  7.3-7.4  and  a  temperature  of  37°  C.  (Prosser  ct  al,  1950). 
Arenicola  hemoglobin  has  an  especially  high  oxygen  affinity,  />r>0  =  2—2.5  mm.  Hg 
at  20°  C.  (Allen  and  Wyman,  1952)  ;  yet,  by  virtue  of  its  extremely  sigmoid  oxy- 
gen dissociation  curve  (n  --  6)  Arenicola  hemoglobin  appears  to  be  more  suitable 
for  oxygen  transport  than  hagfish  hemoglobin. 

The  comparison  between  hemoglobin  solutions  and  erythrocyte  suspensions 
prepared  from  the  blood  of  the  hagfish  indicates  that  no  specific  interaction  occurs 
between  the  hemes  of  adjacent  hemoglobin  molecules  inside  the  cell.  Hence,  these 
data  are  consistent  with — but  do  not  necessarily  establish — the  idea  that  the  molecu- 
lar weight  of  hagfish  hemoglobin  in  situ  corresponds  to  but  one  heme  per  molecule— 
i.e.,  approximately  17,000-18,000. 

Comparison  of  the  results  with  the  introduction  to  this  study  shows  that  hagfish 
hemoglobin  possesses  all  three  of  the  features  of  the  oxygenation  reaction  con- 
sidered to  be  primitive  by  Wald  (1952).  However,  the  properties  of  hagfish 
hemoglobin,  considered  to  be  characteristic  of  storage  hemoglobin  by  Wald,  are 
displayed  by  a  vascular  hemoglobin,  which  one  might  accordingly  assume  to  be 
involved  in  oxygen  transport. 

Living  hagfish  have  been  examined  in  an  attempt  to  see  whether  there  may  be 
a  significant  difference  in  the  color  of  blood  entering  and  leaving  the  tissues  and 
the  gills,  a  condition  which  would  indicate  participation  of  the  hemoglobin  in  oxy- 
gen transport.  Unanesthetized  hagfish  were  pinned  at  the  extreme  caudal  and 
cranial  ends  (but  not  unnaturally  stretched  out)  to  a  board  immersed  in  oxy- 
genated sea  water  at  10-12°  C.  The  animals  struggled  violently  until  both  ends 
were  pinned  down ;  they  then  remained  quiescent  for  the  duration  of  the  experi- 
ment. A  median  ventral  incision  was  made  in  the  vicinity  of  the  liver  and  the 
heart,  care  being  taken  to  avoid  cutting  any  blood  vessels.  The  slime  secretions 
were  periodically  removed.  When  slime  production  ceased,  blood  in  the  dorsal 
aorta  (leaving  the  gills)  and  in  various  veins  (leaving  the  tissues)  was  compared 

-  This  approximate  range  of  />.-„  for  hagfish  blood  at  physiological  temperatures  has  been 
calculated  (Manwell,  1958a)  on  the  basis  of  the  />.-,„  for  erythrocyte  suspensions  at  18°  C.  (see 
Fig.  1)  and  the  heat  of  oxygenation  (AH°)  of  hagfish  hemoglobin  in  solution. 


HEMOGLOBIN  EVOLUTION  233 

visually  with  "reduced"  and  oxygenated  standards  in  hagfish  blood  vessels.  Blood 
in  the  veins  appeared  to  be  almost  de-oxygenated ;  blood  in  the  arteries  was  ap- 
proximately 50%  oxygenated.  This  condition  did  not  change  over  several  hours 
of  continuous  observation.  By  reference  to  the  oxygen  dissociation  curves,  it  can 
be  seen  that  the  internal  oxygen  tensions  were  extremely  low,  although  the  hemo- 
globin was  functional  in  oxygen  transport.  An  improved  physiological  experi- 
mental approach  would  be  highly  desirable ;  however,  the  hagfish — considering  its 
small  size,  its  surprisingly  violent  activity  when  handled,  and  its  copious  slime- 
producing  abilities — is  not  an  especially  suitable  form  in  which  to  determine 
arterial-venous  oxygen  concentrations. 

Wald  (1952)  comments  (p.  367)  :  "The  business  of  a  circulatory  hemoglobin, 
having  combined  with  oxygen  at  the  body  surface,  is  to  release  it  in  the  tissues 
at  high  tensions.  .  .  ."  (Italics  are  those  of  Wald.)  Clearly,  hagfish  hemoglobin  is 
biochemically  unable  to  function  in  such  a  way ;  and,  the  observations  made  on 
living  specimens  tend  to  strengthen  the  idea  of  oxygen  transport  at  lou>  internal 
oxygen  tensions  in  Polistotrcma  stouti.  Redmond  (1955)  has  found  extensive 
evidence  for  oxygen  transport  at  lowr  internal  oxygen  tensions  in  several  decapod 
crustaceans.  Several  studies  indicate  that  such  a  condition  also  exists  in  some 
but  not  all  annelids  (reviewed  by  Eliassen,  1953;  see  also,  Jones,  1954;  Eliassen, 
1955;  Manwell,  1958e).  Adult  spiny  dogfish,  S  quoins  suckleyi,  have  a  hemo- 
globin with  a  hyperbolic  oxygen  dissociation  curve  inside  and  outside  the  erythro- 
cyte  (Manwell,  1958d)  ;  yet,  polarographically  determined  oxygen  tensions  of 
blood  leaving  the  heart  were  never  above  5  mm.  Hg  in  15  resting  dogfish.  Very 
low  venous  oxygen  tensions  have  been  observed  in  some  teleosts — but  not  the 
mackerel  (Black,  1951).  Especially  interesting  in  this  regard  is  the  marked 
suppression  of  heme-heme  interaction  by  the  erythrocytes  of  some  teleosts  and  a 
species  of  holocephalian  (Manwell,  unpublished  data)  ;  although  n  for  clingfish 
Gobicsox  hemoglobin  in  solution  is  2.5-2.6  and  thus  approaches  values  of  n  for 
mammalian  hemoglobins  (2.6-3.0),  inside  the  red  blood  cell  the  oxygen  equi- 
librium of  Gobieso.r  hemoglobin  is  almost  devoid  of  heme-heme  interaction 
(n  -•  1.2-1.4)  ;  this  trend  is  exactly  the  opposite  of  what  would  be  expected  were 
the  sigmoid  oxygen  dissociation  curve  always  so  vital  for  oxygen  transport. 

Under  conditions  where  the  tissues  tolerate — or  require — low  oxygen  tensions 
the  properties  usually  associated  with  a  transport  hemoglobin  would  be  of  little 
selective  advantage.  In  addition,  if  a  large  diffusion  gradient  were  necessary  to 
account  for  movement  of  sufficient  oxygen  across  the  epithelium  of  the  gills  or 
skin,  then  such  properties  as  low  oxygen  affinity  and  large  Bohr  effect  would  pre- 
vent loading  of  the  respiratory  pigment  with  sufficient  oxygen  in  the  organ  of 
external  respiration.  ("Sufficient"  does  not  imply  complete  saturation;  see  Red- 
mond, 1955.)  Partial  use  of  anaerobic  metabolism  could  free  tissues  from  de- 
pendence on  large  internal  oxygen  tension  gradients.  At  the  same  time  as  such 
a  rigorous  dependence  on  oxygen  were  reduced,  however,  so  would  the  metabolic 
efficiency  decline  (aerobic  metabolism  yielding  several  times  more  energy  per  unit 
weight  of  substrate  than  anaerobic  metabolism).  Consequently,  one  might  expect 
large,  very  active  animals  (e.g.,  cephalopods,  some  fishes,  birds,  and  mammals)  to 
have  evolved  increasing  dependence  on  the  more  efficient  aerobic  metabolic  path- 
ways— and  at  the  same  time  oxygen  transport  at  high  internal  oxygen  tensions. 
In  such  cases  the  sigmoid  oxygen  dissociation  curve,  the  low  oxygen  affinity,  and 


234  CLYDE  MANWELL 

the  large  Bohr  effect  would  be  of  the  greatest  selective  advantage  in  increasing  the 
efficiency  of  the  respiratory  pigment.  It  is  well  known  that  squid  hemocyanin, 
mammalian  and  avian  hemoglobins,  and  mackerel  and  trout  hemoglobins  possess 
all  of  these  characteristics  (reviewed  by  Florkin,  1949;  Prosser  et  al.,  1950). 

Phylogenetic 

In  terms  of  Wald's  (1952)  previously  mentioned  theory  on  the  origin  and 
evolution  of  hemoglobin  one  might  be  tempted  to  infer  that  the  primitive  hagfish 
has  retained  in  a  hemoglobin  used  in  oxygen  transport  all  three  oxygenation  prop- 
erties to  be  expected  of  hemoglobin  in  an  earlier  stage  of  evolution — that  repre- 
sented by  an  oxygen  storage  hemoglobin.  However,  some  or  all  of  the  properties 
of  hagfish  hemoglobin  may  represent  specialization  to  a  particular  mode  of  life  far 
different  from  that  of  known  fossil  Agnatha.  The  hagfishes  are,  in  spite  of  some 
primitive  characteristics,  well-adapted,  biologically  successful  animals.  Over  sev- 
eral types  of  ocean  bottom  in  temperate  seas  the  hagfishes  are  among  the  dominant 
scavengers — or  parasites — feeding  on  dead  and  dying  fishes ;  they  are  often  present 
in  such  numbers  as  to  restrict  or  prevent  several  types  of  fishing  operations  (Young, 
1950).  Certain  characteristics  of  the  hagfish,  such  as  the  rasping  tongue,  com- 
plete absence  of  scales  and  bone,  and  the  habit  of  feeding  on  teleost  fishes,  are  not 
properties  of  fossil  Agnatha  (Ostracoderms).  These  features  must  have  evolved 
independently  of  other  aspects  of  early  vertebrate  phylogeny.  The  differences  in 
the  properties  of  sea  lamprey  (Wald  and  Riggs,  1951)  and  hagfish  hemoglobin 
may  be  correlated  with  the  well-known  ecological  observation:  the  hagfish  enters, 
often  in  large  numbers,  the  body  of  its  prey  and  thus  is  often  exposed  to  low  O._, 
and  high  CO2  tensions ;  the  lamprey  remains  attached  to  the  surface  of  its  host, 
thereby  having  well-oxygenated  water  of  low  carbon  dioxide  tension  available  for 
its  respiration  at  all  tim^s.  In  addition,  so  far  as  is  known,  the  hagfish  does  not 
make  any  sustained  active  movement  comparable  to  the  anadromous  migration  of 
the  sea  lamprey. 

Several  other  objections  to  Wald's  (1952)  theory  in  its  present  form  can  be 
raised : 

( 1 )  Cytochrome  oxidase  has  been  considered  the  phylogenetic  precursor  of 
hemoglobin  because:  (a)  it  combines  reversibly  with  CO  and  reacts  with  O2;  and, 
(b)  beef  heart  cytochrome  oxidase  has  an  extremely  high  oxygen  affinity,  no  Bohr 
effect,  and  an  almost  hyperbolic  equilibrium  curve  with  CO — all  properties  that 
a  "primitive"  hemoglobin  ought  to  possess  (Wald  and  Allen,  1957).  Unfortu- 
nately, neither  the  prosthetic  group  (Paul,  1951;  Stotz,  Morrison  and  Marinetti. 
1956)  nor  the  protein  moiety  (Lemberg  and  Legge,  1949)  of  this  respiratory 
enzyme  (or  enzyme  complex)  resembles  the  corresponding  parts  of  hemoglobin  as 
closely  as  might  be  desired.  Cytochrome  r  would  be  a  better,  although  not  entirely 
satisfactory,  hemoglobin  phylogenetic  precursor.  At  least  its  prosthetic  group  is 
the  same  as  that  of  hemoglobin,  although  linked  to  the  protein  differently;  and, 
its  protein  moiety  is  readily  water-soluble,  although  of  lower  molecular  weight 
(one  heme  per  13,000-15,000)  and  higher  isoelectric  point  (pi  - :  10)  (Paleus, 
1955)  than  any  known  hemoglobin.  When  the  heme  of  cytochrome  c  is  not  com- 
pletely protected  by  coordination  of  the  iron  with  the  imidazole  groups  of  two 


HEMOGLOBIN  EVOLUTION  235 

histidine  residues,  the  enzyme  combines  with  CO  and  is  oxidized  by  CX  (Lemberg 
and  Legge,  1949;  Theorell,  1956).  Bartsch  and  Kamen  (1958)  isolated  a  bac- 
terial heme  protein — originally  called  a  "pseudohemoglobin"- —which  resembles 
cytochrome  c  in  many  respects,  although  its  isoelectric  point  (pi  =  5)  is  com- 
parable to  that  of  invertebrate  and  cyclostome  hemoglobins  (Prosser  et  al.,  1950) 
and  it  is  readily  oxidized  by  O2  and  combines  reversibly  with  CO.  The  carbon 
monoxide  reaction  of  this  bacterial  heme  protein  is  not  invariant  to  pH  change- 
in  contrast  to  cytochrome  oxidase  (Wald  and  Allen,  1957).  In  support  of  sonic 
connection  between  the  syntheses  of  cytochrome  and  hemoglobin  is  the  finding  of 
Yeas  (1956)  that  aerobically  grown  yeast  in  the  presence  of  antimycin  produces 
less  cytochrome  a  and  more  hemoglobin  than  controls ;  however,  as  Yeas  suggests, 
this  relation  may  be  explained  by  assuming  that  the  heme  of  hemoglobin  is  a 
precursor  to  the  modified  heme  of  cytochrome  a.  At  present  there  is  so  little 
comparative  biochemical  information  on  the  cytochromes  and  other  heme-containing 
enzymes  that  one  cannot  rule  out  the  possibility  that  the  proteins  of  various  hemo- 
globins have  arisen  from  apoenzymes  of  quite  unrelated  biocatalysts ;  certainly,  the 
protoheme  prosthetic  group  is  always  phylogenetically  available.  Several  proteins 
besides  globin  will  combine  with  heme,  although  none  are  yet  known  that  will 
enable  this  heme  to  combine  reversibly  with  molecular  oxygen  (Lemberg  and« 
Legge,  1949). 

(2)  Wald    (1952)    states    (p.   369):   "The  hemoglobins   that   have   arisen    so 
sporadically  among  invertebrates  of  various  orders  are  all  storage  hemoglobins." 
However,  oxygen  transport  by  hemoglobin  occurs  in  several  annelids    (Johnson, 
1942;   Eliassen,   1955;   reviewed  by  Eliassen,    1953;   Manwell.    1958e),   the  brine 
shrimp  Artcmia    (Gilchrist.   1954),  and  even  such  small  arthropods  as  daphnids 
(Hoshi,  1957).     As  the  experiments  of  Redmond  (1955)  show,  the  presence  of  a 
respiratory  pigment  in  the  blood  of  invertebrates  in  low  concentration  does  not 
rule   out  significant   oxygen   transport   by   that   pigment.     Coelomic   hemoglobins, 
such  as  those  of  Urechis   (Redfield  and  Florkin,   1931)    and   Citcumaria  winiata 
(Manwell,   1958f),  are  usually  assumed  to  function  in  oxygen  storage;  however, 
the  movement  of  coelomic  fluid,  either  by  muscular  contraction  or  cilia,  presents 
the  possibility  of  oxygen  transport  by  the  coelomic  hemoglobin  from  cloacal  diver- 
ticula    (Urechis)    or  respiratory  trees    (Cucumaria)    to  tissues  in  or  adjacent  to 
the  coelom. 

(3)  That  a  hyperbolic  oxygen  dissociation  curve,  high  oxygen  affinity,  and  no 
Bohr  effect  should  represent  primitive  conditions  (Wald,  1952)  requires  comment. 
The  properties  of  the  oxygen  equilibrium   of  the   vertebrate   storage   hemoglobin 
(myoglobin)   rest  on  studies  of  crude  extracts  or  purified  preparations  prepared 
from  the  muscles  of  five  species  of  mammals   (reviewed  by  Lemberg  and  Legge, 
1949;  see  also,  Rossi-Fanelli  and  Antonini.  1958).     As  Lemberg  and  Legge  point 
out,   the   oxygen  equilibrium  of  myoglobin   in  situ   in   the   muscle   remains  to  be 
evaluated.     The  findings,  that  n  could  be  as  high  as  1.6  for  oxygen  equilibria  of 
extractions  of  Cryptochiton  myoglobin   (Manwell,  1958c)   and  that  n  could  be  as 
high  as  2.8  in  the  reaction  of  horse  metmyoglobin  with  various  ligands  (Kiese  and 
Kaeske,  1942),  indicate  that  heme-heme  interactions  can  exist  under  certain  con- 
ditions in  tissue  hemoglobins.     One  would  expect  tissue  hemoglobins  to  have  a 
high  oxygen  affinity  because  of  limitations  on  the  intracellular  oxygen  tensions 
imposed  by  the  combination  of  passive  diffusion  of  oxygen  and  aerobic  cellular 


236  CLYDE  MANWELL 

metabolism.  In  the  case  of  Cryptochiton  even  when  the  oxygen  dissociation  curve 
of  the  radular  myoglobin  is  sigmoid,  it  lies  far  to  the  left  of  the  corresponding  curve 
for  the  vascular  hemocyanin ;  hence,  the  presence  of  heme-heme  interactions  in  the 
myoglobin  does  not  interfere  with  the  functional  oxygen  transfer  system  (Manwell, 
1958c).  Interactions  between  oxygen-affine  centers  have  evolved  in  all  four  major 
classes  of  respiratory  pigments  (hemoglobin,  hemocyanin,  chlorocruorin,  and  heme- 
rythrin)  ;  Bohr  effects  are  found  in  all  these  classes  except  hemerythrin  (reviewed 
by  Prosser  ct  al.,  1950). 3 

There  is  reason  to  believe  that  heme-heme  interactions  and  the  Bohr  effect  are 
not  necessarily  specialized  acquisitions  restricted  to  respiratory  pigments  in  an 
advanced  state  of  evolution  but  are  expressions  of  very  basic  properties  found  in 
many  unrelated  proteins.  The  frequently  observed  variation  of  enzyme  kinetics  as 
a  function  of  pH  often  involves  interaction  between  proton-affine  centers  on  the 
protein  moiety  and  the  active  center  (Alberty,  1956).  Heme-heme  interaction, 
likewise,  has  its  parallel  in  the  interaction  between  centers  having  similar  reactivi- 
ties in  proteins  possessing  two  or  more  such  sites  per  molecule.  Such  interactions 
occur  in  the  binding  of  dyes  and  ions  to  some  multivalent  proteins  (Klotz,  1954) 
and  in  the  kinetics  of  some  enzymes  (Botts  and  Morales,  1953). 

Finally,  the  ease  with  which  certain  reagents  (various  mercurials,  formaldehyde, 
and  glutathione)  will  remove  the  heme-heme  interactions,  partially  restore  those 
interactions,  greatly  increase  the  oxygen  affinity,  and/or  modify  the  Bohr  effect 
(Guthe,  1954;  Riggs  and  Wolbach,  1956)  implies  that  these  properties  are  not 
invariant  for  a  particular  hemoglobin  molecule.  In  addition,  the  differences  in  the 
oxygen  equilibrium  of  some  hemoglobins  inside  and  outside  the  red  blood  cell 
(Root,  Irving  and  Black,  1939;  Manwell,  unpublished  data)  indicate  also  that  a 
considerable  lability  exists  with  regard  to  the  properties  of  the  oxygen  equilibrium. 

It  seems  reasonable  to  assume  that  the  phylogenetic  order  of  first  appearances 
was :  heme-containing  respiratory  enzymes,  tissue  hemoglobins,  vascular  hemo- 
globins. However,  the  present  discussion  indicates  the  difficulty  of  knowing 
(a)  if  a  certain  set  of  characteristics  of  the  oxygen-hemoglobin  equilibrium — e.g., 
high  oxygen  affinity,  no  heme-heme  interactions,  and  no  Bohr  effect — is  basically 
primitive,  and  (b)  if  any  particular  component  of  the  cytochrome  system  or  any 
other  heme-containing  enzyme  is  evolutionally  the  forerunner  of  hemoglobin. 

SUMMARY 

1.  Oxygen  equilibria  of  hagfish  hemoglobin  inside  and  outside  the  red  blood 
cell  have  been  obtained  under  a  variety  of  conditions.  The  oxygen  affinity  of  the 
hemoglobin  in  the  erythrocyte  suspensions  is  high  (/>50  =  3-4  mm.  Hg  at  18°), 
although  it  is  even  higher  in  hemoglobin  solutions  (/>r>0  --  1.8  mm.  Hg  at  18°  C.). 
There  is  no  interaction  between  hemes  (n--  1.00)  and  virtually  no  Bohr  effect. 
The  effect  of  temperature  on  the  oxygen  equilibrium  of  hagfish  hemoglobin  is 

3  Absence  of  the  Bohr  effect  has  been  confirmed  for  various  sipunculid  coelomic  hemeryth- 
rins  (Manwell,  1958a,  and  unpublished  studies  on  Dendrostomutn  zostericolum  and  Siphonosoma 
ing  ens)  ;  however,  the  coelomic  hemerythrin  of  the  brachiopod  Linc/nla,  a  form  that  is  morpho- 
logically essentially  unchanged  since  the  Cambrian  period,  has  a  Bohr  effect  that  is  two-thirds 
the  magnitude  of  that  observed  for  human  adult  hemoglobin  (Manwell,  1958,  unpublished 
experiments)  ! 


HEMOGLOBIN  EVOLUTION  237 

similar  to  that  observed  in  recent  experiments  on  other  hemoglobins  (AH°         -9.3 
kcal.  for  hagfish  hemoglobin). 

2.  Several  aspects  of  Wald's  (1952;  see,  also,  Wald  and  Allen,  1957)  theories 
on  the.  evolution  and  function  of  hemoglobin  are  criticized  in  view  of  these  data  on 
hagfish  hemoglobin  and  on  the  basis  of  information  in  the  literature.  It  is  concluded 
that:  (1)  At  present  there  is  no  reason  to  favor  cytochrome  oxidase  as  the  phylo- 
genetic  precursor  of  hemoglobin.  (2)  Many  invertebrate  hemoglobins  function 
in  oxygen  transport.  (3)  If  the  internal  oxygen  tensions  are  sufficiently  low,  a 
respiratory  pigment  participating  in  oxygen  transport  does  not  need  to  possess  a 
low  oxygen  affinity,  a  sigmoid  oxygen  dissociation  curve,  and  a  marked  Bohr 
effect.  (4)  It  is  impossible  to  say  if  a  particular  set  of  properties  of  the  oxygen 
equilibrium  is  basically  "primitive."  (5)  Physiological  conclusions  on  hemoglobin 
should  be  made  upon  studies  of  the  pigment  in  the  natural  condition — i.e.,  myoglobin 
in  the  muscle,  or  intracellular  vascular  hemoglobin  in  the  erythrocyte. 

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BLACK,  E.  C,  1951.     Respiration  in  fishes.     Univ.   Toronto  Studies  Biol.  No.  59:  91-111. 
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TOXIC  EFFECTS  OF  NORMAL  SERA  AND  HOMOLOGOUS 
ANTISERA  ON  THE  CHICK  EMBRYO1 

ALTON  M.  MUN 

Department  of  Zoology,  Indiana  University,  Bloomington,  Indiana  ->  3 

The  possibility  of  identifying  embryonic  antigens  of  unique  function,  of  localiz- 
ing their  sites  of  origin  and  action,  and  of  manipulating  them  experimentally  in 
order  to  analyze  their  developmental  significance  by  the  use  of  specific  toxic  sera 
at  lethal  or  sub-lethal  doses  was  advanced  by  Nace  (1955).  By  modifying  the 
normal  function  of  an  antigen  with  sub-lethal  doses  of  a  toxic  antiserum,  a  specific 
anomaly  may  be  produced,  affording  a  key  to  the  localization  and  the  time  and 
nature  of  the  action  of  the  antigen.  Similar  arguments  have  been  advanced  by 
those  who  have  sought  to  block  the  growth  of  tumors  with  specific  antisera  (re- 
viewed by  Ross,  1957;  Wissler  and  Flax,  1957).  However,  before  this  approach 
can  be  employed  critically  in  studying  the  synthesis  of  specific  antigens  and  their 
role  in  development,  the  following  questions  must  be  considered :  ( 1 )  Are  the 
proteins  and  other  macromolecules  of  the  embryo  antigenic  ?  Or  does  the  embryo 
contain  a  population  of  molecules  capable  of  reacting  with  antibody  produced 
against  adult  antigens  but  incapable  of  eliciting  antibody  production?  The  dis- 
tinction must  be  made  between  the  occurrence  in  embryos  of  combining  groups 
identical  with  those  of  adult  antigens  and  the  occurrence  of  embryonic  antigens 
(Ebert,  1958a).  (2)  What  are  the  effects  of  antisera  on  the  embryo?  Does  the 
reaction  between  antigen  and  antibody,  in  vivo,  result  in  measurable  modifications 
of,  or  interference  with,  biological  function?  As  a  general  rule,  tissue-specific 
molecules  exhibit  species-specificity  to  some  degree,  making  analysis  by  immuno- 
chemical  techniques  possible ;  the  principal  advantage  of  these  methods  is  their 
exquisite  sensitivity,  which  makes  possible  the  analysis  of  the  rate  of  synthesis  and 
accumulation  and  site  of  localization  of  proteins  or  other  macromolecules  present 
in  embryos  in  trace  amounts.  The  principal  difficulty,  one  which  is  often  not 
appreciated,  is  that  antigenic  specificity  depends  upon  relatively  small  determinant 
groups  rather  than  on  the  complete  structure  of  the  molecule,  and  that  the  mole- 
cule may  contain  more  than  one  kind  of  determinant  group.  Little  is  known  of 
the  kind,  number,  and  size  of  determinant  groups  of  natural  proteins.  The  anti- 
genically  active  groups  and  physiologically  active  groups  of  a  molecule  may  not  be 

1  This  investigation  was  supported  in  part  by  grants  to  Professor  James  D.  Ebert  from  the 
National  Science  Foundation,  the  American  Heart  Association,  the  National  Heart  Institute  of 
the    National    Institutes   of   Health   of   the   United   States    Public    Health    Service    (grant    No. 
H-1709),  and  the  Indiana  University  Foundation  Research  Division. 

2  From   work   reported   in   a   thesis   submitted   to   the   Faculty   of   the   Graduate    School   in 
partial  fulfillment  of  the  requirements  for  the  degree,  Doctor  of  Philosophy,  in  the  Department 
of  Zoology,  Indiana  University,  July,   1956.     Eigenmann  Fellow  in  Zoology,   1954-1955.     Con- 
tribution number  648  from  the  Department  of  Zoology. 

3  Present  address  :  Department  of  Poultry  Science,  State  College  of  Washington,  Pullman, 
Washington. 

239 


240  ALTON  M.  MUN 

identical.  Similarity  in  immunochemical  properties  of  natural  molecules  of  diverse 
origin  may  result  from  ( 1 )  identity  of  one  or  more  antigenic  determinant  groups, 
(2)  a  degree  of  structural  similarity,  or  (3)  the  presence  of  one  as  an  undetected 
trace  contaminant  in  the  other,  for,  inasmuch  as  traces  of  antigen  may  evoke  large 
amounts  of  antibody,  such  contaminants  tend  to  be  revealed  (Ebert,  1958b). 

The  antigenicity  of  embryonic  tissues  has  been  demonstrated  in  several  species, 
for  example  in  amphibians  (Cooper,  1948,  1950;  Flickinger  and  Nace,  1952; 
Clayton,  1953)  and  in  the  sea  urchin  (Perlmann,  1954;  Perlmann  and  Perlmann, 
1957).  Much  of  the  earlier  literature  has  been  reviewed  by  Tyler  (1955,  1957). 

The  ability  of  the  chick  embryo  to  react  with  antisera  against  tissue-specific 
components  of  adult  chickens  was  shown  by  Burke  et  al.  (1944),  Nace  and  Schecht- 
man  (1948),  Ebert  (1950,  1951),  and  Ebert  et  al.  (1955).  For  example,  spe- 
cific effects  of  antibodies  against  adult  antigens  on  the  development  of  chick 
embryos  were  reported  by  Ebert  (1950).  Saline  extracts  of  heart,  brain,  and 
spleen  from  adult  chickens  were  used  as  antigens.  Rabbit  antiserum  against 
chicken  brain  affected  chiefly  nervous  tissue,  and  anti-heart  and  anti-spleen  sera 
affected  primarily  mesodermal  elements  in  early  chick  blastoderms  cultivated  in 
vitro.  Moreover,  comparing  the  effects  of  anti-heart  and  anti-spleen  preparations, 
it  was  clear  that  the  former  antisera  affected  the  development  of  the  heart,  whereas 
the  latter  did  not.  A  striking  extension  of  this  approach  is  found  in  the  report  by 
Langman  et  al.  (1957)  who  have  demonstrated  specific  effects  of  antisera  devel- 
oped in  rabbits  against  antigens  of  chick  lens,  purified  alpha  crystallin,  and  myosin. 
Anti-lens  and  anti-alpha  crystallin  sera  prevented  the  formation  of  the  lens  from 
ectoderm-optic  cup  combinations  in  vitro,  w^hereas  anti-myosin  sera  permitted  nor- 
mal lens  development  but  inhibited  mesenchyme  formation.  These  studies  clearly 
demonstrate  the  existence  of  reactive  or  combining  groups  in  the  embryo  capable 
of  reacting  with  antibodies  to  adult  tissue  antigens.  However,  the  antigenicity 
of  the  chick  embryo,  i.e.,  its  ability  to  elicit  the  production  of  precipitating  anti- 
bodies in  the  rabbit,  cannot  be  inferred  from  these  studies. 

After  complete  absorption  with  adult  laying  hen  serum,  antisera  against  the 
serum  of  the  10-day  chick  embryo  showed  positive  precipitating  activity  with  the 
homologous  antigens  (Schechtman  ct  al.,  1954).  Moreover,  Levi  and  Schecht- 
man  (1954)  concluded  from  similar  studies  that  the  12-day  embryo  contained 
distinct  embryonic  red  blood  cell  antigens.  Nettleship  (1953)  injected  1-day, 
2-day,  and  6-day  chick  embryos  (p.  325),  "emulsified  in  normal  saline  by  syringe 
suction  and  expulsion  or  grinding  in  a  mortar  without  abrasive,"  into  hamsters. 
The  hamster  anti-chick  sera  were  dropped  or  injected  into  or  near  the  "embryo 
site"  of  unincubated  eggs.  The  titers  of  the  antisera  used  were  not  determined, 
nor  were  the  antisera  against  the  various  stages  tested  with  the  homologous  em- 
bryos. The  hamster  anti-chick  embryo  serum  (p.  326)  "placed  in  proximity  to 
the  preincubated  chick  embryo  stops  the  development  of  these  embryos  at  a  time 
which  corresponds  to  the  time  the  embryo  antigen  was  obtained."  The  results 
were  interpreted  as  pointing  strongly  (p.  327)  "to  the  development  of  qualitatively 
different  protein  complexes  in  the  embryo  concurrent  with  the  embryo's  growth." 
These  results  are  consistent  with  the  studies  of  Cooper  (1950),  Spar  (1953), 
Nace  (1953),  and  Flickinger  and  Nace  (1952)  showing  changes  in  the  antigenic 
constitution  of  the  developing  embryo.  However,  critical  absorption  techniques, 
such  as  those  of  Cooper  (1950)  and  Spar  (1953)  in  which  antisera  to  later  stages 


TOXIC  EFFECTS  OF  ANTISERA  241 

were  absorbed  with  antigens  of  the  earlier  stage,  thus  separating  those  antibodies 
peculiar  to  the  later  stages,  were  not  reported.  Tyler  (1957)  remarked  (p.  356) 
that  "For  an  unabsorbed  antiserum  of  this  type  to  produce  a  highly  specific  effect 
does  seem  surprising,  and  one  wonders  whether  or  not  this  might  be  due  to  for- 
tuitous variation  in  the  antibody  content  of  the  various  antisera." 

The  present  experiments  were  undertaken  with  two-fold  objectives:  (1)  To 
establish  whether  or  not  the  chick  embryo  contains  specific  antigens  (as  opposed 
to  combining  groups  capable  of  reacting  with  antibodies  to  adult  tissues)  by  in- 
jecting whole  chick  embryos  or  embryo  extracts  into  rabbits,  and  (2)  to  examine 
the  effects  of  the  antisera  thus  produced  on  the  development  of  the  homologous" 
embryo.  However,  because  fresh  rabbit  serum  was  found  to  be  highly  toxic  to 
the  chick,  it  became  necessary  first  to  study  the  known  heat-labile  and  heat-stable 
factors  in  fresh  rabbit  serum  in  order  to  devise  means  of  reducing  or  removing 
false  reactions,  after  which  the  action  of  antisera  could  be  explored. 

MATERIALS  AND  METHODS 
Preparation  of  antigens 

The  72-hour  chick  embryo  (stages  16  to  18,  Hamburger  and  Hamilton,  1951) 
was  used  for  the  preparation  of  antigens  because  of  the  significant  number  of  well 
defined  histogenetic  and  morphogenetic  processes  wyhich  occur  during  this  period, 
e.g.,  morphogenesis  and  the  growth  of  the  limb  buds,  the  deposition  of  the  pigment 
in  the  eyes.  Another  factor  in  selecting  the  72-hour  embryo  was  its  larger  size 
and  higher  content  of  protein  nitrogen  as  compared  with  that  of  the  48-hour 
embryo.  Even  in  using  the  72-hour  embryo,  a  substantial  number  of  embryos  was 
required  for  the  preparation  of  the  antigens.  For  example,  it  required  approxi- 
mately twenty  72-hour  embryos  to  furnish  material  for  one  injection  into  a  single 
rabbit. 

The  embryos  were  cut  from  the  yolk  and  transferred  immediately  into  a  dish 
containing  ice  cold  0.15  M  NaCl.  The  adhering  yolk  was  trimmed  off  with  sharp- 
ened steel  needles,  and  all  membranes  were  removed  with  sharpened  jewelers 
forceps.  The  embryos  were  rinsed  several  times  in  ice  cold  saline  and  stored  in 
the  freezer  (—20°  C.)  until  used. 

Saline  extracts  of  the  72-hour  embryo  were  prepared  by  permitting  the  frozen 
embryos  to  thaw  at  room  temperature,  after  which  they  were  homogenized  with 
ice  cold  saline  in  a  chilled  Ten  Broeck  grinder.  Approximately  1  ml.  of  saline 
was  added  for  each  5  embryos.  The  cloudy  suspension  was  refrigerated  for  10 
to  12  hours,  after  which  it  was  centrifuged  at  3000  RPM  (1200  RCF)  for  30 
minutes  at  0  to  4°  C.  The  protein  nitrogen  content  of  the  resultant  translucent 
extract  was  approximately  0.2  mgN/ml.,  as  determined  by  semi-micro  Kjeldahl 
method. 

Preparation  of  antibodies 

In  preliminary  experiments,  6  white  rabbits  weighing  2  to  3  kilograms  were 
injected  intravenously  with  the  saline  extract  and  intraperitoneally  with  homoge- 
nized 72-hour  chick  embryos.  Although  injections  and  booster  shots  were  given 
repeatedly,  antisera  with  workable  titers  were  not  obtained.  In  subsequent  experi- 


242  ALTON  M.  MUN 

ments,  10  rabbits  were  injected  with  antigens  in  adjuvant;  72-hour  chick  embryos 
and  equal  amounts  of  Falba,  paraffin  oil  and  heat-killed  tubercle  bacilli  were  ground 
together  in  a  mortar  without  abrasive  and  emulsified  by  syringe  suction  and  expul- 
sion. Each  rabbit  received  approximately  10  to  12  embryos  (at  least  10  mg.  of 
protein)  in  each  of  three  injections  administered  subcutaneously  in  the  region 
of  the  neck,  one  week  aj  art.  The  rabbits  were  bled  from  the  marginal  vein  of  the 
ear  one  month  after  the  first  injection  and  one  week  after  an  intravenous  booster 
shot  of  10  to  20  embryos  homogenized  in  a  small  amount  of  saline.  This  method 
yielded  antibodies  with  titers  of  1 :  32  to  1  :  128.  By  standards  conventionally 
employed  for  antibodies  against  purified  antigens,  these  titers  are  low.  As  will  be 
made  clear,  however,  they  proved  to  be  valuable  tools. 

Tests  of  antibody  content 

The  presence  of  antibodies  was  detected  by  the  use  of  interfacial  "ring"  tests 
in  which  0.1  ml.  of  the  test  serum  was  overlaid  with  0.1  ml.  of  the  serially  diluted 
chick  embryo  extract.  The  tests  were  performed  in  6  X  50  mm.  culture  tubes  and 
test  materials  were  delivered  with  measuring  pipettes  ground  and  fitted  with  hypo- 
dermic needles  at  the  tip.  After  the  appearance  of  the  rings  was  noted,  the  tubes 
were  mixed  and  placed  in  the  refrigerator  overnight.  The  next  morning  the 
tubes  were  tapped  gently  and  the  presence  of  precipitate  was  detected  as  a  thin 
white  spiral  rising  from  the  bottom  of  the  tube. 

The  titer  of  the  antiserum  used  was  determined  by  precipitin  tests  in  which 
0.1  ml.  of  the  embryo  extract  was  added  to  1  ml.  of  the  serially  diluted  antiserum. 
The  tubes  were  then  incubated  in  a  water  bath  at  37°  C.  for  30  minutes.  The 
tubes  were  read  after  incubation  and  again  after  being  refrigerated  overnight. 

Operative  procedures 

A  technique  for  the  study  of  the  effects  of  antisera  on  the  chick  embryo  was 
described  by  Witebsky  and  Neter  (1935),  who  added  serum  drop  by  drop  to  the 
embryo.  A  modification  of  this  technique  was  adopted  in  this  study.  New  Hamp- 
shire eggs  obtained  from  a  local  hatchery  were  incubated  for  72  hours  at  37.5  to 
38°  C.  Preparatory  to  operation,  the  egg  was  swabbed  with  70%  alcohol.  Then 
a  square  window  1  cm.  X  1  cm.  was  cut  in  the  shell  with  a  sharpened  hack  saw 
blade.  The  cut  piece  of  shell  was  removed,  and  the  shell  membrane  was  cut  off. 
After  the  embryo  was  in  position  immediately  under  the  window,  a  small  hole 
was  made  in  the  vitelline  membrane  just  anterior  to  the  heart,  after  which  0.05  to 
0.1  ml.  of  the  test  serum  was  inserted.  The  material,  especially  if  colored  and 
dense,  could  be  seen  to  envelop  the  embryo  and  remain  in  position  for  several 
hours.  The  window  was  sealed  with  cellophane  tape,  and  the  egg  was  returned 
to  the  incubator. 

All  test  materials  were  sterilized  by  autoclaving  or  by  Seitz  filtration.  After 
the  latter  procedure  the  titer  of  each  serum  was  checked  because  of  the  report 
(Dilks  and  Wolfe,  1949)  that  significant  decreases  in  titer  result  from  Seitz 
filtration.  In  the  present  study,  decreases  in  titer  were  minimized  by  filtering  large 
volumes  of  undiluted  serum. 

More  than  50  experiments  were  conducted.  Each  experiment  consisted  of  at 
least  25  to  35  embryos,  including  sham-operated  or  saline  controls,  and  normal  or 


TOXIC  EFFECTS  OF  ANTISERA 


243 


absorbed  serum  controls.  Each  embryo  was  numbered;  the  time  of  operation, 
the  stage  of  the  embryo,  and  the  amount  of  the  test  substance  administered 
recorded.  All  observations  were  recorded  following  examination  of  the  specimens 
under  a  binocular  dissecting  microscope,  after  which  the  embryos  were  removed 
and  dissected  or  fixed  in  calcium  formol  for  further  histological  studies. 

In  the  interest  of  objectivity,  frequently  the  assistance  of  a  second  person  was 
enlisted  to  code  the  randomly  numbered  treated  eggs  and  to  record  the  observa- 
tions made  by  the  experimenter. 

THE  EFFECTS  OF  NORMAL  RABBIT  SERUM  ON  THE  72-HouR  CHICK  EMBRYO 

In  preliminary  experiments,  it  was  found  that  sera  from  both  uninjected  and 
injected  rabbits  were  toxic  to  the  embryo.  Within  a  few  minutes  after  the  appli- 
cation of  fresh  rabbit  serum  the  blastoderm  begins  to  shrivel,  the  embryo  gradually 
sinks  and  the  heart  stops  beating.  The  toxicity  of  fresh  rabbit  serum  has  been 
encountered  by  others.  Witebsky  and  Neter  (1935)  described  its  effects  on  the 
chick  embryo.  Bernheimer  and  Harrison  (1940)  observed  the  ability  of  normal 
rabbit  serum  to  immobilize  Paramecium.  Green  (1946)  observed  that  normal 

TABLE  I 

The  effect  of  heating  on  the  toxicity  of  fresh  normal  rabbit  serum  (NRS) 


Serum 

Effects  on  72-hour  chick  embryos 

No.  embryos 
treated 

Normal 

Abnormal 

Dead 

Unheated  NRS 

117 

3 

3 

111 

NRS  heated  at  37°  C. 

30  minutes 

8 

0 

0 

8 

NRS  heated  at  42°  C. 

30  minutes 

8 

1 

0 

7 

NRS  heated  at  50°  C. 

30  minutes 

10 

5 

0 

5 

XRS  heated  at  56°  C. 

30  minutes 

17 

17 

0 

0 

rabbit  serum  interfered  with  the  rapid  growth  of  cancer  cells,  and  Imagawa  et  al. 
(1954)  observed  that  normal  rabbit  serum  inhibited  the  proliferative  capacity  of 
mouse  mammary  cancer  cells.  A  spermicidal  factor  in  fresh  human,  bovine, 
rabbit,  and  rat  sera  was  reported  by  Chang  (1947).  Nace  has  described  normal 
rabbit  sera  which  were  toxic  to  the  Rana  piplens  embryo  (1955;  see  also  Nace 
and  Inoue,  1957). 

Witebsky  and  Neter  (1935)  reported  that  the  toxic  effects  of  fresh  rabbit 
serum  were  removed  by  heating  at  56°  C.  for  30  minutes,  an  observation  confirmed 
in  the  present  study.  Partial  inactivation  was  obtained  by  heating  at  50°  C.  for 
30  minutes,  but  below  50°  C.  inactivation  did  not  occur  (Table  I).  What  is  the 
nature  of  the  heat-labile  substance?  Is  it  complement  which  is  defined  in  part  on 
the  basis  of  its  destruction  by  heating  at  56°  C.  for  30  minutes?  Is  it  propcrdin, 
the  heat-labile  substance  recently  found  in  normal  serum  of  a  number  of  animals 
(Pillemer  et  al.,  1954),  or  is  it  another  heat-labile  substance  as  yet  undescribed? 
Other  questions  may  be  asked,  among  them :  does  the  heat-labile  factor  act  inde- 
pendently, or  does  it  require  the  presence  of  heat-stable  and/or  other  heat-labile 
factors  for  its  action?  What  is  the  mechanism  of  its  action? 


244 


ALTON  M.  MUN 


TABLE  II 

The  toxicity  of  heated  normal  rabbit  serum  (HNRS)  coupled  with  unheated 
chicken  serum  (CS)  or  unheated  guinea  pig  serum  (GPS) 


Serum 

Effects  on  embryos 

No.  embryos 
treated 

Normal 

Abnormal 

Dead 

HNRS 

43 

43 

0 

0 

CS 

29 

26 

0 

3 

HNRS 

+  CS  (10  to  50%) 

11 

11 

0 

0 

GPS 

40 

38 

2 

0 

HNRS 

+  GPS  (10  to  50%) 

27 

26 

1 

0 

The  role  of  complement  in  the  toxicity  of  fresh  rabbit  serum  was  examined 
first.  The  sufficiency  of  complement  was  tested  by  adding  complement  in  the  form 
of  fresh  guinea  pig  serum  or  fresh  chicken  serum  to  heated  normal  rabbit  serum. 
The  toxicity  which  wras  characteristic  of  fresh  rabbit  serum  was  not  restored  to 
heated  rabbit  serum  by  the  addition  of  either  fresh  chicken  serum  pooled  from  4  to 
6  chickens,  or  fresh  guinea  pig  serum  pooled  from  4  to  6  guinea  pigs  (Table  II). 

Next,  complement  or  components  of  complement  were  removed  from  rabbit 
serum  by  absorbing  unheated  fresh  rabbit  serum  with  a  nonspecific  precipitate  which 
had  been  prepared  by  combining  beef  serum  albumin  (BSA)  with  heated  homolo- 
gous rabbit  antiserum  (anti-BSA).  The  precipitate  was  washed  three  times  with 
cold  saline,  after  which  10  ml.  of  fresh  normal  rabbit  serum  were  added  to  0.5  ml. 
of  packed  precipitate.  The  mixture  was  refrigerated  (0  to  4°  C.)  for  8  to  12 
hours  with  frequent  stirring.  The  mixture  was  centrifuged ;  next  the  supernatant 
was  poured  into  another  tube  containing  0.5  ml.  of  packed  beef  serum  albumin  pre- 
cipitate. After  three  absorptions,  the  rabbit  serum  was  unable  to  lyse  chicken  red 
blood  cells.  The  fresh  rabbit  serum  absorbed  in  this  manner  was  still  toxic  to  the 
72-hour  chick  embryo.  The  toxicity  was  lost  only  after  heating  for  30  minutes 
at  56°  C.  (Table  III).  These  experiments  clearly  demonstrate  that  the  heat- 
labile  substance,  complement,  which  can  be  absorbed  by  a  nonspecific  precipitate, 
is  neither  sufficient  nor  necessary  for  the  toxic  action  of  fresh  rabbit  serum. 


TABLE  III 

The  toxicity  of  normal  rabbit  serum  (NRS)  absorbed  in  the.  cold  with  beef  serum 
albumin  (BSA)  precipitate  and  chicken  red  blood  cells  (RBC) 


Effects  on  embryos 

Serum 

No.  embryos 

treated 

Normal 

Abnormal 

Dead 

NRS 

BSA 

70 

37 

5 

28 

NRS 

BSA  then  heated 

33 

32 

0 

1 

NRS 

RBC 

36 

6 

3 

27 

NRS 

RBC  then  heated 

30 

28 

2 

0 

NRS 

BSA  and  RBC 

16 

6 

4 

6 

NRS 

BSA  and  RBC  then  heated 

10 

10 

0 

0 

TOXIC  EFFECTS  OF  ANTISERA  245 

To  determine  whether  or  not  heat-labile  factors  were  capable  of  acting  inde- 
pendently, heat-stable  substances  found  in  normal  rabbit  serum  were  removed  by 
absorption.  Several  substances  can  be  used  for  absorption  purposes,  among  them, 
chicken  red  blood  cells  which  were  selected  because  they  contain  Forssman  anti- 
gens (Boyd,  1956).  In  this  manner,  Forssman  antibodies,  as  well  as  other  sub- 
stances absorbable  by  chicken  red  blood  cells,  can  be  removed.  To  minimize  the 
destruction  of  heat-labile  substances  during  the  absorption  process,  the  procedure 
was  conducted  in  the  cold  (0  to  4°  C.).  Red  blood  cells  were  obtained  from  the 
pooled  blood  of  4  to  6  adult  New  Hampshire  chickens.  Approximately  2  ml.  of 
packed  red  blood  cells  were  used  in  the  absorption  of  each  10  ml.  of  serum.  The 
red  blood  cells  and  serum  were  thoroughly  mixed  by  frequent  stirring.  After  8 
to  12  hours,  the  cells  were  removed  by  centrifugation  at  1200  RCF  for  30  minutes. 
The  serum  was  poured  into  another  tube  containing  2  ml.  of  packed  red  blood  cells. 
The  process  was  repeated  until  no  further  agglutination  of  red  blood  cells  was 
observed  under  the  microscope.  The  cold-absorbed  rabbit  serum  was  still  highly 
toxic  to  the  72-hour  chick  embryo. 

Since  lysis  frequently  occurred  during  the  long  course  of  absorption  in  the  cold, 
it  was  necessary  to  inactivate  complement  by  removing  cations  before  absorbing 
with  red  blood  cells  either  by  filtering  the  serum  through  a  column  of  cation  ex- 
change resin  (IRC-50,  Rohm  and  Haas  Company,  Philadelphia,  Pa.),  as  described 
by  Levine  ct  al.  (1953),  or  by  adding  Versene  (sodium  ethylene  diamine  tetra- 
acetate)  to  the  serum.  The  latter  method  was  found  to  be  more  successful.  Upon 
completion  of  absorption,  calcium  and  magnesium  ions  were  reconstituted  to  a  final 
concentration  of  0.00015  M  and  0.0005  M,  respectively  (Mayer  and  Levine,  1954). 

The  cold-absorbed  serum  which  contained  Versene  and  an  insufficient  amount 
of  calcium  and  magnesium  ions  was  found  to  be  toxic  to  the  72-hour  chick  embryo. 
The  picture  of  toxicity,  however,  differed  from  that  produced  by  fresh  rabbit 
serum.  Upon  the  injection  of  absorbed  serum  containing  Versene  (a  6  millimolar 
solution  of  Versene  in  serum),  the  embryo  dies  within  a  few  minutes.  The  heart 
is  engorged  writh  blood  and  becomes  bright  red  in  appearance.  However,  the 
puckering  of  the  blastoderm  and  the  sinking  of  the  embryo,  which  is  characteristic 
of  the  effect  of  fresh  rabbit  serum,  is  not  observed.  The  toxic  effects  of  Versene 
are  observed  only  when  insufficient  calcium  and  magnesium  ions  are  present. 

To  determine  whether  or  not  complement  was  still  present  in  the  cold-absorbed 
rabbit  serum,  its  ability  to  lyse  chicken  red  blood  cells  was  tested.  The  cold- 
absorbed  serum  was  unable  to  lyse  chicken  red  blood  cells.  However,  when  a 
sufficient  amount  of  heated  but  unabsorbed  rabbit  serum  was  added  ( 1 :  1 ) ,  lysis 
occurred  readily.  Thus,  complement,  which  is  dependent  on  the  presence  of  heat- 
stable  substances,  was  not  destroyed  in  the  process  of  absorption  in  the  cold. 
Again,  the  toxic  effects  of  cold-absorbed  rabbit  serum  can  be  removed  by  heating 
(Table  III).  This  result  demonstrates  the  presence  of  a  toxic  heat-labile  sub- 
stance in  fresh  rabbit  serum,  a  substance  not  absorbed  by  chicken  red  blood  cells. 

Absorption  of  fresh  rabbit  serum  with  beef  serum  albumin  precipitates  followed 
by  chicken  red  blood  cells  in  the  cold  also  failed  to  remove  the  toxicity  of  the 
serum  (Table  III).  It  thus  may  be  concluded  that  complement  is  neither  sufficient 
nor  necessary  for  the  toxic  action  of  fresh  normal  rabbit  serum  which  is  evoked  in 
the  absence  of  substances  absorbable  with  chicken  red  blood  cells.  Moreover,  since 


PLATE  I 

Photographs  were  taken  in  ovo  through  the  cut  window  20-24  hours  after  treatment. 

246 


TOXIC  EFFECTS  OF  ANTISERA 


247 


the  action  of  properdin  requires  both  complement  and  magnesium  ions   (Pillemer 
et  al.,  1954),  it  is  suggested  that  this  heat-labile  substance  is  not  properdin. 

THE  EFFECTS  OF  ANTISERA  AGAINST  THE  72-HouR 
CHICK  EMBRYO  ON  THE  HOMOLOGOUS  EMBRYO 

It  is  clear  from  the  foregoing  experiments  that  to  remove  nonspecific  toxic 
factors,  normal  rabbit  sera  and  antisera  must  be  heated  for  30  minutes  at  56°  C. 
When  heated  rabbit  antiserum  against  the  72-hour  chick  embryo  was  placed  on  the 
embryo,  the  immediate  puckering  of  the  blastoderm,  together  with  its  sinking, 
which  was  characteristic  of  fresh  rabbit  serum,  was  not  observed.  However,  a 
number  of  the  embryos  died  after  6  to  8  hours ;  in  some  cases  the  embryos  did  not 
show  any  visible  effects  until  15  to  18  hours  after  the  operation,  at  which  time 
slight  abnormalities  were  detected.  Usually  no  further  changes  appeared  in  the 
surviving  embryos  after  18  to  20  hours.  Occasionally,  some  of  the  embryos  with 


TABLE  IV 

The  effects  of  heated  rabbit  antiserum  against  the  72-hour  chick 
embryo  (HA72)  coupled  with  guinea  pig  serum  (GPS) 


Graded  effects  on  72-hour  chick  embryos 

Serum 

No.  embryos 
treated 

1 

2 

3 

4 

5 

HNRS 

60 

59 

1 

0 

0 

0 

A72 

51 

1 

0 

0 

0 

50 

HA72 

57 

28 

7 

6 

16 

0 

HNRS  +  GPS 

27 

25 

1 

1 

0 

0 

HA72  +  GPS 

69 

19 

3 

8 

6 

33 

slight  visible  abnormalities  appeared  to  recover  completely.  The  toxic  effects  of 
the  sera  on  the  chick  embryo  arbitrarily  are  divided  into  five  different  groups 
(Table  IV;  Plate  I). 

PLATE  I 

Croup  1.  The  embryos  appear  essentially  normal  with  good  cole  r,  as  compared 
with  unoperated  embryos  of  the  same  stage  (Fig.  1). 

Group  2.  The  embryos  appear  essentially  normal  in  stage  and  color  but  show 
slight  morphological  abnormalities,  e.g.,  the  trunk  may  be  turned  ventrad,  instead 
of  to  the  left  in  embryos  in  stage  22.  These  abnormalities  may  be  detected  15  to 
18  hours  after  the  operation  (Fig.  2). 

FIGURE  1.     A  group  1  embryo  which  is  alive  and  appears  normal   (6X). 

FIGURE  2.     A  group  2  embryo  with  accumulation  of  blood  in  trunk  region   (6  X). 

FIG.  3.  A  group  3  embryo  which  is  alive  with  its  head  beneath  the  puckered  portion  of  the 
blastoderm  (6  X). 

FIGURE  4.  A  group  4  embryo  which  is  dead.  The  embryo  lies  on  top  of  the  blastoderm 
which  is  smooth  in  appearance  (6X). 

FIGURE  5.  A  group  5  embryo  which  is  dead  and  partially  hidden  by  the  puckered  blasto- 
derm (6X). 


248  ALTON  M.  MUN 

Group  3.  The  embryos  are  alive  but  show  distinct  abnormalities,  e.g.,  the  trunk 
may  be  turned  to  the  left  or  even  doubled  back  upon  itself.  The  embryos  are 
usually  pale  in  color.  These  abnormalities  may  be  detected  10  to  12  hours  after 
operation  (Fig.  3). 

Group  4.  The  embryos  are  dead  and  appear  quite  small  and  shrunken ;  blood 
vessels  are  not  distinct.  These  embryos  usually  die  5  to  8  hours  after  the 
operation  (Fig.  4). 

Group  5.  The  embryos  are  dead.  The  blastoderm  appears  puckered  or  pursed. 
The  blastoderm  may  be  seen  to  begin  to  shrivel  3  to  5  minutes  after  the  operation. 
The  red  blood  cells  may  be  seen  to  clump  in  the  blood  vessels  in  a  few  minutes  and 
then  cease  to  flow  in  the  smaller  vessels.  The  heart  may  stop  beating  as  soon  as 
5  minutes  after  the  operation  (Fig.  5). 

This  classification  of  the  extent  of  the  toxic  action  on  the  embryo  does  not 
imply  the  expression  of  basically  different  mechanisms  or  functions  in  each  of  the 
five  groups,  nor  does  it  indicate  distinct  and  separate  stages  or  steps  of  a  single 
mechanism  or  function.  The  embryos  earlier  described  as  "normal"  fall  into 
either  group  1  or  2.  Embryos  described  as  "abnormal"  are  similar  to  those  in 
group  3,  whereas  embryos  described  as  "dead"  fall  into  either  group  4  or  5.  The 
low  sensitivity  of  the  system,  probably  owing  in  part  to  the  low  titer  of  the  anti- 
sera  employed  (1 :  32  to  1  :  128),  as  well  as  to  the  heating  of  the  antisera,  increased 
the  possibility  of  introducing  false  negative  reactions.  Methods  were  sought, 
therefore,  to  increase  the  sensitivity  of  the  system.  From  the  foregoing  dis- 
cussion, it  is  apparent  that  methods  to  achieve  this  end  are  available ;  vis.,  the 
expedient  of  adding  back  those  substances  which  are  destroyed  by  heating,  such 
as  complement  and  properdin,  but  which  do  not  contribute  to  the  toxicity  of  normal 
rabbit  serum. 

THE  EFFECTS  OF  GUINEA  PIG  AND  RAT  SERUM  ON  THE  ACTIVITY 
OF  RABBIT  ANTISERUM  AGAINST  THE  72-HouR  CHICK  EMBRYO 

The  role  of  complement  in  vivo  is  not  fully  understood.  It  is  needed  in  addi- 
tion to  antibody  for  bactericidal  and  hemolytic  reactions  of  immune  sera,  as  well 
as  for  other  toxic  effects  (Boyd,  1956).  Witebsky  and  Neter  (1935)  found  that 
adding  fresh  guinea  pig  serum  to  heated  rabbit  antiserum  against  sheep  red  blood 
cells  restored  the  toxic  activity  of  the  antiserum  but  not  that  of  normal  rabbit 

j 

serum.  Imagawa  ct  al.  (1954)  showed  that  antisera  produced  in  guinea  pigs 
against  mouse  cancer  cells  when  heated  lost  the  ability  to  inactivate  mammary 
cancer  cells  but  that  this  activity  could  be  restored  by  the  addition  of  fresh  guinea 
pig  complement.  Therefore,  because  previous  experiments  showed  that  comple- 
ment was  neither  sufficient  nor  necessary  for  the  toxicity  of  fresh  rabbit  serum,  in 
an  attempt  to  increase  the  effectiveness  of  the  heated  antiserum,  complement  was 
returned  to  the  heated  rabbit  antiserum  in  the  form  of  fresh  guinea  pig  serum. 

Fresh  unheated  guinea  pig  serum,  obtained  from  the  pooled  blood  of  4  to  6 
guinea  pigs,  had  no  visible  effects  on  the  72-hour  chick  embryo.  Fresh  guinea 
pig  serum,  when  mixed  with  heated  normal  rabbit  serum  in  varying  proportions, 
also  showed  no  visible  effects.  However,  a  mixture  of  guinea  pig  serum  and 
heated  antiserum  against  the  72-hour  chick  embryo  was  quite  toxic  to  the  72-hour 
chick  embryo,  resulting  in  embryos  in  the  group  5  condition  (Table  IV). 


TOXIC  EFFECTS  OF  ANTISERA  249 

The  effects  of  the  addition  of  a  second  heat-labile  substance,  properdin,  were 
examined  next.  Pillemer  et  al.  (1954)  found  properdin  in  high  concentration  in 
the  rat  (25-50  units  properdin/ml.  serum),  in  intermediate  concentration  in  the 
rabbit  (4-8  units  properdin/ml.  serum),  and  in  low  concentration  in  guinea  pig 
serum  (1-2  units  properdin/ml.  serum).  Therefore,  rat  serum  was  chosen  as  the 
source  of  properdin.  Fresh  rat  serum  was  obtained  from  the  pooled  blood  obtained 
by  cardiac  punctures  from  4  to  6  large  white  rats.  Fresh  rat  serum  alone  was 
highly  toxic  to  the  72-hour  chick  embryo,  producing  the  striking  vascular  phe- 
nomena described  previously  at  all  concentrations  above  4%.  Heating  for  30 
minutes  at  56°  C.  removed  all  observable  toxic  effects.  Preliminary  experiments 
in  which  the  embryos  were  examined  5  minutes  to  4  hours  after  treatment  showed 
that  4%  fresh  rat  serum  mixed  with  heated  normal  rabbit  serum  was  extremely 
toxic  to  the  72-hour  chick  embryo. 

In  the  case  of  rat  serum,  the  titer  of  complement  is  low  but  the  concentration 
of  properdin  was  shown  to  be  high,  whereas,  in  the  case  of  the  guinea  pig  serum, 
the  titer  of  complement  is  high  but  the  concentration  of  properdin  is  low ;  there- 
fore a  study  of  the  combination  of  rat  and  guinea  pig  serum,  together  with  rabbit 
serum,  was  conducted.  A  mixture  of  fresh  rat  serum  and  guinea  pig  serum,  at  a 
dilution  in  which  neither  was  capable  of  eliciting  toxic  effects  alone,  was  quite  toxic 
to  the  chick  embryo.  The  toxicity  of  this  mixture  was  also  increased  when  heated 
normal  rabbit  serum  was  added  to  this  mixture.  The  toxicity  was  lessened  when 
the  concentration  of  the  heated  normal  rabbit  serum  was  reduced  by  dilution  with 
saline  (1:2  to  1:4).  These  experiments  suggest  the  possible  interaction  of  heat- 
labile  substances  in  guinea  pig  and  rat  sera  with  heat-stable  substances  in  guinea 
pig,  rat.  and  normal  rabbit  serum.  Thus,  the  following  absorption  studies  were 
conducted  to  remove  nonspecific  heat-stable  substances. 

ABSORPTION  STUDIES 

Forssman  antigen  is  reported  to  be  present  in  the  tissues  of  the  chick  embryo 
from  the  beginning  of  its  development.  Heated  rabbit  antiserum  against  sheep 
red  blood  cells  mixed  with  guinea  pig  serum  evoked  the  characteristic  vascular 
phenomenon  in  the  early  chick  embryo,  whereas  heated  normal  rabbit  serum  mixed 
with  guinea  pig  serum  would  not  (Witebsky  and  Neter,  1935).  Therefore,  it 
appeared  imperative  that  Forssman  type  antibodies  formed  as  a  result  of  the 
injection  of  chick  embryos  into  the  rabbit  be  removed  by  absorption  with  chicken 
red  blood  cells  (RBC).  This  procedure  increased  the  specificity  of  the  reaction 
but.  owing  to  the  concomitant  dilution,  decreased  the  sensitivity.  When  heated 
antiserum  against  72-hour  chick  embryos  was  absorbed  with  chicken  red  blood 
cells  at  37°  C.,  the  proportion  of  embryos  showing  the  group  4  condition  was  de- 
creased (Table  V). 

Adding  fresh  guinea  pig  serum  increased  the  toxicity  of  the  heated  and  ab- 
sorbed antiserum.  Several  embryos  in  the  group  5  condition  were  observed. 
Absorption  of  the  fresh  guinea  pig  serum  with  chicken  red  blood  cells  in  the  cold 
in  the  presence  of  Versene  decreased  the  action  of  the  heated  and  absorbed  anti- 
serum  and  absorbed  guinea  pig  serum  combination  (Table  V).  This  result  may 
have  been  due  to  some  inactivation  of  complement  during  the  process  of  absorption. 

As  shown  previously,  fresh  rat  serum  was  extremely  toxic  to  the  72-hour  chick 


250 


ALTON  M.  MUN 


embryo  at  concentrations  above  4%.  After  absorption  with  chicken  red  blood  cells 
in  the  presence  of  Versene  in  the  cold  (0  to  4°  C.),  the  rat  serum  was  no  longer 
toxic  to  the  embryo  at  concentrations  below  10%.  A  mixture  of  6%  absorbed  rat 
serum  and  94%  heated  and  absorbed  rabbit  antiserum  was  without  effect  on  the 
embryo,  as  was  a  mixture  of  6%  rat  serum  and  94%  heated  and  absorbed  rabbit 
antiserum  (Table  VI).  This  is  in  strong  contrast  to  the  boosting  effect  of  the 
addition  of  guinea  pig  serum  to  the  heated  rabbit  antiserum.  However,  this  find- 
ing is  not  unexpected,  because  properdin  acts  only  in  conjunction  with  complement 
and  magnesium  ions  (Pillemer  et  a/.,  1954)  and  the  concentration  of  comple- 
ment in  rat  serum  is  low  (Hegediis  and  Greiner,  1938). 

A  mixture  of  guinea  pig  serum  and  rat  serum  at  a  dilution  in  which  neither 
could  elicit  toxic  effects  was  shown  to  be  extremely  toxic  to  the  72-hour  chick 
embryo.  After  absorption  of  the  rat  serum  in  the  cold  with  RBC  following  filtra- 
tion through  a  cation  exchange  (IRC-50)  column,  the  toxic  activity  of  the  rat 
and  guinea  pig  serum  mixture  was  decreased. 

TABLE  V 

The  effects  of  absorption  on  the  toxicity  of  heated  rabbit  antiserum  against  72-hour  chick 
embryos  (PIA72)  and  unheated  guinea  pig  serum  (GPS)  combinations 


TVT/\ 

Graded  effects  on  embryos 

Serum 

Absorbed 

with 

Combined  with 

IN  O. 

embryos 

Vv  1  L  1  1 

treated 

1 

2 

3 

4 

5 

HNRS 

RBC 

None 

52 

40 

9 

3 

0 

0 

HA72 

None 

None 

57 

28 

7 

6 

16 

0 

HA72 

RBC 

None 

45 

30 

2 

6 

7 

0 

HNRS 

RBC 

GPS 

19 

18 

1 

0 

0 

0 

HA72 

RBC 

GPS 

63 

5 

7 

8 

3 

40 

HA72 

RBC 

GPS,  heated 

23 

10 

6 

2 

5 

0 

|  GPS 

RBC 

None 

7 

7 

0 

0 

0 

0 

HNRS 

RBC 

Absorbed  GPS 

14 

13 

1 

0 

0 

0 

HA72 

RBC 

Absorbed  GPS 

17 

3 

3 

1 

4 

6 

The  toxicity  of  a  mixture  of  6%  fresh  rat  serum,  10%  guinea  pig  serum,  and 
84%  heated  normal  rabbit  serum  mixture  was  also  reduced  or  removed  altogether 
by  the  absorption  of  the  guinea  pig  serum  and  rat  serum  at  0  to  4°  C.  with  chicken 
red  blood  cells  in  the  presence  of  Versene,  and  by  the  absorption  of  the  heated 
normal  rabbit  serum  at  37°  C.  with  chicken  red  blood  cells.  On  the  other  hand, 
a  similar  mixture  of  10%  absorbed  guinea  pig  serum,  6%  absorbed  rat  serum, 
with  84%  heated  and  absorbed  rabbit  antiserum  was  toxic  to  the  embryo.  Imme- 
diate vascular  effects  were  observed,  followed  by  the  cessation  of  heart  contractions 
within  30  minutes  (Table  VI).  The  proportion  of  embryos  exhibiting  toxic 
effects  was  greater  in  this  rabbit  antiserum  mixture  containing  both  guinea  pig 
serum  and  rat  serum  than  that  in  rabbit  antiserum  mixtures  containing  either 
guinea  pig  serum  or  rat  serum  alone.  Hence,  a  method  is  available  to  increase 
the  effectiveness  of  antisera.  Since  rat  serum  enhanced  the  effect  of  the  antiserum 
only  in  the  presence  of  complement,  it  is  suggested  that  a  factor  or  factors  analo- 
gous to  properdin  may  be  involved.  Need  for  further  experiments  using  purified 
properdin  is  indicated. 


TOXIC  EFFECTS  OF  ANTISERA 


251 


Properdin  can  participate  in  such  diverse  activities  as  the  destruction  of  bac- 
teria, the  neutralization  of  viruses,  and  the  lysis  of  certain  red  blood  cells  (Pillemer 
et  al.,  1955).  Although  the  presently  reported  experiments  suggest  the  interaction 
of  "the  properdin  system"  with  this  specific  antibody  in  the  serum  (A72),  it  is 
possible  that  the  rat  serum  acts  by  supplementing  the  components  of  complement 
which  are  low  in  both  rabbit  and  guinea  pig  sera,  e.g.,  the  C'l  component  (HegediAs 
and  Greiner,  1938).  The  application  of  quantitative  techniques  for  handling  com- 
plement and  the  use  of  purified  components  of  complement,  C'l,  C'2,  C'3,  and 
C'4,  may  elucidate  this  aspect  of  the  problem. 

The  absorption  of  the  antiserum  against  72-hour  chick  embryos  with  the  hom- 
ologous antigen  removed  most  of  the  toxic  effects  of  the  antiserum  on  the  72-hour 
chick  embryo.  The  antiserum  was  first  heated  and  absorbed  with  chicken  red 
blood  cells  in  the  manner  described  previously,  and  then  mixed  with  a  slight  excess 
of  minced  and  homogenized  72-hour  chick  embryos.  The  suspension  was  placed 


TABLE  VI 

The  effects  of  the  addition  of  absorbed  rat  scrum  (RAT-RBC)  and  absorbed  guinea  pig  serum 

(GPS-RBC)  to  heated  and  absorbed  antiserum  against  the  72-hour 

chick  embryo  (HA72-RBC) 


No. 

Graded  effects  on  embryos 

Serum 

embryos 

treated 

1 

2 

3 

4 

5 

RAT-RBC  (100%) 

10 

0 

1 

1 

2 

6 

RAT-RBC  (6  to  10%) 

8 

8 

0 

0 

0 

0 

HNRS-RBC  (04%)  +  RAT-RBC  (6%) 

9 

9 

0 

0 

0 

0 

HNRS-RBC  (84%)  +  GPS-RBC  (10%) 

+  RAT-RBC  (6%) 

16 

14 

2 

0 

0 

0 

HA72-RBC  (100%) 

45 

30 

2 

6 

7 

0 

HA72-RBC  (94%)  +  RAT-RBC  (6%) 

18 

9 

8 

0 

1 

0 

HA72-RBC  (84%)  +  GPS-RBC  (10%) 

+  RAT-RBC  (6%) 

32 

5 

2 

2 

5 

18 

in  the  water  bath  for  two  to  three  hours  at  37.5°  C.  A  dense  white  precipitate  was 
usually  observed  after  15  to  30  minutes.  The  tube  was  placed  in  the  refrigerator 
at  0  to  4°  C.  for  12  hours  and  later  centrifuged  at  1200  RCF  for  30  minutes.  The 
supernatant  was  poured  off  into  another  tube  containing  a  slight  excess  of  homoge- 
nized 72-hour  chick  embryos.  A  smaller  amount  of  precipitate  was  observed  after 
the  second  absorption.  The  process  was  repeated  until  a  negative  interfacial 
"ring"  test  was  obtained. 

When  guinea  pig  and  rat  sera  were  added  to  this  heated  antiserum  which  had 
been  absorbed  with  both  chicken  red  blood  cells  and  72-hour  chick  embryos,  the 
toxic  effects  of  the  antiserum  were  not  found  to  be  completely  removed  (Table  VII) . 
The  failure  of  the  absorption  of  the  antiserum  by  the  homologous  antigen  is  sur- 
prising but  not  without  precedent.  Ebert  (1950)  reported  the  failure  of  absorp- 
tion by  homologous  antigen  to  remove  the  striking  lethal  and  growth  inhibitory 
powers  of  anti-organ  sera.  This  non-absorption  of  one  fraction  of  the  antiserum 
was  attributed  to  individual  differences  in  the  organ  antigens  used  in  injections 


252 


ALTON  M.  MUN 


TABLE  VII 

The  toxicity  of  heated  antiserum  against  the  72-hour  chick  embryo  (HA  72)  absorbed 

with  the  homologous  antigen,  singly,  and  in  combination  with  absorbed  guinea 

pig  serum  (GPS)  and  absorbed  rat  serum  (RA  T) 


No. 

Graded  effects  on  embryos 

Serum 

Absorbed  with 

embryos 

treated 

1 

2 

3 

4 

5 

HA72 

RBC 

11 

8 

1 

0 

2 

0 

HA72 

RBC,  72-hour  chick 

embryos 

23 

21 

2 

0 

0 

0 

HA72  (90%)  +  GPS  (10%) 

RBC 

57 

3 

5 

7 

3 

39 

HA72  (90%)  +  GPS  (10%) 

RBC,  72-hour  embryos 

34 

21 

6 

2 

4 

1 

HA72  (94%)  +  RAT  (6%) 

RBC 

23 

14 

8 

0 

1 

0 

HA72  (94%)  +  RAT  (6%) 

RBC,  72-hour  embryos 

12 

8 

0 

1 

3 

0 

HA72(84%)+GPS(10%) 

+  RAT  (6%) 

RBC 

32 

5 

2 

2 

5 

18 

HA72  (84%)  +  GPS  (10%) 

+  RAT  (6%) 

RBC,  72-hour  embryos 

14 

7 

1 

1 

1 

4 

and  absorptions.  Although  large  numbers  of  embryos  were  used  in  both  injections 
and  absorptions,  a  long  course  of  injections  was  given.  Such  treatment  often  re- 
sults in  antisera  of  reduced  specificity.  This  result  may  be  even  more  pronounced 
in  animals  receiving  adjuvant.  However,  the  injection  of  adjuvant  with  heterolo- 
gous  antigen  was  insufficient  to  evoke  a  nonspecific  response  in  the  rabbit.  Beef 
serum  albumin  (BSA)  combined  with  adjuvant  was  injected  into  three  rabbits. 
Tests  of  heated  anti-BSA,  and  heated  anti-BSA  absorbed  with  BSA  were  nega- 
tive (Table  VIII).  The  number  of  different  kinds  of  antibodies  may  be  so  great 
as  to  be  incompletely  absorbed  by  the  antigen,  even  though  an  excess  of  antigen 
was  used  in  absorptions  and  negative  interfacial  "ring"  tests  were  obtained  after 
the  final  absorption.  This  is  not  to  say,  however,  that  antibodies  with  new  and 
different  specificities  are  formed.  The  in  vivo  system  employed  here  may  be  so 
sensitive  as  to  respond  strongly  to  these  weaker  or  less  "avid"  antibodies.  That 
embryonic  proteins  may  be  unique  in  their  behavior  in  precipitin  reactions  was 
reported  by  Schechtman  (1952),  who  found  an  unusual  result  when  antiserum 
against  the  plasma  from  the  10-day  embryo  was  reacted  with  adult  and  10-day 

TABLE  VIII 

Effects  of  antiserum  against  beef  serum  albumin  (A BSA)  on  the  72-hour  chick  embryo 


No. 

Effects  on  embryos 

Treatment  of  serum 

embryos 

treated 

1 

2 

3 

4 

5 

ABSA  imheated 

3 

0 

0 

0 

0 

3 

ABSA  heated 

7 

6 

1 

0 

0 

0 

ABSA  heated  and  absorbed  with 

RBC  +  GPS 

15 

15 

0 

0 

0 

0 

ABSA  heated  and  absorbed  with 

RBC  and  BSA  +  GPS 

6 

6 

0 

0 

0 

0 

TOXIC  EFFECTS  OF  ANTISERA  253 

serum.  He  wrote  (p.  95),  "This  antiserum  forms  higher  (antigen-antibody  pre- 
cipitation) curves  with  the  heterologous  antigen,  adult  serum.  The  antiserum  is 
obviously  not  lacking  in  antibody  since  it  produces  heavy  precipitates  with  adult 
material."  He  concluded  that  the  embryonic  serum  forms  antigen-antibody  com- 
plexes with  inferior  light-scattering  properties  or  that  it  contains  substances  in- 
hibitory to  the  precipitin  reaction. 

DISCUSSION 

The  toxicity  of  fresh  rabbit  serum  to  the  early  chick  embryo  was  destroyed  by 
heating  at  56°  C.  for  30  minutes.  The  above  experiments  show  clearly  that  the 
toxic  substance  in  fresh  rabbit  serum  is  not  complement ;  nor  is  it  dependent  on 
complement  for  its  activity.  In  view  of  the  latter  observation,  it  is  also  probably 
not  properdin.  The  following  questions  remain  to  be  answered :  ( 1 )  What  are 
the  physicochemical  properties  of  this  toxic  heat-labile  substance?  (2)  Is  is  com- 
posed of  one  or  many  substances?  Can  substances,  other  than  complement,  be 
separated  or  isolated  from  this  heat-labile  fraction  which  would  further  enhance 
the  action  of  heat-stable  fractions,  as  was  shown  above  for  complement  and  proper- 
din  or  properdin-like  substances?  (3)  What  is  the  mechanism  of  action  of  this 
heat-labile  substance?  Is  it  similar  to  that  brought  about  by  heat-stable  fractions? 
It  was  observed  that  the  toxic  effects  of  fresh  normal  rabbit  serum  in  general 
resembled  those  produced  by  the  action  of  heated  rabbit  antiserum  to  the  72-hour 
chick  embryo  coupled  with  fresh  guinea  pig  serum  and  rat  serum.  Witebsky  and 
Neter  (1935)  also  described  similar  toxic  effects  on  the  early  chick  embryo  of 
heated  rabbit  anti-sheep  red  blood  cell  serum  plus  complement.  Pomerat  (1949) 
reported  similar  results  with  rabbit  anti-chick  spleen  serum.  However,  although 
the  final  picture  appears  to  be  the  same,  the  mechanisms  involved  may  not  be 
similar.  The  development  and  use  of  more  specific  antisera  to  embryonic  anti- 
gens may  reveal  more  definitive  and  specific  morphological  expressions  than  those 
elicited  by  toxic  factors  in  fresh  rabbit  serum.  The  present  study  has  demon- 
strated that  the  72-hour  chick  embryo  is  antigenic,  i.e.,  capable  of  eliciting  the 
production  of  precipitating  antibodies. 

The  presently  reported  investigation  also  demonstrated  the  fact  that  comple- 
ment and  properdin  or  properdin-like  substances  can  play  an  active  role  in  the 
action  of  the  antiserum  in  vivo.  The  demonstrated  ability  of  complement  and 
properdin  or  properdin-like  substances  to  increase  the  magnitude  of  the  action  of 
the  antiserum  will  permit  the  observation  of  the  effects  of  weaker  but  perhaps  more 
specific  antisera  which  otherwise  would  go  unnoticed.  Thus,  the  manner  in  which 
antisera  act  to  block  development  or  modify  the  normal  function  of  reactive  groups 
in  the  embryo  may  be  studied  more  readily.  The  use  of  purified  properdin  or 
related  substances,  together  with  the  components  of  complement,  C'l,  C'2,  C'3, 
and  C'4,  may  contribute  to  our  understanding  of  the  mechanism  of  action  of  the 
toxic  antiserum  in  vivo. 

I  wish  to  express  my  sincere  appreciation  to  Professor  James  D.  Ebert  for  his 
encouraging  interest  and  valuable  advice  throughout  the  course  of  this  investiga- 
tion, and  to  Dr.  Royal  F.  Ruth  for  his  many  helpful  suggestions.  I  also  wish  to 
thank  Dr.  Joseph  F.  Albright  and  Mr.  Lowell  M.  Duffey  for  expert  technical  aid. 


254  ALTON  M.  MUN 

SUMMARY 

1.  The  specific  objectives  of  the  present  investigation  were  at  first  two-fold: 
(1)  to  determine  the  antigenicity  of  the  early  chick  embryo,  and  (2)  to  study  the 
effects  of  homologous  antisera  on  the  chick  embryo.     However,  because  at  the 
outset  a  profound  toxic  action  of  fresh  normal  rabbit  serum  was  encountered,  it 
became  imperative  to  describe  the  toxic  factor. 

2.  The  toxic  action  of  normal  rabbit  serum,  characterized  by  the  puckering  of 
the  blastoderm,  the  sinking  of  the  embryo  and  its  ultimate  death,  was  removed  In- 
heating  at  56°  C.  for  30  minutes.     The  toxic  action  was  not  restored  by  adding 
fresh  guinea  pig  serum  to  heated  rabbit  serum.     The  toxicity  was  not  removed 
by  absorption  in  the  cold  with  nonspecific  antigen-antibody  precipitates  and/or 
thicken  red  blood  cells.     These  results  are  interpreted  as  indicating  that  comple- 
ment is  neither  necessary  nor  sufficient  for  the  toxic  action  of  fresh  rabbit  serum. 
The  toxic  heat-labile  substance  can  also  act  independently  of  heat-stable  substances 
which  are  removed  by  absorption  with  chicken  red  blood  cells. 

3.  The  antigenicity  of  the  72-hour  chick  embryo  was  demonstrated  by  its  ability 
to  elicit  the  production  of  precipitating  antibodies  in  the  rabbit.     Heated  rabbit 
antiserum  against  the  72-hour  chick  embryo   evoked   a   weak  but   definite   toxic 
response  when  placed  on  the  homologous  embryo. 

4.  In  an  attempt  to  decrease  the  probability  of  false  negative  reactions,  methods 
were  sought  to  increase  the  effectiveness  of  the  antisera.     Substances  which  may 
have   been   inactivated   by   heat   were   returned   to   the   antiserum    singly    and   in 
combination. 

5.  The  toxic  action  of  heated  rabbit  antiserum  was  partially  enhanced  by  the 
addition  of  fresh  guinea  pig  serum,  rich  in  complement. 

6.  The  toxic  action  of  the  heated  rabbit  antiserum  was  not  increased  by  adding 
fresh  rat  serum,  reported  to  contain  large  amounts  of  properdin,  but  was  enhanced 
by  a  mixture  of  guinea  pig  serum  and  rat  serum. 

7.  The    results   suggest   the    interaction    of   complement   and   properdin    or    a 
properdin-like  factor  in  the  action  of  the  antiserum  on  the  chick  embryo. 

LITERATURE  CITED 

BERNHEIMER,  A.  W.,  AND  J.  A.  HARRISON,  1940.     Antigen-antibody  reactions  in  Parameciitm  : 

the  aurelia  group.     /.  Immunol.,  39:  73-83. 
BOYD,  W.   C.,   1956.     Fundamentals  of   Immunology.     Third   Edition.     Interscience   Publishers, 

Inc.,  New  York. 
BURKE,  V.,  N.  P.  SULLIVAN,  H.  PETERSEN  AND  R.  WEED,  1944.     Ontogenetic  change  in  antigenic 

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256  ALTON  M.  MUN 

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THE  DYNAMICS  OF  A  DIATOM  BLOOM  1 

1.  H.  RYTHER,  C.  S.  YENTSCH,  E.  M.  HULBURT  AND  R.  F.  VACCARO 
IVoods  Hole  Occanographic  Institution,  Woods  Hole,  Mass. 

Phytoplankton  cells  respond  so  rapidly  to  their  environment  that  conventional 
methods  of  studying  their  populations  fail  to  reveal  many  of  the  more  subtle  and 
more  interesting  aspects  of  their  dynamic  ecology.  This  is  particularly  true  of 
surveys  in  which  observations  are  made  at  intervals  of  weeks  or  months,  where 
the  very  use  of  the  term  "the  phytoplankton  population,"  when  carried  over  from 
one  set  of  observations  to  the  next,  implies  more  knowledge  than  is  available.  It 
is  also  true  of  productivity  measurements  made  over  24  hours  or  the  daylight  por- 
tion of  a  day,  since  Rodhe  (personal  communication).  Doty  and  Oguri  (1957), 
Yentsch  and  Ryther  (1957)  and  others  have  shown  that  the  plants  vary  in  their 
composition  and  react  differently  to  their  environment  at  different  times  of  the 
day.  To  study  such  phenomena  it  is  obviously  necessary  to  make  intensive  obser- 
vations at  very  frequent  intervals  throughout  one  or  more  days  on  single,  isolated 
populations. 

This  type  of  study  presents  obvious  difficulties.  The  average  natural  population 
is  sparse  enough  so  that  its  properties  can  be  measured  only  with  rough  accuracy, 
and  the  errors  of  such  measurements  may  be  larger  than  the  changes  in  the 
organisms  and  their  environment  which  are  under  investigation.  Some  insight 
into  these  problems  may  be  had  by  studying  cultures,  but  the  difficulties  of  growing 
organisms  under  completely  natural  conditions  need  no  elaboration  here.  A  com- 
promise may  be  reached,  however,  by  working  with  a  dense  phytoplankton  bloom. 
Here  natural  populations  may  be  studied  under  their  natural  growing  conditions 
and  very  rapid  responses  of  the  organisms  to  changes  in  their  physical  or  chemical 
environment  may  be  detected  and  measured. 

The  authors  encountered  such  a  diatom  bloom  in  a  small  tidal  creek  on  the 
south  shore  of  Long  Island,  N.  Y.,  in  June,  1957.  The  following  report  will 
describe  the  studies  of  this  bloom  which  were  made  over  a  40-hour  period  including 
two  days  and  one  night. 

DESCRIPTION  OF  THE  AREA 

Senix  Creek  is  approximately  one  mile  long,  tapering  from  a  width  of  about  300 
meters  at  its  mouth  to  less  than  10  meters  at  its  upper  end  in  the  town  of  Center 
Moriches.  Our  observations  were  made  about  halfway  up  its  length  where  the 
water  depth  is  approximately  one  meter.  Underlying  this  shallow  body  of  water 
is  a  thick  deposit  of  black,  organic  muck  which  discharges  H2S  gas  when  disturbed. 

There  is  no  river  or  other  obvious  source  of  fresh  water  to  Senix  Creek  except 
for  local  runoff.  The  latter  was  negligible  in  the  early  summer  of  1957  due  to 

1  Contribution  No.  978  from  the  Woods  Hole  Oceanographic  Institution.  This  study  was 
made  with  the  assistance  of  research  grants  from  the  National  Science  Foundation. 

257 


258  RYTHER,  YENTSCH,  HULBURT  AND  VACCARO 

abnormally  low  rainfall  (none  whatever  during  the  month  of  June).  This  drought 
was  undoubtedly  an  important  contributory  factor  to  the  existence  of  the  diatom 
bloom,  since  it  helped  to  maintain  the  salinity  at  a  moderately  high  level  in  the 
estuary  and  also  reduced  flushing  action  so  that  the  latter  did  not  greatly  influence 
the  population  at  the  time  and  place  of  our  observations. 

While  many  of  the  cributaries  to  Moriches  Bay  receive  large  quantities  of  pollu- 
tants from  duck  farms  which  line  their  shores  (see  Ryther,  1954),  there  is  no  such 
direct  source  of  enrichment  to  Senix  Creek.  The  origin  of  the  nutrients  which 
gave  rise  to  the  phytoplankton  bloom  in  question  was  not  investigated.  They 
presumably  resulted  either  from  domestic  pollution  from  Center  Moriches  and  the 
residences  located  along  the  banks  of  the  creek,  or  from  an  invasion  of  water  from 
one  of  the  other,  heavily  polluted  estuaries  via  Moriches  Bay  (i.e.,  Forge  River 
located  just  V<y  mile  from  Senix  Creek  at  their  respective  mouths). 

METHODS  AND  PROCEDURE 

The  studies  were  initiated  at  sunrise  on  June  26,  with  the  plan  to  make  obser- 
vations hourly  during  the  day  and  less  frequently  at  night  for  24  hours.  Unfor- 
tunately, the  day  was  foggy  and  partially  overcast,  and  it  seemed  doubtful  that  we 
would  be  able  to  observe  phenomena  associated  with  high  incident  light  intensities. 
Consequently  we  continued  the  study  for  the  daylight  portion  of  a  second  day, 
when  the  sky  was  clear. 

Incident  radiation  was  measured  at  one-hour  intervals  throughout  the  day  with 
a  GE  radiation  meter.  Light  penetration  was  measured  with  a  submarine  pho- 
tometer constructed  from  a  Weston  photronic  cell.  Water  level,  measured  with 
an  improvised  tide  gage,  was  also  recorded  hourly  during  the  day,  less  frequently 
at  night.  At  these  same  time  intervals  water  samples  were  collected  in  a  bottle 
equipped  with  a  siphon  which  permitted  the  sample  to  enter  through  a  tube  run- 
ning to  the  bottom  of  the  bottle  and  the  air  to  escape  through  a  tube  extending 
to  the  surface  of  the  water.  In  this  way  samples  for  gas  analysis  were  not  con-- 
taminated  by  bubbles.  As  the  bottle  filled  it  was  slowly  lowered  from  the  surface 
to  the  bottom,  thereby  obtaining  an  integrated  sample  of  the  one-meter  water 
column.  Immediately  after  collection,  the  water  temperature  was  taken  and  an 
aliquot  of  the  sample  was  siphoned  into  a  150-ml.  glass-stoppered  bottle  and  ana- 
lyzed for  dissolved  oxygen  using  the  Pomeroy-Kirschman-Alsterberg  modification 
of  the  Winkler  method  (see  APHA,  1955).  The  pH  of  the  sample  was  measured 
with  a  Coleman  pH  meter,  and  a  100-ml.  aliquot  \vas  withdrawn  and  millipore- 
filtered  for  subsequent  pigment  analysis  using  the  method  of  Richards  with  Thomp- 
son (1952)  as  modified  by  Creitz  and  Richards  (1955).  Pigments  were  computed 
using  the  nomographs  prepared  by  Duxbury  and  Yentsch  (1956). 

Every  two  hours  during  the  day,  additional  aliquots  wrere  siphoned  into  four 
150-ml.  bottles,  one  of  which  was  darkened  with  black  tape.  These  were  then 
suspended  in  the  water,  the  three  transparent  bottles  at  depths  of  0,  0.5  and  1.0 
meter.  After  two  hours,  the  bottles  were  removed  and  their  dissolved  oxygen 
concentration  determined. 

At  high  and  low  water  each  day,  as  determined  from  the  tide  gage,  additional 
samples  were  collected  from  the  surface  and  from  a  few  centimeters  above  the 
bottom.  These  were  returned  to  the  laboratorv  where  thev  were  analvzed  for 


DYNAMICS  OF  A  DIATOM  BLOOM  259 

salinity  and  used  for  total  phytoplankton  counts.  At  10:30  on  June  27  a  single 
sample,  taken  from  the  whole  water  column,  was  frozen  and  subsequently  analyzed 
for  phosphorus  and  nitrogen  fractions.2 

OBSERVATIONS  AND  RESULTS 
a.  The  phytoplankton 

The  phytoplankton  population  in  Senix  Creek  consisted  predominantly  of  cen- 
tric diatoms,  principally  Chactoccros  simplex,  Thalassiosira  nana,  and  Skeletoncuia 
costatum.  Other  species  present  in  abundance  were  the  naviculoid  diatom  Phaco- 
dactylum  tricornutum  (Nitsschia  closterium  forma  minutissima) ,  the  green  flagel- 
late Carteria  excavata,  and  the  dinoflagellate,  Pr or o centrum  minimum.  The  con- 
centration of  diatoms  alone  ranged  from  61  to  109  million  cells  per  liter.  In  addi- 

TABLE  I 
The  vertical  distribution  of  salinity  and  diatoms  in  Senix  Creek 

Salinity  Diatoms 

(%o)  (lOVHter) 

June  26     High  tide 

surface  11.94  92.8 

bottom  13.48  80.6 

Low  tide 

surface  10.22  109.0 

bottom  13.10  82.0 

June  27     High  tide 

surface  11.73  82.8 

bottom  14.54  61.3 

Low  tide 

surface  11.64  79.5 

bottom  16.53  61.3 

tion  there  were  observed  large  numbers  of  small  coccoid  cells,  1-2  ^  in  diameter, 
which  were  not  identified  but  were  either  bacteria,  blue-green  or  green  algae. 
They  bore  some  resemblance  to  the  green  alga,  Nannochloris  at  omits,  which  was 
formerly  present  throughout  Moriches  Bay  and  its  tributaries  in  concentrations 
exceeding  1010  cells  per  liter  prior  to  the  opening  of  Moriches  Inlet  in  1954.  At 
that  time  the  growth  of  Nannochloris,  which  virtually  replaced  the  normal  estuarine 
plankton  flora,  was  attributed  to  high  concentrations  of  pollutants  originating  from 
the  duck  farms,  low  salinities,  and  high  temperatures  (Ryther,  1954).  These 
conditions  still  persist  near  the  sites  of  pollution  in  the  estuaries  of  Moriches  Bay 
(for  example  in  the  Forge  River  and  Seatuck  Cove),  where  the  phytoplankton  was 
dominated  by  green  algae  and  the  water  was  a  distinct  green  color  in  contrast  to 
the  rich  brown  color  of  the  water  in  Senix  Creek. 

The  small  microorganisms  in  Senix  Creek,  though  about  ten  times  as  numerous 

2  Analyses  were  made  by  methods  described  in  the  following  references :  inorganic  phos- 
phorus (Robinson  and  Thompson,  1948)  ;  total  phosphorus  (Harvey,  1948)  ;  ammonia  (Riley, 
1953)  ;  nitrite  (Rider  and  Mellon,  1946)  ;  nitrate  (Mullin  and  Riley,  1955). 


260 


RYTHER,  YENTSCH,  HULBURT  AND  VACCARO 


i i    i    i    i    i    i    i    i 


. — i — =»a ii  i i      •     L^-*—  -i 

3       15        17       19      21         23 1     I          3        5         7         9 


JUNE     27 


FIGURE  1.  Variables  measured  in  Senix  Creek  between  05:00,  June  26  and  20:00,  June  27. 


DYNAMICS  OF  A  DIATOM  BLOOM 


261 


as  the  diatoms,  were  probably  insignificant  in  terms  of  total  biomass  since  their 
cell  volume  is  several  hundreds  of  times  smaller  than  that  of  the  average  diatom. 

b.  The  physical  environment 

Figure  1A  shows  the  incident  radiation  for  the  two  days.  The  total  daily 
radiation,  obtained  by  integration  of  these  curves,  was  300  gram-calories/cm2,  on 
June  26  and  740  gram-calories/cm-,  on  June  27. 

MOi-i. 


. 
UJ 


100 


co 


UJ 

o 


O 

CO 

z 
o 


CO 

^. 
o 


90 


80 


70 


60 


50 


10 


12 


13 


14 


15 


16 


17 


SALINITY    %<> 


FIGURE  2.  The  relation  between  salinity  and  total   diatom   concentration,   showing 
least  squares  line   (R  =  —  .875).     Data  from  Table  I. 


The  tidal  influence  in  Senix  Creek  is  extremely  small.  The  range  between 
high  and  low  water  on  both  days  \vas  approximately  seven  inches.  Salinities 
ranged  from  10-15  %0  and  showed  no  obvious  correlation  with  the  stage  of  the  tide. 
However,  salinities  at  the  bottom  were  slightly  higher  than  those  at  the  surface, 
and  salinities  were  higher  the  second  day  of  observations  than  on  the  first  (Table  I). 

Diatom  counts  were  slightly  lower  at  the  bottom  than  at  the  surface  and  lower 
at  both  depths  on  the  second  day.  Again  there  was  no  obvious  relation  to  the  tide, 


262  RYTHER,  YENTSCH,  HULBURT  AND  VACCARO 

but  there  was  a  good  inverse  correlation  (r  —  —0.875)  between  the  diatom  concen- 
tration and  the  salinity  (Fig.  2).  This  correlation  suggests  that  the  diatom  popu- 
lation did  not  change  over  the  two-day  period  as  a  result  of  growth  or  death,  but 
that  the  population  was  being  diluted  slowly  with  water  from  Moriches  Bay,  where 
the  salinity  ranged  from  20  to  25  %0  and  diatom  concentrations  were  generally  less 
than  one  million  cells  per  liter. 

c.  Dissolved  oxygen  and  pH 

Both  pH  and  dissolved  oxygen  behaved  in  essentially  the  same  manner,  as  may 
be  seen  by  comparing  Figures  IB  and  C.  However,  the  high  pH  attained  in  the 
late  afternoon  of  both  days  was  maintained  for  several  hours  whereas  the  oxygen 
concentration  reached  its  peak  at  the  same  time  but  then  began  to  decline  imme- 
diately. Water  temperatures  (which  are  not  shown)  ranged  from  25°  C.  to  28°  C. 
during  the  two-day  period.  Assuming  a  mean  salinity  of  I2r/Cc,  the  water  was 
approximately  90%  saturated  with  oxygen  at  daybreak,  about  270%  saturated  at 
14 :00  on  June  27.  Despite  this  supersaturation,  there  did  not  appear  to  be  a  sig- 
nificant loss  of  oxygen  to  the  air  by  diffusion  since  the  decrease  in  oxygen  concen- 
tration at  night  by  respiration  appears  to  have  occurred  at  a  constant  rate.  If 
appreciable  loss  by  diffusion  had  occurred,  this  would  have  been  dependent  upon 
the  oxygen  concentration,  and  the  decrease  due  to  both  causes  (respiration  and 
diffusion)  would  have  been  non-linear. 

The  pH  reached  a  minimum  of  about  8.5  early  in  the  morning  and  a  maximum 
of  almost  9.5  in  the  afternoon.  Presumably  at  its  maximum,  no  free  CO.,  was 
available  and  any  further  photosynthesis  was  dependent  upon  bicarbonate  or  car- 
bonate ions.  Unfortunately  no  measurements  were  made  of  CO2  in  any  of  its 
fractions,  nor  may  these  values  be  calculated  from  pH,  salinity,  and  temperature 
for  these  estuarine  conditions  as  they  may  for  either  fresh  or  sea  water.  Again 
the  regular  behavior  of  the  pH  curve  with  time,  shown  in  Figure  1C,  indicates  that 
CO2  diffusion  from  air  to  water  was  negligible  in  comparison  to  the  changes  caused 
by  photosynthesis  and  respiration. 

d.  Plant  pigments 

Figure  ID  shows  the  concentration  of  chlorophyll  a  in  the  composite  samples 
taken  during  the  two-day  period.  Since  the  cell  counts  \vere  not  made  on  the 
same  samples,  it  is  not  possible  to  represent  chlorophyll  on  a  cellular  basis.  The 
chlorophyll  concentration  in  the  water  ranged  from  116  to  245  mg./m3..  about  a 
two-fold  variability.  Although  the  highest  concentrations  coincided  on  both  days 
with  low  water,  the  connection  between  these  factors  is  probably  fortuitous.  Cer- 
tainly the  variations  in  the  pigment  concentration  are  far  greater  than  the  observed 
differences  in  the  diatom  counts,  caused  by  tidal  fluctuations  or  otherwise.  Despite 
the  somewhat  erratic  distribution  of  the  pigment  concentration,  it  is  still  obvious 
that  the  chlorophyll  increased  gradually  throughout  the  day,  reaching  its  peak  at 
about  sunset,  after  which  it  decreased  rapidly  throughout  the  night  until  daybreak. 

Similarly  the  plant  carotenoid  pigments  increased  during  the  day  and  decreased 
at  night  (Fig.  IE).  Both  chlorophyll  and  carotenoids  increased  to  higher  values 
on  the  second  day,  which  differed  from  the  first  primarily  in  the  amount  of  incident 


DYNAMICS  OF  A  DIATOM  BLOOM 


263 


o 

tr 
< 
o 


01 
_j 

X 

o 


-O 


I       I       I       I 


I       I       I       I       I       1       I       i 


.5  1.0 

LIGHT  INTENSITY  (g  CAL/CM2/MIN  ) 


1.5 


FIGURE  3.     The  relation  between  incident  radiation  and  the  ratio  chlorophyll  a: carotenoid 
pigments  in  the  diatom  population.     Open  circles,  June  26.     Closed  circles,  June  27. 

radiation.     The  carotenoid  pigments  ranged  on  June  27  from  a  minimum  of  30 
SPU  3  at  05  :45  to  95  SPU  at  17  :45,  more  than  a  three-fold  variation. 

The  ratio  chlorophyll  a: carotenoid  pigments  (Fig.  IF)  decreased  throughout 
the  daylight  periods  of  both  days  from  maximum  values  observed  at  sunrise,  the 
more  rapid  decrease  on  June  27  again  correlated  with  the  greater  incident  radiation 

3  The  spectrophotometric  analysis  of  carotenoid  pigments  has  not  been  standardized  in 
absolute  units  and  they  are  reported  in  specific  pigment  units  after  Richards  with  Thompson 
(1952).  One  SPU,  however,  is  closely  equivalent  to  one  milligram  of  pigment. 


264  RYTHER,  YENTSCH,  HULBURT  AND  VACCARO 

on  that  day.  Figure  3  shows  the  inverse  relationship  which  was  found  between 
the  intensity  of  solar  radiation  and  the  chlorophyll :  carotenoid  ratio.  The  varia- 
tions in  this  ratio  are  the  result  of  a  differential  effect  of  light  on  pigment  synthesis 
and  decomposition  where  the  chlorophyll  changes  are  of  much  greater  magnitude 
than  are  the  carotenoid  changes  (Yentsch  and  Scagel,  unpublished).  The  signifi- 
cance and  interpretation  of  the  magnitude  and  changes  in  this  ratio  will  be  discussed 
in  the  final  section  of  this  paper. 

c.  Primary  production 

Primary  production  was  calculated  by  the  following  three  methods :  ( 1 )  the 
in  situ  changes  of  oxygen  in  the  water,  (2)  the  "light-and-dark-bottle"  oxygen 
measurements,  (3)  the  "chlorophyll-radiation"  method  of  Ryther  and  Yentsch 
(1957).  The  inability  to  measure  or  calculate  total  CO2  prevented  the  use  of  pH 
changes  or  the  C14  method  for  this  purpose. 

On  June  26,  the  dissolved  oxygen  in  the  integrated  sample  collected  over  the 
one-meter  water  column  increased  from  a  minimum  of  4.5  ml. /liter  at  07  :00  hours 
to  a  maximum  of  11.8  ml./liter  at  17:00  hours,  a  difference  of  7.3  ml./liter.  If  an 
assimilatory  quotient  of  1.25  is  used,  this  change  in  oxygen  is  equivalent  to  a  carbon 
fixation  of  3.15  grams/m2./day.  As  mentioned  earlier,  the  decrease  in  oxygen  at 
night  appears  to  have  been  due  almost  entirely  to  respiration.  This  loss  was 
equivalent  to  0.5  ml.  oxygen/liter/hour.  During  the  10  hours  of  daylight,  if 
respiration  occurred  at  the  same  rate,  this  wrould  account  for  a  total  of  5.0  ml.  O2/ 
liter  or  2.15  grams  carbon/m2./day.  Adding  this  respiratory  loss  to  the  observed 
net  production  of  3.15  grams  carbon/m2./day  gives  a  total  or  gross  production  of 
5.20  grams  carbon/m2./day. 

In  the  same  way  production  was  calculated  for  June  27,  the  net  change  in 
oxygen  being  equivalent  to  assimilation  of  3.8,  the  respiration  loss  1.7  and  the  gross 
production  5.5  grams  carbon/m2./day. 

The  two-hour  "light-and-dark-bottle"  experiments  which  were  described  above 
were  also  used  to  calculate  gross  and  net  production.  The  differences  between  the 
oxygen  concentration  of  the  light  bottles  at  0,  0.5,  and  1.0  meter  and  that  of  the 
accompanying  dark  bottle  over  the  two-hour  experimental  periods,  converted  to 
carbon  assimilation  as  above,  are  shown  in  Figure  1G.  The  carbon  equivalent  of 
respiration  for  the  same  two-hour  periods  was  obtained  from  the  difference  be- 
tween the  oxygen  content  of  the  water  at  the  beginning  of  the  two-hour  period 
and  that  of  the  dark  bottle.  These  curves  were  integrated  to  obtain  daily  photo- 
synthesis at  each  depth  and  daily  respiration.  These  values  in  turn  were  plotted 
against  depth  and  integrated  to  give  daily  photosynthesis  and  respiration  beneath 
a  square  meter  of  surface.  Gross  photosynthesis  for  June  26  calculated  by  this 
method  was  3.5,  respiration  was  1.7  and  net  photosynthesis  1.8  grams  carbon/m2./ 
day.  On  June  27  the  values  were  6.2,  2.8  and  3.4  grams  carbon/m2. /day  for  gross 
production,  respiration  and  net  production,  respectively. 

The  in  situ  oxygen  changes  at  night  appeared  to  indicate  a  constant  respiration 
rate  of  0.5  ml.  O2/liter/hour,  and  this,  as  described  above,  was  used  to  correct  the 
net  in  situ  change  observed  in  daylight  to  give  gross  production.  An  examination 
of  the  two-hour  bottle  experiments  during  the  day  shows  that,  when  measured  in 
this  way,  respiration  was  by  no  means  constant  but  varied  roughly  in  proportion 
to  the  rate  of  photosynthesis.  On  June  27,  for  instance,  the  respiratory  rate  ranged 


DYNAMICS  OF  A  DIATOM  BLOOM 


265 


from  0.06  ml./liter/hour  in  the  early  morning  to  about  1.00  ml./liter/hour  at  mid- 
day. These  measurements,  though  somewhat  crude,  emphasize  the  need  for  a 
reconsideration  of  the  tacit  assumption  made  by  most  ecologists  that  respiration 
measured  at  night,  or  for  long  periods  in  dark  bottles,  is  the  same  as  that  which 
occurs  in  the  light  in  conjunction  with  photosynthesis. 

The  third  method  for  estimating  production  is  that  developed  by  Ryther  and 
Yentsch  (1957).  This  method  requires  measurement  of  the  concentration  of 
chlorophyll  a,  the  total  daily  incident  radiation,  and  the  extinction  coefficient  of 
visible  light  in  the  water.  The  latter  was  determined  by  the  measurement  of  light 
penetration  to  one  meter  with  a  submarine  photometer  at  13:30  hours  on  June  26. 
The  extinction  coefficient  (k)  so  determined  was  4.0.  Use  of  this  method  required 
an  obvious  over-simplification,  since  the  chlorophyll  a  concentration,  as  has  been 
pointed  out,  varied  throughout  the  day.  A  mean  value  of  200  mg.  chla/m3.  was 
used  for  the  calculation  for  both  days,  and  this  was  assumed  to  be  uniformly 
distributed  over  the  one-meter  water  column.  The  resulting  values  for  gross 
production  were  3.2  and  5.1  grams  carbon/m2./day  f°r  June  26  and  27,  respectively. 

The  results  obtained  by  these  three  methods  are  summarized  in  Table  II.  They 
show  rather  good  agreement  except  for  the  values  obtained  by  in  situ  oxygen 
changes  on  June  25  which  are  almost  twice  as  high  as  those  obtained  by  the  other 

TABLE  II 

Primary  production  in  Senix  Creek  on  June  26  and  June  27,  as  measured  by  three  methods 

(grams  carbon  assimilated /m?/ day) 


Method 

Gross 

Net 
(day) 

Net 
(24  hrs.) 

Gross 

Net 
(day) 

Net 
(24  hrs.) 

In  situ  O2 

5.3 

3.15 

0 

5.5 

3.8 

0 

L-D  bottle  O2 

3.5 

1.76 

— 

6.2 

3.4 

— 

Chlorophyll 

3.2 

— 



5.1 



1 

two  methods.  It  should  be  pointed  out  that  the  net  production  values  which  have 
been  discussed  refer  to  this  process  during  the  daylight  hours  only.  The  only 
estimates  over  a  24-hour  period  which  can  be  made  are  based  upon  the  in  situ  oxy- 
gen changes  (and  pH  changes)  which  clearly  reflect  a  net  production  for  this 
period  of  zero.  Finally,  the  net  changes  observed  in  situ  and  in  vitro  are  acknowl- 
edged as  representing  changes  brought  about  by  the  whole  community  including 
animals  and  bacteria,  and  do  not  characterize  the  plant  population  alone. 

The  efficiency  of  production  on  the  two  days  may  be  roughly  estimated  by 
taking  the  median  of  the  values  obtained  by  the  three  methods  for  daily  gross  produc- 
tion, 3.5  and  5.5  grams  carbon/m2.  on  June  26  and  27,  respectively.  If  the  assump- 
tion is  made  that  50%  of  the  photosynthetic  production  is  carbon  and  has  a  heat 
of  combustion  of  5.5  k  cal./gr.  (see  Krogh  and  Berg,  1931),  and  further  that  half 
the  incident  radiation  may  be  used  for  photosynthesis,  the  efficiency  may  be  cal- 
culated as : 

3.5  X  2  X  5,500 
a)     June  26     • 


b)     June  27 


1,500,000 

5.5  X  2  X  5,500 
3,700,000 


=  2. 


=  1-6% 


266  RYTHER,  YENTSCH,  HULBURT  AND  VACCARO 

/.  The  physiology  of  the  bloom 

There  are  several  indications  that  the  diatom  population  in  Senix  Creek  was 
a  non-growing  one  which  had  exhausted  its  supply  of  available  nutrients  and  was 
able  to  subsist  at  a  basal  level,  photosynthesizing  just  enough  during  the  day  to 
compensate  for  its  metabolic  requirement  over  a  24-hour  period.  This  is  best 
illustrated  by  the  in  situ  oxygen  and  pH  values,  in  which  the  net  oxygen  produced 
and  CO2  assimilated  during  the  day  are  exactly  compensated  by  the  reverse  proc- 
esses at  night.  Further  evidence  of  this  is  the  fact  that  the  concentration  of  diatoms 
remained  unchanged  over  the  48  hours  of  observation  except  where  such  changes 
are  attributable  to  tidal  flushing. 

The  evidence  that  the  bloom  was  nutrient-limited  is  somewhat  sparse  and  in- 
direct, but  rather  convincing.  At  10 :30  hours  on  June  27  an  integrated  water 
sample  was  collected  and  frozen.  This  was  later  analyzed  for  nitrogen  and  phos- 
phorus fractions  at  the  Woods  Hole  Oceanographic  Institution.  The  results  of 
these  analyses  are  given  below. 

/jg  Atoms/liter 

NO2-  +  NO3-  3.40 

NH3+  1.49 

PO4  3.80 

Total  P  16.0 

A  photosynthetic  rate  of  5.5  grams  carbon/m2./day  in  a  one-meter  water  column 
represents  a  requirement  of  460  jugA  carbon/liter/day.  As  Redfield  (1934)  and 
others  have  pointed  out,  marine  phytoplankton  assimilate  carbon,  nitrogen  and 
phosphorus  at  an  atomic  ratio  closely  approaching  100:  15  :  1.  This  rate  of  carbon 
assimilation  is  therefore  equivalent  to  a  daily  requirement  of  71  /xg A/liter  of  nitro- 
gen and  4.6  /^gA/liter  of  phosphorus.  Thus,  the  concentrations  of  these  elements 
in  the  mid-morning  of  June  27  represented  no  more  than  a  fraction  of  a  day's  supply 
of  either  nitrogen  or  phosphorus.  These  calculations  were  based  upon  the  require- 
ment of  normal  cells.  Photosynthesis  may  of  course  continue  after  nitrogen  and 
phosphorus  are  exhausted  with  the  storage  of  carbohydrates  and  lipids.  This  is 
presumedly  what  was  happening  in  this  population,  the  cells  using  these  stored 
materials  to  satisfy  their  metabolic  requirements  at  night.  Further  studies  of  this 
type  of  population,  with  emphasis  placed  upon  the  diurnal  cycle  of  nutrients,  would 
be  particularly  interesting. 

The  behavior  of  the  plant  pigments  is  a  further  indication  of  the  physiological 
condition  of  the  population.  The  fact  that  both  chlorophyll  a  and  the  carotenoids 
were  synthesized  during  the  day  and  decomposed  at  night  signifies  that  the  plants 
were  drawing  upon  their  cellular  reserves  to  maintain  themselves  in  the  dark. 
When  nutrients  are  available,  this  does  not  occur ;  in  fact,  chlorophyll  may  be  syn- 
thesized in  the  dark  under  favorable  growing  conditions  if  the  cells  have  sufficient 
respiratory  reserves  (Harvey,  1953). 

Experiments  in  this  laboratory  (Ketchum  ct  a/.,  1958)  and  elsewhere  have 
shown  that  both  chlorophyll  a  and  the  carotenoid  pigments  decrease  in  diatoms  in 
response  to  nitrogen,  phosphorus  or  iron  deficiency  or  excessive  illumination.  This 
nutritional  chlorosis  results  in  a  more  rapid  decomposition  of  chlorophyll  than 
carotenoid  pigments.  As  the  day  progressed  the  pigment  ratio  decreased,  presum- 
ably in  part  because  of  nutrient  exhaustion  which  was  hastened  by  greater  demands 
of  photosynthesis  at  high  light  intensities  (Fig.  3). 


DYNAMICS  OF  A  DIATOM  BLOOM  267 

The  picture  which  emerges  from  these  various  bits  of  evidence,  then,  is  that  of  a 
static  diatom  bloom  of  great  magnitude,  its  nutrient  supply  exhausted  or  at  least  re- 
duced to  the  level  where  growth  could  not  occur.  Yet  it  was  not  a  dying  population, 
except  insofar  as  physical  forces  tended  to  disperse  it.  It  was  capable  of  carrying 
out  organic  synthesis  at  a  rate  some  10-100  times  that  of  normal  plankton  commu- 
nities, drawing  upon  these  materials  for  its  metabolic  requirements  much  the  same 
as  a  mature  animal  maintains  a  balance  between  its  assimilation  and  metabolism. 

It  would  appear,  then,  that  populations  of  phytoplankton  such  as  we  have  de- 
scribed here,  though  not  actively  growing,  are  not  necessarily  dying  either.  They 
are  merely  living  in  a  different  growth  phase,  a  condition  in  which  they  may  per- 
sist for  long  periods  of  time  if  they  are  not  destroyed  or  dispersed  by  external 
factors.  Perhaps  in  diatom  populations,  as  elsewhere,  the  bloom  of  maturity  may 
outlast  the  bloom  of  youth. 

SUMMARY 

1.  A  dense  population  of  planktonic  diatoms  was  studied  over  a  40-hour  period 
in  a  small  tidal  creek  on  the  south  shore  of  Long  Island,  New  York. 

2.  Measurements  were  made  at  frequent  intervals  of  incident  radiation,  light 
penetration,  salinity,  temperature,  dissolved  oxygen,  pH,  concentration  of  diatom 
cells  and  their  pigments,  and  dissolved  inorganic  nutrients.     Photosynthesis  and 
respiration  were  measured  by  oxygen  changes  in  bottle  experiments  and  estimated 
from  in  situ  oxygen  changes  and  from  chlorophyll  a  and  radiation. 

3.  The  plankton  community  appeared  to  be  nutrient-limited  and  consisted  of 
a  static,  non-growing  diatom  population  which  was  being  slowly  diluted  by  tidal 
action.     This  was  indicated  by  the  diatom  counts,  the  behavior  of  their  pigments 
(which  increased  throughout  the  day  and  decreased  during  the  night)    and  the 
concentration  of  available  plant  nutrients. 

4.  Rates  of  primary  production  measured  by  three  methods  showed  good  agree- 
ment, the  values  ranging  from  3.2  to  5.3  grams  carbon  assimilated/m2./day  on 
June  26,  from  5.1-6.2  on  June  27.     Total  incident  radiation  for  the  two  days  was 
300  and  740  gram  calories/cm2. /day,  respectively,  and  the  efficiency  of  the  photo- 
synthetic  utilization  of  visible  radiation  for  the  two  days  was  estimated  at  2.6% 
and  1.6%,  respectively. 

LITERATURE  CITED 

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sewage,  and  industrial  wastes.  Tenth  Edition.  American  Public  Health  Assoc.,  Inc., 
New  York.  259  pp. 

CREITZ,  G.  I.,  AND  F.  A.  RICHARDS,  1955.  The  estimation  and  characterization  of  plankton 
populations  by  pigment  analyses.  III.  A  note  on  the  use  of  "millipore"  membrane 
niters  in  the  estimation  of  plankton  pigments.  /.  Mar.  Res.,  14:  211-216. 

DOTY,  M.  S.,  AND  M.  OGURI,  1957.  Evidence  for  a  photosynthetic  daily  periodicity.  Limnol. 
and  Occanogr.,  2:  37-40. 

DUXBURY,  A.  C.,  AND  C.  S.  YENTSCH,  1956.  Plankton  pigment  nomographs.  /.  Mar.  Res., 
15  :  92-101. 

HARVEY,  H.  W.,  1948.  The  estimation  of  phosphate  and  total  phosphorus  in  sea  waters. 
/.  Mar.  Biol.  Assoc.,  27:  337-359. 

HARVEY,  H.  W.,  1953.  Synthesis  of  organic  nitrogen  and  chlorophyll  by  Nitsschia  closterium. 
J.  Mar.  Biol.  Assoc.,  31 :  477-478. 

KETCHUM,  B.  H.,  J.  R.  RYTHER,  C.  S.  YENTSCH  AND  N.  CORWIN,  1958.  Productivity  in  rela- 
tion to  nutrients.  Rapp.  et  Proc.  Verb.  Cons.  Internal.  Explor.  Mcr.,  144:  132-140. 


268  RYTHER,  YENTSCH,  HULBURT  AND  VACCARO 

KROGH,  A.,  AND  K.  BERG,  1931.  Uber  die  chemische  Zusammensetzung  des  Phytoplanktons  aus 
dem  Fredriksborg-Schlossee  und  ihre  Bedeutung  fiir  die  Maxima  der  Cladoceren. 
Int.  Rev.  Gesamt.  Hydrobiol.  u  Hydrog.,  25 :  205-218. 

MULLIN,  J.  B.,  AND  J.  P.  RILEY,  1955.  The  spectrophotometric  determination  of  nitrate  in 
natural  waters,  with  particular  reference  to  sea  water.  Analyt.  Chim.  Acta,  12: 
464-480. 

REDFIELD,  A.  C.,  1934.  On  the  proportions  of  organic  derivatives  in  sea  water  and  their  rela- 
tion to  the  composition  of  plankton.  James  Johnstone  Memorial  Vol.,  The  University 
Press,  Liverpool.  Pp.  176-192. 

RICHARDS,  F.  A.,  WITH  T.  G.  THOMPSON,  1952.  The  estimation  and  characterization  of  plank- 
ton populations  by  pigment  analysis.  II.  A  spectrophotometric  method  for  the  esti- 
mation of  plankton  pigments.  /.  ]\Iar.  Res.,  11 :  156-172. 

RIDER,  B.  F.,  AND  M.  G.  MELLON,  1946.  Colorimetric  determination  of  nitrites.  Ind.  Engin. 
Chem,  Anal.  Edit.,  18 :  96-99. 

RILEY,  J.  P.,  1953.  The  spectrophotometric  determination  of  ammonia  in  natural  waters  with 
particular  reference  to  sea  water.  Analyt.  Chim.  Acta,  9:  575-589. 

ROBINSON,  R.  J.,  AND  T.  G.  THOMPSON,  1948.  The  determination  of  phosphates  in  sea  water. 
/.  Mar.  Res.,  7 :  33-41. 

RYTHER,  J.  H.,  1954.  The  ecology  of  phytoplankton  blooms  in  Moriches  Bay  and  Great  South 
Bay,  Long  Island,  New  York.  Biol.  Bull,  106:  198-209. 

RYTHER,  J.  H.,  AND  C.  S.  YENTSCH,  1957.  The  estimation  of  phytoplankton  production  in  the 
ocean  from  chlorophyll  and  light  data.  Limnol.  and  Occanogr.,  2  :  281-286. 

YENTSCH,  C.  S.,  AND  J.  H.  RYTHER,  1957.  Short-term  variations  in  phytoplankton  chlorophyll 
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THE  FORMATION  OF  SUBNUCLEAR  AGGREGATES  IN  NORMAL 
AND  SYNCHRONIZED  PROTOZOAN  CELLS1 

OTTO   H.   SCHERBAUM,  ALLAN   L.   LOUDERBACK  AND   THEODORE   L.   JAHN 

Department  of  Zoology,   University  of  California,  Los  Angeles  24,  Cal. 

Since  Biitschli  (1876)  established  the  nuclear  dualism  in  ciliates,  there  has 
been  much  speculation  about  the  biological  role  of  the  micro-  and  macronucleus. 
The  mitotic  behavior  of  the  micronucleus,  with  its  delicate  apparatus  for  chromo- 
somal segregation,  led  to  the  generally  accepted  view  of  the  importance  of  the 
micronucleus  in  inheritance  and  reproduction.  The  macronucleus,  on  the  other 
hand,  which  was  found  to  divide  "simply"  by  pinching  in  two,  was  considered  to  be 
concerned  "only"  with  the  regulation  of  metabolic  functions  in  the  cell. 

This  concept  of  the  duality  of  nuclear  function  as  formulated  by  Hertwig  in 
1889,  was  substantiated  by  Goldschmidt  (1904)  and  Popoff  (1908).  These 
authors  distinguished  between  the  genetically-active  idiochromatin  and  the  tropho- 
chromatin,  which  was  concerned  exclusively  with  the  cellular  metabolism.  In  the 
uninuclear  protists  both  types  of  chromatin  \vere  considered  to  be  present  in  one 
nucleus,  while  in  ciliates  the  idiochromatin  was  confined  to  the  micronucleus  and 
the  trophochromatin  was  found  in  the  macronucleus  only. 

The  view  of  the  dualistic  function  of  the  nuclei  in  ciliates  was  abandoned  after 
experimental  data  accumulated  showing  the  controlling  role  the  macronucleus 
plays  in  the  processes  of  cell  division  and  regeneration  (Grell,  1950).  The  genetic 
importance  of  the  macronucleus  in  ciliates  stimulated  cytological  studies  of  its 
structure.  A  considerable  body  of  evidence  has  been  accumulated  during  the  past 
30  years  showing  Feulgen-positive  bodies  in  the  cytoplasm,  which  could  not  be 
accounted  for  by  "macronuclear  fragmentation,"  the  process  of  disintegration  of 
the  macronucleus  upon  conjugation  of  two  cells.  These  bodies  often  have  a  spheri- 
cal shape,  and  after  Feulgen  staining  show  the  homogeneous  appearance  of  micro- 
nuclei.  Very  often  these  bodies  have  been  erroneously  described  as  micronuclei, 
a  fact  which  was  pointed  out  by  Kidder  (1933).  Diller  (1936)  observed  simple 
fragmentations  of  the  macronucleus  in  Paraniecium  aurelia,  and  he  used  the  term 
"hemixis"  to  denote  such  autonomous  changes  of  the  macronucleus  which  are  not 
related  to  sexual  phenomena  or  binary  fission. 

It  is  believed  now  that  the  macronucleus  of  the  ciliates  consists  of  many  diploid 
subnuclei  (Sonneborn,  1947).  We  therefore  propose  the  term  "subnuclear  aggre- 
gates" (SNA's)  for  the  Feulgen-positive  material  lost  or  expelled  from  the  macro- 
nucleus  into  the  cytoplasm.  The  formation  of  SNA's  may  occur  1)  by  simple 
extrusion  of  Feulgen-positive  material  from  the  macronucleus,  or  2)  by  loss 
during  the  process  of  binary  fission  of  the  macronucleus.  An  example  of  extrusion 
of  chromatin  masses  from  the  macronuclear  anlagen  in  the  exconjugates  of  Ancis- 
truma  isseli  was  described  by  Kidder  (1933),  and  a  spontaneous  "budding"  of 

1  This  work  was  supported  by  grant  G-2490  of  the  National  Science  Foundation. 

269 


270  SCHERBAUM,  LOUDERBACK  AND  JAHN 

macronuclei,  independent  of  cell  division,  in  Ichthyophthirius  multifiliis  was  found 
by  Haas  (1933).  In  the  course  of  binary  fission  of  the  macronucleus  of  Colpidium 
colpoda  Kidder  and  Diller  (1934)  described  how  some  of  the  nuclear  material 
is  left  behind  in  the  fission  plane.  This  material  becomes  condensed  and  finally 
disappears.  A  similar  phenomenon  was  described  by  Furgason  (1940)  in  the 
amicronucleate  strain  T  of  Tctrahyincna  pyriformis  and  by  McDonald  (1958)  in 
Tetrahymena  pyriformis  H. 

These  frequently  observed  chromatin  extrusions  from  the  macronucleus  led 
Kidder  and  Diller  (1934)  to  the  suggestion  of  a  presumptive  role  for  this  phe- 
nomenon. It  wras  thought  extrusion  might  be  a  manifestation  of  a  universal 
principle  of  nuclear  reorganization  which,  in  turn,  could  account  for  a  high  division 
rate.  However,  no  quantitative  studies  have  so  far  been  carried  out  on  the  forma- 
tion of  the  SNA's.  their  frequency  of  formation,  and  their  absolute  size  in  various 
phases  of  population  growth. 

The  system  of  synchronous  cell  division  in  Tetrahymena  pyriformis,  strain  GL, 
as  worked  out  by  Scherbaum  and  Zeuthen  (1953,  1955),  was  used  for  the  study 
of  this  phenomenon.  During  the  induced  synchrony  about  85  per  cent  of  the 
cells  are  in  the  visible  stage  of  fission  and  all  stages  of  SNA  formation  can  readily 
be  found. 

METHOD 

The  amicronucleate  strain  GL  of  Tetrahymena  pyriformis  was  grown  princi- 
pally as  described  earlier  (Scherbaum  and  Zeuthen,  1955).  The  growth  medium 
was  two  per  cent  proteose  peptone  (Difco)  with  0.5  per  cent  glucose  and  0.1  per 
cent  liver  fraction  L  (Wilson  Laboratories)  in  glass-distilled  water.  Salts  were 
added  as  in  the  basal  medium  A  of  Kidder  and  Dewey,  except  that  phosphates 
were  omitted.  The  medium  was  filtered  and  autoclaved  at  15  pounds  for  15 
minutes.  One  ml.  of  a  three-day-old  stock  culture  (approximately  2  X  105  cells 
per  ml.)  was  used  for  the  inoculation  of  150  ml.  of  culture  medium  in  a  500-ml. 
culture  flask.  The  flask  was  submerged  in  a  temperature-controlled  water  bath, 
which  was  mounted  on  a  shaker. 

Samples  of  5  ml.  were  removed  from  the  experimental  flask  at  regular  intervals 
for  counting  (Scherbaum,  1957)  and  for  nuclear  preparations.  For  the  latter, 
the  samples  were  concentrated  by  centrifugation  in  a  hand  centrifuge  and  the 
supernatant  removed  by  suction.  The  concentrated  cell  suspension  wras  fixed  in 
one  per  cent  aqueous  osmic  acid  for  two  minutes.  The  cells  were  removed  from 
the  fixative  by  centrifugation,  washed  in  water,  and  passed  through  alcohol  (30 
per  cent  to  100  per  cent).  The  cells  were  then  pipetted  onto  albuminized  cover- 
slips,  slightly  dried  to  affix  the  cells  to  the  glass  surface,  and  transferred  to  absolute 
alcohol  for  ten  minutes.  The  cover  slips  wrere  stored  in  70  per  cent  alcohol.  For 
the  Feulgen  reaction  the  samples  were  hydrolyzed  in  1  N  HC1  at  60°  C.  for  12 
minutes  and  exposed  to  the  Schiff  reagent  for  one  hour. 

RESULTS 

At  an  approximate  population  density  of  5  X  103  cells  per  ml.  the  first  sample 
was  removed.  This  served  as  the  control  for  normal  exponential  multiplication. 
The  second  sample  was  removed  during  the  synchronous  division.  For  the  indue- 


SUBNUCLEAR  AGGREGATES  IN  PROTOZOA 


271 


A 


FIGURE  1.  Photomicrographs  of  Feulgen-stained  cells  from  various  growth  phases:  Normal 
exponential  multiplication  (A),  after  temperature  treatment  (B),  during  and  after  the  first 
synchronous  division  (C  to  K),  and  in  maximum  stationary  phase  (L).  The  distance  between 
the  two  lines  on  the  scale  in  (A)  is  10  /*;  "n"  denotes  a  subnuclear  aggregate  (SNA). 
Further  explanation  in  the  text. 


272 


SCHERBAUM,  LOUDERBACK  AND  JAHN 


tion  of  synchrony  the  culture  was  exposed  to  seven  temperature  cycles.  The 
temperature  was  changed  every  half  hour  between  28°  and  33.9°  C.  One  hour 
and  15  minutes  after  the  end  of  the  seventh  cycle  80  to  85  per  cent  of  the  cells  were 
in  the  visible  stage  of  fission.  The  third  sample  was  removed  after  48  hours  of  sub- 
sequent growth  when  the  cells  were  in  the  early  stationary  phase. 

Figure  1  shows  photomicrographs  of  cells  in  various  stages  of  population  growth 
and  of  the  formation  of  the  SNA's  during  division.  In  A  the  deeply  stained  SNA 
(n)  is  close  to  the  macronucleus  and  resembles  the  micronucleus  as  shown  by  Holz, 
Scherbaum  and  Williams  (1957)  in  mating  type  1,  variety  1,  of  Tetrahymcna  pyri- 
formis.  Figure  1,  B  shows  a  typical  enlarged  cell  and  nucleus  after  the  end  of 
the  temperature  treatment.  No  SNA  from  a  previous  division  can  be  seen,  al- 
though some  were  found  in  other  preparations.  In  C  the  macronucleus  elongates 
during  the  onset  of  synchronous  division.  A  distinct  portion  of  the  nucleus  seems 
to  be  "suspended"  between  the  macronuclear  halves  pulling  apart  amitotically.  In 

TABLE  I 
Size  and  number  of  subnuclear  aggregates  (SNA's)  in  various  growth  phases 


Sample  No. 

Number  of  cells 
with  SNA  (%) 

Mean  macronuclear 
volume  in  p? 

Mean  volume 
of  SNA  in  fi3 

SNA/macronucl. 
volume  ratio  in  % 

1 
Control  exponent, 
multiplication 

16 

265.0 

1.9 

0.72 

2 
Prior  to  synchr.  division 

22 

1063.0 

X 

X 

3 
After  synchr.  division 

55 

430.0 

12.2 

2.84 

4 
Max.  station,  phase 

6 

122.0 

1.8 

1.48 

In  order  to  determine  the  average  percentage  cells  with  SNA's,  100  cells  of  each  type  were 
examined.  The  macronuclear  volume  for  each  growth  stage  is  the  average  for  the  100  cells 
measured.  The  mean  volume  given  for  the  SNA  is  the  average  of  50  measurements;  "x"  denotes 
that  no  measurements  were  made. 

D  to  G  this  macronuclear  remnant  can  be  seen  at  various  stages  of  cell  division. 
In  these  phases  of  division  the  fragment  still  shows  the  typical  granular  compo- 
sition of  the  macronucleus.  However,  somewhat  later,  when  the  fibrous  connection 
between  the  macronucleus  and  the  fragment  disappears,  the  fragment  tends  to  be- 
come spherical,  the  granular  structure  disappears,  and  the  fragment  becomes  a 
dense  homogeneous  mass,  resembling  the  micronucleus  in  this  respect  (I-L). 
Figure  1,  J  and  K  shows  cells  immediately  after  division.  In  cells  of  the  early 
stationary  phase  of  growth,  SNA's  were  also  found  (L). 

For  a  quantitative  estimation  of  the  size  and  number  of  the  SNA's  the  experi- 
mental culture  was  sampled  in  various  growth  phases.  The  result  is  shown  in 
Table  I. 

The  number  of  cells  with  SNA's  is  relatively  constant  in  exponentially  growing 
cultures  (16  per  cent)  and  increases  slightly  in  the  course  of  the  heat  treatment. 


SUBNUCLEAR  AGGREGATES  IN  PROTOZOA  273 

However,  after  synchronous  division  SNA's  were  found  in  55  per  cent  of  the  cells. 
On  the  assumption  that  the  SNA's  observed  in  the  cells  prior  to  division  are  carried 
through  the  synchronous  division  step,  one  can  calculate  that  in  approximately 
45  per  cent  of  the  cells  undergoing  division  new  formation  of  SNA's  took  place. 

The  mean  volume  of  the  SNA's  is  relatively  constant  in  the  logarithmic  phase 
and  stationary  phase  of  growth.  It  is  approximately  2.0  /A  This  value  is  0.7  per 
cent  and  1.5  per  cent  of  the  macronuclear  volume  at  these  two  growth  phases, 
respectively.  After  the  synchronous  division  the  average  SNA  volume  is  12  //,3, 
showing  a  six-fold  increase  as  compared  to  normal  values. 

EVALUATION  OF  THE  RESULTS  AND  DISCUSSION 

In  almost  all  cells  examined  only  one  SNA  was  found,  but  in  some  cases  two 
or  three  SNA's  could  be  observed  in  one  cell.  From  the  frequency  with  which 


FIGURE  2.  Photomicrograph  of  a  Feulgen-stained  cell  during  the  first  division  after  the 
heat  treatment.  The  distance  between  the  two  lines  on  the  scale  is  10  /JL.  Further  explanation 
in  the  text. 

the  SNA's  occur  at  various  growth  phases  it  seems  as  if  that  they  are  broken  down 
to  Feulgen-negative  material  or  are  extruded  from  the  cell.  However,  there  is 
no  evidence  which  might  serve  to  evaluate  either  of  these  possibilities.  The  ab- 
normally large  nuclei  of  synchronized  cells,  together  with  the  larger  size  of  the 
SNA's  of  synchronized  cells,  might  suggest  that  the  size  of  the  SNA's  depends 
to  some  degree  on  the  volume  of  the  parent  macronucleus.  However,  the  size  of 
the  newly  formed  SNA's  may  vary,  as  can  be  seen  in  Figure  1,  E-H.  Further- 
more, the  size  appears  to  be  a  function  of  the  age  of  the  SNA,  since  when  first 
formed  it  is  granular,  similar  to  the  macronucleus,  and  it  then  becomes  homo- 
geneous and  smaller,  apparently  by  condensation,  before  it  disappears.  In  the 
present  analysis  we  followed  the  formation  of  the  SNA's  with  the  Feulgen  method 
for  DNA  only.  However,  nothing  is  known  about  the  concentration  of  the  basic 
proteins  in  these  bodies.  Basic  proteins  are  normally  found  to  be  associated  with 


274  SCHERBAUM,  LOUDERBACK  AND  JAHN 

the  DNA  in  the  nucleus.  A  difference  in  stainability  of  the  basic  proteins  in  the 
micro-  and  macronucleus  was  observed  by  Alfert  and  Goldstein  (1955)  in  mating 
types  I  and  II  of  Tetrahymcna.  One  could  imagine  that  the  original  DNA  basic 
protein  ratio,  as  characteristic  for  the  macronucleus  and  for  the  young  SNA's  in 
Tetrahymcna  GL,  could  change  by  preferential  degradation  of  DNA  in  the  course 
of  the  presumed  condensation  process  occurring  after  formation  of  the  SNA's. 

Although  the  extrusion  of  macronuclear  material  in  non-dividing  cells  and  the 
formation  of  SNA's  have  been  observed  in  various  protozoan  cells  (see  introduc- 
tion), there  is  no  conclusive  evidence  concerning  the  role  which  these  phenomena 
play  in  the  metabolism  of  the  cells.  Following  the  concept  of  strict  equal  distri- 
bution of  parental  DNA  to  the  daughter  cells  one  might  be  somewhat  puzzled  by 
this  phenomenon.  However,  the  high  degree  of  polyploidy  in  the  macronucleus 
suggests  that  such  an  equal  distribution  may  not  be  a  "conditio  sine  qua  non." 

A  slight  imbalance  in  timing  of  nuclear  and  cell  division  could  cause  this  loss 
of  DNA  in  the  fission  plane,  and  the  failure  for  it  to  be  incorporated  into  the 
daughter  nuclei.  In  rare  instances  an  "imbalance"  of  nuclear  and  cell  division 
was  observed  in  synchonized  cells.  For  instance,  Figure  2  shows  a  cell  during 
synchronous  division,  dividing  into  three  instead  of  two  daughters.  In  the  right 
part  of  the  cell,  nuclear  division  is  completed,  while  cellular  division  lags  slightly 
behind.  In  the  left  part  of  the  cell  the  macronucleus  is  in  division,  while  cyto- 
plasmic  division  is  far  more  advanced  than  in  a  normal  cell  with  a  comparable 
nuclear  figure.  That  such  irregularities  hardly  affect  the  viability  of  the  cells  is 
not  surprising  in  view  of  the  fact  that  the  protozoan  macronucleus  is  a  highly 
polyploid  system.  Sonneborn  (1947)  concludes,  from  genetic  evidence,  that  the 
macronucleus  of  Paramecium  aurclia  must  contain  about  40  diploid  "subnuclei." 
These  observations  suggest  the  interesting  problem  of  to  what  extent  this  high 
polyploidy  of  the  macronucleus  could  be  reduced  experimentally.  For  instance,  in 
starving  cultures  of  Tetrahymcna  pyrifonnis  strain  S,  Weis  (1954)  found  a  re- 
duction in  cell  size  to  less  than  10  per  cent  of  the  normal  volume.  These  cells 
"regulated"  back  to  their  normal  size  upon  addition  of  nutrients  to  the  culture 
medium.  If  one  assumes  an  almost  constant  nucleo/cytoplasmic  ratio  and  40 
diploid  "subnuclei"  (as  found  for  Paramecium'),  one  might  expect  the  starved 
cells  to  carry  only  4  diploid  "subnuclei." 

Opposed  to  the  view  that  the  loss  of  subnuclei  during  binary  fission  is  an 
arbitrary  phenomenon,  based  on  mere  chance,  is  the  idea  which  attributes  a  strict 
regulatory  function  to  these  processes.  Findings  by  Kidder  and  Claff  (1938) 
seem  to  substantiate  this  point  of  view.  These  authors  investigated  the  life  cycle 
of  Colpoda  cue  nil  us  and  described  chromatin  extrusion  following  each  division  in 
regular  and  predictable  fashion.  This  "budding"  of  the  macronuclei  occurs  almost 
synchronously  in  the  two  daughter  cells.  In  contrast  to  the  loss  of  DNA  during 
the  fission  process,  as  described  for  synchronized  cells  of  Tetrahymena,  we  have  in 
Colpoda  cuciillus  an  example  of  active  regulation  or  reorganization  of  some  sort 
after  the  daughter  cells  are  formed. 

The  authors  wish  to  thank  Prof.  W.  H.  Furgason,  Department  of  Zoology, 
U.C.L.A.,  for  his  criticism  and  advice  in  preparation  of  the  manuscript.  Prof.  W. 
Balamuth,  Department  of  Zoology,  University  of  California,  Berkeley,  Prof.  Fur- 
gason and  Dr.  J.  Loefer,  O.N.R.,  Pasadena,  kindly  supplied  samples  of  Tetra- 
hymena pyrifonnis,  strain  GL. 


SUBNUCLEAR  AGGREGATES  IN  PROTOZOA  275 

SUMMARY 

1.  The  formation   of   "subnuclear   aggregates"    (SNA's)    is   studied   quantita- 
tively in  synchronously-dividing  cells  of  Tetrahymena  pyriformis  strain  GL. 

2.  In  normal  cultures  approximately   16  per  cent  of  the  cells  were  found  to 
contain  SNA's.     This  value  rises  to  55  per  cent  after  synchronous  division.     The 
SNA/macronuclear  volume  ratio  is  0.72  per  cent  in  normal  cells  and  2.8  per  cent 
in  cells  after  synchronous  division. 

3.  The  possible  significance  of  the  formation  of  SNA  is  discussed. 

LITERATURE  CITED 

ALFERT,  M.,  AND  N.  O.  GOLDSTEIN,  1955.  Cytochemical  properties  of  nucleoproteins  in  Tetra- 
hymena pyriformis ;  a  difference  in  protein  composition  between  macro  and  micronuclei. 
/.  E.rp.  ZooL,  130:  403-419. 

BUTSCHLI,  O.,  1876.  Studien  iiber  die  ersten  Entwicklungsvorgange  der  Eizelle,  der  Zellteilung 
und  die  Conjugation  der  Infusoricn.  Abh.  Scnckenbcrg.  natitrforsch.  Gcs.  Frank  jurt, 
10:  1-250. 

DILLER,  W.  F.,  1936.  Nuclear  reorganization  processes  in  Paramccium  aurelia,  with  descrip- 
tions of  autogamy  and  "hemixis."  /.  Morphol.,  59:  11-67. 

FURGASON,  W.  H.,  1940.  The  significant  cytostomal  pattern  of  the  "Glaucoma-Colpidium  group" 
and  a  proposed  new  genus  and  species,  Tetrahymena  gelcii.  Arch.  f.  Protist.,  94: 
224-266. 

GOLDSCHMIDT,  R.,  1904.     Die  Chromidien  der  Protozoen.     Arch.  f.  Protist.,  5  :   126-144. 

GRELL,  K.,  1950.     Der  Kerndualismus  der  Ciliaten  und  Suctorien.     Natuntnss.,  37 :  347-356. 

HAAS,  G.,  1933.  Beitrage  zur  Kenntnis  der  Cytologie  von  Ichthyophthirius  multifiliis.  Arch, 
f.  Protist.,  81 :  88-137. 

HERTWIG,  R.,  1889.  Uber  die  Konjugation  der  Infusorien.  Abhandl.  Bayr.  Akad.  Wiss.,  17: 
150-233. 

HOLZ,  G.  G.,  O.  H.  SCHERBAUM  AND  N.  WILLIAMS,  1957.  The  arrest  of  mitosis  and  stomato- 
genesis  during  temperature  induction  of  synchronous  division  in  Tetrahymena  pyri- 
jormis,  mating  type  1,  variety  1.  E.rp.  Cell  Res.,  13:  618-621. 

KIDDER,  G.  W.,  1933.  On  the  genus  Ancistruma  Strand  II.  The  conjugation  and  nuclear  re- 
organization of  A.  isscli.  Arch.  f.  Protist.,  81  :  1-18. 

KIDDER,  G.  W.,  AND  C.  L.  CLAFF,  1938.  Cytological  investigations  of  Colpoda  cncitllus.  Biol. 
Bull.,  74:  178-197. 

KIDDER,  G.  W.,  AND  W.  F.  DILLER,  1934.  Observations  on  the  binary  fission  of  four  species  of 
common  free-living  ciliates,  with  special  reference  to  the  macronuclear  chromatin. 
Biol.  Bull.,  67:  201-219. 

MCDONALD,  BARBARA  B.,  1958.  Quantitative  aspects  of  deoxyribose  nucleic  acid  (DNA) 
metabolism  in  an  amicronucleate  strain  of  Tetrahymena.  Biol.  Bull.,  114:  71-94. 

POPOFF,  M.,  1908.  Die  Gametenbildung  und  die  Konjugation  von  Carchesium  polypinum  L. 
Zcitschr.  iviss.  ZooL,  89:  478-524. 

SCHERBAUM,  O.,  1957.  The  application  of  a  standard  counting  method  in  estimation  of  growth 
in  normal  and  heat-treated  cultures  of  Tetrahymena  pyriformis.  Acta  path,  microbiol. 
scand.,  40:  7-12. 

SCHERBAUM,  O.,  AND  E.  ZEUTHEN,  1953.  Induction  of  synchronous  cell  division  in  mass  cul- 
tures of  Tetrahymena  pyriformis.  E.rp.  Cell  Res.,  6 :  221-227. 

SCHERBAUM,  O.,  AND  E.  ZEUTHEN,  1955.  Temperature  induced  synchronous  divisions  in  the 
ciliate  protozoon  Tetrahymena  pyriformis  growing  in  synthetic  and  proteose-peptone 
media.  Exp.  Cell  Res.,  Suppl.  3:  312-325. 

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STUDIES  ON  DIGENETIC  TREMATODES  OF  THE  GENERA 
GYMNOPHALLUS  AND  PARVATREMA 

HORACE  W.   STUNKARDi  AND  JOSEPH  R.   UZMANN  2 
U.  S.  Fisli  and  Wildlife  Service 

HISTORICAL  REVIEW 

The  genus  Gymnophallus  was  erected  by  Odhner  (1900)  to  contain  Distomum 
deliciosum  Olsson,  1893  and  other  small  species  from  the  gall  bladder,  intestine 
and  bursa  Fabricii  of  shore-birds.  Subsequent  observations  have  shown  that  the 
asexual  generations  of  these  worms  occur  in  bivalve  mollusks  and  that  the  cercariae, 
which  are  produced  .in  sporocysts,  belong  to  the  Dichotoma  group  of  furcocercous 
larvae.  Typically,  these  cercariae  have  eye-spots  and  short  bifid  tails,  although 
either  or  both  may  be  reduced  or  absent.  Cercaria  dichotoma  emerges  and  swims 
as  a  furcocercous  larva.  In  certain  species  the  tail  undergoes  regression  and  is  lost 
before  the  larva  emerges  from  the  sporocyst  whereas,  in  others,  apparently  no  tail 
is  formed.  On  emergence  from  the  first  intermediate  host,  the  cercariae  attach  to 
the  mantle  or  body  wall  of  bivalve  or  gastropod  mollusks  where  as  unencysted 
metacercariae,  they  develop  to  almost  definitive  size.  The  metacercariae  may  pro- 
duce lesions  on  the  mantles  of  their  hosts  and  such  injuries  stimulate  proliferation 
of  tissues,  especially  of  the  secreting  layer  of  the  mantle,  and  deposition  of  nacreous 
material.  Despite  the  observations  of  many  investigators  over  a  period  of  more 
than  fifty  years,  no  complete  life-history  has  yet  been  worked  out  and  the  specific 
relations  between  particular  cercariae,  metacercariae,  and  sexually  mature  worms 
remain  undetermined. 

The  presence  of  pearly  formations  in  the  mantle  of  Mytilus  edulis  has  been 
known  for  at  least  three  hundred  years.  According  to  Giard  (1907),  they  were 
reported  by  Olaus  Worm  in  1655  from  mussels  taken  at  Roeskild,  near  Copen- 
hagen. Robert  Garner  observed  them  in  M.  edulis  from  the  English  coast  and  he 
(1872)  recognized  that  they  were  formed  as  a  reaction  by  the  mollusk  to  a  small 
distome  parasite  on  the  mantle.  Baron  d'Hamonville  (1894)  found  pearls  (sans 
valeur}  in  M.  edulis  at  Billiers  (Morbihan)  France,  although  the  infection  was 
limited  to  the  area  of  the  port.  Giard  (1897)  reported  small  distomes,  often  asso- 
ciated with  irregularly  shaped  calcareous  deposits,  between  the  mantle  and  shell 
of  Dona.v  trunculus  L.,  Tcllina  fabiila  Gronov,  Tcllina  tennis  DaCosta  and  Telllna 
solidula  (=  T.  balthica  L.,  ex  parte}  from  Boulogne-sur-Mer.  The  worms  were 
0.5  mm.  long,  with  rudiments  of  testes,  but  no  ovary,  and  Giard  suspected  that  they 
might  be  stages  in  the  life-cycle  of  Brachycoelium  luteum  (van  Beneden),  a  para- 
site of  the  common  dogfish,  Scyllium  canlcula.  The  larger  specimens  were  often 
less  active,  more  opaque,  and  filled  with  sporozoans  (Glugidees).  Dubois  (1901) 
studied  the  parasites  of  M.  edulis  at  Billiers  and  found  them  in  reddish  brown  spots, 

1  The  American  Museum  of  Natural  History,  New  York  24,  N.  Y. 

2  Fisheries  Center,  University  of  Washington,  Seattle  5,  Wash. 

276 


DIGENETIC  TREMATODES  277 

which  were  the  loci  of  pearl  formation.  The  worms  measured  0.4-0.6  mm.  in 
length  and  for  them  he  proposed  the  name,  Distomum  margaritarum.  He  found 
the  same  or  a  similar  parasite  in  Mytilus  gallopr  ovine  ialis  from  the  coast  of 
Provence. 

Jameson  also  studied  the  parasites  at  Billiers;  he  (1902)  described  and  figured 
the  worms  which  he  stated  closely  resembled  Distomum  somateriae,  whose  mature 
stages  had  been  described  by  Levinsen  (1881)  from  the  intestine  of  the  eider 
duck,  Somateria  mollissima,  taken  near  Egedesminde,  Greenland.  He  recalled 
that  Mobius  (1857)  had  reported  a  trematode  associated  with  pearls  in  the  pearl 
oyster,  Margaritifera  margaritifcra,  from  the  west  coast  of  N.  America.  Follow- 
ing the  action  of  Stossich  (1899),  Jameson  referred  the  species  from  M.  edulis  to 
Lecithodendrium  Looss,  but  misspelled  the  name,  Lcucithodendriiim.  He  reported 
that  he  had  found  larvae,  similar  to  those  in  M.  cdulis,  in  sporocysts  in  Tapes 
decussatus  and  that  he  had  infected  M.  edulis  with  these  sporocysts.  The  larvae 
in  the  sporocysts  were  tailless ;  they  differed  from  those  in  M.  edulis  only  in  their 
smaller  size,  paler  color,  more  distended  excretory  organs  and  empty  gut,  and  in 
the  possession  of  special  sense  organs  and  eyes.  The  sporocysts  were  present  in 
all  of  almost  200  Tapes  examined,  located  chiefly  in  the  margin  of  the  mantle  where 
it  is  attached  at  the  pallial  line.  The  sporocysts  were  spherical  to  oval,  about  0.5 
mm.  in  diameter,  <with  six  to  ten  cercariae  in  each.  Jameson  also  reported  that 
M.  cdulis  from  Piel,  near  Barrow,  Lancashire,  England,  were  heavily  infected 
with  distome  larvae,  similar  to  those  at  Billiers,  but  that  Tapes  was  not  present  at 
Piel.  The  sporocysts  at  Piel  were  found  in  Cardium  edule.  Mytilus  edulis  at 
Piel  were  exposed  with  specimens  of  Tapes  decussatus  from  Billiers,  with  an  ap- 
parent increase  in  the  number  of  parasites.  Returning  to  Billiers,  Jameson  found 
every  specimen  of  Oidemia  nigra  heavily  infected  with  L.  somateriae.  The  worms 
were  present  throughout  the  length  of  the  intestine.  He  described  and  figured  the 
adult  stage  of  L.  somateriae;  the  worms  were  0.2  to  0.55  mm.  in  length,  about  one- 
half  the  size  of  the  larvae  in  M.  edulis,  and  Jameson  stated  that  the  striking  like- 
ness except  for  size,  between  the  larvae  from  M.  cdulis  and  L.  somateriae  and  the 
occurrence  of  the  latter  in  two  species  of  birds  that  are  known  to  feed  par  excel- 
lence on  mussels  is  almost  sufficient  to  prove  their  identity  without  the  feeding 
experiment.  He  described  the  manner  in  which  the  mantle  of  M.  cdulis  forms  a 
sac  around  the  trematode  larva  and  deposits  the  nacreous  material.  Giard  (1903) 
confirmed  the  observations  of  Jameson  on  pearl  formation  and,  since  pearls  are  the 
sarcophagi  of  trematode  larvae,  proposed  their  production  by  artificial  means. 

The  first  report  of  the  asexual  generations  of  Gymno  phallus  was  an  incidental 
footnote  by  von  Siebold  (1837)  who  found  the  sporocysts  and  cercariae  in  Macorna 
balthica.  Lebour  (1904)  described  the  sporocysts  in  the  liver  and  about  the  intes- 
tine of  Cardium  edule  as  (p.  83)  "oval,  pointed  at  one  extremity,  with  two  con- 
spicuous black  eyes,  the  other  end  is  rounded  and  the  whole  body  is  covered  with 
cilia  which  are  constantly  in  motion  in  the  living  animal.  The  sporocyst  may  con- 
tain three  different  elements,  (1)  spherical  masses  full  of  smaller  spheres,  (2)  the 
same  but  with  two  of  these  masses  in  one  envelope,  (3)  minute  sporocysts  exactly 
like  itself  and  containing  small  spheres."  Miss  Lebour  regarded  the  "sporocysts" 
as  the  first  larval  stage  of  an  echinostome  species  whose  metacercariae  she  found 
encysted  in  the  foot  of  the  mollusk  but  whose  cercarial  stage  wras  not  discovered. 
In  a  footnote  she  added  (p.  84),  "since  writing  this  I  have  discovered  another  stage 


278  H.  W.  STUNKARD  AND  J.  R.  UZMANN 

of  the  worm  in  the  liver  of  the  cockle,  of  which  particulars  will  be  given  later."  In 
the  report  for  1905,  Miss  Lebour  recognized  that  the  "sporocysts"  from  the  liver  of 
C.  ednle  were  not  related  to  the  echinostome  larvae  in  the  foot,  for  which  she  had 
postulated  the  life-cycle.  She  stated  that  C.  cdule  harbors  the  sporocyst  stage  of 
three  species  of  trematodes ;  the  one  she  reported  in  1904,  the  second  is  "the  sporo- 
cyst and  cercaria  stage  of  the  'pearl  trematode'  which  is  the  cause  of  pearls  in  the 
mussel,"  and  the  third  species  was  described  as  very  contractile  and  of  various 
shapes,  without  eyes  and  not  ciliated.  The  report  contains  a  brief  description  and 
figure  of  a  "cercaria"  from  Tcllina  tennis  and  Dona.v  vittatns  from  the  Alnmouth 
sands.  From  the  figure,  it  is  clear  that  the  supposed  "cercaria"  is  a  gymno- 
phallid  metacercaria. 

Johnstone  (1905),  in  C.  cdulc  at  Piel,  found  a  fork-tailed  larva  which  he  de- 
scribed and  figured  as  Cercaria  fissicanda  La  Valette.  It  was  probably  not  the 
tailless,  ocellate  species  that  had  been  reported  by  Jameson  and  was  possibly  Cer- 
caria dichotoma  Miiller.  This  latter  species,  found  free  in  the  Mediterranean  near 
Nice  by  Miiller  and  described  in  La  Valette  (1855),  was  reported  from  the  marine 
lamellibranch,  Scrobicula  tennis  by  Villot  (1875),  but  Villot  (1878)  assigned  the 
larvae  to  Cercaria  fissicanda  La  Valette,  1855.  As  noted  by  Pelseneer  (1906), 
C.  fissicanda  was  described  from  a  fresh-water  gastropod,  Lymnaca  stagnalis,  and 
Villot's  (1878)  designation  was  obviously  an  error.  The  cercaria  described  and 
figured  as  C.  fissicanda  by  Villot  (1878)  was  identified  as  C.  dichotoma  by  Pel- 
seneer. Pelseneer  described,  as  C.  dichotoma,  a  species  which  he  found  in  Tellina 
solidnla  from  deep  water  near  Boulogne-sur-Mer.  The  cercariae  developed  in 
short,  nodose,  colorless  sporocysts  and  closely  resembled  the  figures  of  C.  dichotoma 
as  given  by  La  Valette  and  that  of  the  species  by  Villot  (1878)  which  he  desig- 
nated as  C.  fissicanda,  but  which  he  previously  (1875)  had  identified  as  C.  dich- 
otoma. Pelseneer  also  referred  to  C .  dichotoma  an  unnamed  cercaria  described  by 
Huet  (1888).  Huet  found  this  species  in  Cardinm  ednle  on  the  coast  of  Normandy 
and  his  description  clearly  contravenes  its  identity  with  C.  dichotoma.  According 
to  Huet,  the  sporocysts  were  short,  compact,  spherical  to  pyriform,  with  the  an- 
terior end  elongated  in  the  form  of  a  neck.  The  sporocysts  were  covered  with  cilia 
and  swam  freely.  Immature  sporocysts  were  0.2  mm.  in  length  and  grayish  in 
color;  older  ones,  0.3  mm.  in  length,  were  yellowish  and  the  birth-pore,  situated 
at  the  anterior  end,  was  surrounded  by  five  tubercules,  below  which  there  was  a 
crown  of  verrucosities  and  the  intervening  area  was  covered  with  fine,  stiff  bristles. 
Mature  sporocysts,  0.5  mm.  long,  were  yellow  and  contained  a  few  distomid  cer- 
cariae with  bifid  tails,  but  on  completion  of  development  the  tails  were  shed  and 
the  cercariae  emerged  from  the  sporocysts.  The  figures  of  Huet  show  that  the 
cercaria  is  a  gymnophallicl  larva,  but  it  is  not  C.  dichotoma,  which  emerges  and 
swims  as  a  furcocerous  larva.  Pelseneer  also  described  a  new  species,  Cercaria, 
syndosmyac,  from  Syndosmya  alba  at  Boulogne-sur-Mer.  The  sporocysts  were 
0.75  to  1.0  mm.  in  length,  cylindrical,  without  constrictions.  The  cercariae  were 
elongate,  the  acetabulum,  near  the  middle  of  the  body,  was  slightly  larger  than  the 
oral  sucker ;  the  ceca  were  short,  globose,  and  the  cephalic  glands  were  straight, 
moderately  long.  The  furcae  were  longer  than  the  stem  of  the  tail.  Young 
distomes,  of  corresponding  morphology,  were  found  often  between  the  mantle  and 
shell  of  Dona.r  and  other  bivalves  in  the  same  locality.  Odhner  (191  la)  postu- 
lated that  C.  syndosmyac  is  a  larval  stage  of  Haplocladns  minor.  This  suggestion 


DIGENETIC  TREMATODES  279 

appears  more  credible  since  Cable  (1953)  included  the  Haplocladinae  and  Gymno- 
phallinae  as  subfamilies  in  the  family  Fellodistomatidae. 

Meanwhile  Odhner  had  studied  the  trematodes  of  marine  birds.  He  (1900) 
re-described  and  figured  Distomum  delidosum  Olsson,  1893,  from  the  gall  bladder 
of  three  species  of  Larus,  which  he  named  as  type  of  a  new  genus,  Gyinno phallus. 
In  the  new  genus  he  included  G.  somateriac  (Levinsen,  1881)  from  the  intestine 
of  Somateria  mollissima,  G.  micropharyngeus  (Liihe,  1898)  from  the  gall  bladder 
of  a  North  African  flamingo,  and  two  new  species,  G.  choledochus  from  the  gall 
bladder  of  Vulpanscr  tadorna,  and  G.  bursicola  from  the  bursa  Fabricii  of  Soma- 
teria mollissima.  In  a  later  paper,  Odhner  (1905)  re-described  G.  somateriac  and 
commented  on  the  findings  of  Jameson,  in  which  the  metacercariae  from  M.  cdulis 
were  identified  as  larval  stages  of  G.  somateriae.  Since  the  larvae  from  M.  cdulis 
were  much  larger  than  the  sexually  mature  G.  somateriae,  Odhner  contended  that 
they  could  not  be  identical  and  that  the  material  of  Jameson  represented  two  closely 
related  species.  The  larger  species,  whose  larvae  induced  pearl  formation  in  M. 
cdulis,  was  referred  to  G.  bursicola  since  in  morphology  it  agreed  completely  with 
the  description  of  that  species.  The  smaller  mature  worms  from  Oidemia  nigra, 
according  to  Odhner.  belonged  to  a  different  and  unnamed  species.  The  meta- 
cercariae described  by  Jameson  from  M.  edulis  agreed  completely  with  others 
found  by  Odhner  in  the  original  material  of  Sa.vicava  mgosa  (  =  Hiatclla  nigosa] 
collected  by  Levinsen  at  Egedesminde,  Greenland  and  which  as  noted  by  Levinsen, 
differed  from  G.  somateriae  in  total  size  and  size  of  suckers.  Odhner  noted  differ- 
ences between  G.  somateriae  and  the  mature  worms  found  by  Jameson  in  Oidemia 
nigra  at  Billiers,  which  he  postulated  belong  to  a  new  and  as  yet  unnamed  species. 
Discussing  the  genus  Gymno phallus,  Odhner  stated  that  it  could  not  be  included  in 
any  of  the  previously  recognized  subfamilies,  and  it  was  designated  as  type  of  a 
new  subfamily,  Gymnophallinae. 

Nicoll  (1906)  examined  Cardiitm  ednlc  at  St.  Andrews,  Scotland;  from  May 
to  July  no  infection  was  noted  in  several  hundred  individuals ;  later,  sporocysts 
similar  to  those  reported  by  Jameson  in  Tapes  were  found  abundantly  in  the  mid- 
line,  dorsally,  just  over  the  posterior  border  of  the  liver.  Individual  sporocysts 
appeared  as  yellow  spots  of  various  sizes  in  an  opaque,  whitish  mass  which  meas- 
ured about  four  by  three  millimeters.  Each  sporocyst  contained  from  two  to  fifty 
cercariae.  The  cercariae  were  tailless,  0.11  to  0.28  mm.  in  length,  ocellate ;  the 
oral  sucker  (with  minute  papillae)  about  twice  the  size  of  the  acetabulum  which 
was  situated  just  anterior  to  the  bifurcation  of  the  excretory  vesicle.  The  testes 
were  lateral,  somewhat  behind  the  acetabulum.  Nicoll  kept  cockles  (C.  cdulc} 
and  mussels  (M.  cdulis}  in  the  same  tank  for  some  time,  but  no  infection  of  the 
mussels  was  noted. 

Giard  (1907)  reviewed  previous  work  on  the  trematodes  which  induce  pearl 
formation  in  marine  lamellibranchs ;  he  reported  that  a  worm,  similar  to  the  one 
described  by  Pelseneer,  occurs  frequently  in  M .  edulis  at  Wimereux,  near  Boulogne- 
sur-Mer ;  that  this  species,  named  D.  margaritarum  by  Dubois  (1901)  is  probably 
identical  with  the  one  found  in  Saxicava  rugosa  by  Levinsen  and  a  stage  in  the 
life-cycle  of  G.  bursicola.  Giard  also  reported  another  and  different  distome  which 
occurs  abundantly  "dans  les  Dona.v  et  les  Tellines  du  Port  de  Boulogne"  and  which 
agrees  with  G.  somateriae  and  differs  from  the  parasite  in  Mytilus.  Oidemia 
nigra,  the  pochard,  was  listed  as  the  probable  final  host.  Lebour  (1905)  had  re- 


280  H.  W.  STUNKARD  AND  J.  R.  UZMANN 

ported  and  figured  a  "cercaria"  which  occurred  in  Tcllina  tennis  and  Dona.v  vittatits, 
which  she  identified  as  the  species  described  by  Giard  (1897)  and  regarded  by  him 
as  possibly  a  larval  stage  of  Brachycoelium  lutcnm. 

Nicoll  (1907)  described  Gymnophallus  dapsilis  n.  sp.,  from  the  bursa  Fabricii 
of  the  scoters,  Oidcmia  fnsca  and  Oidcnria  nigra.  This  species  differs  from  G.  bur- 
sicola  and  other  species  of  Gymnophallus,  in  that  the  vitellaria  are  anterior  to  the 
acetabulum.  The  worms  were  small,  0.84  to  1.13  mm.  in  length;  however,  Nicoll 
suggested  that  they  may  be  adults  of  the  species  whose  larvae  occur  in  M .  cdnlis. 

Lebour  (1908)  described  three  gymnophallid  cercariae  (actually  metacercariae)  : 
C.  gland osa  from  Paludestrina  stagnalis  (a  gastropod)  which  measured  0.2  mm. 
in  length  and  had  many  large  glands  in  the  anterior  part  of  the  body;  C.  macomae 
from  Maconia  balthica.  which  measured  0.7  mm.  long;  and  C.  strigata,  the  species 
which  Giard  (1897)  had  regarded  as  the  larva  of  Brachycoelium  hitcnin  and  (1907) 
as  that  of  Gymnophallus  somateriae.  Miss  Lebour  reported  that  it  occurs  very 
commonly  in  Tcllina  tennis  and  rarely  in  Dona.v  vittatits  at  Alnmouth.  This  is  the 
same  species  reported  by  Lebour  in  1905  ;  the  worms  measured  0.3  to  0.4  mm.  in 
length;  the  oral  sucker  was  0.09  mm.  in  diameter,  and  the  acetabulum  0.05  mm. 
Miss  Lebour  noted  that  Pelseneer  had  described  and  figured  what  was  evidently 
the  same  larva,  and  which  he  regarded  as  a  later  stage  of  the  furcocercous  cercaria 
named  by  him,  C.  syndosuiyac.  But  in  C.  syndosuiyac  the  suckers  are  nearly  equal 
in  size  which  renders  their  identity  unlikely,  and  the  larvae  found  in  sporocysts  in 
Cardinal  cdidc  and  Tapes  decnssata  by  Jameson,  Nicoll  and  Lebour  were  tailless. 
Miss  Lebour  stated  that  C.  strigata  is  broader  than  C.  somateriae  and  the  striations 
produced  by  the  rows  of  spines  are  more  conspicuous.  Furthermore,  ducks  seldom 
feed  at  Alnmouth,  and  the  adult  stage  of  these  parasites  is  probably  in  gulls  ;  possibly 
it  is  G.  dcliciosus.  Miss  Lebour  also  described  a  larva  which  she  identified  as 
Cercaria  dielwtoma  Muller,  from  the  small  bivalve,  Scrobicularia  tennis.  The 
figure  shows  that  it  is  the  cercarial  stage  of  a  species  of  Gym-no  phallus  and  Miss 
Lebour  believed  that  the  same  species  had  been  recorded  by  Pelseneer  from  TeHina 
soliditla  (--Maconia  balthica),  by  Johnstone  from  Cardinal  edule  on  the  Lanca- 
shire coast,  and  by  Huet  in  C.  edule  in  Normandy. 

Lebour  (1912),  in  a  review  of  the  British  marine  cercariae,  recognized  five 
gymnophallid  cercariae  (actually  metacercariae):  (1)  Cercaria  aiargaritae,  the 
pearl  trematode  of  Jameson  (1902),  which  occurs  in  sporocysts  in  C.  edule  and 
Tapes  decnssata;  (2)  Cercaria  scrivcnensis  sp.  inq.,  based  on  two  specimens  from 
Tapes  pnllastra  at  Loch  Scriven,  the  Clyde,  but  not  well  characterized;  (3)  Cer- 
caria glandosa  Lebour,  1908;  (4)  Cercaria  inacoinae  Lebour,  1908;  and  (5)  Cerca- 
ria strigata.  Lebour.  1908.  Apparently  Miss  Lebour  was  not  yet  aware  that  the 
larvae  she  regarded  as  cercariae  were  really  unencysted  metacercariae,  since  she 
noted  that  Cercaria  diclwtoma,  which  she  described  and  figured,  is  closely  allied 
in  structure  to  Gymnophallus  but,  because  of  its  forked  tail,  she  placed  it  in  a  sepa- 
rate group.  Cercaria  margaritae  has  eye-spots  and  according  to  Jameson  develops 
in  sporocysts  in  the  edges  of  the  mantle  of  Tapes  decnssatns  and  Cardinal  edule 
whereas  Nicoll  and  Lebour  reported  this  species  from  sporocysts  in  jelly-like 
masses  under  the  umbo  of  C.  edule.  These  differences  suggest  that  two  distinct 
species  were  regarded  as  identical. 

Sinitsin   (1911)   described  sporocysts  from  Syndosmya  alba  of  the  Black  Sea 


DIGENETIC  TREMATODES  281 

as  Cercaria  discursata  n.  sp.  The  sporocysts  were  simple,  saccate,  in  the  liver 
and  gonad ;  the  cercariae  0.06  mm.  long  with  a  bifid  tail  somewhat  longer,  normally 
shed  in  the  sporocyst,  but  vised  in  sluggish  swimming  when  the  cercariae  are  re- 
leased from  the  sporocyst.  According  to  Sinitsin,  some  of  the  larvae  remain  as 
unencysted  metacercariae  in  5.  alba,  while  others  leave  and  migrate  to  various 
species  of  mollusks.  They  attack  the  tissues  but  do  not  encyst  and  increase  about 
four  times  in  size.  In  the  same  paper  Sinitsin  described  Adolescaria  pcrla  from 
various  mollusks,  mostly  near  the  gill  plates  of  M.  edulis  and  unidentified  species 
of  Venus.  From  the  descriptions  and  figures  of  Sinitsin,  both  of  these  species  can 
be  identified  as  gymnophallids,  and  indeed,  they  may  be  stages  in  the  life-history 
of  a  single  species. 

Stafford  (1912)  reported  sporocysts  and  cercariae  in  Mya  arcnaria  of  the 
Gaspe  Bay  region  of  Canada.  The  venter  of  the  clam  was  distended,  soft,  trans- 
lucent, pale  greenish  yellow-  in  color  with  hundreds  of  sporocysts,  each  containing 
about  twenty  fully-formed,  forked-tailed  cercariae ;  the  body  of  one  cercaria  meas- 
ured 0.138  by  0.082  mm.  From  the  surface  of  the  mantle  of  M.  arcnaria  and 
M.  edulis,  Stafford  reported  metacercariae  which  measured  0.078  by  0.056  mm. 
These  specimens  were  similar  to  those  reported  by  Levinsen  from  the  intestine  of 
Somateria  mollissima  which  occurs  on  the  Gaspe,  but  since  they  were  smaller  than 
the  cercariae  from  M.  arcnaria,  Stafford  concluded  that  they  did  not  belong  to  the 
same  species. 

Jameson  and  Nicoll  (1913)  reviewed  the  question  of  pearl  formation  in  M. 
edulis  and  the  identity  and  life-history  of  the  trematode  larvae  which  are  the  incit- 
ing agents.  Jameson's  earlier  experiments  were  not  controlled ;  the  form  described 
by  him  (1902)  as  G.  (Lcucithodendrium)  somatcriac  was  now  described  as  Gymno- 
phallus  ocdcmiac.  Since  the  gravid  worms  are  smaller  than  the  metacercariae  in 
M.  edulis,  these  authors  reasoned  that  they  cannot  be  the  adult  stage  of  that  species. 
They  stated  that  the  metacercariae  in  M.  edulis  may  be  G.  bursicola  as  believed  by 
Odhner  or  possibly  G.  dapsilis  Nicoll.  They  postulated  that  the  sporocysts  and 
cercariae  in  Tapes  dccitssatus  are  G.  bursicola  ;  that  those  in  C.  cdule  are  G.  dap- 
silis ;  and  that  metacercariae  of  both  may  occur  in  M.  edulis.  Although  specimens 
of  Oideinia  nigra  were  naturally  infected  with  a  species  of  Gyrnno  phallus,  metacer- 
cariae from  M.  edulis  did  not  develop  in  Oidemia  nigra  or  Fuligula  (Nyroca) 
fcrina.  Jameson  and  Nicoll  recognized  six  species;  G.  bursicola  Odhner;  G.  dap- 
silis  Nicoll;  and  four  new  species;  G.  oedcniia,  G.  affinis,  G.  macroporus  and 
G.  oz'oplcnus.  all  from  the  intestine  of  0.  nigra. 

Dollfus  (1923)  studied  the  metacercariae  from  Mytilus  galloprovincialis  de- 
scribed earlier  by  Dubois  (1901).  He  found  the  worms  to  measure  0.23  to  0.27 
mm.  in  length,  with  the  oral  sucker  about  twice  the  size  of  the  acetabulum.  They 
were  similar  to  the  Adolescaria  pcrla  of  Sinitsin  and  to  others  which  Dollfus  had 
observed  between  the  mantle  and  shell  of  Tapes  pullastra  on  the  English  channel. 
For  them  he  proposed  the  name,  Gymnophallus  duboisi  n.  sp. 

Odhner  (1900)  had  described  Gymnophallus  choledochus  n.  sp.  on  the  basis 
of  sketches  made  by  Jagerskiold,  of  a  single  specimen  (then  lost)  from  the  gall 
bladder  of  Vulpanscr  tadorna.  He  (1905)  re-described  the  species  from  speci- 
mens taken  from  the  gall  bladder  of  Somateria  mollissima  and  Somateria  spcctabi- 
lis  from  East  Greenland.  The  worms  were  pyriform,  0.9  to  1.1  mm.  long,  0.35  to 


H.  W.  STUNKARD  AND  J.  R.  UZMANN 

0.50  mm.  wide,  with  the  acetabulum  near  the  middle  of  the  body,  ceca  which  ex- 
tended to  the  middle  of  the  body,  and  testes  lateral,  postcecal.  Isaitchikow  (1924), 
apparently  unaware  of  Odhner's  re-description,  reported  G.  cJiolcdochits  from  the 
gall  bladder  of  AytJia  (Nyroca)  ferina  from  the  Crimea.  He  noted  that  the  dis- 
tribution of  the  species  is  not  restricted  to  Scandinavia,  but  extends  to  the  Black 
Sea.  He  found  that  the  coils  of  the  uterus  may  fill  the  body  from  the  oral  sucker 
to  the  reproductive  glands,  that  the  topography  of  the  genital  organs  is  not  fixed, 
the  variations  are  numerous  and  very  inconstant ;  eight  different  types  were  depicted 
by  figures. 

Palombi  (1924)  re-described  Ccrcaria  margaritarnm  Dubois,  1901  (actually 
a  metacercaria)  and  stated  that  G.  duboisi  Dollfus,  1923  is  a  synonym.  Later 
Palombi  (1934)  described  and  figured  the  metacercariae  of  Gymno phallus  strigata 
(Lebour)  and  Gymnophallus  uicgacocla  n.  sp.  In  an  article  by  Fujita  (1925), 
Dollfus  described  Gymnophalloides  tokiensis  n.g.,  n.  sp.,  a  gymnophallid  metacer- 
caria from  the  surface  of  the  mantle  of  the  Japanese  oyster,  Ostrea  gigas.  The 
text  was  translated  by  R.  Ph.  Dollfus.  who  added  notes,  diagnoses,  and  a  bibli- 
ography. In  this  species  the  genital  pore  is  located  some  distance  anterior  to  the 
acetabulum  and  for  it  Dollfus  provisionally  proposed  a  new  genus,  Gymnophal- 
loides. In  the  paper,  Dollfus  described  a  second  species,  Gymnophalloides  tapetis, 
a  metacercaria  from  Tapes  pullastra  at  Saint  Vaast-la-Hougue  (Manche),  which 
he  noted  is  very  similar  to  Adolcscaria  pcrla  Sinitsin. 

Cole  (1935)  reported  an  "orange  sickness  of  mussels"  at  the  Conway  statiqn 
on  the  coast  of  North  Wales.  According  to  him  (p.  276),  "The  infected  speci- 
mens were  found  without  difficulty  as  the  mantle  is  a  vivid  marigold  to  blood- 
orange  due  to  the  presence  of  innumerable  sporocysts."  The  sporocysts  were 
present  also  throughout  the  body  of  the  mollusk.  They  were  oval,  bright  orange, 
thin-walled,  moderately  contractile  and  measured  about  1.1  mm.  by  0.45  mm. 
They  contained  tailless  cercariae  which  were  described  as  a  new  species.  Ccrcaria 
tcnuans.  The  cercaria  measured  up  to  0.3  mm.  in  length  when  extended ;  the  oral 
sucker  was  0.05  mm.  and  the  acetabulum  0.07  mm.  in  diameter.  The  cercariae 
were  without  spines  and  died  soon  after  emergence,  which  indicates  an  immature 
condition.  In  one  mussel,  Cole  reported  similar  sporocysts  in  the  digestive  gland, 
but  they  contained  daughter  sporocysts  and  no  cercariae.  This  observation  led  to 
the  suggestion  that  M.  cdnlis  acquires  the  infection  by  way  of  the  intestine.  Cole 
predicated  that  the  species  described  by  him  is  probably  identical  with  the  one 
reported  by  Miss  Atkins  (1931)  in  2.16  per  cent  of  over  ten  thousand  mussels 
from  the  estuary  of  the  Camel,  near  PadstowT. 

Young  (1936)  described  fork-tailed  cercariae  taken  in  a  tow  net  in  the  Bering 
Sea,  15-65  kilometers  from  the  nearest  land.  The  larvae  were  without  eyes  or 
spines,  0.4  mm.  long ;  the  oral  sucker  was  0.085  mm.  and  the  acetabulum  0.09  mm. 
in  diameter.  The  specimens  were  reported  to  agree  best  with  C.  syndosmyae 
Pelseneer,  and  they  may  be  a  species  of  Gymnophallus. 

Markowski  (1936)  described  Cercaria  baltica,  a  furcocercous  species  from 
sporocysts  in  Macoma  balthica.  The  sporocysts  were  long,  cylindrical,  whitish, 
slowly  motile;  1.2  by  0.15  mm.,  each  contained  from  two  to  fifty  cercariae. 
The  cercariae  measured  in  microns;  body  length,  133;  tail-stem,  90;  furcae,  38; 
oral  sucker,  11;  acetabulum,  44  by  30;  cecal  length,  52.  The  measurements  as 
given  do  not  agree  with  his  Figure  6,  in  which  the  oral  sucker  is  approximately 


DIGENETIC  TREMATODES  283 

the  same  size  as  the  acetabulum.  In  C.  baltica  the  entire  surface  of  the  body,  in- 
cluding the  tail-stem  and  furcae,  is  spined,  there  are  four  pairs  of  glands  near  the 
oral  sucker,  and  the  ceca  extend  to  the  anterior  edge  of  the  acetabulum  which  is 
slightly  behind  the  middle  of  the  body:  Markowski  recognized  four  species  in  the 
Dichotoma  group  of  cercariae ;  C.  dichotoma  Muller,  C.  syndosmyae  Pelseneer, 
C.  discursata  Sinitzin,  and  C.  baltica  n.  sp.  From  the  same  host  Markowski  de- 
scribed two  metacercariae :  Metacercaria  inornla,  in  which  a  large  number  of  larvae 
were  enclosed  in  a  single  cyst;  and  Metaccrcaria  iniitabilis  which  are  about  three 
times  as  large  as  the  former  species  and  occur  unency steel  on  the  mantle  or  in 
folds  of  it. 

Cole  (1938)  reported  a  brown  mass  below  the  hinge  of  C.  cdulc,  situated  in  a 
wedge-shaped  cavity  which,  when  torn,  liberated  thick-walled,  colorless,  immobile 
sporocysts  and  brown  granules.  The  tailless  cercariae,  named  C.  cambrensis,  were 
spined  and  measurements  in  millimeters  were  :  length,  0.27-0.32 ;  oral  sucker,  0.04 ; 
acetabulum,  0.03;  pharynx.  0.015.  The  acetabulum  was  about  one-third  of  the 
body  length  from  the  posterior  end ;  the  ceca  were  reported  to  extend  to  the  level 
of  the  posterior  border  of  the  acetabulum  but  are  preacetabular  in  the  figure.  The 
cercaria  was  similar  to  C.  margaritae  of  Jameson  (1902)  as  described  by  Lebour 
(1912)  and  Jameson  and  Nicoll  (1913),  but  differed  in  the  absence  of  eye-spots 
and  sensory  papillae. 

Rees  (1939)  re-described  Cercaria  strigata  Lebour,  1908,  which  was  actually 
a  metacercaria  found  by  Miss  Lebour  in  Tellina  tennis  and  Dona.v  vittatus  at 
Alnmouth,  Northumberland.  Presumably  it  was  identical  with  the  unnamed  larva 
found  by  Giard  (1907)  from  the  same  hosts.  Miss  Rees  found  the  cercariae  in 
thin-walled,  colorless  sporocysts  in  the  digestive  gland  of  C.  cdulc.  The  cercariae 
were  not  described  other  than  the  statement  that  they  were  the  same  as  the  meta- 
cercariae. The  excretory  vesicle  extended  to  the  region  of  the  oral  sucker  and  the 
anterior  ends  were  bifid.  The  flame-cell  formula  was  2[  (2  +  2)  +  (2  +  2)]. 
Cercaria  strigata  was  distinguished  from  C.  margaritae  of  Jameson  by  absence  of 
eye-spots  and  bristles  and  location  of  sporocysts,  which  in  the  former  were  just 
below  the  hinge.  It  differs  from  C.  cambrensis  which  develops  among  brown 
granules  in  thick-walled  sporocysts.  According  to  Rees,  C.  strigata  could  be  the 
larva  of  Gymnophallns  macroporous  Jameson  and  Nicoll,  1913  from  Oidemia  nigra 
but  more  likely  is  the  larva  of  G.  dcliciosus  which  was  found  abundantly  in  the  gall 
bladders  of  gulls  in  the  region.  She  suggested  that  C.  cambrensis  is  the  cercarial 
stage  of  the  metacercaria  in  M.  cdulis  and  identical  with  Gymnophallus  margari- 
tarum  (Dubois). 

Yamaguti  (1939)  described  Gymnophallus  macrostoma  from  the  intestine  of 
Melanitta  (Oidemia}  nigra  americana  (Swainson)  from  Korea.  According  to 
Yamaguti,  the  new  species  differs  from  G.  affinis  and  G.  macroporus  in  size  of 
eggs  and  position  of  the  ovary;  it  resembles  Gymnophalloidcs  tokicnsis  Fujita, 
1925,  a  metacercaria,  but  identity  must  await  experimental  evidence.  Yamaguti 
re-described  G.  bnrsicola  from  Melanitta  fusca  stcjnegeri  and  Melanitta  nigra 
americana :  variations  in  morphology  led  him  to  regard  G.  dapsilits  Nicoll  as  prob- 
ably identical  with  G.  bursicola. 

Ogata  (1944)  found  metacercariae  in  Paphia  (Rnditapes)  philippinarum,  Lat- 
ernida  kamakurama  and  Tellina  spp.  which  were  raised  to  adults  in  cats  and  mice. 
The  mature  worms  were  identified  as  G.  bnrsicola. 


284  H.  W.  STUNKARD  AND  J.  R.  UZMANN 

Uzmann  (1952)  described  Ccrcaria  inyac  from  the  gonads  and  digestive  gland 
of  Mya  arenaria  at  Newburyport,  Massachusetts.  The  sporocysts  were  clavate, 
unpigmented,  motile,  with  thin  walls  and  apical  birthpores ;  they  measured  0.21  to 
0.60  mm.  in  length.  He  suggested  that  this  is  the  same  species  reported  by  Staf- 
ford (1912)  from  the  same  host  in  Canada.  The  body  of  the  cercaria  is  0.12  to 
0.25  mm.  long ;  the  tail-stem  is  about  one-third  the  body  length  with  furci  slightly 
longer  than  the  stem  and  spined  at  the  tips. 

Dubois,  Baer  and  Euzet  (1952)  described  Ccrcaria  inathiasi,  a  furcocercous 
cercaria  from  the  Mediterranean,  which  was  identified  as  a  species  of  Tcrgcstia. 
The  cercaria  was  included  in  a  group  with  Cercaria  haswelli  Dollfus,  1927,  which 
Odhner  (1911b)  had  recognized  as  a  species  of  Tcrgcstia,  and  C.  dichotoma 
La  Valette.  Although  C.  dichotonia  has  been  widely  accepted  as  a  gymnophalline 
larva,  the  relation  to  Tcrgcstia  does  not  appear  unlikely  since  Cable  (1953)  has 
included  the  Haplocladinae  (which  includes  Tergestia)  and  the  Gymnophallinae 
in  the  family  Fellodistomatidae. 

Hutton  (1952)  studied  the  gymnophallid  parasites  of  C.  cdnlc  at  Plymouth, 
England.  He  described  a  new  species,  Ccrcaria  julbrighti,  which  developed  in 
motile,  irregularly  shaped  sporocysts  with  birthpores  at  the  tips  of  snout-like  pro- 
trusions. The  sporocysts  were  found  in  the  digestive  gland,  gonad,  and  dorsal 
part  of  the  foot.  Young  cercariae  in  sporocysts  have  forked  tails,  which  degenerate 
when  the  larvae  are  about  one-half  grown.  The  cercariae  lack  eye-spots,  are 
spined,  while  the  excretory  vesicle  extends  parallel  to  the  ceca  and  is  not  lyre- 
shaped.  The  mature  cercaria  agrees  closely  with  G.  choledochits  and  differs  from 
C.  ruargaritae  which  has  eye-spots  and  in  wrhich  the  excretory  vesicle  is  lyre- 
shaped  and  extends  to  the  oral  sucker.  He  reported  that  the  excretory  system  has 
thirteen  flame-cells  on  each  side  of  the  body.  It  is  possible  that  the  cilia  in  the 
common  collecting  duct  were  counted  as  the  thirteenth  flame-cell.  Hutton  differ- 
entiated between  C.  julbrighti  and  C.  cauibrcnsis  which  he  found  in  the  same  host 
species.  He  noted  resemblances  between  C.  julbrighti  and  C.  dichotonia  as  de- 
scribed by  Pelseneer  (1906)  and  Lebour  (1908),  but  certain  differences  led  him 
to  regard  them  as  distinct  species. 

Hutton  (1953)  described  Ccrcaria  rccsi  from  Hiatclla  arctica  and  Hiatclla 
striata  taken  at  Drake's  Island  and  Plymouth  Sound.  The  sporocysts  were  ovoid 
to  sausage-shaped,  0.32  to  0.80  mm.  long  and  0.196  to  0.352  mm.  wide.  The  cer- 
cariae were  fork-tailed,  non-oculate,  with  two  pairs  of  penetration  glands.  The 
flame-cell  formula  was  2[(2  +  2)  +  (2  +  2)],  a  total  of  16  cells,  the  same  as  Rees 
(1939)  had  found  in  C.  strigata.  The  oral  sucker  was  surrounded  by  two  pairs 
of  protrusible  spines  and  six  tubercules.  Hutton  stated  that  C.  rccsi  closely  re- 
sembles C.  discnrsata  Sinitsin  and  C.  invac  Uzmann,  1952,  and  that  all  are  probably 
members  of  the  genus  GymnopliaUus. 

Brinkmann  (1956)  in  a  study  of  the  Trematoda  of  Iceland,  reported  a  single 
unidentified  specimen  of  Gynmophallus  from  the  gall  bladder  of  Soniatcria  niollis- 
sinui  and  another  from  the  gall  bladder  of  Clangula  hyemalis  which  he  described  as 
a  new  species,  Gvninophallus  bilis.  He  noted  that  except  for  the  location  of  the 
genital  pore  the  latter  worm  agrees  entirely  with  Levinsen's  (1881)  description 
of  G.  somateriae,  which  differs  in  essential  respects  from  Odhner's  re-description  of 
that  species,  based  on  new  material  and  possibly  on  a  different  species  of 
Gynmophallus. 


DIGENETIC  TREMATODES  285 

Gymnophallid  trematodes  have  been  known  for  more  than  a  century.  The 
first  of  these  worms  to  be  discovered  were  metacercariae  from  the  mantle  of  Mytilns 
eduhs,  and  sporocyst  stages  were  reported  by  von  Siebold  (1837)  in  Macoma 
balthica  from  the  coast  near  Danzig.  Sexually  mature  specimens  were  described 
and  named  by  Levinsen  (1881)  from  the  digestive  tract  of  eider  ducks,  Somateria 
molhssima.  Subsequently,  worms  have  been  reported  from  the  gall  bladder,  in- 
testine, ceca,  and  bursa  Fabricii  of  different  species  of  birds.  Twelve  specific 
names  have  been  applied  to  these  sexually  mature  specimens  but  the  species  are 
not  clearly  distinguished  and  the  validity  of  certain  of  them  is  doubtful.  During 
this  time,  cercarial  stages  have  been  described  from  a  variety  of  bivalve  mollusks, 
but  frequently  cercarial  and  metacercarial  stages  have  been  mistaken  for  one  an- 
other and  there  is  no  reliable  information  to  identify  any  cercaria  with  its  meta- 
cercarial or  adult  stage.  Metacercariae  occur  frequently  in  bivalve  and  rarely  in 
gastropod  mollusks,  but  knowledge  of  their  previous  larval  or  final  sexually  mature 
stages  is  completely  lacking.  The  situation  is  chaotic  and  one  of  utter  confusion. 

Even  the  systematic  position  of  the  subfamily  Gymnophallinae  is  equivocal.  It 
was  erected  by  Odhner  (1905)  who  postulated  relationship  to  the  Heterophyidae 
but  admitted  (p.  314),  "Die  Frage,  wo  diese  Unterfamilie  zu  placieren  ist,  kann 
dagegen  nur  der  Gegenstand  sehr  unsicher  Vermutungen  sein."  Fuhrmann  (1928) 
included  it  in  the  family  Acanthostomidae ;  Dawes  (1946)  in  the  family  Micro- 
phallidae ;  and  Uzmann  (1952)  suggested  its  probable  relationship  to  the  family 
Brachylaemidae.  Cable  (1953)  transferred  the  Gymnophallinae  to  the  family 
Fellodistomatidae,  in  the  superfamily  Brachylaemoidea.  This  action  was  based 
on  the  discovery  of  the  first  life-cycle  in  the  subfamily,  that  of  Parvatrenia  bo- 
rinquenac,  a  new  genus  and  species  from  Puerto  Rico.  The  unencysted  meta- 
cercariae from  the  snail,  CcritJiidea  costata,  developed  to  maturity  in  baby  chicks 
and  since  sandpipers,  plovers,  terns  and  herons  of  the  region  did  not  harbor  gymno- 
phalline  trematodes,  Cable  postulated  that  the  natural  hosts  were  migrant  ducks. 
Small,  furcocerous  cercariae,  produced  in  sporocysts  in  Gcunna  pnrpnrea,  were 
recognized  as  the  antecedent  larval  stage  of  the  species.  In  the  same  paper.  Cable 
suggested  that  Metaccrcaria  month  of  Markowski  (1936)  may  be  a  cercaria,  and 
that  Cercaria  baltica  may  be  conspecific  with  Metaccrcaria  iinttabilis  from  the 
same  host.  This  species  is  probably  the  one  found  by  von  Siebold  in  the  same  host 
a  century  before. 

PRESENT  PROJECT 

For  some  years  the  U.  S.  Fish  and  Wildlife  Service  has  been  concerned  with 
the  attrition  in  stocks  of  the  soft-shelled  clam,  J\tya  arenaria,  along  the  New  Eng- 
land coast.  Reduction  in  numbers  of  these  clams  was  reported  by  Smith  (1950), 
Smith  and  Chin  (1951),  and  Glude  (1955).  The  junior  author  began  a  study 
of  the  significance  of  parasitism  in  relation  to  this  problem  at  Newburyport,  Massa- 
chusetts and  continued  it  at  Milford,  Connecticut.  He  (Uzmann,  1952)  published 
a  description  of  the  sporocysts  and  cercariae  found  in  M.  arenaria  at  Newburyport, 
Massachusetts.  The  larvae,  named  Cercaria  utyac.  emerge  from  the  sporocysts 
and  swim  in  the  sea ;  they  have  forked  tails  but  no  eye-spots.  The  amount  of 
living  material  was  limited  and  no  infection  experiments  were  attempted.  Uzmann 
also  found  sporocysts  and  cercariae  in  Hiatella  arctica  of  the  Boothbay  Harbor  area 
of  Maine,  but  they  were  not  reported.  In  addition,  Uzmann  (1953)  described 


286 


H.  W.  STUNKARD  AND  J.  R.  UZMANN 


i 


3 


5 


PLATE  I 


DIGENETIC  TREMATODES  287 

sporocysts  and  cercariae  from  Mytilus  edulis  taken  in  Long  Island  Sound,  New 
York ;  this  species,  named  Cercaria  milfordcnsis,  was  referred  tentatively  to  the 
genus  Proctocccs.  Metacercariae  had  been  found  in  lesions,  and  associated  with 
pearly  deposits,  in  the  mantle  of  M.  edulis  collected  in  the  region  of  Newburyport, 
Massachusetts. 

In  collaboration  with  the  senior  author,  attempts  were  made  to  determine  the 
adult  stage  of  the  metacercariae  from  M.  edulis.  Specimens  were  fed  on  February 
24,  1951  to  a  hamster  and  the  animal  was  sacrificed  on  March  3.  1951.  Sexually 
immature  worms  (Fig.  4)  were  recovered,  which  manifested  diagnostic  character- 
istics of  the  genus  Gymnophallus  and  it  appeared  that  the  natural  definitive  hosts 
were  mollusk-eating  birds.  Accordingly,  metacercariae  (Fig.  9)  were  fed  to 
domestic  chicks  and  ducklings,  but  no  infection  was  obtained.  Eggs  of  eider  ducks, 
sent  from  Boothbay  Harbor,  Maine,  were  incubated  in  the  laboratory  at  New  York 
University  and  metacercariae,  fed  June  1,  1951  to  recently  hatched  eider  chicks, 
developed  during  ten  days  in  the  intestine  to  sexual  maturity  (Fig-  5).  These 
worms  belong  to  the  genus  Gymnophallus,  but  because  specific  descriptions  are  so 
inadequate,  identification  is  tentative.  Subsequently,  the  junior  author  was  trans- 
ferred to  the  Seattle,  Washington,  Laboratory  of  the  U.  S.  Fish  and  Wildlife 
Service  and  the  senior  author,  on  retirement  from  teaching  at  New  York  Uni- 
versity, was  assigned  to  a  study  of  the  parasites  of  clams  and  of  their  predators. 
The  green  crab,  Carcinides  maenas,  an  important  predator,  harbors  the  metacer- 
carial  stages  of  a  digenetic  trematode  whose  life-history  was  worked  out  (Stunkard, 
1957)  and  the  adults  were  identified  as  Microphallus  similis  (Jagerskiold,  1900). 
Returning  to  the  investigation  of  the  sporocysts  and  cercariae  in  M.  arcnaria,  a 
study  of  the  gymnophallid  trematodes  was  resumed.  For  whole-hearted  coopera- 
tion and  for  material,  we  are  indebted  to  Walter  R.  Welch,  Chief  of  Clam  Investi- 
gations, Boothbay  Harbor,  Maine  and  members  of  his  staff.  In  the  summer  of 
1957  sporocysts  and  cercariae  were  found  by  the  senior  author  in  the  digestive 
gland  of  Gemma  gemma  taken  at  Boothbay  Harbor  and  unencysted  metacercariae 
from  the  mantle  of  G.  gemma  developed  to  sexual  maturity  in  the  intestine  of 
Somateria  mollissima . 

Most  of  the  previous  work  on  gymnophallid  trematodes  has  been  done  in 
Europe ;  the  parasites  have  been  reported  from  different  locations  in  different 
hosts,  but  specific  determination  on  the  basis  of  existing  descriptions  is  virtually 
impossible.  A  satisfactory  solution  of  the  difficulties  requires  controlled  experi- 
ments to  discover  and  relate  successive  stages  in  the  life-cycles  of  individual  species. 
Certain  questions  are  pertinent  to  a  consideration  of  the  problem  of  specificity : 
(1)  to  what  extent  can  one  species  of  Gymnophallus  infect  different  primary,  sec- 
ondary, and  definitive  hosts;  (2)  are  the  gymnophallids  in  gulls,  eider  ducks,  and 

EXPLANATION   OF   PLATE  I. 

FIGURE  1.  Adult  from  the  bursa  Fabricii  of  S.  mollissima,  Boothbay  Harbor,  Maine. 
Natural  infection,  specimen  1.0  mm.  long,  fixed,  stained  and  mounted,  ventral  view. 

FIGURE  2.  Adult  from  the  gall  bladder  of  v$\  mollissima,  Boothbay  Harbor,  Maine.  Natural 
infection,  specimen  sketched  alive,  1.3  mm.  long,  dorsal  view. 

FIGURE  3.  Same  specimen   shown  in  Figure  2,   fixed,   stained  and  mounted,   1.7  mm.   long. 

FIGURE  4.  Immature  specimen  from  the  mantle  of  M.  edulis,  recovered  from  the  intestine 
of  a  hamster  seven  days  later,  somewhat  flattened,  0.43  mm.  long,  ventral  view. 

FIGURE  5.  Mature  but  not  fully  gravid  specimen,  removed  from  the  mantle  of  M.  cdidis  and 
developed  ten  days  in  a  recently  hatched  chick  of  5*.  mollissima,  0.72  mm.  long,  dorsal  view. 


288 


H.  \V.  STUNKARD  AND  J.  R.  UZMANN 


PLATE  II 

FIGURE  6.  Adult,  flattened  specimen  from  the  intestine  of  S.  inollissima,  experimental  in- 
fection, after  feeding  metacercariae  from  Gemma  gemma.  Specimen  0.24  mm.  long,  ventral 
view. 

FIGURE  7.  Another  specimen,  same  infection  as  before,  worm  not  flattened,  0.22  mm.  long, 
ventral  view. 

FIGURE  8.  Metacercaria  from  Mya  areuaria.  Woods  Hole,  Mass.,  0.9  mm.  long,  excretory 
system  added  from  sketches  made  when  worm  was  alive. 

FIGURE  9.  Metacercaria  from  Mytilits  cditlis,  Xewburyport,  Mass.,  same  species  as  Figures 
4  and  5.  Specimen  0.46  mm.  long. 


DIGENETIC  TREMATODES  289 

other  shore-birds  members  of  the  same  or  different  species;  (3)  are  the  worms  in 
the  gall  bladder,  the  intestine,  the  ceca,  and  bursa  Fabricii  of  eider  ducks  members 
of  the  same  or  different  species;  (4)  do  the  asexual  generations  of  sporocysts  in 
Gemma  gemma,  in  HiatcUa  arctica,  Mytilns  cditlis,  and  Mya  arcnaria  belong  to  the 
same  or  different  species;  (5)  which,  if  any,  of  the  reported  metacercariae  and 
adults  are  later  stages  in  the  life-cycle  of  Ccrcaria  myacf  In  an  attempt  to  find 
answers  to  these  questions  the  following  procedures  were  devised :  ( 1 )  collect  and 
compare  worms  from  the  several  locations  in  eider  ducks  and  herring  gulls  (Larus 
argentatus)  ;  (2)  determine  whether  the  larvae  (miracidia)  in  the  eggs  of  these 
worms  are  mature  and  infective  or  whether  the  eggs  need  to  be  embryonated; 
(3)  try  to  infect  the  different  species  of  bivalve  mollusks  with  eggs  from  worms 
taken  from  different  hosts  and  different  locations;  (4)  attempt  transplantation  of 
metacercariae  from  each  species  of  bivalve  to  each  of  the  others ;  ( 5 )  attempt  in- 
fection of  eider  duck  chicks  with  metacercariae  from  G.  gemma,  H.  arctica  and 
M.  arenaria;  (6)  attempt  infection  of  mammalian  hosts  with  metacercariae  from 
each  of  the  molluscan  hosts. 

The  material  so  far  available  3  consists  of  adult  worms  from  natural  infections 
in  the  bursa  Fabricii  and  gall  bladder  of  Somateria  mollissima  taken  at  Boothbay 
Harbor,  Maine  and  others  from  experimental  infections  of  the  intestine  of  eider 
chicks  after  feeding  metacercariae  from  the  mantle  of  M.  edulis  and  from  the 
mantle  of  G.  gemma. 

Metacercariae  have  been  found  on  the  mantle  of  M.  arcnaria  from  Boothbay 
Harbor,  Maine  and  Woods  Hole,  Massachusetts ;  on  the  mantle  of  HiatcUa  arctica 
and  Gemma  gemma  from  Boothbay  Harbor;  and  on  the  mantle  of  Mytilns  cdiilis 
from  Newrburyport,  Massachusetts  and  Milford,  Connecticut. 

Sporocysts  and  cercariae  from  M.  arcnaria  were  described  by  Uzmann  (1952). 
Further  details  are  given  in  the  present  paper.  Sporocysts  and  cercariae  from 
H.  arctica  and  from  G.  gemma  are  described  in  this  report. 

Laboratory-hatched  eider  ducks,  recently  hatched  herring  gulls,  golden  ham- 
sters, and  white  mice  have  been  used  as  possible  experimental  hosts.  Adult  and 
recently  hatched  herring  gulls  were  provided  by  the  Marine  Biological  Laboratory. 

EXPERIMENTAL  RESULTS 

Attempts  to  infect  hamsters  and  white  mice  by  feeding  metacercariae  from  the 
three  species  of  mollusks  were  not  successful.  Metacercariae  from  Mya  arenaria 
and  HiatcUa  arctica,  although  fed  in  large  numbers,  failed  to  infect  eider  ducks. 
As  noted,  metacercariae  from  Mytilus  cditlis  and  from  Gemma  gemma  developed 
to  sexual  maturity  in  laboratory-raised  and  previously  unexposed  eider  ducks. 
These  worms  were  not  only  specifically  distinct,  but  belong  to  different  genera,  as 
recounted  in  the  descriptive  section  of  the  present  paper. 

3  In  a  personal  communication,  Dr.  John  S.  Rankin,  Jr.,  reported  that  in  1938  he  collected 
about  one  hundred  gymnophallid  trematodes  from  the  ring-necked  plover,  Charadrius  scmi- 
palmatus,  which  it  is  hoped  may  become  available  for  study. 

FIGURE  10.  Metacercaria  from  HiatcUa  arctica,  Boothbay  Harbor,  Maine.  Specimen  0.21 
mm.  long,  pressed  to  study  the  excretory  system. 

FIGURE  11.  Metacercaria  from  Gemma  gemma.  Specimen  0.2  mm.  long,  same  species  as 
Figures  6  and  7. 


290  H.  W.  STUNKARD  AND  J.  R.  UZMANN 

Many  worms  were  taken  from  the  bursa  Fabricii  of  eider  ducks  at  Boothbay 
Harbor.  Eggs  of  these  parasites  are  embryonated  when  passed  in  the  feces  of 
their  hosts,  but  do  not  hatch  in  sea-water.  Accordingly,  it  is  reasonably  certain 
that  the  miracidia  emerge  only  after  the  eggs  reach  the  digestive  tract  of  the  first 
intermediate  host.  Eggs  of  worms,  found  as  natural  infections  in  the  bursa  Fabricii 
'of  eider  ducks,  were  further  embryonated  and  pipetted  into  the  mantle  cavities  of 
Mya  arenaria  and  Mytilus  cdulis.  Other  eggs  were  placed  with  specimens  of 
Hiatclla  arctica  in  a  gallon  jar,  half-filled  with  sea  water  and  agitated  by  a  stream 
of  compressed  air.  The  attempts  to  infect  mollusks  with  eggs  of  the  parasites 
were  entirely  fruitless.  There  is  no  assurance  that  the  eggs  were  actually  ingested 
•although  in  natural  infection  it  is  probable  that  the  eggs  enter  the  mantle  cavity 
with  water  currents,  become  enmeshed  in  the  mucus  that  covers  the  gills,  and  reach 
the  digestive  tract  in  the  stream  of  material  that  is  driven  by  ciliary  action  from 
the  gills  to  the  mouth. 

Metacercariae  were  removed  from  M.  arenaria,  H.  arctica,  and  G.  gemma  and 
introduced  into  the  mantle  cavities  of  each  of  the  other  species.  In  no  instance 
was  successful  transplantation  assured.  Even  attempts  to  transfer  metacercariae 
from  one  M.  arenaria  to  another  gave  uncertain  results.  Often*  the  worms  failed 
to  adhere  to  the  new  host.  Also,  the  recipients  may  have  been  infected  before 
the  introduction  of  the  new  worms  and  a  degree  of  resistance  may  have  been 
developed.  When  experiments  must  be  conducted  on  specimens  that  have  been 
exposed  previously  to  the  same  or  related  parasites,  results  must  be  subjected 
to  rigid  scrutiny. 

DESCRIPTIVE  RESULTS 
Adult  worms 

I.  From  the  bursa  Fabricii  of  Somateria  mollissima  taken  at  Boothbay  Harbor, 
Maine    (Fig.    1) 

About  one  hundred  worms  were  collected ;  some  were  studied  alive,  others 
were  fixed  and  stained  for  morphological  study,  and  the  others  were  dissected  to 
obtain  eggs  for  infection  experiments.  The  worms  are  oval  to  pyriform,  usually 
rounded  anteriorly  and  more  pointed  posteriorly.  When  active  the  sides  may  be 
almost  parallel  and  either  end  may  be  wider.  They  vary  from  0.47  mm.  long  by 
0.30  mm.  wide  to  1.00  mm.  long  by  0.50  mm.  wide,  the  size  of  the  somewhat 
flattened  specimen  shown  in  Figure  1.  The  cuticula  is  covered  with  scale-like 
spines,  somewhat  smaller  posteriorly.  The  acetabulum  is  situated  slightly  behind 
the  middle  of  the  body  and  measures  0.10  to  0.14  mm.  in  diameter.  The  anterior 
portion  of  the  body  contains  many  gland-cells  whose  ducts  open  to  the  surface,  chiefly 
around  the  oral  sucker.  The  oral  opening  is  subterminal,  the  sucker  measures 
0.13  to  0.18  mm.  in  diameter  and  the  pharynx,  which  follows  immediately,  is  0.045 
to  0.055  mm.  in  diameter.  The  esophagus  varies  much  in  length  with  the  extension 
and  retraction  of  the  anterior  portion  of  the  body  and  measures  0.06  to  0.20  mm. 
in  length.  The  ceca  are  relatively  short  and  may.  when  the  body  is  contracted, 
extend  to  the  level  of  the  acetabulum.  The  excretory  vesicle  is  Y-shaped,  with 
long  arms  which  when  filled  may  extend  to  the  level  of  the  pharynx.  They  follow 
the  contour  of  the  lateral  edges  of  the  body,  but  in  living  specimens  the  anterior 
ends  may  become  widened,  flattened,  lobed  or  slightly  bifid,  and  retraction  of  the 
vesicle  along  the  lateral  faces  of  the  ceca  may  produce  the  lyre-shaped  appearance 
figured  in  many  species  of  Gymnoplialhts.  The  flame-cell  pattern  was  not  com- 


DIGENETIC  TREMATODES  291 

pletely  worked  out,  but  insofar  as  could  be  determined,  it  is  2  [(2 +  2 +  2) 
+  (2  +  2  +  2)|,  identical  with  that  in  the  metacercariae  from  M.  arenaria  and 
H.  arctica  (Figs.  8,  10). 

The  testes  are  oval,  and  vary  much  in  size  in  different  individuals.  They 
measure  0.06  to  0.13  mm.  by  0.04  to  0.10  mm. ;  are  lateral,  acetabular  to  completely 
postacetabular,  almost  opposite ;  the  one  on  the  ovarian  side  is  usually  slightly 
more  posteriad.  Sperm  ducts  arise  at  the  anterior  ends,  pass  forward  medially 
and  dorsally  and  join  to  form  the  seminal  vesicle  which  lies  dorsal  and  anterior 
to  the  acetabulum.  From  a  dorsal  or  ventral  view  it  may  appear  ovoid  or  pyriform, 
but  in  lateral  aspect  the  bipartite  character  is  clearly  apparent.  The  vesicle  is 
followed  by  a  cylindrical  duct,  enclosed  in  large  prostate  cells,  which  extends  pos- 
teriad and  ventrad  opening  into  the  small  genital  atrium.  The  common  genital 
pore  is  median,  just  in  front  of  the  acetabulum.  The  ovary  is  lateral,  usually  on 
the  left  side,  at  the  acetabular  level.  It  is  about  the  size  of  a  testis  and  may 
partially  overlap  the  testis  of  that  side  or  the  two  may  be  separated,  the  ovary  its 
own  diameter  in  front  of  the  testis.  The  oviduct  arises  at  the  median  posterior 
aspect  of  the  ovary,  passes  mediad  and  backward  where  it  expands  into  a  fertiliza- 
tion space  from  which  Laurer's  canal  passes  to  the  dorsal  surface  of  the  body. 
Immediately  following,  it  receives  the  short  vitelline  duct  and  expands  into  the 
ootype,  enclosed  in  the  cells  of  Mehlis'  gland.  There  is  no  seminal  receptacle ; 
instead,  the  initial  section  of  the  uterus  is  expanded  and  filled  with  spermatozoa. 
The  course  of  the  uterus  is  not  constant ;  some  of  the  loops  described  later  may  not 
be  present,  and  the  extent  is  determined  in  part  at  least  by  the  number  of  eggs  in 
the  body.  Typically,  from  the  ootype  the  uterus  passes  backward,  forms  a  loop  or 
series  of  coils  and  then  crosses  behind  the  acetabulum  to  the  antovarian  side  of 
the  body.  It  then  makes  a  backward  loop,  sometimes  almost  to  the  posterior  end 
of  the  body,  then  a  loop  or  series  of  coils  forward  almost  to  the  level  of  the  pharynx, 
then  backward  where  behind  the  ceca  it  crosses  to  the  ovarian  side  and  coils  may 
pass  forward  to  the  level  of  the  pharynx,  then  backward  mediad  of  the  ovary,  and 
then  forward  to  the  genital  pore.  The  vitelline  follicles  are  at  the  sides  and  above 
the  acetabulum.  six  to  twelve  indistinct  lobes  on  each  side ;  they  may  extend  through 
the  ovarian  and  testicular  zones ;  ducts  from  each  side  meet  behind  the  acetabulum 
to  form  a  small  receptacle  from  which  the  common  duct  leads  to  the  oviduct.  The 
eggs  are  operculate,  oval,  and  measure  0.021  to  0.025  mm.  by  0.015  to  0.018  mm. 
(average  0.023  by  0.016  mm.). 

II.  From  the  gall   bladder  of  5".   mollissima  taken   at   Boothbay   Harbor,    Maine 
(Figs.  2,  3) 

Two  specimens  were  found  in  the  gall  bladder  of  one  bird ;  the  bladders  of 
seven  other  ducks  were  negative.  The  worms  were  about  the  same  size ;  extended 
they  measured  1.72  mm.  long  and  0.60  mm.  in  width  and  contracted  1.00  mm.  long 
and  0.90  mm.  in  width.  Although  about  twice  as  large  as  the  worms  from  the 
bursa,  these  specimens  were  similar  in  shape  and  moved  in  a  similar  manner. 
However,  as  in  most  trematodes,  the  shape  and  relative  position  of  structures  are 
so  pliable  that  an  account  based  on  a  single  specimen  may  be  very  misleading. 
Figures  2  and  3,  made  from  the  same  specimen,  show  changes  in  shape  when  alive 
and  moderately  relaxed  and  when  fixed  under  coverglass  pressure  with  the  anterior 
portion  retracted  and  the  posterior  portion  extended.  •  Like  the  worms  from  the 


H.  W.  STUNKARD  AND  J.  R.  UZMANN 

bursa,  the  anterior  portion  of  the  body  contains  many  glandular  cells.  The  ace- 
tabulum  measures  0.17  to  0.185  mm.  in  diameter.  The  oral  sucker  is  0.18  to 
0.23  mm.  in  diameter ;  the  diameter  of  the  pharynx  is  about  one-third  that  of  the 
oral  sucker.  The  bifurcation  of  the  digestive  tract  is  about  midway  between  the 
suckers  and  the  ceca  extend  to  the  acetabular  level.  The  excretory  vesicle  is 
almost  identical  with  that  of  the  worms  from  the  bursa. 

The  testes  are  oval,  0.15  to  0.19  mm.  in  diameter,  typically  postacetabular  ;  the 
seminal  vesicle  is  in  part  dorsal  to  the  acetabulum  and  the  prostatic  portion  curves 
ventrad  and  posterad  in  front  of  the  acetabulum  to  open  into  the  genital  atrium. 
The  opening  to  the  exterior  is  immediately  anterior  to  the  aperture  of  the  sucker 
and  may  appear  below  the  front  portion  of  the  acetabulum.  The  ovary  is  about 
the  same  size  as  the  testes,  lateral,  sinistral  in  both  specimens,  at  the  acetabular 
level  or  slightly  posteriad.  The  relations  of  the  oviduct,  ootype,  and  associated 
structures  are  quite  similar  to  those  in  the  worms  from  the  bursa.  The  course  of 
the  uterus  is  similar  also,  with  coils  that  extend  almost  to  the  posterior  end  of  the 
body  and  others  to  the  region  of  the  pharynx.  Some  of  the  coils  that  initially  were 
more  median  in  position  were  pushed  laterally  by  pressure  of  the  coverglass.  The 
vitelline  follicles  are  lateral  and  posterior  to  the  acetabulum ;  they  extend  through 
the  ovarian  and  part  of  the  testicular  zone,  but  are  in  large  part  postacetabular. 
The  eggs  are  about  the  same  size  as  those  in  the  worms  from  the  bursa  and  average 
0.023  by  0.017  mm. 

III.  From  the  intestine  of  6".  inollissiina  ;  experimental  infection  after  feeding  meta- 
cercariae  from  Mytilns  ednlis.  Long  Island  Sound,  New  York  (Figs.  4,  5) 

As  noted  earlier,  sexually  immature  worms  were  recovered  from  the  intestine 
of  a  hamster,  seven  days  after  feeding  metacercariae  from  M.  cdidis.  The  largest 
worm,  shown  in  Figure  4,  is  not  quite  sexually  mature  but  measures,  fixed  and 
stained,  0.43  mm.  long  and  0.235  mm.  wide.  Feeding  experiments  were  carried 
out  later  with  eider  chicks  hatched  in  the  laboratory  from  eggs  sent  from  Boothbay 
Harbor,  Maine.  Feeding  began  when  the  birds  were  one  day  old.  After  ten  suc- 
cessive days  of  feeding  metacercariae,  a  series  of  worms  was  taken  from  the 
intestine  of  one  bird.  One  worm,  which  had  just  begun  egg-production,  with 
seven  eggs  in  the  initial  part  of  the  uterus,  is  only  slightly  larger  than  the  one  from 
the  hamster.  The  largest  specimens,  which  measure  0.57  to  0.72  mm.  in  length, 
are  not  completely  gravid,  although  coils  of  the  uterus  extend  posteriad  about  one- 
half  the  distance  from  the  acetabulum  to  the  end  of  the  body  and  forward  to  the 
pharynx.  The  worm  which  was  just  attaining  maturity  is  0.44  mm.  long  and 
0.25  mm.  wide.  The  acetabulum  is  0.08  mm.  in  diameter ;  the  oral  sucker  0.092 
mm.  in  diameter,  and  the  pharynx  is  0.057  mm.  wide  and  0.045  mm.  long.  The 
seminal  vesicle  is  0.056  mm.  in  diameter ;  the  right  testis  is  0.09  by  0.06  mm.,  the 
left  testis  0.09  by  0.056  mm. ;  the  ovary  0.080  by  0.074  mm.,  and  the  eggs  0.027  by 
0.020  mm.  One  of  the  largest  specimens,  shown  in  Figure  5,  somewhat  flattened, 
is  0.72  mm.  long  and  0.36  mm.  wide.  The  acetabulum  is  0.096  mm.  in  diameter ; 
the  oral  sucker  is  0.12  mm.  wide  and  0.10  mm.  long;  the  pharynx  is  0.057  mm. 
wide  and  0.050  mm.  long.  The  seminal  vesicle  measures  0.090  by  0.062  mm. ;  the 
right  testis  is  0.126  by  0.078  mm.;  the  left  testis  is  0.12  by  0.080  mm.;  the  ovary 
is  0.12  by  0.083  mm.;  and  the  eggs  average  0.026  by  0.019  mm.  It  appears  that 
the  first  eggs  are  slightly  larger  than  those  produced  later. 


DIGENETIC  TREMATODES  293 

IV.  From  the  intestine  of  S.  mollissima  ;  experimental  infection  after  feeding  meta- 
cercariae  from  Gemma  gemma  taken  at  Boothbay  Harbor,  Maine  (Figs.  6,  7) 

Laboratory-raised  specimens  of  S.  mollissima,  never  exposed  previously  to  in- 
fection, were  fed  metacercariae  on  alternate  days  from  October  13  to  October  23, 
1957,  a  total  of  five  feedings.  More  than  100  worms,  most  of  them  sexually  mature, 
were  recovered  from  the  intestine.  Gravid  specimens  measured  0.15  to  0.33  mm. 
in  length  and  0.09  to  0.16  mm.  in  width.  Juvenile  worms  were  only  slightly 
smaller.  The  cuticula  bears  flat,  scale-like  spines.  The  acetabulum  is  0.029  to 
0.037  mm.  in  diameter,  only  about  one-half  the  size  of  the  oral  sucker.  It  is  situ- 
ated slightly  posterior  to  the  middle  of  the  body.  The  oral  sucker  is  0.057  to  0.080 
mm.  in  diameter;  the  posterior  half  of  the  sucker  contains  many  unicellular  glands 
which  in  living  worms  appear  as  yellowish  columns,  and  the  sucker  when  com- 
pressed has  lateral  ear-like  projections  (Figs.  6,  7).  The  pharynx  is  0.027  to 
0.033  mm.  in  diameter,  almost  as  large  as  the  acetabulum.  The  esophagus  is 
variable  in  length ;  the  ceca  may  be  entirely  preacetabular  or  approach  the  level 
of  the  gonads.  The  excretory  vesicle  is  V-shaped  with  long  arms  which  when 
filled  may  extend  to  the  level  of  the  pharynx.  The  flame-cell  pattern  could  not  be 
resolved  since  the  worms  were  so  filled  with  eggs  that  they  recalled  the  figure  of 
GymnophaUus  ovoplenus  as  given  by  Jameson  and  Nicoll  (1913).  However, 
since  the  worms  are  almost  fully  grown  in  the  metacercarial  stage  (Fig.  11),  it  is 
reasonably  certain  that  the  excretory  system  does  not  undergo  change  with  repro- 
ductive maturity  and  the  production  of  eggs.  The  testes  are  oval,  0.025  to  0.035 
mm.  by  0.035  to  0.050  mm.  in  diameter,  situated  on  opposite  sides  in  the  posterior 
one-third  to  one-fourth  of  the  body.  Sperm  ducts  lead  forward  and  mediad  to 
open  into  a  large,  clavate  seminal  vesicle  which  extends  from  a  level  only  slightly 
anterior  to  the  testes  almost  to  the  bifurcation  of  the  digestive  tract.  Anteriorly 
it  becomes  continuous  with  an  ejaculatory  duct,  surrounded  by  secretory  cells, 
which  opens  into  the  shallow  genital  atrium.  The  genital  pore  is  median,  some 
distance  anterior  to  the  acetabulum,  often  below  the  posterior  edge  of  the  pharynx. 
The  ovary  is  lateral,  either  left  or  right,  slightly  smaller  and  immediately  anterior 
to  or  overlapping  the  testis  of  the  ovarian  side.  The  oviduct  arises  at  the  median, 
posterior  portion  of  the  ovary,  passing  mediad  where  it  enters  the  ootype.  A  semi- 
nal receptacle  and  Laurer's  canal  were  not  observed,  but  may  be  present.  The 
vitellaria  are  composed  of  compact  follicles,  forming  reniform  glands,  dorsal,  lateral 
and  somewhat  posterior  to  the  acetabulum.  Vitelline  ducts  pass  mediad  and  form 
a  common  duct  which  opens  into  the  initial  portion  of  the  ootype.  Mehlis'  gland 
is  present  but  consists  of  a  small  number  of  cells.  The  coils  of  the  uterus  may 
almost  fill  the  body  from  the  level  of  the  oral  sucker  to  the  posterior  end.  The  eggs 
measure  0.015  to  0.017  by  0.010  to  0.011  mm. 

Metacercariae 

I.  From  M\a  arcnaria,  taken  at  Boothbay  Harbor,  Maine  and  at  Woods  Hole, 
Massachusetts  (Fig.  8).  Uniformly  light  infection,  1-12  worms  per  clam; 
about  25%  of  the  clams  infected 

Specimens  vary  greatly  in  size  as  the  unencysted  metacercarial  stage  is  an  im- 
portant growth  phase  in  the  life  of  the  species.  The  worms  actively  ingest  material 


294  H.  W.  STUNKARD  AND  J.  R.  UZMANN 

from  the  mantle  of  the  host  and  develop  from  a  size  hardly  larger  than  a  cercaria 
(0.12  to  0.25  mm.  in  length  when  retracted  and  extended)  to  almost  definitive 
size  (0.60  to  1.20  mm.,  corresponding  measurements  of  length).  Ordinarily  the 
width  is  about  one-half  the  length,  but  specimens  may  contract  until  the  length 
and  width  are  equal  or  elongate  until  the  width  is  less  than  one-fourth  the  length. 
During  the  metacercarial  period  the  larvae  increase  about  five  times  in  length  and 
the  organs,  except  the  gonads  and  reproductive  structures,  attain  almost  full 
growth.  There  are  six  papillae  around  the  acetabular  opening.  The  acetabulum 
measures  0.13  to  0.14  mm.  in  diameter  in  the  large  individuals.  The  cuticular 
spines  are  pointed  and  sharp.  The  anterior  end  bears  several  small  papillae,  each 
tipped  by  a  short,  stiff  bristle.  The  anterior  region  of  the  body  contains  a  large 
number  of  unicellular  glands  which  open  around  the  oral  sucker.  The  oral  sucker 
is  0.147  to  0.16  mm.  in  diameter  in  large  specimens.  The  digestive  ceca  are  lined 
with  large  cells  and  yellow  granules  are  conspicuous  in  the  cytoplasm  of  the  cells 
and  in  the  lumen  of  the  ceca.  The  yellow  material  appears  like  that  in  the  digestive 
gland  of  the  clam  and  may  be  taken  by  the  parasite  from  the  vascular  fluid  of  the 
mollusk.  The  excretory  system  has  been  worked  out  and  the  Arrangement  of 
the  tubules  and  flame-cells  is  shown  in  Figure  8.  The  formula  is  2  [(2  +  2  +  2) 
+  (2  +  2  +  2)  |;  the  common  duct,  which  leads  from  the  junction  of  the  anterior 
and  posterior  collecting  ducts,  bears  tufts  of  cilia  which  may  simulate  flame  cells. 

II.  From  Mytilus  cditlis,  taken  at  Milford,  Connecticut  and  Newburyport,  Massa- 
chusetts  (Fig.  9).     Uniformly  light  infection,  1-10  worms  per  mussel;  about 
20%  of  the  mollusks  infected 

The  metacercariae  in  M.  cditlis  produce  lesions  in  the  mantle  and  body-wall, 
which  may  result  in  the  deposition  of  nacreous  material.  The  larvae  attain  a  length 
of  0.4  to  0.6  mm.  and  a  width  of  0.2  to  0.3  mm.  In  a  fixed  and  stained  specimen, 
0.45  mm.  long  and  0.275  mm.  wide,  the  acetabulum  is  0.078  by  0.07  mm. ;  the  oral 
sucker  is  0.088  mm.  and  the  pharynx  is  0.030  mm.  in  diameter.  Metacercariae 
from  M .  cdulis  developed  to  sexual  maturity  in  a  ten-day-old  eider  duck  (rf.  Figs. 
4,  5). 

III.  From  the  mantle  of  Hiatclla  arctica,  taken  at  Boothbay  Harbor,  Maine  (Fig. 
10).     Uniformly  light  infection,  10  to  8  worms  per  clam;  about  20%  of  mol- 
lusks infected 

The  metacercariae  in  H.  arctica  are  similar  in  shape  to  those  in  Mya  arcnaria ; 
the  smallest  individuals  of  the  two  species  are  about  the  same  size,  0.12  to  0.175 
mm.  in  length,  but  the  largest  worms  from  H.  arctica  do  not  exceed  0.3  mm.  in 
length  and  accordingly  are  only  about  one-fourth  as  large  as  those  from  M.  arcnaria. 
The  suckers,  however,  are  relatively  larger ;  the  acetabulum  measures  0.050  to  0.058 
mm.  in  diameter  in  large  specimens.  The  anterior  end  of  the  body  does  not  con- 
tain the  large  number  of  glandular  cells  so  characteristic  of  the  worms  from  M. 
arcnaria  and  the  digestive  ceca  do  not  contain  the  yellow  material  so  conspicuous 
in  that  species.  The  oral  sucker  of  large  individuals  measures  0.054  to  0.064  mm. 
in  diameter.  Some  of  the  measurements  were  made  while  the  worms  were  under 
considerable  pressure  from  the  coverglass  while  the  details  of  the  excretory  system 


DIGENETIC  TREMATODES  295 

were  studied.     The  excretory  system  is  identical  with  that  in  the  larva  from  M. 
arenaria  and  the  flame-cell  formula  is  2  [(2  +  2  +  2)  +  (2  +  2  +  2)]. 

IV.  From  the  mantle  of  Gemma  gemma  at  Boothbay  Harbor,  Maine  (Fig.  11). 
Uniformly  light  infection;  1  to  10  worms  per  clam;  about  W%  of  the  clams 
infected 

These  metacercariae  were  fed  to  an  eider  duck  and  became  mature  (cf.  Figs. 
6,  7).  Specimens  from  G.  gemma  measure  0.13  to  0.23  mm.  in  length  and  0.06 
to  0.12  mm.  in  width.  The  anterior  end  contains  many  glandular  cells  that  open 
around  the  oral  sucker.  The  cuticula  bears  flat,  scale-like  spines.  The  acetabu- 
lum  is  just  posterior  to  the  middle  of  the  body  and  is  0.028  to  0.033  mm.  in  diameter. 
The  oral  sucker  measures  0.05  to  0.07  mm.  in  diameter  in  large  individuals.  The 
posterior  half  of  the  sucker  contains  yellowish  columns  as  described  for  the  adult 
and  when  compressed,  the  sucker  has  lateral  ear-like  projections.  The  pharynx 
is  0.026  to  0.030  mm.  in  diameter  and  the  ceca  usually  extend  into  the  postacetabu- 
lar  zone  and  often  to  the  gonads.  The  reproductive  structures  are  well  developed. 
The  testes,  ovary  and  vitelline  glands  are  almost  as  large  as  in  the  sexually  mature 
individuals  and  the  terminal  portions  of  the  ejaculatory  duct  and  metraterm  are 
shown  in  Figure  11.  The  excretory  vesicle  is  typical  of  the  gymnophallids  but 
the  flame-cell  pattern  is  simpler;  it  is  shown  in  Figure  11  and  the  formula  is 
2  [(2  +  2)  +  (2)].  Such  a  difference  probably  has  considerable  taxonomic 
significance. 

Sporocysts  and  cercariae 

I.  From  Mya  arenaria  taken  at  Newburyport,  Massachusetts  and  Boothbay  Harbor, 

Maine  (Fig.  12) 

This  species,  described  by  Uzmann  (1952)  from  M.  arenaria.  taken  at  Newbury- 
port, Massachusetts,  has  been  found  in  the  same  host-species  from  Boothbay  Har- 
bor, Maine.  The  incidence  of  infection  is  low  also  in  Maine ;  only  three  infections 
have  been  observed  in  the  dissection  of  over  1000  clams.  No  cercariae  were  found 
in  water  in  which  the  clams  were  kept,  perhaps  because  the  larvae  were  carried 
away  in  the  current  of  water  flowing  over  the  mollusks.  The  cercariae  were  de- 
scribed and  figured  by  Uzmann  who  noted  that  a  similar  and  probably  identical 
larva  had  been  described  by  Stafford  (1912).  Uzmann  also  noted  the  close 
morphological  agreement  between  the  cercaria  from  M.  arenaria  and  Cercaria  dis- 
cursata  Sinitsin,  1911.  Allison  (1943)  assigned  Leucochloridioniorpha  constan- 
tiae  (Mueller,  1935),  which  has  a  furcocercous  cercaria,  to  the  family  Brachy- 
laemidae  and  suggested  that  C.  discursata  may  belong  to  the  same  family.  It  now 
appears  that  the  resemblance  between  the  cercaria  of  L.  constantiae  and  Cercaria 
discursata  is  merely  superficial  and  that  both  C.  discursata  and  C.  myae  should  be 
placed  in  the  subfamily  Gymnophallinae. 

II.  From  Hiatella  arctica  taken  at  Boothbay  Harbor,  Maine  (Figs.  13,  14) 

The  description  of  this  species  is  based  entirely  on  material  collected  by  Uzmann 
in  1953.  Of  13  specimens  examined  on  March  18th,  2  were  infected ;  of  136  ex- 


296 


H.  W.  STUNKARD  AND  J.  R.  UZMANN 


12 


14 


PLATE  III 

FIGURE  12.  Sporocyst,  0.67  mm.  long,  and  cercariae  from  Mya  arcnaria.  This  is  Ccrcaria 
myac  Uzmann,  1952. 

FIGURE  13.  Sporocyst,  0.80  mm.  long,  and  cercariae  from  Hiatclla  arctica.  This  species  is 
identified  as  Ccrcaria  rccsi  Hutton,  1953. 


DIGENETIC  TREMATODES  297 

aminecl  May  12th,  6  were  infected.  Several  hundred  specimens  of  H.  orctica, 
taken  from  the  same  area  where  Uzmann  made  his  collections,  have  been  examined 
in  the  summers  of  1956  and  1957  without  finding  the  parasite. 

The  sporocysts  are  oval  to  pyriform,  usually  with  a  neck-like  extension  which 
may  be  one-fourth  or  even  one-third  of  the  total  length  of  the  sporocyst  and  which 
bears  the  birth-pore  at  the  end.  The  largest  fixed  and  stained  sporocyst  is  0.96 
mm.  long  and  0.37  mm.  wide,  but  most  of  them  are  much  smaller.  The  one  shown 
in  Figure  13  is  0.8  mm.  long.  The  cercariae  have  forked  tails,  spined  cuticula  and 
no  eye-spots;  the  body  is  0.12  to  0.175  mm.  in  length  and  0.05  to  0.068  mm.  wide. 
The  acetabulum  is  0.036  to  0.043  mm.  in  diameter  and  the  oral  sucker  is  approxi- 
mately the  same  size.  The  tail-stem  is  variable  in  length,  0.02  to  0.052  mm.,  and 
the  furci  are  somewhat  longer  when  fully  extended,  but  they  may  contract  to  less 
than  one-half  the  fully  extended  length.  The  pharynx  is  0.015  to  0.022  mm.  in 
diameter,  spherical  to  oval,  and  longer  than  broad  when  the  anterior  end  is  pro- 
truded. The  cephalic  glands  and  ducts  could  not  be  resolved  with  certainty.  The 
excretory  vesicle  is  shown  in  Figure  14,  but  since  the  supply  of  living  material 
was  limited,  the  flame-cell  formula  was  not  worked  out. 

III.  From  Gemma  gemma,  taken  at  Boothbay  Harbor,  Maine  (Figs.  15,  16) 

No  infection  by  this  species  was  discovered  by  isolation  of  the  clams,  perhaps 
because  the  larvae  swim  well  and  were  carried  away  in  the  running  water  that 
bathed  the  mollusks.  Of  824  G,  gemma-  dissected,  three  were  infected.  The 
sporocysts  were  dispersed  in  the  interlobular  spaces  of  the  digestive  gland.  They 
are  cylindrical  to  oval  to  pyriform  to  clavate,  with  a  narrow  end  in  which  the  birth- 
pore  is  situated.  The  smaller  ones  are  motile  while  the  larger  ones  lose  motility 
as  they  become  filled  with  cercariae.  They  may  extend  to  a  length  of  about  0.50 
mm.  The  cercariae  emerge  from  the  clam  and  swrim  vigorously.  In  swimming, 
the  tail  is  turned  ventrad.  the  furci  are  extended  and  lash  from  side  to  side  while 
the  anterior  end  wobbles  back  and  forth.  Otherwise,  the  tail  manifests  nervous 
twitching  movements  while  the  furci  separate  and  then  come  together.  When 
killed  by  adding  hot  AFA  (alcohol-formol-acetic  acid)  solution  to  a  beaker  con- 
taining a  small  amount  of  swirling  sea-water  in  which  they  are  suspended,  the 
cercariae  are  very  uniform  in  size  and  shape  (Fig.  15).  The  cuticula  bears  spines 
on  both  the  body  and  tail.  The  body  is  0.12  to  0.14  mm.  in  length  and  0.04  to 
0.05  mm.  in  width.  The  tail-stem  is  0.04  to  0.05  mm.  in  length,  0.015  to  0.017  mm. 
wide  at  the  base  and  0.013  to  0.015  mm.  wide  at  the  bifurcation  of  the  tail.  The 
furci  are  0.060  to  0.068  mm.  long  and  0.011  mm.  wide  at  the  base.  Alive,  the  cer- 
carial  body  varies  from  0.08  to  0.2  mm.  in  length  and  0.03  to  0.07  mm.  in  width ; 
the  tail  varies  from  0.05  to  0.14  mm.  in  length,  the  stem  from  0.025  to  0.06  mm.  and 
the  furci  from  0.025  to  0.13  mm.  There  are  no  eye-spots.  The  acetabulum,  situ- 
ated just  posterior  to  the  middle  of  the  body  protrudes  slightly  and  measures  0.027 
to  0.030  mm.  in  diameter.  The  oral  sucker  is  the  same  size  as  the  acetabulum ; 
there  is  a  short  prepharynx,  and  the  pharynx  measures  0.014  to  0.019  mm.  in 

FIGURE  14.  Ccrcaria  rccsi  Hutton,   1953   from   Hiatclla   arctica,   Boothbay   Harbor,   Maine. 

FIGURE  15.  Cercaria  from  Gemma  i/cnuua,  Boothbay  Harbor,  Maine. 

FIGURE  16.  Cross-section  of  G.  gemma  through  the  pedal  ganglion,  mantle  and  part  of  the 
gill  of  one  side  removed,  to  show  location  and  extent  of  the  infection.  At  this  level,  the  sporo- 
cysts are  more  numerous  than  the  follicles  of  the  digestive  gland. 


298  H.  W.  STUNKARD  AND  J.  R.  UZMANN 

diameter.  The  esophagus  varies  in  length  as  the  body  elongates  and  contracts ;  in 
fixed  specimens  it  is  about  as  long  as  the  diameter  of  the  pharynx.  The  ceca  are 
ovate,  wider  anteriorly.  They  may  be  entirely  preacetabular  or  extend  posteriad 
to  the  level  of  the  middle  of  the  acetabulum ;  their  walls  are  composed  of  large  cells. 
A  pair  of  lobed  unicellular  glands  is  situated,  one  on  either  side,  at  the  level  of  the 
intestinal  bifurcation  and  their  ducts  pass  forward  to  open  above  the  mouth.  The 
excretory  vesicle  consists  of  a  dorsal,  pouch-like  expansion  from  which,  on  the 
ventral  side,  the  arms  extend  forward  to  the  pharyngeal  level.  They  are  ventral 
to  the  digestive  ceca  and  are  filled  with  concretions,  0.005  to  0.006  mm.  in  diameter. 
On  each  side  there  is  a  flame-cell  at  the  level  of  the  pharynx,  and  the  capillary 
from  it  divides  at  the  level  of  the  intestinal  bifurcation ;  one  branch  leads  to  the 
vesicle  but  the  other  could  not  be  followed  with  certainty.  It  may  extend  back  to 
a  flame-cell  located  at  the  level  of  the  vesicular  pouch,  but  the  duct  from  that  cell 
was  hidden  at  the  acetabular  level  by  concretions  in  the  excretory  vesicle. 

DISCUSSION 

The  present  account  includes  descriptions  of  sporocysts,  cercariae,  and  meta- 
cercariae  from  marine  bivalves  and  adult  worms  from  the  eider  duck.  Somateria 
mollissima.  Specific  identification  is  so  uncertain  that  we  prefer  to  list  the  worms 
by  host  and  location  rather  than  propose  names  that  might  further  confuse  the 
taxonomic  situation. 

The  adult  forms  I,  II,  and  III  are  members  of  the  genus  Gymno phallus. 
Worms  from  the  bursa  Fabricii  of  S.  mollissima.  Adult  No.  I,  although  they  are 
somewhat  smaller,  may  be  identical  with  G.  bursicola  Odhner,  1900  or  G.  dapsilis 
Nicoll,  1907,  if  indeed  these  species  are  actually  distinct.  It  is  interesting  to  note 
that  Jameson  and  Nicoll  (1913)  reported  G.  dapsilis  from  the  intestine  as  well  as 
the  bursa.  Our  Adult  No.  II,  from  the  gall  bladder,  may  be  identical  with  G.  dc- 
liciosus  (Olsson,  1893)  or  G.  cholcdochus  Odhner,  1900,  if  the  latter  of  these 
species  is  really  valid.  The  account  of  Isaitchikow  (1924),  if  it  dealt  with  a 
single  species  and  if  that  species  was  G.  cJwledochns,  would  suggest  that  the  re- 
ported differences  between  G.  chofedochus  and  G.  deliciosus  are  not  significant. 
The  single  specimen  from  the  gall  bladder  of  S.  mollissima  taken  in  Iceland  and 
described  as  a  new  species,  G.  bilis,  by  Brinkmann  (1956)  is  similar  to  G.  bursicola 
and,  despite  reported  differences,  may  belong  to  that  species.  The  naming  of  a 
new  species  on  a  single  specimen  is  not  recommended.  Our  Adult  No.  Ill,  from 
the  intestine  of  an  eider  chick,  the  sexually  mature  stage  of  the  metacercaria  in 
Mytilns  cdulis,  is  larger  than  G.  somateriae  (Levinsen,  1881)  and  may  be  G.  bus- 
sic  ola,  which  had  not  yet  settled  in  the  bursa  and  reached  full  size. 

Our  Adult  No.  IV  is  clearly  a  member  of  the  genus  Parz'atrema  Cable,  1953. 
The  worms  are  very  similar  to  Pari'atrcma  borinqucnac  Cable,  1953,  the  morpho- 
logical differences  are  minor,  the  chief  differences  are  in  geographical  location  and 
in  primary  and  secondary  hosts.  The  cercariae  from  Gemma  pur  pur  ea,  described 
by  Cable  as  larvae  of  P.  borinqitenae,  are  smaller,  the  tail  is  relatively  much  smaller 
and  the  cercaria,  according  to  Cable,  "is  a  poor  swimmer"  when  compared  with  the 
cercariae  from  Gemma  gemma  at  Boothbay  Harbor,  Maine.  Because  of  these 
differences,  we  recognize  the  worms  we  have  described  as  a  new  and  distinct  species 
for  which  we  propose  the  name  Parvatrema  borealis.*  Cable  noted  the  possible 


DIGENETIC  TREMATODES 

identity  of  Parvatrema  and  Gymnophalloides.  Both  Yamaguti  (1939)  and  Cable 
(1953)  accredit  this  genus  to  Fujita  (1925)  although  it  appears  from  the  footnotes 
in  his  French  translation  of  the  text  and  from  his  accompanying  note,  that  Dollfus 
claims  credit  for  the  generic  name.  The  worms  described  as  Gynmopliallus  ovo- 
plcnus  by  Jameson  and  Nicoll  (1913)  differ  distinctly  from  Gymnophallus  and 
although  the  morphology  is  very  imperfectly  known,  it  appears  from  the  figure 
and  account  of  Jameson  and  Nicoll  that  the  worms  agree  better  with  the  character- 
istics of  Parvatrema  and  accordingly  we  transfer  the  species  to  the  latter  genus  as' 
Parvatrema  ovoplenus  (Jameson  and  Nicoll,  1913). 

Metacercaria  I  is  common  in  Mya  arenaria  but  it  did  not  persist  or  develop  in 
mice,  hamsters,  eider  clucks  or  herring  gulls,  and  other  stages  in  the  life-cycle  are 
yet  unknown.  Only  when  the  cercarial  and  adult  stages  become  available  will  the 
taxonomic  position  of  the  species  be  clarified. 

Metacercaria  II  is  common  in  Mytilus  cdulis;  it  developed  in  the  eider  chick 
and  as  noted,  may  be  the  asexual  stage  of  G.  bursicola. 

Metacercaria  III  is  relatively  common  in  Hiatclla  arctica  but  like  Metacer- 
caria I,  it  did  not  persist  or  develop  in  experimental  animals  and  its  status  is 
yet  uncertain. 

Metacercaria  IV,  from  Gemma  gemma,  is  the  asexual  stage  of  Parvatrema 
borealis  n.  sp.,  which  developed  in  large  numbers  in  the  intestine  of  5".  mollissima. 
Whether  other  birds  also  serve  as  final  hosts  is  unknown  but  probable. 

The  sporocysts  and  Cercaria  I,  which  may  cause  the  condition  known  by  clam 
diggers  as  "waterbelly,"  were  described  by  Uzmann  (1952).  Although  the  species 
has  received  further  study,  later  stages  in  the  life-cycle  of  the  parasite  are  still 
unresolved. 

The  sporocysts  and  Cercaria  II,  from  Hiatclla  arctica  at  Boothbay  Harbor, 
agree  in  detail  with  the  corresponding  stages  of  Cercaria  recsi  as  described  by 
Hutton  (1953)  from  H.  arctica  and  Hiatella  striata  at  Plymouth,  England.  They 
are  referred  to  that  species. 

The  sporocysts  and  cercariae  III,  from  G.  gemma  at  Boothbay  Harbor  may  be 
identical  with  the  metacercariae  from  the  same  host-species  which  developed  to 
maturity  in  S.  inoUissima  and  which  we  describe  as  Parvatrema  borealis  n.  sp.  If 
so,  the  oral  sucker  must  double  in  diameter  while  the  acetabulum  remains  essen- 
tially unchanged. 

Present  literature  concerning  gymnophalline  trematodes  discloses  descriptions 
of  twelve  species  of  adult  worms  from  the  gall  bladder,  intestine,  ceca  and  bursa 
Fabricii  of  various  shore-birds  ;  an  even  larger  number  of  metacercariae  from  marine 
mollusks ;  and  a  score  of  different  sporocysts  and  cercariae  have  been  recorded  from 
various  marine  bivalves.  Yet  there  is  no  agreement  on  the  number  of  valid  species 
and  specific  relations  between  particular  cercariae,  metacercariae  and  sexually 
mature  worms  remain  undetermined.  As  yet  there  are  no  precise  data  concerning 
host  specificity,  i.e.,  the  ability  of  one  species  to  infect  different  primary,  secondary, 
and  definitive  hosts.  Furthermore,  information  concerning  organ  specificity  is 
equally  meager,  and  it  is  uncertain  whether  or  not  members  of  a  given  species  can 
persist  in  more  than  one  location  in  or  on  a  given  host.  Comparison  of  specimens 
from  different  hosts  and  different  locations  does  not  provide  satisfactory  answers; 

4  Holotype  and  paratype  deposited  in  U.  S.  Nat.  Mus.,  Helminth.  Coll.    No.  56235. 


300  H.  W.  STUNKARD  AND  J.  R.  UZMANN 

the  selected  site  in  each  instance  may  be  determined  by  physiological  adjustments. 
The  extent  of  morphological  variation  that  may  result  from  development  in  different 
hosts  or  different  locations  is  quite  unknown.  Only  when  life-cycles  have  been 
discovered  and  controlled  experiments  permit  tests  on  host  and  organ  specificity, 
will  it  be  possible  to  determine  the  extent  of  variation  in  individual  species  and  the 
validity  of  the  several  named  adult  and  larval  forms. 

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THE  REPETITION  OF  PATTERN  IN  THE  RESPIRATION 

OF  UCA  PUGNAX 

H.   MARGUERITE  WEBB   AND   FRANK   A.   BROWN,   JR.1 

Department  of  Biological  Sciences,  Gonchcr  College,   Toivson  4,  Md.; 

Department  of  Biological  Sciences,  Northzvcstern  University,  Evanston,  Illinois; 

Marine  Biological  Laboratory,   Woods  Hole,  Massachusetts 

Persistent  rhythms  of  O2-consumption  for  two  species  of  fiddler  crabs,  Uca 
pugna.v  and  Uca  pugilator,  were  described  by  Brown,  Bennett  and  Webb  in  1954. 
Analyses  of  the  data  revealed  rhythms  of  several  different  periods  including  diur- 
nal, semi-lunar  and  lunar  ones.  Of  particular  interest,  from  the  point  of  view  of 
the  mechanism  by  which  biological  rhythms  are  maintained,  is  the  observation  that 
two  rhythms  of  such  similar  periods  as  24.0  hours  (diurnal)  and  24.8  hours  (lunar- 
day)  persist  in  an  organism  under  constant  conditions.  The  present  investigation 
has  been  carried  out  in  an  attempt  to  characterize  these  two  rhythms  in  terms  of 
the  regularity  of  period  and  of  form,  and  to  investigate  the  persistence  of  the  lunar- 
day  rhythm  under  conditions  in  which  the  ordinary  tidal  effects  were  absent  from 
the  environment. 

MATERIALS  AND  METHODS 

All  of  the  animals  used  in  these  experiments  were  specimens  of  Uca  pugna.v 
collected  at  Chappoquoit  Beach,  Cape  Cod,  Mass.  The  animals  were  transported 
to  the  Marine  Biological  Laboratory  in  open  containers  and  were  kept  there  in 
enamel  pans  with  a  small  amount  of  sea  water. 

Oo-consumption  was  measured  by  means  of  Brown  respirometers  (Brown, 
1954),  modified  as  described  by  Brown  (1957).  Four  respirometer  vessels  were 
attached  to  a  recording  unit  and  the  whole  assembly  placed  in  a  sealed  barostat. 
The  barostat  was  evacuated  to  a  pressure  of  28.5  inches  of  mercury,  which  was 
somewhat  below  the  expected  minimum  barometric  pressure.  A  maximum  of 
six  such  units  was  in  operation  at  any  one  time.  The  barostats  were  opened  at 
approximately  three-day  intervals  at  which  time  the  ammonia  and  CO2  absorbents 
were  changed,  the  oxygen  supply  was  replenished,  and  fresh  animals  were  placed 
in  the  vessels.  The  barostats  themselves  were  contained  in  water  baths  kept  at  a 
constant  temperature  of  24°  C.  and  they  were  located  in  a  room  without  windows 
and  provided  with  constant  illumination  such  that  the  illumination  within  the  baro- 
stats was  less  than  one  foot-candle.  The  lever  system  of  the  recording  units  was 
such  that  the  recording  arms  were  displaced  1.2  mm.  for  each  gram  of  weight 
increase  of  the  respirometer  vessels.  The  ink-writing  pen  of  the  recorder  traced 
on  millimeter  graph  paper  which  was  marked  off  in  hours  after  being  removed 
from  the  drum.  The  displacement  values  for  each  hour  were  then  recorded. 

The  methods  by  which  the  data  so  obtained  were  analyzed  are  described  in  the 
following  section. 

1  These  studies  were  aided  by  a  contract  between  the  Office  of  Naval  Research,  Department 
of  the  Navy,  and  Northwestern  University,  NONR-122803. 

303 


304 


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


RESULTS  AND  ANALYSES 

Hourly  rates  of  oxygen  consumption,  when  calculated  as  mean  values  for 
single  days,  reveal  a  range  for  the  summer  of  1957  of  from  28  to  69  ml./kg./hr. 
The  mean  rate  of  oxygen  consumption  for  the  first  lunar  period  of  1956  was  found 
to  be  32.4  ±  8.4  ml./kg./hr. ;  for  the  second  lunar  period  it  was  36.0  ±  6.9  ml. /kg./ 


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6  12  18 

TIME  OF  SOLAR   DAY 


24 


FIGURE  1.  The  mean  diurnal  variation  in  Os-consumption  of  Uca  pu//na.r  for  three  29-day 
periods,  (A)  July  15  to  Aug.  13,  1955,  (B)  July  13  to  Aug.  11,  1956^  and  (C)  July  14  to 
Aug.  12,  1957. 


hr.     In  the  summer  of  1957  the  comparable  values  were  41.9  ±  9.4  ml./kg./hr.  and 
44.6  ±  11.9  ml./kg./hr. 

In  Figure  1  are  seen  the  average  diurnal  curves  for  representative  periods  in 
three  successive  years.  Each  curve  represents  the  mean  hourly  values  ( expressed 
as  deviations  from  the  mean)  for  a  period  of  29  days.  Figure  1A  represents  data 


PATTERNS  IN  RESPIRATION 


305 


from  two  recording  units  for  the  period  July  15  to  August  13,  1955.  Figure  IB 
shows  similar  values  from  five  recorders  for  the  period  July  13  to  August  11, 
1956.  Figure  1C  shows  the  mean  daily  curve  for  the  period  July  14  to  August 
12,  1957.  In  this  year  six  recorders  were  used  with  a  minimum  of  two  on  any 
one  day. 


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12  18 

TIME  OF    LUNAR    DAY 


24 


FIGURE  2.  The  primary  lunar  rhythm  in  O2-consumption  of  Uca  pugnax  for  three  29-day 
periods.  (A)  July  15  to  Aug.  13,  1955,  (B)  July  13  to  Aug.  11,  1956,  and  (C)  July  14  to 
Aug.  12,  1957. 

In  all  three  cases  the  diurnal  curve  is  characterized  by  a  single  maximum 
between  2  AM  and  6  AM,  then  a  broad  minimum  extending  from  about  noon  to 
7  PM,  after  which  there  is  an  increase  in  rate  that  continues  until  midnight.  In 
1956  (Fig.  IB)  the  amplitude  is  greater  than  in  the  other  two  years,  but  the  form 
and  phase  relations  appear  to  be  essentially  the  same  in  all  three.  The  amplitude 


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

of  the  mean  diurnal  rhythm  can  be  described  by  the  ratio  of  maximum  to  minimum 
value.  Expressed  in  these  terms,  the  mean  amplitude  obtained  in  1956  was  1.4, 
while  that  for  1957  was  1.2. 

In  Figure  2  are  shown  the  primary  lunar  curves  for  the  periods  represented  in 
Figure  1.  The  curves  in  Figure  2  were  obtained  by  rendering  random  the  diurnal 
variations  while  events  of  a  lunar  frequency  were  kept  constant  (see  Brown,  Ben- 
nett and  Webb,  1954).  The  points  are  plotted  in  such  a  way  that  lunar  zenith 
is  at  12  hours  and  lunar  nadir  is  at  24  hours.  Figure  2A  represents  the  data  from 
1955,  Figure  2B  those  from  1956,  and  Figure  2C  those  from  1957.  The  similarity 
among  the  primary  lunar  curves  for  these  three  years  is  even  more  striking  than 
that  exhibited  by  the  diurnal  curves,  since  the  likeness  now  includes  amplitude  as 
well  as  form  and  phase  relationships.  The  ratio  of  maximum  to  minimum  for  the 
lunar  rhythm  remains  at  about  1.4  in  all  three  years. 

All  of  the  curves  in  Figure  2  show  two  maxima  ancl  two  minima.  The  peak 
rates  of  oxygen  consumption  are  seen  to  occur  at  approximately  lunar  zenith  and 
lunar  nadir.  Both  maxima  are  about  the  same  height  and  there  is  similarly  little 
difference  between  the  two  minima  in  a  lunar  day.  Since  there  appears  to  be 
little  difference  between  events  occurring  at  the  time  of  lunar  zenith  and  those  at 
lunar  nadir  the  effect  is  of  a  rhythm  with  a  period  of  about  12  hours.  Further, 
since  the  amplitude  of  the  lunar  rhythm  is  at  least  as  great  as  that  of  the  diurnal 
one  (and  for  1957  it  is  considerably  greater),  one  would  expect  that  the  curves 
for  respiration  on  single  days  would  exhibit  the  lunar  component  rather  prominently 
and  that  the  form  of  the  daily  curves  would  tend  to  repeat  at  approximately 
15-day  intervals. 

A  direct  and  elementary  test  for  15-day  repetition  of  form  is  possible  from  the 
data  presented  in  Figure  3.  In  this  figure  each  point  represents  the  average  of 
all  machines  recording  on  the  particular  day.  The  ordinate  values  are  the  dis- 
placement in  mm.  of  the  recording  levers.  The  number  of  measurements  con- 
tributing to  each  point  ranges  from  three  to  six.  All  of  the  data  are  from  the 
summer  of  1957  and  the  days  represented  are  as  follows :  Curve  A,  for  June  24, 
is  the  third  day  before  new  moon;  Curve  B,  for  July  2.  the  fifth  day  after  new 
moon;  Curve  C,  July  8,  the  third  day  before  full  moon;  Curve  D,  July  16,  the  fifth 
day  after  full  moon ;  Curve  E,  July  23,  the  third  day  before  new  moon ;  Curve  F, 
July  31,  the  fifth  day  after  new  moon;  Curve  G,  August  7,  the  third  day  before 
full  moon;  Curve  H,  August  15,  the  fifth  day  after  full  moon;  Curve  I.  August  22, 
the  third  day  before  new  moon ;  and  Curve  J,  August  30,  the  fifth  day  after  new 
moon.  Thus,  reading  across  the  figure,  Curves  A,  E,  and  I  are  synchronous  with 
respect  to  lunar  period ;  each  represents  the  third  day  before  new  moon.  Curves 
B,  F,  and  J  are  synchronous,  each  representing  the  fifth  day  after  new  moon. 
Curves  C  and  G  both  represent  the  third  day  before  full  moon,  while  Curves  D 
and  H  represent  the  fifth  day  after  full  moon.  If  one  takes  a  semi-lunar  rather 
than  a  lunar  period,  then  alternate  curves  throughout  the  figure  are  synchronous. 
Thus,  Curves  A,  C,  E,  G,  and  I  are  effectively  synchronous  in  semi-lunar  periods. 

Examination  of  Figure  3  shows  that  on  each  of  the  days  represented  there  are 
fluctuations  such  that  maximal  values  represent  two  to  three  times  the  minimal 
values  for  the  day.  Two  maxima  and  two  minima  occur  daily,  dividing  the  day 
roughly  into  quarters.  It  is  also  obvious  that  the  curves  can  readily  be  divided 


PATTERNS  IN  RESPIRATION 


307 


into  two  classes :  those  which  exhibit  a  maximum  in  the  hours  between  6  AM  and 
12  N  and  a  minimum  between  12  N  and  6  PM,  and  those  which  show  a  minimum 
between  6  AM  and  12  N  and  a  maximum  between  12  N  and  6  PM.  Curves  A, 
C,  E,  G,  and  I  fall  into  the  first  category  and  all  of  the  others  into  the  second. 
In  the  period  immediately  preceding  new  moon  (e.g.,  curve  A)  lunar  zenith  occurs 
in  the  late  morning  hours,  while  as  full  moon  approaches  (e.g.,  curve  C)  lunar 
zenith  will  be  in  the  late  evening  hours.  The  data  presented  in  Figure  3  give  no 
evidence  that  there  is  any  consistent  difference  between  a  semi-lunar  period  in- 
cluding new  moon  and  one  including  full  moon.  The  relative  heights  of  respiratory 
maxima  on  any  day  seem  not  to  be  greatly  affected  by  the  time  of  day  at  which 
lunar  zenith  occurs.  These  data  support  the  description  derived  from  the  mean 
lunar  day  curves.  The  times  of  lunar  zenith  and  lunar  nadir  are  clearly  indicated 
in  the  respiratory  data  for  single  days,  and  these  times  are  indicated  by  major 


w 


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z 
o 
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24 


6  12  18 

TIME    OF   SOLAR    DAY 


24 


12 


18 


24 


FIGURE  3.  Variation  in  Oa-consumption  of  Uca  pugnax  on  single  days  during  the  summer 

of   1957.     See  text  for   further  explanation. 

maxima.     The    respiratory    pattern    is    thus    repeated    at    approximately    15-day 
intervals. 

Although  the  major  maxima  and  minima  are,  in  general,  readily  distinguished 
in  Figure  3,  it  is  seen  that  on  many  days  single  points  appear  which  deviate  widely 
from  the  trend  of  the  series  of  points  around  them.  Such  points  may  make  difficult 
the  comparison  of  the  form  of  curves,  especially  on  days  on  which  the  amplitude 
of  fluctuation  is  low.  Another  aspect  of  the  data  that  might  contribute  to  the 
difficulty  of  analysis  of  form  is  the  day-to-day  variation  in  the  level  at  which  O2- 
consumption  occurs.  Such  variation  might  permit  a  single  day  with  high  values 
to  contribute  disproportionately  to  the  form  of  a  mean  curve  for  periods  of  several 
days  taken  together.  The  first  difficulty  can  be  minimized  by  the  use  of  successive, 
overlapping  three-hour  averages  to  obtain  the  hourly  values.  The  effect  of  varia- 


308 


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


tion  in  level  of  O2-consumption  can  be  reduced  by  using  the  ratio  of  hourly  values 
to  mean  value  for  the  day. 

Evidence  that  the  use  of  three-hour  overlapping  averages  preserves  the  form 
and  phase  relations  of  fluctuations  occurring  over  a  period  of  four  or  more  hours, 
while  eliminating  the  irregularities  of  single  hours,  is  presented  in  Figure  4.  In 
this  figure  are  plotted  (A)  the  primary  lunar  curve  for  the  period  July  14  to 
August  12  obtained  by  converting  the  raw  hourly  data  to  the  ratio  of  hourly  value 
to  mean  for  the  day  and  then  rendering  random  the  diurnal  variations  in  the  man- 


LJ 


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6  12  18 

TIME  OF  LUNAR   DAY 


24 


FIGURE  4.  The  primary  lunar  rhythm  in  O^-consumption  of   Uca  f>ncina.r.     Curve   A   presents 
raw  data.  Curve  B  presents  data  smoothed  by  use  of  overlapping  3-hour  averages. 

ner  previously  described.  Curve  B.  Figure  4,  shows  the  primary  lunar  curve 
obtained  by  use  of  overlapping  three-hour  averages  for  hourly  values.  The  data 
are  for  the  same  period  as  those  in  Curve  A.  (Figure  4B  is  the  same  curve  as 
was  seen  in  Figure  2C.) 

As  might  reasonably  have  been  expected.  Curve  B  is  smoother  than  Curve  A— 
that  is,  there  is  some  loss  of  sharpness  of  definition  so  far  as  points  of  transition 
are  concerned.     It  is  quite  clear,  however,  that  if  one  is  concerned  with  the  form 


PATTERNS  IN  RESPIRATION 


309 


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12  18 

TIME  OF   SOLAR   DAY 


24 


FIGURE  5.  Variations  in  O2-consumption  of  Uca  pugnax  for  two  successive  7-day  periods, 
(A)  June  23  to  June  29,  and  (B)  June  30  to  July  6,  1957.  The  ordinate  scale  used  throughout 
the  figure  is  that  indicated  for  the  top  curves. 


310 


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


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B-3 


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B-3 


6  12  18 

TIME    OF     SOLAR    DAY 


24 


FIGURE  6.  Variations  in  pattern  of  Os-consumption  throughout  semi-lunar  period  as  shown 
by  the  recurrence  of  form  on  comparable  days.  The  ordinate  scale  used  throughout  the  figure 
is  that  indicated  for  the  top  curve. 


PATTERNS  IN  RESPIRATION  311 

and  placement  of  major  fluctuations  occurring  over  periods  of  several  hours  Curve 
B  is  adequate.  If  one  is  concerned  with  the  precise  difference  between  adjacent 
hours  Curve  A  would  be  preferable.  Since  we  are  in  the  present  work  interested 
only  in  fluctuations  with  periods  of  12  to  24  hours  we  shall  use  overlapping  three- 
hour  means  in  the  analyses  of  the  data. 

The  day-to-day  changes  in  pattern  associated  with  the  overt  lunar  rhythm  are 
illustrated  in  Figure  5  which  shows  hourly  data  for  successive  single  days.  Each 
point  represents  the  mean  hourly  value  for  all  recorders  operating  on  that  day, 
the  values  being  expressed  as  deviations  from  the  mean  for  that  day. 

In  Figure  5A  are  seen  the  CX-consumption  data  for  the  seven  days  beginning- 
June  23,  1957  (top  curve)  and  ending  June  29,  1957  (lowest  curve).  The  di- 
agonal lines  indicate  progression  of  maxima  and  minima  across  the  day.  It  can 
be  seen  that  both  maximum  and  minimum  have  advanced  6  hours  in  the  7  days 
illustrated.  This  is  a  rate  of  51.4  minutes  per  day.  This  is  to  be  compared  with 
about  50  minutes  per  day,  the  rate  of  lunar  progression.  In  the  period  represented, 
new  moon  occurred  on  the  fifth  day. 

In  Figure  5B  are  seen  data  for  the  seven  days  immediately  following  those  in 
5A,  i.e.,  June  30  to  July  6  inclusive.  It  is  seen  that  only  the  first  four  of  these 
curves  are  clearly  bimodal.  Moreover  in  these  first  four  there  does  not  appear 
the  clear  progression  of  a  peak  through  the  day  that  was  observed  in  the  preceding 
seven  days.  In  Curve  A,  Figure  5B,  two  maxima  are  obvious,  one  at  about  5  AM 
and  the  second  at  2  PM.  Both  maxima  seem  to  have  disappeared  in  the  last  three 
curves  of  the  series.  The  minimum  present  in  Curve  A  can  be  identified  with  one 
present  in  each  of  the  last  three,  as  is  indicated  by  the  diagonal  line.  The  rate  of 
progression  of  this  minimum  is  such  that  in  7  days  it  has  moved  5  hours,  or  about 
43  minutes  per  day.  In  trie  two  7-day  periods  represented  in  Figure  5,  a  single 
minimum  has  progressed  11  hours  in  14  days,  at  the  over-all  rate  of  47.2  minutes 
per  day,  which  is  quite  good  agreement  with  the  average  rate  of  lunar  progression. 

To  demonstrate  the  recurrence  of  characteristic  forms  of  the  daily  pattern  of 
respiration  at  comparable  times  in  a  semi-lunar  period,  Figure  6  has  been  prepared. 
For  convenience  of  description  and  labelling  a  lunar  period  is  divided  into  units 
of  7  or  8  days  each  as  follows :  June  23  to  June  29  is  called  an  A  period,  consists  of 
7  days  of  which  the  fifth  is  the  day  of  new  moon.  The  period  from  June  30 
through  July  6  is  a  B  period  and  is  7  days  long.  From  July  7  to  13  inclusive  is 
again  an  A  period,  is  7  days  long,  and  full  moon  occurs  on  the  fifth  day.  Con- 
tinuing through  the  summer  in  the  same  manner,  A  periods  are  always  7  days 
long  and  new  or  full  moon  occurs  on  the  fifth  day.  The  intervening  days  are  in- 
cluded in  the  B  periods  which  may  be  either  7  or  8  days,  depending  on  the  number 
of  days  available.  In  this  way  any  given  day  of  a  semi-lunar  period  can  be  iden- 
tified by  a  letter  and  a  number.  The  days  so  represented  in  Figure  6  are:  A-l, 
A-4,  A-7,  and  B-3.  Each  group  of  days  synchronous  with  respect  to  semi-lunar 
period  is  indicated  by  two  vertical  lines  connecting  members  of  a  group  at  maxima 
and  minima. 

The  first  group  of  curves  in  Figure  6,  representing  four  A-l  days  during  the 
summer  of  1957,  shows  quite  clearly  the  resemblance  among  A-l  days  even  though 
separated  in  time  by  as  much  as  two  months.  All  of  the  other  groups  also  reveal 
great  internal  similarity.  Moreover,  any  member  of  A-l  is  more  nearly  like  any 
member  of  A-4  than  like  any  member  of  A-7  or  B-3,  regardless  of  the  absolute 


312 


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


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6  12  18  24 

TIME  OF   SOLAR  DAY 


FIGURE  7.  Comparison  of  changes  in  pattern  of  respiration  throughout  mean  semi-lunar 
period  as  obtained  in  1956  (upper  member  of  each  pair)  with  those  obtained  in  1957  (lower 
member  of  each  pair).  The  ordinate  scale  used  throughout  the  figure  is  that  indicated  for 
the  top  curve. 


PATTERNS  IN  RESPIRATION 


313 


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1     2    3   4    5    6    7    8    9    10  II    12  13  14  15    16  17  18  19  20 

DAYS  AFTER   COLLECTION 


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DATE    OF    RECORDING 

FIGURE  8.  Coefficients  of  correlation  of  simultaneously  recorded  values  of  O2-consumption 
for  different  groups  of  Uca  pugnax,  (A)  as  a  function  of  duration  of  stay  in  laboratory,  (B)  as 
a  function  of  date  of  collection,  (A  and  B  for  1957),  and  (C)  as  a  function  of  date  of  collection 
(data  from  1956). 

time  difference  between  the  curves  compared.  The  progression  of  maxima  and 
minima  throughout  the  day  seen  in  Figure  5  for  a  single  semi-lunar  period  is  seen, 
in  Figure  6,  to  have  been  repeated  throughout  the  entire  summer. 

The  striking  similarity  of  the  form  of  curves  representing  comparable  times 


314 


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


of  the  semi-lunar  period  is  further  illustrated  by  a  comparison  of  the  results  ob- 
tained in  different  years.  In  Figure  7  are  plotted  the  mean  curves  for  every 
second  day  of  a  semi-lunar  period.  The  points  were  calculated  as  deviations  from 
daily  means.  The  top  curve  of  each  pair  represents  the  mean  of  all  the  indicated 
days  from  1956,  the  lower  member  of  each  pair  the  mean  of  comparable  days  from 
1957.  With  respect  to  form  of  the  curve  and  the  placement  of  major  maxima  and 
minima  the  curves  for  the  two  years  are  practically  indistinguishable.  It  is  thus 
clear  that  a  characteristic  pattern  of  fluctuations  in  respiratory  rate  is  exhibited  by 
Uca  pugna.r  and  that,  although  the  pattern  varies  in  a  regular  manner  with  the 
semi-lunar  period,  both  the  basic  pattern  and  its  regular  variations  were  almost 
identical  in  the  summers  of  1956  and  1957. 

With  the  establishment  of  the  existence  of  a  regularly  repeating  pattern   of 
respiration  it   became  of  interest  to   investigate  the  extent  of  agreement   within 

TABLE  I 

Comparison  of  simultaneously  recorded  respiratory  values 


Collection 
date 

Coefficients  of  correlation 
Days  after  collection 

1 

3 

4 

5 

7 

8 

15-17 

6/20 

+0.779 

+0.652 

+0.700 

+0.625 

+0.565 

6/26 

+0.825 

+0.849 

+0.666 

+0.226 
±0.12 

7/10 

+0.808 

+0.256 
±0.11 

-0.464 
±0.11 

-0.022 
±0.12 

7/21 

+0.120 
±0.10 

+0.125 
±0.10 

+0.185 
±0.10 

8/3 

+0.600 

+0.726 

+0.490 
±0.06 

+0.545 

+0.618 

8/20 

+0.915 

+0.602 

groups  of  animals  at  any  one  time.  For  this  purpose  correlations  of  simultaneous 
hourly  values  for  Oo-consumption  of  different  groups  of  animals  were  performed. 
The  coefficients  of  correlation  were  calculated  for  limited  periods  of  one  to  three 
days  and  were  calculated  separately  for  data  from  animals  collected  at  different 
times.  This  method  of  analysis  permitted  comparison  of  results  in  terms  of  the 
actual  dates  of  recording,  the  duration  of  time  in  the  laboratory,  and  the  dates  of 
collection.  The  resulting  comparisons  are  presented  in  Figure  8  and  in  Table  I. 

In  Figure  8A  the  coefficients  of  correlation  for  all  of  the  data  obtained  in  the 
summer  of  1957  are  plotted  as  ordinate  values  with  the  number  of  days  in  the 
laboratory  along  the  abscissa.  There  is,  in  general,  a  decrease  in  correlation  with 
time  in  the  laboratory,  such  that  after  eight  days  the  values  approach  a  random 
distribution.  It  is  also  obvious  that  there  is  a  high  degree  of  scattering  of  values 
even  when  the  number  of  days  in  the  laboratory  is  small. 


PATTERNS  IN  RESPIRATION  315 

Figure  8B  shows  the  coefficients  of  correlation  for  simultaneous  hourly  values 
of  O2-consumption  obtained  within  the  first  seven  days  after  collection.  These 
values  are  plotted  as  a  function  of  the  date  on  which  respiration  was  recorded.  In 
this  graph  the  low  values  are  clearly  grouped  in  the  last  two  weeks  of  July  while 
strong  positive  correlations  are  found  during  the  rest  of  the  summer. 

In  Figure  8C  are  plotted  similar  coefficients  of  correlation  calculated  from  the 
data  for  the  summer  of  1956.  During  that  summer  animals  were  collected  weekly 
and  none  were  used  after  being  in  the  laboratory  for  seven  days.  Here,  too,  there 
is  a  drop  in  the  value  of  the  coefficients  of  correlation  during  the  same  period  for 
which  it  was  found  in  1957.  During  1956  there  was,  in  addition,  a  reduction  in 
correlation  in  the  latter  part  of  August.  Unfortunately  there  are  insufficient  data 
for  these  dates  in  1957  to  confirm  or  deny  the  existence  of  a  similar  reduction 
in  that  year. 

TABLE  II 
Correlation  of  hourly  values  for  successive  semi-lunar  periods 

Coefficients  of  correlation 
Day  of  semi-lunar  period  1956  1957 

A-l  +0.232±0.10  +0.550±0.08 

A-2  +0.618±0.06  +0.485±0.08 

A-3  +0.568±0.07  +0.667±0.05 

A-4  +0.075±0.14  +0.530±0.07 

A-5  +0.455±0.09  +0.040±0.10 

A-6  +0.610±0.06  +0.402±0.08 

A-7  +0.150±0.12  +0.428±0.09 

B-l  +0.518±0.08  +0.430±0.09 

B-2  +0.390±0.12  +0.502±0.09 

B-3  +0.660±0.06  +0.360±0.09 

B-4  +0.230±0.11  +0.610±0.06 

B-5  +0.440±0.08 

B-6  +0.100±0.11  +0.020±0.10 

B-7  +0.440±0.09  +0.430±0.08 

B-8  +0.440±0.12  +0.740±0.06 

In  Table  I  are  recorded  values  for  the  coefficients  of  correlation  from  each 
collection  made  during  1957.  The  coefficients  are  listed  as  obtained  for  various 
short  intervals  after  collection  up  to  eight  days  and  for  the  fifteenth  and  seventeenth 
days  after  collection.  A  comparison  of  the  values  for  the  third  day  after  collection 
shows  that  the  only  group  of  animals  not  showing  a  strong  positive  correlation, 
highly  significantly  different  from  zero,  was  the  one  collected  on  July  21.  It  is  also 
of  considerable  interest  that  the  group  collected  on  July  10,  although  showing  the 
usual  strong  positive  correlation  on  the  third  day,  no  longer  shows  a  significant 
degree  of  correlation  by  the  seventh  day  and  actually  shows  a  significant  negative 
correlation  on  the  eighth  day.  This  is  in  marked  contrast  with  the  collections  of 
June  20  and  August  3.  In  this  latter  case  a  strong  positive  correlation  was  still 
found  at  seventeen  days  after  collection.  It  appears,  then,  that  considerable  varia- 
tion in  both  the  initial  coefficient  of  correlation  and  the  rate  of  decrease  of  the 
coefficient  is  found  among  the  various  groups  of  animals.  The  variations  observed 
are  not  obviously  related  either  to  phase  of  moon  or  to  treatment  related  to  main- 
tenance in  the  laboratory.  The  only  factor  that  seems  to  give  any  system  to  these 
variations  is  the  date  of  recording  respiration. 


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

Coefficients  of  correlation  were  also  calculated  for  the  hourly  values  on  one  day 
of  a  semi-lunar  period  with  the  hourly  values  for  each  of  the  comparable  days  of 
successive  semi-lunar  periods  throughout  the  summer.  These  provide  a  measure 
of  the  similarity  between  comparable  days  in  successive  semi-lunar  periods  for  five 
such  periods  for  the  summer  of  1957.  These  values  are  presented  in  Table  II 
from  which  it  is  seen  that  for  thirteen  of  the  fifteen  days,  coefficients  ranging  from 
0.358  to  0.740  were  obtained.  For  the  other  two  days  the  coefficients  of  correlation 
were  found  to  be  not  significantly  different  from  zero.  The  two  days  for  which 
significant  positive  correlations  did  not  obtain  were  the  day  of  new  or  full  moon 
(day  A-5)  and  the  eighth  day  after  new  or  full  moon  (day  B-6).  Similar  co- 
efficients of  correlation  were  calculated  from  the  data  for  1956  and  are  included  in 
Table  II.  It  will  be  observed  that  of  the  days  for  which  sufficient  data  were  avail- 
able to  permit  making  the  correlations,  days  B-6,  A-4,  and  A-7  yielded  coefficients 
not  significantly  different  from  zero. 

DISCUSSION 

Two  apparently  conflicting  characteristics  of  the  behavior  of  Uca  pugna.v 
emerge  from  the  results  reported  in  this  paper.  We  find  that  when  population 
samples,  ranging  in  size  from  8  to  24  animals,  are  compared  at  15-,  30-,  and  45-day 
intervals  the  pattern  of  respiration  is  being  reproduced  almost  identically  every  15 
days.  When  data  from  somewhat  larger  numbers  of  animals  are  compared  from 
year  to  year  the  same  precise  reproduction  of  pattern  is  observed.  However,  when 
a  group  of  four  animals  is  compared  with  two  to  five  other  groups  of  four  animals 
at  the  same  time,  as  was  done  by  the  correlation  of  simultaneous  hourly  values, 
extreme  variability  is  found.  It  should  be  emphasized  that  there  are  at  least  three 
possible  situations,  all  of  which  would  result  in  a  lack  of  good  correlation.  One 
possibility,  of  course,  is  that  there  are  in  fact  only  negligible  or  random  fluctuations 
in  the  respiratory  rate  of  all  animals.  This  possibility  can  almost  certainly  be  ex- 
cluded. In  the  latter  part  of  July  when  conspicuously  low  coefficients  of  correla- 
tion were  obtained,  the  mean  daily  curves  were  of  normal  amplitude  and  phase 
relations.  An  example  of  this  can  be  seen  in  Figure  3,  curve  E,  the  points  of  which 
represent  the  mean  hourly  values  for  July  23. 

A  second  situation  that  would  lead  to  poor  correlation  among  simultaneous 
hourly  values  would  occur  if  one  or  two  machines  were  recording  the  normal  rhyth- 
mic pattern  while  three  or  four  were  producing  non-rhythmic  fluctuations  or  no 
significant  fluctuations. 

The  third  possibility  leading  to  lack  of  correlation  is  that  different  samples, 
while  still  rhythmic,  have  become  out  of  phase.  Examination  of  the  individual 
records  revealed  one  two-day  period  (July  18-19)  when  two  recorders  showed 
typical  high-amplitude  fluctuations  but  with  one  recorder  almost  precisely  in  oppo- 
site phase  to  that  of  the  other.  This  situation  is  reflected  in  the  large  negative 
correlation  (—0.464)  recorded  in  Table  I  for  the  collection  of  July  10.  In  no 
other  case  was  a  situation  of  this  type  obvious.  However,  it  is  recognized  that  if 
the  individuals  making  up  a  sample  of  four  were  out  of  phase  with  each  other  the 
record  would  be  indistinguishable  from  that  produced  by  four  non-rhythmic  indi- 
viduals. The  multiple  peaks  evident  in  Figure  5B,  Curves  D  and  E,  may  indicate 
such  a  lack  of  synchrony  among  individuals  within  samples. 


PATTERNS  IN  RESPIRATION  317 

Even  though  there  is  no  way  of  distinguishing  between  a  loss  of  rhythm  by  part 
of  the  population  and  a  loss  of  synchrony,  the  fact  remains  that  a  sufficiently  great 
proportion  of  the  population  retains  a  rhythm  in  normal  phase  relations  to  impart 
to  the  mean  daily  curves  a  striking  regularity.  The  question  of  the  basis  for  this 
regularity  represents  one  of  the  fundamental  problems  in  biological  rhythms. 

For  the  study  of  this  basic  problem  there  are  certain  distinct  advantages  to  be 
gained  by  examining  rhythms  with  periods  other  than  twenty-four  hours.  If  the 
rhythm  being  studied  is  clearly  of  semi-lunar  (or  tidal)  frequency  there  can  be  no 
question  of  induction  by  fluctuations  in  environmental  factors  associated  with 
solar  day-night.  There  is  similarly  little  probability  that  the  normal  activity  in  the 
laboratory  where  the  animals  are  kept  will  exhibit  tidal-frequency  fluctuations.  We 
are  faced  then  with  the  situation  that  a  predominant  part  of  the  population  can 
maintain  the  rhythm  of  CX-consumption  with  almost  absolute  precision  for  as  long 
as  16  days  away  from  the  ordinary  tidal  influences.  This  occurred  while  the  ani- 
mals simultaneously  exhibited  a  diurnal  rhythm  of  O, -consumption.  Either  one 
must  attribute  a  remarkable  precision  to  a  biological  system  in  a  situation  not 
constant  but  quite  different  from  the  normal  habitat  of  the  animals,  or  one  must 
invoke  environmental  factors  not  universally  accepted  as  constituting  stimuli  for 
the  organisms  concerned.  The  first  alternative  implies,  on  the  part  of  the  organism, 
a  degree  of  detachment  from  the  environment  that  is  not  entirely  acceptable  to 
modern  biological  thought.  The  second  alternative  permits  an  acceptable  degree 
of  dependence  upon  the  environment  but  requires  the  recognition  of  hitherto  un- 
suspected stimulating  factors. 

SUMMARY 

1.  The  form  of  the  mean  diurnal  rhythm  of  Oo-consumption  of  Uca  pugna.v  is 
described  and  found  to  be  practically  identical  for  the   summers  of   1955,    1956, 
and  1957. 

2.  The  mean  lunar-day  rhythm  of  Oo-consumption  is  described  for  the  summers 
of  the  same  three  years.     The  curve  for  any  one  of  these  years  is  indistinguishable 
from  that  for  either  of  the  other  two  years. 

3.  The  mean  lunar-day  rhythm  consists  of  two  maxima,  of  equal  magnitude, 
occurring  approximately  at  lunar  zenith  and  at  lunar  nadir ;  between  the  maxima 
are  two  minima  symmetrical  with  respect  to  time  of  occurrence  and  magnitude. 

4.  For  the  primary  lunar  rhythm  the  ratio  of  maximum  to  minimum  is  about 
1.4;  for  the  diurnal  rhythm  the  ratio  of  maximum  to  minimum  is  1.2  in  1955,  1.4 
in  1956,  and  1.2  in  1957. 

5.  Because  of  the  amplitude  of  the  lunar  component  of  the  rhythm,  the  data  for 
single  days  reveal  clearly  the  progression  of  lunar  maxima  and  minima. 

6.  Because  of  the  equality  in  amplitude  of  fluctuations  correlated  in  time  with 
lunar  zenith  and  with  lunar  nadir,  the  overt  rhythm  is  one  with  a  period  of  12.4 
hours.     There  is  a  pattern  of  fluctuations  characteristic  of  each  day  in  a  semi-lunar 
period. 

7.  The  reproducibility  of  the  daily  pattern  in  successive  semi-lunar  periods,  and 
in  successive  years,  is  demonstrated. 

8.  In  general  a  strongly  positive  correlation   is   found   between   simultaneous 
hourly  values  for  different  groups  of  animals  during  the  first  seven  days  after  col- 


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

lection.     There  is  a  general  decrease  in  the  extent  of  these  correlations  with  time 
in  the  laboratory. 

9.  There  is  strong  evidence  for  a  time-dependent  variable  affecting  the  size  of 
the  coefficients  of  correlation  for  simultaneous  hourly  values  obtained  during  a 
wide  range  of  times  in  the  laboratory. 

10.  Hourly  values  for  single  days  of  a  semi-lunar  period  were  correlated  with 
the  hourly  values  for  comparable  days  of  successive  semi-lunar  periods.     The  co- 
efficients  of  correlation   were   positive   and   significantly   different  from   zero   for 
thirteen  of  the  fifteen  days  of  1957,  the  exceptions  being  the  eighth  day  after  new 
or  full  moon  and  the  day  of  new  or  full  moon  for  which  the  coefficients  were  not 
significantly  different  from  zero.     For  1956  the  exceptions  were  the  day  before 
new  or  full  moon,  and  the  second  and  eighth  days  after ;  the  coefficients  for  these 
three  days  were  not  significantly  different  from  zero.     For  the  other  twelve  days 
the  correlations  were  strongly  positive. 

11.  The  relevance  of  these  findings  to  an  understanding  of  the  phenomenon  of 
biological  rhythmicity  is  discussed. 

LITERATURE  CITED 

BROWN,  F.  A.,  JR.,  1954.     A  simple,  automatic,  continuous-recording  respirometer.     Rev.  Sci. 

Instr.,  25:  415-417. 
BROWN,  F.  A.,  JR.,  1957.     Response  of  a  living  organism,  under  "constant  conditions"  including 

barometric    pressure,    to    a    barometric-pressure-correlated    cyclic,    external    variable. 

Biol.  Bull.,  112:  288-304. 
BROWN,  F.  A.,  JR.,  M.  F.  BENNETT  AND  H.  M.  WEBB,  1954.     Persistent  daily  and  tidal  rhythms 

of  O2-consumption  in  fiddler  crabs.     /.  Cell.  Comp.  Physio!.,  44 :  477-506. 


ABSTRACTS   OF   PAPERS   PRESENTED  AT 
THE  MARINE  BIOLOGICAL  LABORATORY 

1958 

ABSTRACTS  OF  SEMINAR  PAPERS 

JULY  1,  1958 
Complete  reconstitution  from  ectoderm  in  Cordylophora.     EDGAR  ZWILLING. 

When  ectoderm  from  Cordylophora  coenosarc  was  cut  into  a  number  of  small  fragments 
and  allowed  to  fuse  with  an  equal  amount  of  fragmented  endoderm,  reconstitution  was  very 
rapid;  the  tissues  of  both  layers  retained  their  integrity,  quickly  separated  into  two  layers  and 
formed  a  hydranth  within  36  hours.  When  pure  ectoderm  was  isolated  or  left  with  a  small 
trace  of  endoderm  reconstitution  of  an  individual,  complete  with  two  layers,  occurred  in  many 
cases  after  5-7  days.  Characteristically  such  masses  formed  a  small  sphere  whose  wall  was 
composed  of  small  cells  and  whose  center  was  filled  (within  ten  hours)  with  the  debris  from 
disintegrated  cells.  All  of  the  large  cells  (including  the  endoderm  when  it  was  present) 
were  involved  in  this  disintegration.  An  inner  layer  then  formed  slowly  and  was  evident  after 
four  days.  In  order  to  be  certain  of  elimination  of  all  possible  contaminating  endoderm  cells 
a  number  of  masses  of  ectoderm  were  set  up ;  the  spheres  were  opened  after  10  hours  (when 
the  central  mass  had  broken  down),  the  central  mass  was  cleaned  out  and  the  small-celled 
walls  from  several  spheres  were  cut  up  and  allowed  to  fuse  together.  All  five  of  the  "second 
generation"  reconstitution  masses  formed  an  inner  layer  after  5-7  days  and  one  of  them  formed 
a  complete  hydranth.  Without  specifying  the  particular  cell  involved,  this  evidence  reveals 
that  endoderm  may  form  from  ectoderm  in  Cordylophora. 

Neural  and  mesodermal  hierarchies  in  chick  development.  MAXWELL  H.  BRAVERMAN. 

Specific  inhibition:  The  injection  of  one-day-old  chick  eggs  with  extracts  of  adult  organs, 
as  suggested  by  P.  Lenicque,  demonstrates,  within  a  few  days,  the  specific  inhibitory  effect  of 
these  extracts  on  the  corresponding  embryonic  organ  system.  (Details  of  extraction  will  be 
found  elsewhere  in  this  volume.)  Neural  fractions  affect  the  neural  system;  mesodermal  deriva- 
tives their  corresponding  systems.  Eggs  receiving  injections  of  the  normal  saline  carrier  of 
the  extracts  develop  an  insignificant  number  of  specific  defects. 

Cumulative  inhibition:  A  pattern  of  neural  inhibition  can  be  seen  if  the  effects  of  extracts 
of  different  parts  of  the  brain  are  compared.  Fore-brain  extracts  affect  only  fore-brain  forma- 
tion. Extracts  of  whole  brains  inhibit  normal  formation  of  the  entire  brain  structure.  Spinal 
cord  extracts,  in  addition  to  affecting  spinal  cord,  exert  an  inhibitory  influence  on  the  whole 
brain.  Thus  there  is  indicated  a  tendency  for  neural  structures  to  inhibit  not  only  homologous 
tissues  but  also  any  tissue  forming  anteriorly  in  the  neural  system. 

Mesodermal  inhibition:  Tests  made  using  extracts  of  heart,  blood  vessels,  ureter  and  kidney 
show  that  these  extracts  can  inhibit  formation  of  structures  more  dorsal  in  the  mesodermal 
hierarchy  of  Yamada.  Diminution  of  somite  number  was  the  most  frequent  defect ;  however, 
some  animals  injected  with  either  blood  vessel  or  heart  extract  lacked  all  or  almost  all  meso- 
dermal structures. 

Contradiction:  Some  structures,  such  as  neural  ganglia  and  the  infundibulum,  form  normally 
even  when  surrounding  regions  are  severely  inhibited,  suggesting  that  these  receive  their  de- 
velopmental cue  from  outside  the  neural  system.  Unlike  other  brain  parts,  they  do  not  differen- 
tiate as  hierarchical  alternatives  in  a  self-limited  system. 

319 


320         PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

A  feed-back  mechanism  of  growth  control  in  tadpoles.     S.  MERYL  ROSE. 

Several  workers  have  demonstrated  that  the  growth  of  aquatic  organisms  can  be  limited 
by  exhaustion  of  nutrients  and  by  accumulation  of  relatively  specific  products  in  the  medium. 

Five  to  10  Rana  pipiens  tadpoles,  each  weighing  0.5  gram,  growing  in  one  liter  of  water 
previously  conditioned  by  snails,  fish,  plants  and  a  microflora  and  microfauna  add  something 
to  the  water  within  24  hours  which  greatly  reduces  the  growth  rate  of  assay  tadpoles  weighing 
0.1  to  0.2  gram  apiece.  This  material  can  be  removed  by  heating  to  60°  C,  by  freezing  and 
thawing,  by  centrifugation  at  2000  X  g  and  in  other  ways  as  demonstrated  by  Richards. 

Growth  of  R.  pipiens  tadpoles  is  increased  by  culturing  them  with  other  animals :  Tritnrus, 
-Nectunts,  Lebistes  and  Physa.  R.  pipiens  are  inhibited  by  R.  catcsbiana.  Culture  water  from 
starved  R.  pipiens  is  not  inhibitory.  As  the  number, of  tadpoles  cultured  in  12-liter  aquaria  is 
increased  from  14  to  53,  the  average  maximum  weight  decreases  from  5.1  grams  to  4.2  grams. 
However,  the  maximum  weight  of  the  largest  14  is  the  same  whether  there  are  14,  16,  37  or  53. 
The  differences  in  averages  arise  because  in  the  more  crowded  conditions  some  are  greatly 
inhibited.  These,  if  removed  to  uncrowded  conditions,  can  resume  rapid  growth.  When  four 
0.5-gram  tadpoles  are  cultured  with  four  0.1-gram  tadpoles  the  smaller  ones  all  stop  feeding 
and  die  in  approximately  15  days.  The  inhibitory  effects  of  culture  water  from  equal  weights 
of  large,  medium  and  small  tadpoles  tested  on  small  tadpoles  is  proportional  to  size. 

The  indication  is  that  growth  of  tadpoles  in  limited  volumes  of  water  is  controlled  by  a 
feed-back  mechanism  involving  the  production  of  relatively  specific  materials  capable  of  being 
metabolized  away  by  other  organisms. 

JULY  8,  1958 

The  nature  of  chromato graphic  ainylose  and  amylopectin  fractions.     FREDERICK  A. 
BETTELHEIM. 

Fractionation  of  amylose  on  a  double  column  of  aluminum  oxide  of  which  the  upper  part 
is  acidic  (pH  4.5)  and  the  lower  part  is  basic  (pH  7.8)  yields  three  fractions.  Two  of  these 
can  be  obtained :  I,  by  eliminating  the  basic  part  of  the  column ;  II,  by  eluting  the  remainder 
with  acetate  buffer  (pH  5.8).  The  third  fraction  cannot  be  eluted  and  its  properties  might  be 
inferred  only.  Similarly,  three  fractions  can  be  obtained  by  chromatographing  amylopectin. 
Viscosity,  osmotic  pressure  measurements,  light  scattering  of  the  solutions  of  the  fractions  were 
performed,  together  with  enzymatic  digestions  coupled  with  biochemical  analyses.  The  experi- 
mental data  indicate  that  fraction  I  of  the  amylose  is  a  fairly  rigid  rod-like  structure  with  the 
possibility  of  induced  positive  surface  charges  which  accounts  for  its  passage  through  acidic 
adsorbent.  Fractions  II  and  III  of  amylose  are  polymers  of  a  more  flexible  nature  with  in- 
creasing random  type  of  coiling.  Fraction  I  of  the  amylopectin  which  passes  through  acidic 
adsorbent  is  composed  of  compact  spherical  bodies  with  small  percentage  of  outer  branches 
and  large  number  of  branching  points.  Amylopectins  II  and  III  have  an  increasingly  open 
shape  in  that  order,  i.e.,  the  molecular  radius  of  gyration  is  increasing  and  so  does  the  per- 
centage of  outer  branches,  while  the  number  of  branching  points  decreases  in  this  order. 

In  general,  the  shape  of  the  molecules  in  this  type  of  chromatography  has  a  greater  influence 
upon  the  adsorption  characteristics  than  the  molecular  weight. 

Coordination  of  ciliary  motion  and  muscular  contractions  in  the  gills  of  Crassostrea 
z'irginica.     PAUL  S.  GALTSOFF. 

Ciliary  motion  along  the  isolated  plicae  of  the  gills  of  bivalves  continues  for  many  hours 
after  the  severance  of  tissues  from  the  body.  This  fact  leads  to  general  conclusion  that 
ciliary  activity  is  not  under  the  control  of  nerve  ganglia.  Observations  made  by  using  oysters 
with  gills  exposed  by  partial  removal  of  shell,  but  otherwise  intact,  show  that  the  ciliary  motion 
along  the  terminal  groove  of  the  gill  and  of  frontal  cilia  frequently  stops  following  spontaneous 
contraction  of  the  adductor  muscle.  The  cessation  of  ciliary  motion  may  be  general,  involving 
all  demibranchs,  or  may  be  limited  to  a  small  segment  of  one  terminal  groove.  The  cessation 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY         321 

may  be  complete,  lasting  from  a  fraction  of  a  minute  to  almost  an  hour,  or  it  may  continue 
but  at  greatly  slower  rate. 

All  observations  were  made  on  large,  adult  oysters,  partially  or  completely  spawned.  AU 
readings  were  made  with  low  power  binocular.  Oysters  were  kept  under  strong  illumination 
in  frequently  renewed  sea  water.  Rate  of  ciliary  motion  was  measured  by  recording  with  a 
stop-watch  the  time  necessary  for  a  small  particle  of  chalky  shell  substance  to  pass  a  known 
distance  along  the  edge  of  the  demibranch.  Records  were  taken  at  one-minute  intervals  for 
periods  lasting  from  fifteen  to  fifty  minutes. 

Electric  shock  and  pricking  of  the  gill  surface  and  mantle  produced  no  effect  on  ciliary 
rhythm.  Conclusion  is  reached  that  in  an  intact  gill  there  is  direct  connection  between  spon- 
taneous contractions  of  the  adductor  muscle  and  the  inhibition  of  the  ciliary  motion. 


JULY  15,  1958 
Factors  and  genes  in  Mormoniella.     P.  W.  WHITING  AND  SARAH  B.  CASPARI. 

Among  several  different  eye-color  loci,  one,  called  R,  has  two  factors,  O  and  S,  which 
mutate  to  colorless,  oy,  "oyster"  and  to  scarlet,  st,  respectively.  Much  less  frequently  these  two 
factors  may  mutate  to  intermediate  colors.  Rarely  mutations  occur  in  two  further  factors,  M 
and  N,  giving  dark  red  eyes.  All  mutant  colors  are  recessive  to  wild-type  brown.  The  mutant 
genes  are  designated  by  the  color  with  a  number  or  with  the  initials  of  the  finder.  The  factors 
in  the  gene  formulae  are  arranged  arbitrarily  in  order  O-S-M-N  with  symbols  indicating  the 
color.  Thus  gene  o^'-DR  is  oy+,  st-DR  is  +'st,  mahogany-605  is  +-mh  and  dahlia-846  is 
H — \--da.  These  are  unifactorial  genes  with  one-factor  difference  from  wild  type.  Oy-NH 
oyst,  st-426,  da-st,  oj-848,  oyinh  and  orange-806,  +-st-tn!i,  are  bifactorial  and  peach-333.5, 
pc -st- -\--nih,  and  rfa-838,  rdh-  +  •  da •  rdh  (rdh  =  reddish)  are  trifactorial.  Compound  females 
are  wild  type  if  the  mutant  factors  of  each  gene  have  their  wild-type  alternatives  in  the  allelic 
gene,  as  ofa-846//u'-333.5  (H — \--da-+/pe-st-  +  -mh) ,  but  they  are  mutant  type  if  the  same  factor 
is  mutant  in  both  alleles,  as  c/a-GF/tinged-277  (da-+/ti-st).  The  states  or  conditions  of  the 
different  factors  form  subseries  of  alternatives  within  the  series  of  alleles. 

Mutation  of  factors  essential  to  fertility  and  viability  has  occurred  in  some  eye-color  genes. 
Thus,  scarlet  female  st-689/oy-423  (  +  -st-fsa/oyst-fsa)  and  wild-type  female  da-83S/st-689 
(-) — | — \--da-la/+fst'-\ — \--fsa)  are  sterile  having  an  impairment,  fsa,  of  factor  A  which  is 
dominant  over  lethal-a,  la.  However,  scarlet  female  ^-841/0^-423  (+'St'  +  'fsb/oyst-fsa-+)  is 
fertile  heterozygous  for  female  sterility  in  different  factors,  A  and  B.  The  stock  with  these  last 
two  genes  is  balanced  with  fertile  males,  sterile  homozygous  females.  (Work  done  under  U.  S. 
Atomic  Energy  Commission  contract  AT  (30-1) -1471.) 

Tail  regeneration  in  lengthened  and  shortened  earthworms.     SEARS  CROWELL. 

These  experiments  were  designed  to  determine  whether  in  the  earthworm,  Eisenia  foetida, 
the  number  of  segments  which  regenerate  following  amputation  is  controlled  by  the  total  number 
of  segments  remaining  (or  some  other  feature  of  the  worm-as-a-whole)  or  by  some  local  con- 
dition at  the  place  of  amputation.  The  plan  of  the  experiment  was  to  cut  two  earthworms,  one 
at  segment  50,  the  other  at  segment  70.  The  longer  anterior  end  of  one  was  sutured  to  the 
longer  posterior  end  of  the  other;  and  the  shorter  ends  were  similarly  joined.  This  gave  two 
worms,  one  abnormally  long  by  20  segments,  the  other  short  by  20  segments.  Ten  to  15  seg- 
ments were  later  removed  from  the  posterior  end  and  the  subsequent  regeneration  observed. 
After  no  further  segments  were  being  added  the  same  worm  was  tested  again  by  removing  the 
regenerate  plus  a  few  of  the  old  segments.  The  operations  themselves  were  performed  by 
Mr.  Reisberg  and  Mr.  Buell. 

To  date  we  have  19  cases  of  regeneration  by  shortened  worms  and  7  by  lengthened  worms. 
In  both  shortened  and  lengthened  worms  the  number  of  segments  which  regenerated  was  a 
few  less  than  the  number  removed.  In  25  of  the  26  cases  the  length  of  the  regenerate  conformed 
to  the  expectation  based  on  the  hypothesis  that  the  local  level  of  the  amputation  determines  the 
amount  of  regeneration. 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

Motion  pictures  of  some  changes  in  cells  induced  by  x-ray  treatments  of  tadpoles 
and  tetrahymenae.1     CARL  CASKEY  SPEIDEL. 

Modifications  of  cell  division  in  tadpoles  were  induced  by  single  whole-body  x-ray  treat- 
ments. In  the  regenerating  tail  tip  of  the  tadpole  a  few  days  after  suitable  irradiation  (5-10  kr), 
many  cells  reached  the  mitotic  stage.  Some  completed  their  division  with  subsequent  differ- 
entiation of  the  daughter  cells.  Others  failed  to  complete  their  division,  the  chromatin  often 
remaining  in  the  prophase  stage.  Degenerative  changes  followed.  In  one  case  a  dividing 
myoblast  suddenly  ceased  its  activity  shortly  after  the  metaphase  stage. 

The  protozoan  ciliate  Tctrahymcna  corlissi,  a  facultative  parasite  of  tadpoles,  was  very 
resistant  to  irradiation.  Some  individuals  survived t  single  treatments  of  400-700  kr.  The 
survivors  exhibited  conspicuous  temporary  effects,  such  as  sluggishness,  change  to  a  spheroidal 
shape,  and  decrease  in  feeding  activity  and  in  rate  of  reproduction.  Rapid  recovery  ensued. 
Repeated  treatments  caused  permanent  loss  of  the  micronucleus,  a  structure  rich  in  deoxyribo- 
nucleic  acid. 

Suitable  irradiation  of  tadpole-tetrahymena  combinations  favored  tetrahymenal  invasion. 
While  the  radio-sick  tadpoles  became  progressively  weaker  after  the  treatment,  the  more  radio- 
resistant  tetrahymenae  were  not  noticeably  affected.  They  multiplied  and  thrived  at  the  expense 
of  the  host  tadpole  tissues. 

Radiation-induced  strains  of  amicronucleate  tetrahymenae,  even  after  they  had  received 
cumulative  doses  totaling  several  million  roentgens,  were  still  able  to  invade  and  parasitize 
weakened  tadpoles.  They  seemed  somewhat  less  vigorous  in  their  attack  on  the  tadpoles,  how- 
ever, as  compared  with  unirradiated  normal  micronucleate  tetrahymenae. 

JULY  22,  1958 

The  uptake  of  radiosnlplinr  during  tJic  in  vitro  induction  of  cartilage."     JAMES  W. 
LASH  3  AND  HOWARD  HOLTZER. 

It  has  been  shown  previously  that  the  formation  of  embryonic  vertebral  cartilage  is  de- 
pendent upon  the  presence  of  the  embryonic  spinal  cord  or  notochord,  in  both  in  vivo  and  in 
vitro  development.  If,  in  tissue  culture,  embryonic  spinal  cord  is  placed  on  one  side  of  a  milli- 
pore  filter  with  somites  on  the  other  side,  cartilage  forms  in  the  somites  on  the  fourth  day  of 
culture.  If  the  spinal  cord  is  removed  from  the  filter  after  12  hours  of  culture,  the  somites 
will  still  form  cartilage.  This  demonstrates  that  the  spinal  cord  factor  has  passed  through  the 
filter  and  acted  upon  the  somites  within  the  first  12  hours  of  culture,  even  though  cartilage 
does  not  appear  for  another  three  days.  If  chemical  differentiation  precedes  morphological 
differentiation  during  chondrogenesis,  chemical  evidence  of  chondroitin  sulphate  might  occur 
any  time  after  induction  prior  to  the  appearance  of  cartilage.  In  order  to  detect  the  appearance 
of  chondroitin  sulphate  in  minute  quantities,  radioactive  sulphur  (Na235SO4)  was  added  to 
notochord-somite  cultures  and  the  cultures  were  analyzed  for  the  appearance  of  radioactive 
chondroitin  sulphate  at  intervals  during  the  culture  period  of  6  days.  The  chondroitin  sulphate- 
protein  complex  was  extracted  with  the  method  of  Einbinder  and  Schubert  and  the  activity  of 
incorporated  35S  was  determined.  Chondroitin  sulphate  was  found  only  when  cartilage  was 
visible  in  culture.  Within  the  limits  of  detection  thus  employed,  the  appearance  of  chondroitin 
sulphate  and  of  morphological  cartilage  are  concomitant  in  occurrence. 

Mode  of  action  of  choline  esters.     Substrate  specificity  of  their  "receptor-protein." 
MENACHEM  WURZEL. 

A  series  of  choline  esters  applied  to  a  variety  of  responsive  tissues  (striated  and  smooth 
muscle,  heart,  salivary  gland,  blood  pressure)  demonstrated  a  potency  relative  to  acetylcholine 

1  This  investigation  was  supported  by  a  research  grant  (PHS  RG-4326  C)  from  the  Na- 
tional Institutes  of  Health,  Public  Health  Service. 

-  This  research  was  supported  in  part  by  a  grant  to  Dr.  H.  Holtzer  from  the  National 
Institutes  of  Health,  Public  Health  Service  (B-493-C4). 

3  Fellow  of  the  Lalor  Foundation. 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY         323 

(ACh),  that  followed  one  of  two  orders.  (1)  Butyrylcholine  (ButCh)  >  propionylcholine 
(PropCh)  >  ACh  >  benzoylcholine  (BenzCh)  >  acetyl-b-methylcholine  (MeCh).  This  is  also 
the  order  of  their  relative  rates  of  hydrolysis  by  pseudocholinesterase  (pseudoChE)  taken  at 
pSopt..  (2)  ACh  >  MeCh  >  PropCh  >  ButCh  >  . . .  >  >  BenzCh  and  this  is  the  order  of  hy- 
drolysis rates  with  true  cholinesterase  (true  ChE)  at  pSopt.,  except  MeCh  and  PropCh  being 
interchanged.  We  called  the  first  order  of  activity  "pseudo  ChE  pattern,"  the  second  "true 
ChE  pattern."  We  never  met  a  different  order  of  potency,  although  MeCh  in  a  true  ChE- 
patterned  uneserinised  organ  may  sometimes  be  more  potent  than  ACh,  but  not  if  the  organ 
was  eserinised. 

The  above  patterns  could  be  simply  accounted  for  if  the  requirement  for  biological  equi- 
potency  would  be  a  given  constant  reaction  velocity  of  an  enzymatic  reaction  v  =  k.E.S  catalysed 
by  pseudo  ChE  or  true  ChE.  This  means  that  if  the  affinity  of  that  enzyme  for  one  particular 
substrate  is  small,  then  a  higher  S  substrate  concentration  is  needed  to  give  the  same  v  —  const, 
necessary  to  one  given  effect.  A  common  end-product  of  this  ester  hydrolysis  is  H+.  This 
way  equipotent  choline  ester  concentrations  would  be  those  producing  a  given  amount  of  H+ 
per  unit  of  time. 

To  make  legitimate  comparison  between  the  enzymatic  rates  of  hydrolysis  and  biological 
potencies,  we  determined  enzymatic  hydrolysis  curves  at  low  substrate  concentrations  down 
to  10~8  M,  in  the  range  of  biological  effects.  Suitable  reaction  mixtures  of  enzymes  plus  sub- 
strates were  made  up  and  samples  taken  at  time  intervals  measured  on  a  calibrated  frog 
rectus  muscle,  guinea  pig  intestine  or  other  sensitive  organs.  Curves  obtained  in  this  way  for 
human  serum  pseudo  ChE,  Torpedo  true  ChE,  eel  true  ChE  were  presented.  It  was  seen  that 
the  order  of  concentrations  giving  the  same  H+  production  rate  gave  the  orders  of  their  bio- 
logical potency.  The  interchanged  order  of  PropCh-MeCh  in  true  ChE  patterned  organs  was 
discussed  and  this  relatively  minor  divergence  attributed  to  molecular  structure  differences 
which  act  on  some  factor  independent  of  the  enzyme. 

JULY  29,  1958 

Ecological  isolation  and  independent  speciation   of   the  alternate  generations   of 
plants.     ALBERT  J.  BERNATOWICZ. 

Hutchins  (1947)  theorized  that,  since  temperature  affects  both  reproduction  and  survival, 
at  least  four  combinations  of  limiting  temperatures  must  be  considered  in  comparing  the  north- 
south  distribution  of  species.  From  the  viewpoint  of  plant  biology  two  complications  arise : 
1 )  vegetative  reproduction  is  so  common  that  re-population  may  be  possible  wherever  survival 
of  individuals  is  possible,  even  if  critical  temperatures  for  sexual  or  sporic  reproduction  do  not 
occur  in  some  parts  of  the  range;  2)  it  is  possible  that  gametophyte  and  sporophyte  generations 
of  a  species  may  have  different  temperature  tolerances.  The  first  possibility  implies  that  some 
plant  species  which  are  basically  of  Hutchins'  zonal  types  2,  3,  or  4  will  become  established 
only  as  gametophytes  or  as  sporophytes  at  one  or  both  ends  of  their  ranges,  since  vegetative 
reproduction  usually  does  not  produce  the  alternate  phase.  Likewise,  the  second  possibility 
suggests  that  only  one  phase  of  a  species  may  persist  in  an  area  if  reproduction  produces  an 
alternate  generation  which  cannot  survive  the  local  temperatures.  Similar  reasoning  for  salinity 
as  an  isolating  mechanism  can  be  applied  to  those  algae  of  the  Baltic  and  Black  Seas  which 
persist  in  only  one  of  their  phases,  and  photoperiod  or  other  ecological  factors  may  operate  in 
like  manner  for  certain  species.  An  implication  of  this  hypothesis  of  ecologically  isolated 
generations  is  that  genetic  divergence  or  "speciation"  of  the  separated  phases  can  occur  if 
somatic  mutations  accumulate  and  result  in  distinct  clones.  This  would  explain  the  puzzling- 
situation  in  the  algal  genus  Dcrbesia,  of  which  there  are  apparently  more  "species"  of  sporo- 
phytes than  of  gametophytes. 

A  technique  for  the  study  of  the  effects  of  "Iwst-jactor"  on  the  behavior  of  com- 
mensal polychaetcs  and  Crustacea.     DEMOREST  DAVENPORT. 

A  new  technique  is  described  whereby  it  is  possible  to  analyze  the  behavior  of  the  indi- 
vidual commensal  polychaete  or  crustacean  when  influenced  by  "host-factor."  By  this  tech- 


324         PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

nique  one  can  investigate  the  specificity  of  response  of  commensals  and  at  the  same  time  accrue 
enough  quantitative  data  to  demonstrate  the  presence  or  absence  of  chemotaxes,  rheotaxes  and 
kineses. 

The  development  of  scJwoling  behavior  in  the  genus  Menidia.     EVELYN  SHAW. 

Field  and  laboratory  investigations  show  that  schooling  in  Menidia  develops  gradually. 
Schooling  begins  between  two  and  three  weeks  after  hatching  following  a  period  during  which 
a  characteristic  developmental  pattern  of  approach  and  orientation  can  be  observed.  Young 
fish,  5-7  mm.  in  length,  approach  species  mates  closely  and  quickly  part  without  orientation. 
Fish,  8-10  mm.  in  length,  approach  closely,  assume  a-  parallel  orientation  and  swim  together 
for  a  short  distance.  Fish,  12  mm.  in  length  (about  the  seventeenth  day  after  hatching), 
approach,  orient  parallel  to  one  another  and  maintain  a  rather  constant  orientation  while  swim- 
ming for  long  distances  and  long  periods  of  time.  In  this  early  schooling,  the  fish-to-fish 
distance  is,  at  first,  highly  uneven,  ranging  from  5  to  35  mm.  The  members  of  the  school  do 
not  move  at  uniform  speeds  and  they  do  not  always  swim  parallel  to  one  another.  As  the 
members  of  the  school  grow  the  fish-to-fish  distance  becomes  less  variable,  the  swimming  speed 
is  more  uniform  and  orientation  is  more  precisely  parallel.  An  explanation  for  certain  aspects 
of  the  gradual  development  of  this  uniform  and  precise  behavior  may  be  that  some  experience 
is  required  to  perfect  orientation  within  the  school.  In  order  to  evaluate  the  importance  of 
experience,  attempts  were  made  to  rear  fish  in  physical  and  visual  isolation.  Out  of  400  fish, 
only  four  grew  to  12  mm.  in  length.  When  these  four  were  presented  to  a  school  of  fish  of 
the  same  size  they  joined  the  group  immediately.  However,  initially,  they  seemed  unable  to 
maintain  their  position  in  the  school ;  they  occasionally  swam  away  from  the  school  and  they 
often  bumped  into  species  mates.  At  the  end  of  four  hours,  however,  fish  reared  in  isolation 
could  not  be  distinguished  from  those  reared  in  groups.  The  results  with  these  four  fish  agree 
with  the  proposition  that  the  precision  of  orientation  within  the  school  is,  in  part,  a  learned 
phenomenon. 

AUGUST  5,   1958 

Effects  of  sperm  extract  and  oiher  agents  on  the  egg  membranes  in  relation  to  spenn 
entry  in  Hydroides  lie.vagoniis.1     ARTHUR  L.   COLVVIN  AND  LAURA  HUNTER 

COLWIN. 

Electron  microscope  studies  of  thin  sections  show  that  the  outer  covering  of  the  egg, 
sometimes  described  as  a  thick,  refractive  "vitelline  membrane,"  is  in  fact  composed  of  at  least 
three  distinct  entities:  (1)  a  thin  outer  border  layer  which  is  complex  in  structure,  (2)  a 
thick  middle  layer,  constituting  the  major  portion  of  the  membrane,  and  (3)  a  thin  inner  border 
layer  whose  underlying  structure  is  somewhat  similar  to,  but  denser  than  that  of  the  middle 
layer.  The  appearance  of  both  these  layers  differs  greatly  from  that  of  the  outer  border  layer. 

Observations  and  photomicrographs  of  living  material  now  clearly  confirm  that  a  hole 
or  space  remains  in  the  vitelline  membrane  after  the  sperm  head  has  completed  its  passage 
into  the  egg  proper.  Following  previous  electron  microscope  studies  it  was  suggested  (Colwin 
and  Colwin,  1957a)  that  the  spermatozoon  produced  a  lytic  agent  which  could  dissolve  the 
membrane,  thus  forming  these  holes.  It  was  then  found  (Colwin  and  Colwin,  1957b)  that 
extracts  of  frozen-thawed  sperm  could  rapidly  dissolve  material  beneath  the  outer  border 
layer.  Electron  micrographs  of  thin  sections  of  eggs  exposed  to  these  extracts  now  show 
the  extent  of  the  membrane  lytic  action :  only  the  middle  layer  is  dissolved  by  the  extract ; 
the  inner  as  well  as  the  outer  border  layer  appears  to  persist.  The  microvilli  of  the  egg  are  not 
affected  and  are  left  to  project  freely  following  dissolution  of  the  middle  layer  material  which 
normally  surrounds  them. 

Electron  micrographs  show  no  apparent  modification  of  the  components  of  the  egg  mem- 
brane following  fertilization  and  the  membranes  of  fertilized  eggs  were  affected  in  the  same 
way  as  those  of  unfertilized  eggs  when  exposed  to  the  sperm  extract. 

1  Supported  by  a  Grant  (RG-4948)  from  the  National  Institutes  of  Health,  U.  S.  Public 
Health  Service. 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY         325 

Fertilisation  and  agglutination  inhibitors  from  Arbacia.1     CHARLES  B.  METZ. 

In  keeping  with  observations  of  some  others  (e.g.,  E.  B.  Harvey,  1956)  it  is  found  that 
gametes  obtained  from  Arbacia  by  KCl-injection  sometimes  give  reduced  fertilizability.  This 
appears  to  be  related  to  release  of  a  yellow-green  dermal  secretion  which  inhibits  fertilization 
(Oshima,  1921;  Pequegnat,  1948).  Further  study  reveals  that  the  dermal  secretion  has  other 
effects  as  well.  These  include  an  enhancement  of  sperm  motility,  a  two-fold  increase  in  sperm 
respiration  and  inhibition  of  fertilizin  agglutination  of  sperm. 

This  last  effect  suggests  that  further  study  of  the  dermal  secretion  might  provide  informa- 
tion concerning  a  role  of  fertilizin  in  fertilization.  Experiments  show  that  the  dermal  secretion 
destroys  the  agglutinating  action  of  fertilizin.  Thus  the  inhibiting  agent  is  heat-labile  whereas 
fertilizin  is  heat-stable.  Heated  fertilizin-inhibitor  mixtures  neither  agglutinate  sperm  nor 
inhibit  agglutination.  Evidently  the  inhibitor  inactivates  fertilizin  and  is  subsequently  itself 
destroyed  by  the  heat  treatment.  Furthermore,  the  inhibitor  does  not  inactivate  by  merely 
converting  fertilizin  to  the  univalent  form.  It  actually  inactivates  the  combining  sites  of  the 
agglutinin.  Thus,  sperm  washed  from  inhibitor  alone  or  from  inhibitor-fertilizin  mixtures 
agglutinates  on  addition  of  fertilizin,  whereas  sperm  washed  from  fertilizin  alone  fails  to  agglu- 
tinate. Accordingly,  fertilizin  fails  to  block  the  sperm  surface  in  the  presence  of  the  inhibitor. 
This  is  explained  by  assuming  that  the  inhibitor  inactivates  the  combining  sites  of  fertilizin. 

The  question  whether  inactivation  of  fertilizin  is  related  to  the  fertilization-inhibiting  action 
of  the  dermal  secretion  remains  to  be  answered.  Moreover,  the  question  is  complicated  by 
the  fact  that  immunological  analysis  reveals  three  antigens  in  the  dermal  secretion,  and  that 
the  dermal  secretion  appears  to  contain  a  second,  heat-stable  agent  which  inhibits  agglutination 
of  eggs  by  antifertilizin  from  sperm. 

Behaviour   of   metachromatic   grannies   during    cleavage   in   Spisnla.     LIONEL    I. 
REBHUN. 

Eggs  stained  vitally  in  dilute  solutions  of  such  dyes  as  toluidine  blue,  azure  A,  azure  B, 
and  methylene  blue  in  sea  water  possess  small  (about  -^-micron)  granules  which,  in  the  case 
of  the  first  three  dyes  mentioned,  are  metachromatic.  These  granules  appear  in  the  unfertilized 
egg.  After  centrifugation  for  10  minutes  at  10,000  g  against  a  sucrose  barrier,  the  meta- 
chromatic granules  are  seen  in  the  mitochondrial  layer.  If  stained  eggs  are  fertilized  the 
particles  migrate  into  the  asters  at  cleavage  and  after  a  given  cleavage  are  localized  on  the 
peripheral  poles  of  the  individual  blastomere  nuclei.  Just  prior  to  the  succeeding  cleavage  the 
mass  of  particles  divides  into  two  smaller  masses,  each  subsequently  outlining  an  aster  of  the 
forming  spindle.  This  behaviour  continues  until  at  least  the  fifth  cleavage,  beyond  which  we 
have  not  attempted  to  trace  it. 

Neither  the  material  stainable  with  zinc-free  janus  green  B  nor  that  revealed  by  the  Nadi 
reaction  shows  the  same  specific  localization  change  as  the  metachromatic  particles.  This  pre- 
sumptive evidence  that  the  particles  are  not  mitochondria  is  supported  by  electron  micrographs 
which  show  that  the  mitochondria  are  scattered  at  random  in  the  egg  at  cleavage,  being  excluded 
only  from  the  spindle. 

AUGUST  12,  1958 
The  A  band  of  muscle  from  Liiuitlns  polyphcinns.     G.  W.  DE  VILLAFRANCA,  T.  S. 

SCHEINBLUM  AND  D.  E.  PHILPOTT. 

Strips  of  rest-length  muscle  from  the  cephalothorax  of  Limulus  were  tied  to  splints  and 
placed  in  ice-cold  50%  glycerol.  After  deep-freeze  storage  for  about  10  days  they  were  cut 
from  the  splints,  blended  1%  minutes  and  washed  seven  times  in  0.04  M  KC1-0.0067  M  phosphate 
buffer  (pH  7.4)  solution.  In  five  experiments  an  average  of  29.3%  of  the  original  protein  was 
left  after  washing. 

The  washed  fibrils  retained  their  characteristic  morphology  as  judged  by  phase  contrast 
and  electron  microscopy.  One  could  observe  A,  I  and  Z  bands,  but  no  M  lines  or  H  zones 

1  Aided  by  a  grant  from  the  National  Science  Foundation. 


326         PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

which  are  also  absent  in  fresh  or  fixed  material.  Filaments  about  150  A  or  larger  and  fila- 
ments, or  rings,  of  material  about  60  A  wide  were  observed  in  cross-section.  Occasionally 
filaments  were  seen  running  through  the  Z  bands. 

When  the  washed  fibrils  were  extracted  with  solutions  of  high  salt,  (O.48  M  KC1,  0.01  M 
pyrophosphate,  0.1  M  phosphate  buffer  pH  6.5,  and  0.001  M  MgCU;  or,  0.3  M  KC1,  0.15  M  phos- 
phate buffer  pH  6.5  and  0.002  M  ATP)  the  A  band  disappeared  leaving  a  ghost  fibril  with  Z 
band,  Z  band  filaments  and  60  A  filaments  extending  throughout  the  sarcomere,  often  in  dis- 
array. This  is  similar  to  the  effect  of  these  solutions  on  rabbit  psoas  muscle.  There  was, 
however,  a  striking  difference  between  rabbit  and  horseshoe  crab  muscle.  Under  these  condi- 
tions actomyosin,  rather  than  myosin,  was  always  the  chief  protein  extracted  from  the  crab. 
In  10  experiments  an  average  of  38.2%  of  the  washed' fibril  protein  was  removed  (11.2%  of 
the  whole  fiber).  The  extract  had  an  ATP  sensitivity  (viscosity  drop  with  ATP)  of  from 
65.1  to  165.0.  About  70%  of  the  protein  precipitated  upon  dilution  with  10  volumes  of  water 
and  exhibited  both  the  super-precipitation  and  ATPase  reaction  of  other  actomyosins. 

Sperm  cell  models  and  the  question  of  ATP-indiiced  rhythmic  motility.     DAVID  W. 
BISHOP. 

Several  types  of  KCl-glycerine-extracted  cell  systems  which  respond  to  ATP  by  rhythmic 
motility  have  previously  been  demonstrated.  These  systems  include  grasshopper  sperm  and 
Vorticella  stalk  myonemes  (Hoffmann-Berling),  toad  pharyngeal  cilia  (Alexandrov  and  Arrouet) 
and  spermatozoa  of  several  species  of  mammals  (Bishop).  Considerable  evidence  now  indi- 
cates that  rhythmic  flagellation  is  due  to  contraction-relaxation  cycles  inherent  in  the  contractile 
protein  present  in  these  cells.  Two  fundamental  problems  are  posed  by  the  behavior  of  these 
ATP-reactivated  cells:  (1)  How  do  contraction-relaxation  cycles  continue,  in  contrast  to  the 
muscle  fibril  model  which  contracts  only  once  upon  the  addition  of  ATP?  (2)  How  can 
a  coordinated  wave-like  movement  occur  in  cells  whose  permeability,  ionic  balance,  and  meta- 
bolic integrity  have  been  destroyed  ? 

A  partial  answer  to  the  second  question  has  been  found  in  a  comparison  of  the  motility 
of  normal  cells  with  that  of  sperm  models.  The  motility  of  fresh  bull  sperm  consists  of  two 
components  :  a  two-dimensional  vibration  initiated  proximally  and  propagated  along  the  flagel- 
lum,  and  a  superimposed  spin  along  the  longitudinal  axis  due  to  helical  flagellation  of  the 
distal  region  of  the  tail.  Sperm  models,  on  the  other  hand,  display  only  the  two-dimensional 
bending  waves ;  neither  wave  propagation  nor  the  three-dimensional  spiraling  occurs.  The 
lack  of  these  two  elements  of  motion  suffices  to  explain  the  failure  of  cell  models  to  undergo 
forward  progression,  despite  very  vigorous  movements.  Attempts  to  restore  the  missing  com- 
ponents of  flagellation  by  adjustment  of  the  ionic  balance  in  the  models  have  not  proved  suc- 
cessful. It  is  recognized  that  the  disrupted  processes  discussed  here  are  perhaps  more  involved 
with  intracellular  co-ordination  than  with  the  chemistry  of  wave  mechanics,  but  such  a  study 
permits  a  recognition  of  those  aspects  of  motility  which  are  and  are  not  concerned  with  the 
movement  of  reactivated,  extracted  sperm  models. 

ATP — an  energy  source  for  sperm  motility.1     LEONARD  NELSON. 

Morphological  features  of  motile  systems  as  well  as  ATP-ase  activity  and  contractility 
must  be  taken  into  account  in  theories  concerning  mechanisms  of  motility.  Perhaps  more  than 
a  superficial  similarity  exists  between  the  submicroscopic  organization  of  sperm  of  warm- 
blooded animals  and  the  elegant  Huxley-Hansen  interdigitating  creep  arrangement  of  muscle. 
Following  appropriate  fixation  and  staining  of  sperm,  connections  appear  to  link  the  nine 
outer  longitudinal  fibers  with  the  corresponding  nine  inner  fibers.  Reversible  changes  in  align- 
ment of  these  structures  during  generation  of  the  undulatory  wave  would  presumably  be  asso- 
ciated with  release  of  energy.  The  average  power  developed  in  the  propulsion  of  bull  sperm 
through  a  viscous  medium,  calculated  as  a  first  approximation  using  Carlson's  formula,  is  about 
3.15  X  10"8  erg/second.  To  yield  this  amount  of  energy,  an  average  sperm  must  split  1  X  10"19 
M  ATP/second.  At  a  QP  of  150,  the  spermatozoon  can  avail  itself  of  a  "safety  factor"  of  two 
orders  of  magnitude.  Succinic  dehydrogenase  activity  can  regenerate  at  least  twenty  times 

1  Supported  by  grants  from  the  Population  Council,  Inc.,  New  York. 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY         327 

the  amount  of  ATP  utilized.  Electron  microscope  cytochemical  studies  suggest  an  organiza- 
tion which  can  meet  the  requirements  imposed  on  an  undulatory  filament  propelling  itself  by 
three-dimensional  waves.  The  micrographs  reveal  an  association  of  both  ATP-ase  and  suc- 
cinic  dehydrogenase  within  the  nine  outer  fibers.  To  determine  whether  these  fibers  contain 
substances  related  to  known  contractile  proteins,  frozen-dried  sperm  were  incubated  in  solutions 
containing  myosin  antibodies.  Electron  micrographs  consistently  show  increased  density  of 
the  nine  outer  fibers.  Perhaps  the  fibers  bind  the  antibodies  weakly,  since  results  were  ob- 
tained only  when  unorthodox  procedures  were  employed.  If  propulsion  of  mammalian  sperm 
depends  on  sequential  "contraction"  of  the  outer  fibers,  this  may  be  achieved  by  a  lateral 
"creeping"  along  the  inner  fibers.  The  initiation  and  regulation  of  the  propagated  waves  re- 
mains to  be  investigated. 

Polarisation  optical  studies  on  amebae.     ROBERT  D.  ALLEN. 

It  has  been  our  hope  that  analysis  of  the  cytoplasm  of  ameboid  cells  with  polarized  light 
might  yield  useful  information  on  the  molecular  mechanisms  involved  in  ameboid  movement. 
The  sign  and  magnitude  of  birefringence  and  rotation  can  reveal  not  only  molecular  orientation, 
but  sometimes  designate  the  molecules  involved  and  provide  information  on  their  conformation. 
Earlier  attempts  have  been  hindered  by  (1)  lack  of  sufficiently  sensitive  methods  for  measuring 
small  retardations  and  angles  of  rotation,  and  (2)  the  presence  of  crystalline  cytoplasmic 
inclusions  which  scatter  and  depolarize  light.  Recent  advances  in  the  design  of  polarizing 
microscopes  (Inoue)  have  made  it  feasible  to  re-investigate  ameboid  cells  by  a  new  method 
employing  objective  recording  of  small  intensity  differences  in  the  presence  of  scattered  light. 
In  this  method,  suggested  by  Dr.  Inoue,  the  plane  of  polarization  of  the  incident  beam  has 
been  wobbled  by  a  rotating,  tilted,  strain-free,  optically-flat  cover-glass.  When  the  polars  are 
crossed,  the  photomultiplier  records  for  each  revolution  of  the  tilted  cover-glass  four  equal 
intensity  modulations,  the  peaks  of  which  correspond  to  the  positions  in  which  the  axis  of  tilt 
forms  an  angle  of  ±45°  with  the  plane  of  polarization.  Various  model  experiments  have  shown 
that  either  small  retardations  or  small  angles  of  rotation  modify  the  recorded  intensity  pattern. 
In  the  present  experiments  to  measure  birefringence,  retardation  could  be  separated  from  rota- 
tion by  the  orientation  of  the  specimen,  since  a  birefringent  object  shows  opposite  intensity  pat- 
terns at  plus  and  minus  45°  settings.  Using  a  20°  tilt,  retardations  of  0.5  A  could  be  detected 
with  monochromatic  light. 

A  spot  of  wobbling  linearly-polarized  light  was  directed  at  the  anterior  portion  of  a  some- 
what compressed  monopodial  ameba  (Chaos  chaos).  As  the  specimen  advanced,  the  light  beam 
scanned  its  long  axis,  recording  retardations  in  the  front,  middle  and  rear  portions  as  well  as 
a  control  for  the  strain  birefringence  of  the  cover-glasses  (maximum  2  A).  The  endoplasmic 
stream  was  positively  birefringent  (10~u  to  10~5),  with  the  highest  values  recorded  in  the  middle 
and  lowest  near  the  tail.  Although  birefringence  seemed  to  depend  on  the  rate  of  streaming, 
it  would  be  premature  to  call  this  flow  birefringence,  as  it  has  not  yet  been  established  whether 
streaming  or  some  other  factor  associated  with  movement  brought  about  the  inferred  alignment 
of  protein  molecules. 

The  isolation  and  analysis  of  cilia.     FRANK  M.  CHILD. 

In  order  to  speculate  seriously  about  the  mechanism  of  the  movement  of  cilia  and  flagella 
it  is  necessary  to  know  something  about  the  molecular  organization  and  chemical  composition 
of  these  cellular  organelles.  Such  information  is  obtainable  by  isolation  and  analysis  of  the 
structures  themselves. 

Cilia  have  been  isolated  from  the  protozoan  ciliate  Tetrahymcna  pyriformis  in  quantities 
sufficient  for  analysis.  The  cilia  are  isolated  from  the  living  cell  directly  into  20  per  cent 
glycerol.  Electron  micrographs  show  that  isolated  cilia  are  composed  of  the  fibrillar  axonemes. 
Isolated  cilia  are  insoluble  in  water,  KC1  solutions,  Versene,  6  M  urea,  and  within  the  pH 
range  3  to  11.  The  substance  of  cilia  that  have  been  dissolved  at  a  pH  greater  than  11  remains 
in  solution  down  to  pH  5.4.  Dissolved  ciliary  substance  shows  but  one  electrophoretic  and 
ultracentrifugal  component  in  phosphate  buffer,  pH  7.4.  Ciliary  substance  is  largely  protein, 
but  has  a  U.V.  absorption  maximum  at  260  m/i  in  acid  which  is  attributed  to  the  presence  of 
adenine  and  uracil  nucleotides  since  the  material  contains  phosphate,  pentose,  adenine  and  uracil. 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

RNA  is  not  present.  Calculations  based  on  the  pentose  determination  indicate  that  the  amount 
of  the  nucleotide  is  about  2.59c  that  of  the  protein.  The  nucleotide  is  not  separable  from  the 
protein  ingredient  by  precipitation  at  pH  5.4,  nor  by  prolonged  dialysis.  However,  the  nucleo- 
tide does  not  sediment  proportionally  with  the  protein  when  ultracentrifuged.  Isolated  cilia 
possess  enzymatic  activity  which  will  split  phosphate  from  adenosine  triphosphate,  adenosine 
diphosphate,  and  adenosine-5'-monophosphate. 

AUGUST  19,  1958 

A  physical  study  of  the  ground  substance  of  the  Spisula  egg.     L.  V.  HEILBRUNN 
AND  W.  L.  WILSON. 

As  is  well  known,  the  most  widely-used  method  of  measuring  protoplasmic  viscosity  is 
the  centrifuge  method.  In  this  method,  cells  are  centrifuged  and  the  speed  of  movement  of 
protoplasmic  granules  is  determined.  According  to  Stokes'  law,  this  speed  gives  a  measure 
of  the  viscosity.  But  in  determining  relative  values  for  the  viscosity  of  the  hyaline  protoplasm, 
it  is  essential  that  the  size  of  the  granules,  their  specific  gravity  and  their  number  or  concen- 
tration remain  constant. 

It  has  often  been  reported  that  in  immature  invertebrate  eggs,  the  protoplasmic  viscosity 
is  very  high  and  decreases  markedly  following  the  breakdown  of  the  germinal  vesicle.  In  the 
eggs  of  the  clam,  Spisula  solidissima,  after  breakdown  of  the  germinal  vesicle,  granules  move 
through  the  protoplasm  much  more  readily.  However,  when  following  centrifugation,  the 
speed  of  return  of  the  granules  is  measured,  and  Einstein's  formula  for  Brownian  movement 
is  applied,  it  can  be  shown  that  the  viscosity  of  the  hyaline  protoplasm  or  ground  substance 
is  essentially  the  same  before  and  after  germinal  vesicle  breakdown.  Thus  a  series  of  14 
measurements  gave  a  value  of  5.0  centipoises  for  the  viscosity  of  the  ground  substance  before 
germinal  vesicle  breakdown,  whereas  13  measurements  gave  a  value  of  4.7  centipoises  after 
breakdown.  When  the  large  germinal  vesicle  breaks  down,  the  concentration  of  granular 
material  in  the  cytoplasm  becomes  much  less — also  new  smaller  granules  appear  in  the  cyto- 
plasm. Both  these  changes  are  primarily  responsible  for  the  fact  that  the  non-granular 
(hyaline)  zone  of  centrifuged  eggs  appears  much  more  rapidly  when  eggs  are  centrifuged 
after  germinal  vesicle  breakdown. 

According  to  the  equations  of  Einstein  and  of  Simha  for  the  viscosity  of  suspensions,  the 
viscosity  of  the  entire  protoplasm  is  decidedly  greater  in  immature  eggs,  but  this  does  not 
appear  to  be  true  for  the  hyaline  protoplasm  or  ground  substance. 

Physical  properties  of  lobster  nerve  axoplasm.     CARL  FELDHERR. 

The  physical  properties  of  giant  lobster  axons  were  studied  with  the  aid  of  microinjection 
methods.  When  small  drops  of  paraffin  oil  (4-40  M)  were  injected  into  the  axons,  the  drops 
did  not  move  under  the  influence  of  gravity  as  long  as  the  axons  were  capable  of  transmitting 
an  impulse.  However,  when  as  a  result  of  injury  or  aging,  the  axons  had  lost  their  ability 
to  conduct,  oil  drops  did  move  readily  through  the  axoplasm.  This  indicates  that  in  the  normal 
axon  there  are  structural  elements  which  break  down  after  injury.  The  injected  oil  drops 
were  always  spherical,  whereas  according  to  Chambers  and  Kao  oil  drops  injected  into  squid 
axons  assume  an  ovoid  shape.  Apparently  squid  axoplasm  is  more  rigid  than  similar  axo- 
plasm in  the  lobster.  If  large  amounts  of  oil  are  injected  into  lobster  axons,  the  oil  appears 
to  fill  the  entire  width  of  the  axon,  indicating  that  if  a  cortex  is  present,  it  could  be  no  more 
than  a  few  microns  thick.  Electrical  stimulation  of  the  axon  produced  no  change  in  the  shape 
of  injected  oil  drops.  By  injecting  a  relatively  large  amount  of  oil,  changes  in  the  shape  of 
the  spike  could  be  produced.  These  changes  were  usually  found  to  be  reversible  upon  removal 
of  the  oil. 

Studies  were  also  made  with  phase  contrast  microscopy,  and  these  gave  results  similar 
to  those  previously  obtained  by  Tobias  and  Bryant.  Vigorous  Brownian  movement  could 
always  be  observed,  and  there  was  a  vibratile  movement  of  filamentous  structures  scattered 
through  the  axoplasm.  In  some  instances  fibrils  could  be  observed  in  the  center  of  the  axon ; 
these  were  not  as  extensive  as  those  described  by  de  Renyi,  and  their  presence  was  not  necessary 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY         329 

for  conduction,  for  in  those  cases  in  which  no  fibrils  could  be  observed,  the  axons  were  able 
to  transmit  impulses. 

The  nuclear  envelope  as  a  possible  agent  in  specific  synthetic  events  in  the  cytoplasm 
of  sand  dollar  eggs.     ROBERT  W.  MERRIAM. 

Late  in  the  process  of  vitellogenesis  of  Dcndrastcr  ecccniricus  oocytes,  double  membranes 
scattered  singly  in  the  cytoplasm  can  often  be  seen  in  electron  micrographs  in  intimate,  parallel 
association  with  the  envelope  of  the  germinal  vesicle.  The  membranes  resemble  the  nuclear 
envelope  and  often  have  150-200  A  particles  adhering  to  the  surface.  Concentrically  arranged 
double  membranes  of  "yolk  nuclei"  are  morphologically  similar,  including  150-200  A  adherent 
particles.  Such  membranes  show  no  sign  of  regularly  arranged  annuli. 

After  the  maturation  divisions,  the  mature  nucleus  becomes  massively  associated  with 
closely  applied  and  parallel  arrays  of  the  annulate  lamellae  named  by  Swift.  At  the  same  time 
intranuclear  vesicles  can  be  seen  in  structural  continuity  with  double  membranes  parallel  to, 
but  inside,  the  nuclear  envelope.  The  "intranuclear  membranes"  are  always  closely  applied 
to  the  nuclear  envelope  and  are  morphologically  identical  to  it  except  that  no  annuli  are 
present. 

Such  intimate  association  with  the  nuclear  envelope,  coupled  with  their  morphological 
similarity  to  it,  are  taken  as  evidence  suggesting  that  single  granulate  membranes,  perhaps 
"yolk  nucleus"  membranes,  and  annulate  lamellae  are  somehow  formed  around  the  nuclear 
envelope. 

Annulate  lamellae  are  shown  to  contain  regularly  arranged  annuli  consisting  of  electron- 
dense  rims  in  which  relatively  less  dense  spheres  or  vesicles  are  embedded.  Masses  of 
150-200  A,  basophilic  granules  are  found  closely  associated  with  some  of  them  in  the  cytoplasm. 
These  are  the  "heavy  bodies"  of  Afzelius.  "Heavy  bodies"  are  never  observed  to  be  struc- 
turally associated  with  the  nuclear  envelope.  Therefore,  it  is  suggested  that  150-200  A 
particles  are  formed  by  annulate  lamellae  in  the  cytoplasm. 

If  the  150-200  A  particles  of  the  "heavy  body"  are  homologous  to  the  particles  of  Palade, 
the  annulate  lamellae  may  form  nucleoprotein  structures  which  contribute  to  the  cytoplasmic 
synthesis  of  proteins.  This  suggests  that  synthetic  specificity,  perhaps  originally  derived  from 
the  nucleus,  may  reside  in  nuclear  envelope  structure. 

ELECTROBIOLOGY  SEMINARS 

JULY  10,  1958 
Graded  electrical  responses     HARRY  GRUNDFEST. 

Electrically  excitable  electrogenic  cells  that  normally  produce  regenerative  (all-or-none) 
spikes  become  gradedly  responsive  in  early  relative  refractoriness,  during  sustained  depolariza- 
tion, or  after  treatment  with  synapse  inactivator  drugs.  In  the  grasshopper,  Romalea  microp- 
tera,  electrically  excitable  muscle  fibers  have  only  graded  responsiveness.  This  finding  con- 
firms an  earlier  suggestion ;  the  combination  of  different  grades  of  electrically  inexcitable 
postsynaptic  potentials  with  graded  responsiveness  of  electrically  excitable  fibers  of  insect  and 
crustacean  muscles  underlies  the  variety  of  their  electrical  and  mechanical  responses  to  different 
axons  of  their  innervation.  Normal  occurrence  of  graded  responsiveness  indicates  that  current 
theory,  devised  to  account  for  production  of  spikes,  is  inadequate  as  a  general  account  of 
electrically  excitable  electrogenesis.  A  preliminary  hypothesis  views  electrically  excitable 
electrogenic  membrane  as  a  composite  of  unit  areas,  each  endowed  with  a  complement  of 
"electrogenic  elements"  which  may  be  pores,  carriers,  or  other  mechanisms  for  changing  ionic 
conductances  when  appropriately  triggered  by  some  (depolarizing)  electrical  stimulus.  The 
elements  of  a  unit  area  are  considered  to  have  thresholds  requiring  different  intensities  of 
triggering  stimulation.  Distribution  of  this  population  in  a  narrow  range  with  respect  to 
their  thresholds  (equivalent  to  a  high  amplification  factor  in  a  bistable  electronic  analogue), 
leads  to  regenerative  action  within  the  population,  and  to  all-or-none  responses.  Widespread 


330         PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

distribution  of  the  population  on  the  threshold  axis  would  tend  to  eliminate  regenerative  action 
by  electrotonic  losses,  and  could  result  in  graded  responses.  This  also  occurs  in  the  electronic 
analogue  with  reduced  amplification.  Wide-band  distribution  of  electrogenic  elements  is  con- 
sidered to  be  the  normal  case  in  the  invertebrate  muscle  fibers  and  to  be  produced  physiologically 
or  pharmacologically  in  converting  regeneratively  acting  to  gradedly  responding  tissues.  The 
hypothesis  clarifies  experimentally  observed  distinctions,  blurred  by  the  current  ionic  theory, 
between  the  capacity  for  electrogenic  activity  and  the  initiation  of  this  activity  by  appropriate 
stimuli.  It  has  also  predicted  an  experimentally  verified  relation  between  area  of  stimulated 
membrane  and  the  character  of  responses  in  gradedly  responding  eel  electroplaques.  When 
the  whole  electrically  excitable  surface  is  stimulated,  the  response  appears  to  be  a  spike. 


JULY  17,  1958 

The  membrane  potentials  during  rest  and  activity  of  the  electroplate  of  Raia  clavata. 
ROSAMOND  M.  ECCLES  AND  L.  G.  BROCK. 

The  electrical  responses  of  individual  electroplates  were  investigated  with  intracellular 
micropipettes  in  organs  isolated  from  Raia  clavata.  At  high  potassium  concentrations  there 
was  a  linear  relationship  between  the  logarithm  of  the  concentration  and  the  resting  potential, 
with  a  gradient  of  58  mV  for  a  ten-fold  increase  in  potassium  concentration.  The  resting 
potentials  which  were  in  the  range  of  60-70  mV  were  unaffected  by  changes  in  extracellular 
magnesium,  calcium  and  chloride  ions.  The  response  to  stimulation  in  many  ways  resembled 
the  amphibian  endplate  potentials.  At  its  peak  the  voltage  was  usually  close  to  the  zero  poten- 
tial line  (±10  mV).  The  response  reached  its  maximum  in  3-5  msec,  and  declined  to  the 
resting  level  in  about  25  msec.  In  low  concentrations  of  sodium  chloride  there  was  a  marked 
increase  in  the  duration  of  the  response.  Alterations  in  extracellular  chloride  levels  seemed 
to  indicate  that  movement  of  chloride  ions  was  not  important  in  the  recovery  process.  The 
blockage  by  curare  and  the  prolongation  by  prostigmine  lend  support  to  the  belief  that  trans- 
mission at  the  nerve-electroplate  junction  is  cholinergic. 

Electric  organ  clectrogcncsis  in  Malapterurus  clcctricus.     M.  V.  L.  BENNETT,  R. 
D.  KEYNES  AND  H.  GRUNDFEST. 

The  African  catfish  presents  apparently  aberrant  features  among  electric  fishes.  Dis- 
charging electroplax  become  negative  at  their  rostral,  uninnervated  faces.  They  were  con- 
sidered to  be  derived  from  gland,  to  be  electrically  inexcitable,  and  to  hyperpolarize  during 
activity.  This  explanation  appears  unlikely.  The  synaptic  junction  is  at  the  tip  of  a  long 
caudal  stalk.  The  postsynaptic  potential,  electrically  inexcitable  and  non-propagating,  there- 
fore cannot  involve  the  major  part  of  the  electroplaque,  yet,  each  cell  apparently  generates 
a  high  emf,  which  indicates  this  involvement.  Microelectrode  recordings  from  single  electro- 
plax provide  a  more  satisfactory  explanation,  furnishing  also  new  data  for  the  general  theory 
of  bioelectrogenesis.  The  electroplax  are  electrically  excitable,  the  impulse  synaptically  evoked 
in  the  stalk  propagating  into  and  activating  the  cell  body.  Responses  of  the  latter  to  direct 
stimulation  do  not  differ  from  neurally  evoked  activity.  Electrogenesis  of  the  caudal  as  well 
as  the  rostral  face  contributes  to  an  overshooting  spike,  but  the  caudal  activity  is  smaller, 
higher  threshold  and  briefer  (ca.  0.3  msec.)  than  that  of  the  rostral  face,  which  lasts  about 
2  msec.  The  intracellularly  recorded  spike  consequently  has  a  brief  initial  peak,  when  both 
generators  are  active,  followed  by  a  smaller,  longer-lasting  portion  when  only  the  rostral  face 
continues  activity.  However,  the  responses  recorded  outside  the  two  faces,  representing  the 
difference  between  the  two  electrogenic  activities,  have  a  reduced  potential  while  both  faces 
are  responding.  The  rostral  face  is  negative,  the  caudal  positive  during  the  entire  discharge. 
The  neurally  evoked  activity  of  a  single  electroplaque  thus  must  comprise  at  least  four  com- 
ponents :  a  p.s.p.  at  the  stalk-tip,  giving  rise  to  an  electrically  excitable  response  propagating 
in  the  stalk,  the  latter  in  turn  producing  the  two  activities  in  the  rostral  and  caudal  faces  of 
the  electroplaque.  This  electrogenic  diversity,  which  accounts  for  the  peculiarities  of  Malap- 
tcrnnis  electric  organ,  is  greater  than  has  hitherto  been  found  elsewhere. 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY         331 

AUGUST  7,  1958 
Aspects  of  synaptic  transmission  in  the  squid  stellate  ganglion.     S.  H.  BRYANT. 

The  synaptic  excitation  of  the  stellar  giant  axons  and  recent  studies  supporting  a  chemical 
transmission  process  at  the  distal  (giant)  synapses  were  reviewed.  Evidence  for,  and  the 
properties  of,  the  proximal  (accessory)  excitatory  synapses  were  given. 

New  data  were  presented  showing  the  effects  of  osmotic  pressure,  ions,  fatigue  and  tem- 
perature on  synaptic  delay  in  perfused  excised  ganglia.  Different  methods  of  evaluating 
synaptic  delay  were  compared  and  found  to  agree  closely  if  the  same  reference  points  were 
used.  Values  from  peak  of  preaxon  artefact  to  the  beginning  of  the  psp  are  given  here.  Only 
temperature  variation  produced  marked  changes  in  the  delay  at  the  distal  synapse.  In  the  range 
from  35  to  20°  C.  the  delay  was  relatively  constant,  averaging  0.55  msec.  Below  20°  C.  there 
was  an  accelerated  increase  in  delay  up  to  1  to  3  msec,  near  2°  C.  Further  temperature 
decreases  resulted  in  block.  Similar  temperature  effects  were  noted  in  proximal  synapse 
latency.  The  constancy  of  the  delay  in  the  higher  temperature  range  is  consistent  with  the 
notion  of  transmitter  diffusion  time.  The  reason  for  the  large  increases  at  lower  tempera- 
tures is  not  clear.  High  gain  intracellular  post  axon  recordings  of  the  presynaptic  spike 
"leak  through"  in  the  latter  studies  most  often  show  the  distal  to  be  diphasic  and  the  proximal 
triphasic,  resembling  first  and  second  derivatives  of  the  pre  spike,  respectively.  The  amplitude 
of  these  potentials  decreases  rapidly  with  distance  from  the  synaptic  area. 

Further  attempts  to  detect  a  quantal  nature  of  the  distal  p.s.p.  were  unsuccessful.  Fatigue, 
cold,  high  os. p.,  high  calcium-low  magnesium  medium  and  smaller  distal  synapses  were  used 
to  increase  this  effect  if  present. 

Compounds  not  used  previously  were  tested  for  action  on  the  distal  synapse.  FMN,  thiamine, 
asparagine,  hydroxylamine  (all  at  10~3  gm./ml.)  and  ouabain  (10~5)  were  without  effect  in 
30  to  60  minutes.  Picrotoxin  (10~3)  and  choline  chloride  (2  X  10~2)  caused  a  reversible 
non-depolarizing  block  within  20  minutes.  Guanidine  (4  X  10~4)  reversibly  produced  repetitive 
presynaptic  discharges  to  a  single  shock,  prolonged  p.s.p.'s  and  eventual  synaptic  blockade. 

AUGUST  14,  1958 

Electroplaque  activity  in  marine  electric  fishes.     M.  V.  L.  BENNETT,  M.  WURZEL, 
E.  AMATNIEK  AND  H.  GRUNDFEST. 

Microelectrode  recordings  prove  that  electroplax  of  Astroscopus  guttatus,  Narcine  brasili- 
cnsis,  and  Torpedo  occldentalis  respond  only  to  stimulation  of  their  nerves  and  to  chemical 
agents.  Denervated  electroplax  in  A.,  though  activated  by  drugs,  do  not  respond  to  electrical 
stimuli.  A  constellation  of  properties  (most  extensively  studied  in  A.  and  T.}  associated 
with  electrically  inexcitable  electrogenesis  is  found :  i)  stimuli  to  the  innervated  surface  of 
an  electroplaque  (dorsal  in  A.;  ventral  in  N.  and  T.)  evoke  responses  after  an  irreducible 
latency,  about  1  msec.  Responses  develop  only  at  the  innervated  surface,  are  depolarizations 
from  a  resting  potential  of  50-80  mv,  last  5-8  msec.  «)  Responses  may  be  graded  into  several 
discrete  steps,  m)  At  maximum  they  approach  an  "equilibrium  potential"  which  approximates 
zero  membrane  potential,  iv)  The  amplitude  is  graded  without  change  in  latency  by  changing 
membrane  potential,  increasing  with  hyperpolarization.  ?')  Responses  are  inverted  on  reversing 
membrane  potential,  vi)  Different  axons  apparently  innervate  discrete  regions  of  the  large 
surface  (about  1  cm.2  in  A.  and  T.).  Responses  produced  in  one  region  do  not  propagate 
actively  into  other  zones  of  innervation.  Electrotonic  spread  decays  to  half  in  about  0.5 
mm.  (T.).  i'ii)  Potentials  evoked  by  the  same  or  by  different  axons  sum  non-algebraically, 
addition  becoming  limited  as  depolarization  approaches  the  equilibrium  potential,  viii)  Homo- 
synaptic  facilitation  occurs,  but  not  heterosynaptic.  ix)  Without  anticholinesterases,  acetyl- 
choline  depolarizes  only  in  high  concentrations  (10~2  M  in  T.).  When  protected,  its  threshold 
effectiveness  is  at  10.~4  to  10'5  M.  Carbamylcholine  is  equally  effective  without  protection. 
x)  Anticholinesterases  also  depolarize  and  prolong  responses,  while  curare  blocks  activity 
without  depolarizing,  xi)  Quaternary  drugs  act  only  when  applied  to  the  innervated  side, 
electroplax  below  the  exposed  layer  remaining  unaffected.  However,  eserine  acts  from  either 


332         PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

side  and  also  affects  underlying  units.  Since  Raia  electroplax  are  also  electrically  inexcitable 
(Eccles  and  Brock,  above),  the  electrogenic  membranes  of  electroplax  in  all  marine  electric 
fish,  teleost  and  elasmobranch,  respond  only  with  post  synaptic  potentials.  However,  elec- 
trically excitable,  spike  generating  membrane  also  occurs  in  electroplax  of  all  fresh-water 
electric  fishes  studied  thus  far,  5  species  of  Gymnotids  and  Malapterurus. 

AUGUST  21,  1958 

Electrophysiologv  and  pharmacology  of  lobster  muscle  fibers.     H.  GRUNDFEST,  J. 
P.  REUBEN  AND  W.  H.  RICKLES,  JR. 

Studies  with  intracellular  microelectrodes  analyzed  drug-  and  ion-induced  alterations  of 
lobster  muscle  fiber  membrane.  The  component  that  gives  rise  to  inhibitory  postsynaptic 
potentials  (ipsp's)  is  activated  by  GABA  (7-aminobutyric  acid;  threshold  concentration  about 
10~u  M),  /3-alanine,  /3-hydroxy-GABA  and  7-aminocrotonic  acid.  Slight  hyperpolarization, 
four-fold  or  larger  increase  of  membrane  conductance,  and  appropriate  electrochemical  modifi- 
cations of  epsp's  and  ipsp's  evoked  by  stimulating  the  excitatory  and  inhibitory  axons  denote 
this  action  on  the  synaptic  membrane.  Picrotoxin  selectively  inactivates  the  hyperpolarizing 
membrane  without  changing  conductance.  GABA  antagonizes  its  effects.  Thus,  some  degree 
of  inversion  is  evidenced  between  pharmacological  properties  of  crustacean  hyperpolarizing 
synapses  and  depolarizing  synapses  of  cat  cortical  dendrites,  since  GABA  activates  the  former, 
but  blocks  the  latter,  while  picrotoxin  inactivates  the  former  and  excites  the  latter.  On  the 
other  hand,  carnitine  activates  epsp's  of  the  muscle  fibers,  depolarizing  as  well  as  increasing 
membrane  conductance.  GABA  antagonizes  the  depolarization,  but  increases  conductance  fur- 
ther. Therefore,  the  depolarizing  and  hyperpolarizing  synapses  are  pharmacologically  distinct 
and  act  independently.  Serotonin  and  histamine  depolarize,  but  in  high  concentration.  Even 
in  1%  solutions,  other  drugs  that  activate  or  inactivate  many  vertebrate  synapses  are  without 
effect  (e.g.,  acetylcholine,  prostigmine,  curare,  hexa-  and  decamethonium,  strychnine,  and  the 
C6  and  C8  cu-amino  acids).  Substituting  Rb+  for  external  K+  increases  membrane  resistance, 
but  augments  ipsp's  even  more.  Applying  Ba++  (with  depletion  of  Na+)  increases  membrane 
resistance  about  ten-fold.  This  does  not  prevent  subsequent  actions  of  synaptic  drugs  and 
presumably  does  not  affect  synaptic  membrane.  The  IR  drop  resulting  from  the  emf  of  electro- 
genic units  that  are  active  during  the  normal  graded  electrically  excitable  response  should  be 
increased  when  the  resistance  is  increased  by  Ba++.  The  higher  potential  should  activate  more 
units  of  the  electrogenic  population  (Grundfest,  above),  and  should  give  rise  to  regenerative 
involvement  of  the  whole  population.  This  process  satisfactorily  accounts  for  the  conversion, 
in  the  presence  of  Ba++,  of  the  graded  electrically  excitable  responses  of  invertebrate  muscle 
fibers  into  spikes. 

GENERAL  SCIENTIFIC  MEETINGS 
AUGUST  25-28,  1958 

Abstracts  in  this  section  (including  those  of  Lalor  Fellowship  Reports)  are 
arranged  alphabetically  by  authors  under  the  headings  "Papers  Read,"  "Papers 
Read  by  Title,"  and  "Lalor  Fellowship  Reports."  Author  and  subject  references 
will  also  be  found  in  the  regular  volume  index. 

PAPERS  READ 

Retinal  development  and  phototactic  responses  in  developing  Amcinrus  embryos. 
P.  B.  ARMSTRONG. 

The  first  indication  of  the  rods  and  cones  in  developing  Ameiurus  is  seen  at  the  time  of 
hatching  when  small  protoplasmic  buds  form  in  the  cells  of  the  external  nuclear  layer  in  the 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY         333 

central  part  of  the  retina.  The  cells  of  the  pigment  epithelium  are  heavily  charged  with 
melanin  pigment  and  fill  the  area  between  the  external  nuclear  layer  and  the  chorioid. 

Twenty-four  hours  later  the  receptor  layer  is  broader  and  the  protoplasmic  buds  have 
extended  and  enlarged  enough  so  that  it  is  possible  to  differentiate  the  rods  and  cones  from 
each  other.  These  receptor  elements  are  still  broadly  attached  to  their  respective  cell  bodies. 
The  pigment  of  the  pigment  epithelium  is  only  sparsely  distributed  between  the  receptor  ele- 
ments. It  becomes  very  dense  as  one  proceeds  toward  the  chorioid. 

There  is  a  gradual  development  of  the  myoids  of  the  rods  and  cones.  Photomechanical 
changes  with  varying  light  intensities  are  well  developed  shortly  before  the  complete  absorp- 
tion of  the  yolk.  This  includes  not  only  shortening  and  lengthening  of  the  myoids  of  the  rods 
and  cones  but  movement  of  the  pigment  in  the  pigment  cell  layer. 

Accompanying  the  above  developmental  changes  are  changes  in  the  motor  responses  of  the 
developing  embryos  to  light.  Further  investigations  will  be  necessary  to  correlate  the  develop- 
mental picture  and  the  behavioral  responses. 

Electron  microscopic  investigation  of  the  structure  of  hyaluronic  acid  gels  and 
hyaluronic  acid-protein  complexes.  FREDERICK  A.  BETTELHEIM  AND  DELBERT 
PHILPOTT. 

Hyaluronic  acid  (H.A.)  was  isolated  from  human  umbilical  cords.  It  was  purified  pro- 
gressively from  proteins,  neutral  polysaccharides  and  sulfated  polyuronides.  Its  molecular 
weight  (ultracentrifuge)  was  77,000.  It  was  homogeneous,  free  from  contaminations. 

Electron  micrographs  showed  that  the  pure  H.A.  formed  by  precipitation  with  ethanol  is 
a  three-dimensional  network  of  microfibrils  which  organize  themselves  into  platelets.  These 
platelets  combine  to  form  needle-like  superstructure.  X-ray  diffraction  proved  that  the  H.A. 
gel  is  highly  crystalline. 

Electron  microscopic  studies  of  H.A.  gel  in  its  complexed  form  with  intercellular  proteins 
were  performed  on  samples  taken  in  the  different  stages  of  the  purification  of  H.A.  The  data 
reveal  that  the  association  with  collagen  fibers  is  a  loose  one  and  the  real  complexing  of  H.A. 
occurs  with  non-fibrous  proteins.  The  H.A.  platelets  are  sandwiched  between  layers  of  proteins. 

H.A. -protein  complex  model  system  was  constructed  by  using  pure  H.A.  and  blood  albumin. 
Complexes  were  made  at  different  pH's  and  at  different  concentrations.  At  high  pH's  no 
complexing  occurs.  At  physiological  pH  the  complex  has  a  fibrous  structure  which  contracts 
to  a  spherical  shape  when  one  goes  through  the  isoelectric  point  (4.3)  (which  is,  however, 
not  the  isoelectric  point  of  blood  protein).  The  size  of  these  spheres  is  directly  proportional 
to  the  blood  albumin/H.A.  ratio. 

Potassium  contracture  in  a  variety  of  conditions.  D.  M.  CONWAY  AND  A.  I. 
CSAPO. 

The  retractor  penis  muscle  of  the  turtle  (Chryscinys  picta),  suddenly  immersed  in  excess 
K  Ringer  of  20-100  mM/1.,  goes  into  a  reversible  contracture.  Activation  here  is  generally 
explained  by  the  depolarising  effect  of  K,  and  the  [K]  of  25  mM/1.  is  looked  upon  as  the 
threshold  concentration  for  contracture. 

If  the  [K]  of  20-50  mM  is  gradually  rather  than  suddenly  raised,  however,  no  contracture 
develops. 

The  turtle  muscle  can  be  depolarised  suddenly  by  excess  K  =  20  mM  or  gradually  by 
excess  K  >  20  mM  without  any  sign  of  activity,  and  then  activated  later  by  different  means, 
which  involve  no  change  or  an  increase  rather  than  a  decrease  in  membrane  potential.  If  the 
K  is  not  excessive  the  muscle  can  be  stored  in  this  "primed"  condition.  There  is  no  true 
threshold  for  K-contracture,  and  it  will  develop  after  treatment  of  the  resting  muscle  with 
gradually  increasing  K  <  50  mM,  if  the  subsequent  jump  in  [K]  is  large  enough. 

Sudden  treatment  with  excess  K  results  in  more  contracture  tension  in  a  propagating 
than  in  a  non-propagating  muscle.  Lowering  the  temperature  decreases  the  "threshold"  and 
increases  the  magnitude  of  contracture.  Short  treatment  with  Ca-free  Ringer  increases  the 
slope  of  rise  and  the  magnitude  of  contracture  tension,  whereas  pre-treatment  with  high-Ca 
Ringer  has  an  opposite  effect.  This  "priming"  with  excess  K  can  be  substantially  altered 


334         PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

by  shifting  the  Ca  equilibrium  of  muscle,  suggesting  that  the  primary  effect  may  actually 
involve  such  a  shift. 

The  K-contracture  in  turtle  muscle  is  of  considerable  magnitude :  1.5-2.7  kg./cm.2  cross- 
section  area,  comparable  to  that  observed  in  single  fibers,  suggesting  that  the  diffusion  time 
of  K  to  the  extracellular  space  in  this  preparation  does  not  limit  contracture  very  greatly. 

These  observations  support  the  notion  that  depolarisation  as  such  and  by  itself  does  not 
activate  the  contractile  system,  and  that  depolarisation  and  activation  are  not  directly  linked. 
Depolarisation  might  serve  as  a  "priming"  step  for  activation  but  subsequent  events  need  to 
be  looked  for  which  are  more  directly  linked  to  activation  than  is  depolarisation. 

Calcium,  oxytocin  and  the  regulation  of  the  myometrium.     E.  M.  COUTINHO  AND 
A.  I.  CSAPO. 

The  notion  that  Ca  is  the  key  ion  in  the  regulation  of  myometrial  function  is  derived 
from  the  observations  that  Ca-free  Krebs  increases  the  threshold  of  excitation,  and  decreases 
tension  more  drastically  in  the  estrogen  than  in  the  progesterone  dominated  myometrium. 
The  rapidity  of  the  Ca  effects  suggested  alterations  in  membrane  function. 

Oxytocin  was  found  to  decrease  the  threshold  of  excitation,  and  the  promptness  of  its 
effect  pointed  again  to  an  action  on  the  membrane.  Further  experiments  show  that  "oxytocics" 
do  not  stimulate  a  uterus  of  maximum  working  capacity  if  its  intact  membrane  function  is 
temporarily  suspended. 

Repeated  washing  in  Ca-free  Krebs  results  in  a  quick  and  complete  loss  of  myometrial 
tension.  The  changes  which  accompany  and  explain  the  loss  of  tension  are :  increase  in 
threshold,  loss  of  conduction,  and  decrease  in  the  duration  of  the  mechanical  response  (shorten- 
ing of  the  active  state?).  This  condition  of  Ca  deficiency  is  completely  reversed  in  a  graded 
fashion  by  the  gradual  increase  in  the  Ca. 

The  recovery  of  the  Ca-deficient  muscle  is  strongly  temperature  dependent  (Qio>3). 
Oxytocin  completely  eliminates  the  effect  of  low  temperature,  a  finding  which  offers  the  first 
accurate  method  for  determining  minute  quantities  of  oxytocin. 

If  the  uterus  is  estrogen  dominated  the  drop  in  tension  in  Ca-free  Krebs  is  rapid,  whereas 
it  is  slow  if  the  muscle  is  progesterone  dominated.  Maximum  tension  in  the  Ca-depleted 
uterus  is  obtained  at  higher  [Ca]  if  the  muscle  is  dominated  by  estrogen  than  if  dominated 
by  progesterone. 

It  appears  that  under  progesterone  domination  Ca  is  more  firmly  bound  in  the  myometrial 
cell  or  that  the  cell  requires  less  Ca  for  tension  development  than  under  estrogen  domination. 
Oxytocin  seems  to  be  involved  in  "Ca-transport"  or  might  sensitise  the  particular  structure  of 
the  myometrial  cell  on  which  the  Ca  effect  is  exerted. 

Iodide  contracture  in  potassium  treated  muscle.     BRIAN  A.  CURTIS  AND  ARPAD  I. 
CSAPO. 

When  the  retractor  penis  muscle  of  the  turtle  Chrysemys  picta  is  placed  in  a  modified 
Ringer  containing  50%  (60  mM)  sodium  iodide  (replaced  for  sodium  chloride)  and  20  mM 
potassium  rather  than  2.5  mM,  the  muscle  goes  into  contracture.  No  contracture  is  observed 
if  the  iodide  or  potassium  treatments  are  applied  separately.  Furthermore  if  the  muscle  is 
first  depolarized  by  high  potassium  (12-30  mM)  and  the  iodide  Ringer  is  then  added,  the 
muscle  will  go  into  contracture  irrespective  of  the  change,  if  any,  in  the  potassium  concentra- 
tion within  the  above  range.  Thus  the  iodide  has  an  effect  not  only  if  the  membrane  potential 
is  decreasing,  but  even  if  it  remains  unchanged  or  is  increasing. 

If  a  muscle  is  depolarized  by  high  (for  example  20  mM)  potassium,  contractures  can  be 
repeatedly  induced  by  adding  and  removing  the  iodide,  keeping  the  potassium  concentration 
constant. 

The  effect  of  the  iodide  is  on  the  membrane  as  shown  by  the  rapidity  of  the  onset  and 
cessation  of  contracture. 

These  experiments  support  the  general  notion  that  depolarization  is  a  necessary  step  in 
the  series  of  events  leading  to  activation.  But  this  step  need  not  be  followed  by  myoplasmic 
activity,  allowing  us  to  keep  the  muscle  in  a  "primed"  condition  at  rest,  subject  to  later  acti- 
vation. The  instantaneous  effect  of  iodide  suggests  that  the  step  following  depolarization 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY          335 

is  localised  to  the  membrane  itself.     Further,  iodide  not  only  prolongs,  but  under  certain  con- 
ditions can  initiate  the  active  state. 

Some  aspects  of  the  chemical  composition  of  the  aqueous  humour  and  plasma  of 
the  smooth  dogfish.  RUSSELL  F.  DOOLITTLE,  BONNIE  BEDFORD,  CAROLYN 
CHESBROUGH,  CYNTHIA  THOMAS  AND  WILLIAM  STONE,  JR. 

A  comparative  chemical  analysis  of  the  aqueous  humour  and  blood  plasma  of  the  smooth 
dogfish  was  undertaken  as  a  prerequisite  to  further  inquiry  into  the  mechanisms  of  aqueous 
humour  formation  and  maintenance  of  the  intraocular  pressure.  The  elasmobranch  fishes  were 
chosen  since  the  osmoregulation  of  this  class  of  organisms  is  very  dependent  on  the  retention 
of  great  quantities  of  certain  organic  compounds,  namely,  urea  and  trimethylamine  oxide. 
Inasmuch  as  such  molecules  are  known  to  be  slow  crossing  from  the  plasma  to  the  aqueous 
in  mammals,  it  was  theorized  that  a  large  absolute  concentration  gradient  should  exist  across 
the  so-called  "aqueous  barrier."  Consequently,  factors  tending  to  permit  any  such  osmotic 
differential  should  be  proportionately  exaggerated.  It  was  established  in  this  study  that  a 
large  concentration  drop  does  exist  for  both  urea  and  trimethylamine  oxide.  Therefore,  in 
addition  to  urea  and  trimethylamine  oxide,  it  was  decided  to  measure  certain  constituents 
which  were  known  to  be  in  either  excess  or  deficit  in  many  mammalian  aqueous  humours. 
The  ascorbic  acid  content  of  the  aqueous  was  determined  to  be  higher  than  in  the  plasma. 
Total  CO2  was  also  much  higher,  and  preliminary  pH  studies  show  the  pH  to  be  considerably 
more  alkaline.  The  gross  concentration  of  free  amino  acids  was  shown  to  be  much  lower 
in  the  aqueous  than  in  the  plasma.  Dry  weight  determinations  were  done  on  both  aqueous 
and  plasma,  and  a  rough  tabulation  of  total  solids  in  these  fluids  was  made.  Some  preliminary 
measurements  on  sodium  ion  concentration  were  also  made. 

Some  aspects  of  morphogenesis  in  ascidians.     SYLVIA  FITTON  JACKSON. 

Comparative  studies  are  being  made  on  certain  aspects  of  growth  patterns  in  the  ascidians 
dona  intestinalis,  Molgula  manhattensis,  Perophora  listen,  Botryllus  schlosseri  and  Amarou- 
cium  constcllatum:  a  general  survey  has  been  carried  out  on  the  main  fine-structural  charac- 
teristics of  the  different  cell  types. 

In  longitudinal  sections  of  the  tail  of  newly  shed  larvae  of  Amaroucinm  and  Perophora, 
the  myofibrils  of  the  myoblasts  are  well  differentiated  and  the  arrangement  of  the  bands  is 
superficially  similar  to  that  observed  in  vertebrate  skeletal  muscle.  The  dimensions  of  most 
of  the  sarcomeres  seen  in  electron  micrographs  are  as  follows :  the  length  of  the  sarcimere 
averages  1.9  /*,  that  of  the  A  band  1.4  /a,  the  I  band  0.2  n,  the  Z  line  0.1  ^  and  the  H  zone 
0.15  p.  The  larger  filaments  of  the  A  band  region  are  each  about  230  A  wide  and  are  spaced 
about  200  A  apart.  The  banded  structure  of  adjacent  myofibrils  is  usually  in  register  and 
the  junction  between  the  cells  perpendicular  to  the  fibrils  axes  occurs  consistently  at  the 
Z  line  zone,  the  junction  being  composed  of  two  outer  and  two  inner  dense  parallel  lines. 

Investigations  are  being  made  on  the  mechanism  of  architecture  of  the  branchial  basket, 
and  elaboration  of  the  tunic.  The  cilia  around  the  periphery  of  each  gill  slit  are  arranged 
in  7  parallel  rows ;  the  cilia  are  closely  apposed  to  each  other  and  are  interconnected  at  the 
level  of  the  basal  granule  by  a  fibrous  component.  In  regeneration  experiments  three-day-old 
zooids  of  Amaroucinm  have  been  cut  at  the  base  of  the  branchial  basket,  the  latter  has  been 
removed  but  the  surrounding  tunic  has  not  been  detached.  During  the  next  two  days  regrowth 
proceeds  and  tissue  forms  a  stump  just  above  the  oesophagus.  On  the  third  day  primordia 
of  the  gill  slits  lie  horizontal  to  the  long  axis  of  the  zooid,  but  by  the  fifth  day  four  complete 
rows  of  slits  are  present  with  their  long  axes  parallel  to  that  of  the  zooid.  Calculations 
indicate  that  during  this  period  of  regeneration  a  minimum  number  of  50,000  cilia  is  repro- 
duced. The  subsequent  pattern  of  development  of  treated  zooids  appears  similar  to  control 
zooids  which  were  not  cut. 

The  effect  of  the  ovarian  steroids  on  the  membrane  potential  of  the  uterus.     M. 
GOTO  AND  A.  I.  CSAPO. 

Single  cells  of  the  rabbit  uterus,  in  well  defined  endocrine  conditions,  were  impaled  with 
flexible  microelectrodes  at  25°  C.  and  their  membrane  potential  recorded.  The  myometrial 


336         PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

cell  a  few  days  after  parturition  has  a  low  membrane  potential  of  about  35  mV.  Estrogen 
treatment  results  in  an  increase  up  to  48  mV,  whereas  progesterone  treatment  yields  values 
as  high  as  55  mV. 

Between  the  20th  and  29th  day  of  pregnancy  the  membrane  potential  of  the  uterus  at 
placental  implantation  sites  increases  up  to  60  mV,  whereas  the  interplacental  sites  remain  at 
a  value  of  48  mV.  Thus  the  placenta  exerts  a  local  effect  in  the  neighbouring  myometrium. 
Treatment  with  5  mg.  progesterone/day,  between  days  20-25,  increases  the  membrane  potential 
of  interplacental  sites,  and  the  difference  between  different  uterine  portions  disappears,  sug- 
gesting that  the  effective  placental  product  is  progesterone. 

The  membrane  potential  drops  and  the  difference  between  different  uterine  portions  dis- 
appears after  the  29th  day,  resulting  in  a  uniform  potential  of  about  50  mV  at  parturition. 

The  uterine  membrane  potential  is  a  log  function  of  the  K,  as  in  other  excitable  tissues. 
In  Ca-free  Krebs  the  membrane  potential  decreases  more  in  estrogen  than  in  progesterone 
dominated  uteri.  Action  potentials  after  oxytocin  treatment,  at  25°  C,  are  synchronous  and 
regular.  Local  potentials  of  different  frequency  appear  in  the  same  cell,  a  finding  which 
can  be  best  explained  by  intercellular  bridges,  transmitting  the  electrical  activity  of  one 
uterine  cell  to  the  next.  The  myometrial  cell  membrane,  on  which  excitability  and  contractility 
depend,  is  subject  to  endocrine  regulation.  Changes  in  membrane  potential  can  be  well  corre; 
lated  with  myometrial  function. 

Luciferin  and  luciferase  extracts  of  a  fisli,  Af>ogon  marginatns,  and  their  luminescent 
cross-reactions  with  those  of  a  crustacean,  Cypridina  Irilgendorfii.1  YATA 
HANEDA,  FRANK  H.  JOHNSON  AND  EDWARD  H.-C.  SIE. 

Despite  numerous  attempts  to  obtain  extracts  containing  the  relatively  heat-stable  substrate, 
luciferin,  and  heat-labile  enzyme,  luciferase,  respectively,  which  react  with  light  emission  in 
aqueous  solution  (the  "luciferin-luciferase  reaction"),  such  extracts  have  been  obtained  thus 
far  from  less  than  a  dozen  of  the  many  types  of  luminescent  organisms  known.  Moreover, 
until  now,  luminescent  cross-reactions  between  these  components  from  different  organisms 
have  been  found  only  with  extracts  of  fairly  closely  related  types,  such  as  different  families 
of  fireflies  or  different  genera  of  ostracod  Crustacea.  Recently  (1957),  the  first  clear  example 
of  the  luciferin-luciferase  reaction  among  fishes  was  demonstrated,  in  extracts  of  Parapria- 
canthus  bcrycifonnis,  family  Pempheridae.  A  second  example  is  reported  herewith,  in  extracts 
of  either  the  anterior  or  posterior  photogenic  organs  of  Apogon  marginaius,  family  Apogonidae, 
and  these  extracts  are  found  to  produce  light-emitting  cross-reactions  with  partially  purified 
luciferin  and  luciferase  of  the  ostracod  crustacean,  Cypridina  hilgendorfii.  Extracts  of  fresh 
or  desiccated  anterior  and  posterior  Apogon  light  organs  cross-react  with  each  other,  but  not 
with  extracts  of  non-photogenic  tissues  or  of  lanterns  of  the  Japanese  firefly,  Luciola. 

Quantitative  data  in  cross-reactions  between  the  Apogon  and  Cypridina  systems  reveal  that 
(1)  the  rate  of  light  emission  follows  first  order  kinetics;  (2)  doubling  the  enzyme  concen- 
tration doubles  the  rate;  (3)  total  light  is  proportional  to  initial  luciferin  concentration;  and 
(4)  total  light  is  nearly  the  same  with  the  luciferase  of  either  organism  acting  on  equal  aliquots 
of  luciferin  from  one  organism. 

Crude  Apogon  luciferin  dissolves  poorly  in  cool  water  but  readily  in  methanol,  giving  a 
yellow  solution  that  fluoresces  greenish  in  ultraviolet.  Studies  on  spectroscopy,  chromatography, 
and  possible  co-factors  of  the  Apogon  system  are  in  progress. 

Fluorescence,  pJiospJiorescence  and  biolurninescence  in  the  ctenophore,  Mnemiopsis 
leidyi.2  E.  NEWTON  HARVEY  AND  S.  P.  MARFEY. 

The  chemical  bioluminescent  system  of  Mnemiopsis,  located  in  the  radial  canals,  is  unique 
in  several  respects.  No  dissolved  oxygen  is  required  for  light  emission.  Bright  light  inhibits 
the  bioluminescence.  No  fluorescence  of  the  canals  occurs  in  near  ultraviolet  light  (Keese 

1  Aided  in  part  by  the  Office  of  Naval  Research,  National  Science  Foundation,  and  Eugene 
Higgens  Fund. 

2  Aided   by    grants    from    the    National    Science    Foundation    and    the    National    Institutes 
of  Health. 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY         337 

lamp,  3650  A)  in  resting  Mnemiopsis,  but  after  bioluminescence  has  occurred  the  canals  become 
bluish  fluorescent,  an  effect  which  slowly  disappears  if  the  animals  remain  unstimulated.  Such 
a  change  from  non-fluorescent  reductant  to  fluorescent  oxidant  occurs  among  flavins,  but  we 
do  not  imply  that  a  flavin  is  concerned  in  Mnemiopsis  bioluminescence,  as  it  is  in  luminous 
bacteria.  The  fluorescence  along  canals  is  much  brighter  at  acetone-dry  ice  temperature  (ca. 

-78°  C.),  and  there  is  also  a  phosphorescence,  lasting  about  3-6  seconds. 

If  ctenophores  are  placed  in  1%  sucrose  in  sea  water  and  then  held  at  acetone-dry  ice  tem- 
perature, no  marked  change  in  phosphorescence  was  observed.  The  phosphorescence  is  diffuse 
rather  than  sharply  localized  in  the  canal  region.  The  phosphorescence  is  not  connected  with 
the  bioluminescent  system,  because  (1)  it  can  be  observed  in  frozen  intercanal  tissue  of 
Mnemiopsis  in  sucrose  solution,  (2)  it  is  observed  from  frozen  mantle  and  viscera  of  the 
non-luminous  clam  (Mya).  Moreover,  light-adapted  ctenophores,  which  do  not  bioluminesce 
on  stimulation,  show  no  fluorescence  in  radial  canals  at  23°  C.  or  at  —  78°  C.,  but  with  sucrose 
added,  phosphoresce  diffusely  at  —  78°  after  near  ultraviolet  light  exposure.  Bioluminescent 
Chaetopterus  tissue  (rear  segments  with  eggs)  showed  fluorescence  and  phosphorescence  at 

-70°  C.,  whether  sucrose  was  added  or  not.  Far  ultraviolet  light  (Mineralite  lamp,  2735  A) 
does  not  excite  fluorescence  or  phosphorescence  of  stimulated  Mnemiopsis,  with  or  without 
sugar,  in  any  region  at  room  temperature,  or  at  —  70°  C.  Bioluminescence  and  fluorescence 
are  thus  connected,  but  phosphorescence  is  apparently  a  separate  phenomenon.  Mnemiopsis 
is  recommended  as  an  unusually  favorable  form  for  quantitative  study  of  light  effects. 

Evidence  for  the  splitting-off  of  S35-labelled  snlfate  from  the  fertilisin  of  Arbacia 
eggs  upon  the  spontaneous  reversal  of  sperm-agglutination.  RALPH  R.  HATHA- 
WAY l  AND  ALBERT  TYLER. - 

Arbacia  fertilizin  labelled  with  S35  was  obtained  as  described  by  Tyler  and  Hathaway 
(see  separate  abstract)  and  employed  in  a  series  of  sperm-agglutination  experiments.  As  is 
well  known,  the  agglutination  of  sperm  by  fertilizin  in  sea  urchins  reverses  spontaneously  under 
ordinary  conditions,  the  duration  of  agglutination  depending  upon  various  factors  including 
concentration  of  fertilizin,  concentration  of  sperm,  and  temperature.  Under  certain  conditions 
(excessive  washing  and  picric  acid  treatment)  reversal  does  not  occur.  The  present  experi- 
ments show  that  when  S35  fertilizin  is  absorbed  with  20%  sperm  suspensions  under  ordinary 
conditions  of  agglutination  and  reversal,  very  little  of  the  S85  is  removed  from  the  supernatant 
by  the  sperm  (13%,  14%,  23%,  and  28%  in  four  experiments).  On  the  other  hand,  removal 
of  S35  from  supernatants  results  from  absorption  with  dry  sperm  for  a  short  time  and/or  at 
low  temperatures  or  with  sperm  treated  so  as  to  inhibit  spontaneous  reversal.  For  example, 
in  three  experiments  at  room  temperatures  72%,  74%,  and  64%  was  removed  by  dry  sperm. 

At  0°  C.  similar  experiments  gave  95%,  91%,  83%,  83%,  98%,  and  72%  removed.  Simul- 
taneous short  absorption  experiments,  making  use  of  the  same  batches  of  dry  sperm  and 
fertilizin,  showed  81%  S35  removed  from  the  cold  supernatant  and  50%  removed  from  that  at 
room  temperature.  Excessively  washed  and  picric  acid  treated  sperm  removed  94%  and  99%, 
respectively. 

In  these  experiments  the  densities  of  the  mixtures  made  timing  of  the  duration  of  agglutina- 
tion and  reversal  difficult,  but  judging  from  the  intervals  between  mixing  and  centrifugation 
it  appears  that  reversal  is  accompanied  by  loss  of  sulfate  from  fertilizin  absorbed  to  sperm. 
This  implies  the  presence  of  an  active  sulfatase  on  the  sperm  and  that  the  phenomenon  of 
spontaneous  reversal  relates  to  the  liberation  of  sulfate  from  fertilizin. 

'  An  attempted  analysis  of  schooling  behavior  in  the  marine  snail  Nassarius  obsoletus. 
CHARLES  E.  JENNER. 

Schooling  in  mud  snails  in  Barnstable  Harbor  initiates  annually  as  a  result  of  a  behavior 
change  associated  with  the  abrupt  termination  of  reproductive  activity.  This  change  in  be- 

1  Supported  by  a  National  Science  Foundation  grant  to  Dr.  Charles  B.  Metz. 

2  Supported   by   a   research   grant    (C-2302)    from   the    National    Cancer    Institute,    U.    S. 
Public    Health    Service    and    by    AEC    Contract    AT  (30-1) -1343    to    the    Marine    Biological 
Laboratory. 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

havior,  marked  by  greater  locomotor  activity,  results  in  an  aggregated  distribution  pattern 
strikingly  different  from  the  dispersed  pattern  found  during  the  reproductive  phase.  Schooling 
is  most  apparent  when  snails  are  submerged.  With  exposure  at  low  tide,  many  snails  within 
a  schooling  group  will  stop  schooling  and  bury  in  the  sand,  whereas  others  within  the  group 
will  continue  to  school.  High  temperature  and  drying  of  the  substratum,  associated  with 
exposure  at  low  tide,  are  factors  which  deter  schooling.  Current  plays  an  important  role  in 
determining  direction  of  movement,  schooling  in  general  being  either  into  or  with  the  current. 
However,  schooling  can  be  observed  on  occasion  across  the  current  or  where  there  is  no  cur- 
rent. Physical  contact  also  plays  an  important  role  in  promoting  uniform  orientation.  Con- 
verging snails  upon  contact  will  generally  display  a  turning  response  and  then  proceed  along 
parallel  paths.  In  dense  concentrations  of  schooling  snails,  it  would  be  physically  impossible 
for  a  snail  to  move  in  any  direction  other  than  that  of  the  group.  Vision  and  chemical  factors 
may  also  play  a  role  in  schooling  but  this  has  not  yet  been  demonstrated. 

Neural  photosensitivity  in  Mactra.     DONALD  KENNEDY. 

Many  pelecypod  molluscs  which  lack  obvious  photoreceptor  structures  demonstrate  "shadow 
responses,"  in  which  a  diminution  of  light  intensity  incident  upon  the  siphon  causes  its  with- 
drawal. The  present  experiments  have  revealed,  through  oscillographic  recording  from  the 
pallial  nerve  in  Mactra,  the  presence  of  a  single  neuron  which  discharges  at  high  frequency 
(up  to  120/sec.)  upon  cessation  of  a  light  stimulus.  Such  off-discharges — or  the  spontaneous 
activity  in  the  photoreceptor  fibers  which  usually  occurs  in  the  dark — are  inhibited  with  short 
latency  by  re-illumination.  The  degree  of  inhibition  is  proportional  to  the  intensity  of  the 
re-illuminating  flash,  and  the  latency  of  inhibition  inversely  so.  Latency  and  frequency  of  the 
off-discharge  itself,  however,  vary  in  a  complex  fashion  with  respect  to  the  intensity  of  a 
single  stimulus ;  the  discharge  can,  in  fact,  be  treated  as  a  post-inhibitory  rebound  phenomenon. 

The  discharge  may  be  recorded  with  unimpaired  threshold  and  pattern  in  excised  segments 
of  the  pallial  nerve  from  the  siphonal  region.  It  is  thus  apparent  that  the  primary  receptor 
is  a  neural  element.  In  Mya  and  Venus,  off-discharges  occur  in  many  fibers  of  the  siphonal 
nerves,  but  these  originate  in  end-organs  within  the  siphon.  The  nerves  of  Mactra  (but  not 
of  the  other  two  species)  contain  a  pinkish-red  pigment.  Experiments  are  in  progress  to  de- 
termine whether  this  pigment  endows  the  receptor  neuron  with  its  light  sensitivity. 

These  findings  suggest  that  off-responses  do  not  always  arise  as  a  result  of  inhibitory 
synaptic  interactions  between  on-responding  units,  as  is  clearly  the  case  in  several  of  the 
more  complex  photoreceptor  systems.  Such  off-responses  as  those  from  the  distal  cell  layer 
in  the  mantle  eye  of  Pcctcn,  which  were  presumed  by  Hartline  to  be  of  secondary  origin,  may 
in  fact  prove  to  be  primary. 

The   humoral  control   of  feeding   in   Physalia   and   its   evolutionary   significance. 
HOWARD  M.  LENHOPF  AND  HOWARD  A.  SCHNEIDERMAN. 

The  demonstration  of  Loomis  that  reduced  glutathione  (GSH)  induces  the  feed-response 
in  hydra  led  us  to  examine  the  feeding  response  of  a  distantly  related  hydrozoan,  the  siphono- 
phore  Physalia  physalis  L.  During  normal  feeding,  Physalia  draws  up  prey  to  its  gastrozooids, 
the  only  members  of  the  colony  capable  of  ingesting  food.  The  gastrozooids  apply  their 
mouths  to  the  surface  of  the  prey  and  their  lips  spread  out  to  envelop  it.  This  "spreading" 
of  the  mouths  can  be  elicited  in  isolated  gastrozooids  by  fish  blood.  The  active  principle  in 
the  blood  appears  to  be  GSH.  The  sensitivity  to  GSH  is  remarkable:  10~"  M  caused  90% 
of  the  isolated  cylindrical  gastrozooids  to  spread  their  lips  on  a  glass  dish  and  transform 
into  discs,  often  more  than  20  mm.  in  diameter.  Cysteine  did  not  induce  this  response.  The 
behavior  of  isolated  gastrozooids  in  the  presence  of  GSH  is  identical  with  the  normal  feeding 
behavior  of  gastrozooids  in  the  intact  animal.  Hence  it  appears  that  in  the  feeding  process 
of  Physalia,  as  in  hydra,  the  nematocysts  pierce  the  prey,  thus  releasing  body  fluids  containing 
GSH.  This  GSH  induces  the  feeding  response  in  the  gastrozooids.  Consequently,  like  hydra, 
Physalia  only  feeds  on  forms  with  body  fluids. 

The  chemical  similarities  of  the  nematocyst-GSH  feeding  mechanism  of  Physalia  and 
hydra  invite  inquiry  into  evolutionary  relations  among  the  Cnidaria.  Evidence  was  presented 
that  a  hydrozoan  stem-form  using  the  nematocyst-GSH  feeding  mechanism  evolved  along 
with  animals  having  body  fluids  containing  GSH  which  could  be  released  when  pierced  by 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY         339 

nematocysts  ;  only  animals  of  a  higher  grade  of  organization  than  Cnidaria  fit  this  category. 
It  was  also  argued  that  the  ancestral  cnidarian,  from  which  all  Cnidaria  arose,  probably  did 
not  use  the  nematocyst-GSH  feeding  mechanism,  but  was  a  filter  feeder  like  many  present-day 
Anthozoa. 

Genes  controlling  the  movement  of  flag  ell  a  in  Chlamydomonas.     RALPH  A.  LEWIN. 

By  genetic  analysis  of  paralyzed  mutants  of  C.  moewusii,  several  of  the  genes  controlling 
the  movement  of  flagella  have  been  located  in  linkage  groups.  Two  loci,  about  9  units  apart, 
are  situated  on  the  sex  chromosome  ;  one  of  these  is  extremely  close  to  the  mating-type  locus, 
about  11  units  from  the  centromere.  Three  mutations  have  been  located  at  a  single  locus 
close  to  the  centromere  of  a  second  chromosome.  One  of  these  alleles  is  distinguishable  from 
the  others  in  its  degree  of  paralysis.  Two  other  motility  genes  are  situated  on  a  third  and  a 
fourth  chromosome,  respectively  ;  one  is  closely  linked  with  its  centromere,  the  other  is  not. 

Following  ultra-violet  irradiation,  apparent  reversions  of  several  mutants  were  isolated. 
Three  behaved  genetically  like  wild-type,  indicating  reverse  mutation  at  the  original  locus. 
One  proved  completely  sterile.  A  fifth,  when  crossed  with  wild-type,  segregated  irregularly, 
indicating  a  suppressor  gene  or  complex. 

Fractionation  of  Cypridina  luciferin  and  its  bcnsoyl  derivative.1     S.  P.  MARFEY,  L. 
C.  CRAIG  AND  E.  N.  HARVEY. 

Dried,  fat-free  Cypridina  powder  was  used  for  isolation  of  luciferin  by  extraction  with 
methanol  in  purified  nitrogen  at  4°  C.,  arid  subsequent  fractionation  by  countercurrent  distribu- 
tion, paper  chromatography,  column  chromatography,  dialysis,  and  Reineckate  and  flavianate 
precipitation.  The  best  method  proved  to  be  countercurrent  distribution  in  purified  nitrogen 
atmosphere  at  4°  C.  employing  a  variety  of  acidic  solvent  systems  (containing  ascorbic  acid 
which  reduces  the  autoxidation  of  luciferin)  for  the  separation  of  two  active  luciferin  fractions. 
Evidence  was  obtained  from  distribution  data  for  the  transformation  of  one  of  these  fractions. 
The  best  sample  prepared  by  this  method  had  a  chemiluminescent  activity  with  luciferase  at 
least  6000  times  (by  weight)  that  of  fat-free  Cypridina  powder  and  after  total  acid  hydrolysis 
gave  predominantly  four  amino  acids  (Lys,  Asp,  Glu,  lieu)  and  ammonia  together  with 
smaller  amounts  of  several  additional  amino  acids. 

Benzoyl  luciferin,  more  stable  toward  air  oxidation,  fractionated  by  dialysis  and  counter- 
current  distribution  at  25°  C.  yielded  several  active  fractions.  These  were  analyzed  by  two 
dimensional  paper  chromatography,  giving  in  each  case  a  neutral  yellow-colored  blue  fluorescent 
area,  and  after  total  acid  hydrolysis  several  amino  acids  (including  those  found  in  a  larger 
amount  in  native  luciferin)  in  addition  to  an  inactive  chromophore.  Quantitative  infrared 
spectral  analysis  of  these  active  fractions  revealed  a  variable  degree  of  benzoylation  compatible 
with  their  polarity  in  a  distribution  train.  Redistribution  of  one  of  these  fractions  resulted 
in  a  single  band  close  to  a  theoretical  curve  for  one  component. 

These  results  give  an  additional  evidence  for  the  chromopeptide  nature  of  luciferin  and 
indicate  at  least  two  active  types  of  luciferin  separable  by  countercurrent  distribution.  The 
nature  of  their  difference  is  currently  under  investigation. 

Changes  in  efflux  and  influx  of  potassium  upon  fertilization  in  eggs  of  Arbacia 
punctulata,  measured  by  use  of  K*'2.2     ALBERTO  MONROY  3  AND  ALBERT  TYLER. 

A  preliminary  set  of  experiments  in  1956  indicated  an  increase,  upon  fertilization,  in  the 
rate  of  release  of  K42  from  eggs  that  had  been  loaded  with  this  isotope.  The  present  series 
of  experiments  substantiates  this  finding  and  permits  closer  estimate  of  the  magnitude  and 
the  time  course  of  the  change.  The  average  values  for  25  sets  of  experiments  for  %, 


1  Aided    by    grants    from   the    National    Science    Foundation    and    the    National    Institutes 
of  Health. 

2  Supported  by  a  Research   Grant    (C-2302)    from  the   National   Cancer   Institute,   U.    S. 
Public    Health    Service    and    by    AEC    Contract    AT    (30-1)  -1343    to    the    Marine    Biological 
Laboratory. 

3  F.  R.  Lillie  Memorial  Fellow,  Summer,  1958. 


340         PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

2,  4  and  6  minutes  after  fertilization  are  129%,  154%,  102%,  70%  and  58%,  respectively,  for 
the  excess  K42  released  by  fertilized  as  compared  with  unfertilized  eggs.  The  corresponding 
^-values  for  the  statistical  significance  of  the  difference  of  these  values  from  zero,  are  5.1 
(P<0.01),  5.2  (P<0.01),  4.54  (P<0.01),  3.06  (P<0.01)  and  1.88  (P<0.1  and  >0.05). 

The  rate  of  influx  of  K42  was  followed  over  longer  periods  of  time  (up  to  6  hours)  and 
found  to  be  considerably  greater  in  the  fertilized  than  in  the  unfertilized  eggs.  The  calcula- 
tions show  that,  at  6  hours,  K42  in  the  unfertilized  eggs  is  far  below  (about  4:1)  the  estimated 
equilibrium  value  (ca.  20:1  for  Kmsiae/Koutsiae),  suggesting  that  much  of  the  K+  is  not  freely 
diffusible.  The  K42  uptake  from  the  fertilized  eggs  has  reached  the  expected  equilibrium  value 
before  this  time. 

In  general  the  rapid  change  in  rate  of  efflux  of  K+  upon  fertilization  is  consistent  with 
the  expectation  from  earlier  experiments  (Tyler,  Monroy,  Kao  and  Grundfest,  Biol.  Bull.,  Ill, 
1956)  demonstrating  the  existence,  in  the  unfertilized  echinoderm  egg,  of  a  membrane  poten- 
tial that  undergoes  a  transient  drop  during  the  first  minute  after  fertilization  and  which  is 
reversibly  abolished  by  increase  in  external  K+. 

Action  of  enzymes  on  tJie  hyalines  of  the  Arbacia  ccjcj.     A.  K.  PARPART,  ].  CAGLE 
AND  L.  WOOD. 

The  hyaline  layer  (No.  1)  of  the  egg  of  Arbacia  punchilata  is  formed  from  cortical 
granules  shortly  after  fertilization.  At  first  cleavage  another  layer  of  hyaline  (No.  2)  is 
formed  from  blebs  from  the  furrow  surface.  There  are  therefore  at  least  two  hyalines  formed. 

The  action  of  various  enzymes  on  these  two  hyaline  layers  has  given  the  following 
results.  Proteolytic  enzymes,  trypsin,  chymotrypsin  and  papein  have  no  effect.  Lipase  also 
fails  to  alter  these  hyaline  layers.  Amylolytic  enzymes  act  as  follows :  a-amylase  digests  and 
removes  hyaline  No.  1  ;  it  does  not  alter  hyaline  No.  2.  /3-amylase  has  very  slight  digestive 
action  on  the  two  types  of  hyaline.  However,  it  does  cause  a  marked  release  of  hyaline  No.  2 
from  the  egg  at  the  time  of  cleavage.  Hyaluronidase  loosens  but  does  not  digest  hyaline 
No.  1,  while  it  does  digest  most  of  hyaline  No.  2.  The  action  of  the  enzymes  was  studied 
on  eggs  whose  fertilization  membranes  did  not  form  due  to  prior  treatment  with  trypsin. 

These  and  previous  studies  lead  to  the  conclusion  that  hyaline  No.  1  is  a  mucopolysaccharide 
while  hyaline  No.  2  is  composed  of  hyaluronic  acid  and/or  chondroitin  sulfuric  acid. 

The   action    of   certain   chemical   agents    upon   squid   chromatophores.     WILLIAM 

ROSENBLUM  AND  BENJAMIN  Z \VEIFACH. 

The  chromatophores  of  cephalopod  molluscs  may  provide  an  important  tool  for  studying 
the  basic  mechanisms  controlling  smooth  muscle  activity.  These  chromatophores  are  altered 
in  size  by  the  contraction  or  relaxation  of  smooth  muscle  fibers,  each  of  which  is  innervated 
by  branches  from  the  "CNS"  of  the  squid.  Relaxation  of  the  muscle  fibers  constricts  the 
chromatophores ;  contraction  expands  them. 

Substances  dissolved  in  filtered  sea  water  were  injected  subcutaneously  into  the  squid, 
Loligo  pcalii,  placed  alive  in  sea  water  below  10  degrees.  The  following  monamines  were 
found  to  cause  local  constriction  of  the  chromatophores  (relaxation  of  the  muscle  fibers)  : 
tryptamine  HCL,  tyramine  HCL,  and  5-hydroxy-tryptamine  creatinine  sulfate  (serotonin). 
Serotonin  was  effective  in  lower  dosage  (0.2  Mg/ml.)  than  any  of  the  other  substances. 

These  inhibitors  of  monamine  oxidase  also  were  found  to  constrict  the  chromatophores : 
para  isopropyl  hydrazine,  2-benzyl-l  picolinyl  hydrazine,  and  iproniazid. 

Tryptophan  HCL,  5-hydroxy  tryptophan  HCL,  histamine  dihydrochloride,  L-lysine  and 
L-serine  had  no  effect. 

These  data  are  consonant  with  the  theory  that  chromatophore  control  at  the  local  level 
is  achieved  by  the  local  release  of  an  amine  and  its  continual  destruction  by  amine  oxidase. 

Other  agents  which  constricted  the  chromatophores  were :  LSD-25  which,  like  serotonin, 
contains  an  indole  nucleus ;  chlorpromazine,  an  autonomic  blocking  agent ;  and  eserine  sulfate, 
an  inhibitor  of  acetylcholine  esterase.  The  latter  is  effective  in  high  dosage  (1  mg./ml.). 

Acetylcholine  dilated  these  chromatophores.  Serotonin  reversed  this.  It  may  be  that 
chlorpromazine  acts  by  blocking  the  action  of  a  naturally  occurring  dilating  agent,  such  as 
acetylcholine,  and  thereby  allows  the  constricting  agent  to  act  unopposed.  The  effect  of  eserine 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY         341 

may  be  attributed,  as  in  the  case  of  paralysis  of  other  nerve-muscle  systems  by  eserine,  to  inter- 
ference with  the  cholinergic  mechanism. 

It  may  be  significant  that  all  the  constricting  substances  contain  a  resonating  moiety. 

(This  work  was  made  possible  by  a  grant  to  one  of  us — W.  R. — from  The  United  States 
Public  Health  Service.) 

Contraction  without  membrane  potential  change.     T.  SAKAI  AND  A.  I.  CSAPO. 

It  had  been  shown  in  our  laboratory  that  muscles,  rendered  non-propagating  by  treatment 
with  procaine  or  substitution  of  Na  with  choline,  in  excess  K  Ringer,  contract  along  their 
entire  length  (except  the  extreme  anodal  portion)  when  stimulated  in  a  longitudinal  field. 

It  is  of  considerable  significance  to  determine  whether  activation  of  the  myoplasm,  in  rest- 
ing muscle  pre-treated  with  excess  K,  requires  depolarisation  of  the  excitable  membrane  or 
that  currents  flowing  inside  the  muscle  fibers,  as  a  result  of  the  applied  longitudinal  electric 
field,  can  accomplish  this  task.  We  have  measured,  therefore,  with  microelectrodes  the  mem- 
brane potential  changes  along  the  length  of  the  muscle  during  the  application  of  the  longi- 
tudinal electric  field,  under  similar  conditions  in  which  shortening  has  been  previously  deter- 
mined. We  found  that  the  membrane  potential  is  unchanged  in  the  middle  portion  and  is 
increased  in  the  anodal  half  of  the  muscle  where  shortening  does  take  place.  Thus  the  longi- 
tudinal field  can  activate  a  K  depolarised  resting  muscle  without  change  or  even  with  slight 
increase  in  membrane  potential.  Since  the  "priming"  step,  accomplished  by  K  treatment, 
leaves  behind  a  resting  muscle,  it  is  justified  to  conclude  that  steps  subsequent  to  depolarisation 
are  required  to  complete  the  coupling  process.  These  then  are  more  directly  linked  to  activa- 
tion than  is  depolarisation.  Currents,  which  are  known  to  flow  inside  the  muscle  fibers  during 
normal  excitation,  may  well  contribute  to  one  of  these  steps. 

The  unique  significance  assigned  to  depolarisation  in  the  activation  process  is  also  chal- 
lenged by  the  experiment  in  which  the  fiber  membrane  was  removed  by  micro-surgery  and 
the  "naked"  myofibrils  were  activated,  under  oil,  by  longitudinal  current.  The  threshold  cur- 
rent required  for  the  activation  of  naked  myofibrils  is  of  the  same  order  of  magnitude  as  the 
myoplasmic  currents  of  normal  excitation. 

Antibacterial  action  of  Limulus  blood  in  an  in  vitro  system.     MANOHAR  V.  SHIROD- 
KAR,  FREDERIK  B.  BANG  AND  ANNE  WARWICK. 

An  "intact  system"  was  developed  based  on  the  previous  findings  of  Bang  and  Warwick. 
Two  cc.  of  Limulus  blood  in  a  siliconized  syringe  and  needle  were  explanted  sterilely  into 
a  siliconized  roller  tissue  culture  tube  and  kept  in  a  rotating  drum  at  room  temperature.  A 
high  percentage  of  amoebocytes  remained  intact  in  shape  and  granule  content  for  over  30  days 
without  addition  of  nutrient  medium.  No  antibiotics  were  used.  Two  such  tubes  were  kept 
unstoppered  for  a  month  without  showing  bacterial  contamination  or  major  cytological  changes. 
A  "partially  intact"  system  was  similar  in  other  respects  except  that  no  silicone  was  used 
and  the  majority  of  cells  lost  granules  and  changed  to  a  flattened  form.  Use  of  the  intact  and 
partially  intact  systems  was  made  in  demonstrating  the  potent  antibacterial  action  of  Limulus 
blood.  Sterile  artificial  sea  water  without  sodium  bicarbonate  was  used  for  dilutions  and 
samples  were  plated  on  ZoBell's  sea  water  agar.  A  number  of  timed,  quantitative  experi- 
ments were  performed  with  bacterium  No.  5,  a  gram-negative,  motile  rod  isolated  from  oysters. 
Within  6  hours'  incubation  at  room  temperature  in  the  intact  system,  24,000,000  bacteria  were 
eliminated,  no  viable  bacterium  being  recoverable  even  after  120  hours.  The  partially  intact 
system  showed  some  antibacterial  activity  up  to  24  hours,  after  which  the  bacteria  reappeared 
and  grew  successfully.  Other  experiments  showed  that  an  intact  system  could  not  inhibit 
growth  of  some  bacteria  except,  perhaps,  at  very  high  dilutions.  Good  to  intermediate  anti- 
bacterial activity  was  demonstrable  against  6  of  the  8  different  bacteria  tested  in  the  intact 
system.  Current  experiments  have  thus  far  failed  to  show  antibacterial  activity  in  the  serum 
fraction,  alone,  of  Limulus  blood. 

Amino  acid  uptake  in  marine  invertebrates.    G.  C.  STEPHENS  AND  R.  A.  SCHINSKE. 

These  observations  were  carried  out  to  extend  our  previous  report  of  the  uptake  of  amino 
acids  from  sea  water  by  ciliary-mucoid  filter  feeding  animals.  Thirty-five  species,  representing 


342         PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

12  animal  phyla,  were  used.  Glycine  was  used  in  all  cases ;  sometimes  additional  amino  acids, 
such  as  alanine,  methionine,  glutamic  acid,  arginine,  phenylalanine  and  tyrosine  were  employed. 
After  placing  an  animal  in  2  mM  amino  acid,  periodic  samples  of  the  solution  showed  a 
quantitative  decrease  of  the  acid  when  measured  by  means  of  a  ninhydrin  colorimetric  technique. 

The  addition  of  suitable  concentrations  of  penicillin,  streptomycin  or  tetracycline  had  no 
apparent  effect  on  the  uptake  of  amino  acid.  This,  together  with  the  negative  results  for 
arthropods,  indicates  that  bacteria  are  not  responsible  for  amino  acid  removal.  Such  removal 
also  occurs  from  artificial  sea  water.  Finally,  it  was  possible  to  demonstrate  directly  a  3%-8% 
drop  in  amino  acid  concentration  when  water  collected  from  the  excurrent  siphon  of  the  clam 
Splsula,  was  compared  with  that  collected  from  the  incurrent  siphon. 

The  concentration  of  amino  acids  used  in  most  of  these  observations  was  approximately 
two  orders  of  magnitude  above  estimated  content  of  organic  material  in  naturally  occurring 
sea  water.  Consequently  experiments  were  done  using  0.02  mM  amino  acid  solutions.  By 
means  of  concentration  and  subsequent  acetone-HCl  extraction,  uptake  at  these  levels  could 
be  detected.  The  time  for  total  clearance  in  those  cases  where  it  was  observed  was  not 
strikingly  different  despite  this  hundred-fold  difference  in  concentration. 

No  effort  was  made  to  confine  attention  to  ciliary-mucoid  filter  feeders.  Several  detritus 
feeders  and  large  particle  feeders  were  included  among  the  species  manifesting  this  capacity 
for  amino  acid  removal. 

Synthesis  of  ribonucleic   odd   by   nuclcoli.     W.    S.   VINCENT,   B.   BENSAM   AND 
ARLENE  BENSAM. 

Although  the  nucleolus  is  known  to  contain  RNA  there  has  been  no  satisfactory  demon- 
stration that  it  actually  synthesizes  this  material.  In  the  experiments  described  below  we 
present  evidence  which  indicates  that  the  starfish  oocyte  nucleolus  either  synthesizes  or  accumu- 
lates newly  synthesized  RNA. 

If  cells  are  incubated  in  the  presence  of  a  supply  of  radiophosphate,  the  pools  of  5'  nucleo- 
tides  will  be  labelled  and  subsequently  incorporated  into  the  RNA  polynucleotide  chain.  If 
synthesis  of  RNA  occurs,  isolation  and  hydrolysis  of  the  RNA,  followed  by  separation  of  the 
nucleotides,  will  reveal  the  presence  of  radiophosphate  in  all  four  of  the  constituent  nucleotides. 
Lack  of  label  in  nucleotides  is  indicative  of  no  new  synthesis  of  RNA. 

When  starfish  ovaries  were  incubated  in  sea  water  containing  P32  for  1%  hours,  the  RNA 
from  the  nucleoli  contained  considerable  radioactivity  which  was  tightly  bound  to  the  poly- 
nucleotide. After  hydrolysis  by  alkali  and  separation  of  the  nucleotides  by  paper  electrophoresis 
no  radioactivity  could  be  detected  in  any  of  the  nucleotides.  All  of  the  label  formerly  asso- 
ciated with  the  RNA  was  found  as  inorganic  phosphate.  Other  experiments  revealed  that 
this  phosphate  could  not  be  removed  from  the  RNA  by  7-minute  hydrolysis  in  acid,  indicating 
that  it  was  not  bound  by  a  pyrophosphate  linkage. 

When  incubation  time  was  extended  to  6  hours  all  of  the  nucleotides  were  found  to  contain 
the  radioisotope,  indicating  the  presence  of  newly  synthesized  RNA.  Considerable  amounts 
of  the  additively  bound  phosphate  found  in  the  iVs-hour  experiments  were  still  present. 

These  experiments  are  interpreted  as  demonstrating  an  initial  binding  by  nucleolar  RNA 
of  phosphate  entering  the  nucleolus.  Subsequently  either  this  bound  phosphate,  or  phosphate 
from  other  sources,  is  found  in  newly  synthesized  RNA  in  the  nucleolus. 


PAPERS  READ  BY  TITLE 

Methyl  green  "vital  staining"  in  Arbacia  eggs.     WALTER  AUCLAIR. 

E.  B.  Harvey  reported  that  methyl  green  gives  a  distinctive  purple  stain  to  the  mitochondria 
of  centrifuged  unfertilized  eggs  and  this  has  been  confirmed  in  the  present  experiments.  In 
addition,  it  was  found  that  single  spherical  granules  situated  each  in  one  of  the  echinochrome 
vesicles,  and  each  having  a  diameter  about  Vs  that  of  the  vesicle,  take  an  intense  blue-black 
color  when  eggs  are  placed  in  methyl  green-sea  water  solutions  (1:10,000)  for  5-10  minutes. 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY         343 

Such  stained  granules  are  also  found  in  uncentrifuged  eggs,  both  fertilized  and  unfertilized, 
and  in  fertilized  eggs  centrifuged  with  and  without  pressure  (6000  lbs./in.2)  at  41,000  X  gravity. 
After  a  thorough  washing  of  the  eggs  in  sea  water,  the  staining  persists  throughout  subsequent 
development.  Equivalent  staining  of  the  granules  in  the  pigment  vesicles  takes  less  time 
(3-5  minutes)  after  centrifugation  than  without  centrifugation(8-10  minutes). 

Seen  at  high  magnification  (900  X),  the  movement  of  these  methyl  green-stained  granules 
appears  to  be  distinctive.  Each  maintains  continuous  contact  with  the  wall  of  the  pigment 
vesicle  in  which  it  lies  and  yet  all  the  granules  continually  display  a  sort  of  dancing  movement. 
This  implies  that  each  vesicle  may  display  a  haphazard  sort  of  rotational  movement,  but  this 
tends  to  diminish  as  the  successive  cleavages  ensue.  In  unfertilized  eggs  the  echinochrome 
vesicles  display  sudden,  short,  straight-line  excursions  in  addition  to  the  rotational  movement. 

Cleavage  of  stained  eggs  is  somewhat  abnormal.  Often  the  first  cleavage  is  aborted  and 
two  or  even  three  nuclei  may  be  seen  in  the  single  cell.  At  the  time  when  second  cleavage 
normally  occurs,  many  of  the  stained  eggs  divide  into  two,  or,  more  frequently,  into  three 
blastomeres.  Subsequently  the  blastomeres  are  apt  to  be  of  different  sizes,  and  tend  not  to 
adhere  together,  but  about  30  per  cent  develop  to  swimming  blastulae  (mainly  abnormal). 

Antibacterial  activity  of  Phascolosoma  govldii  blood.1     F.  B.  BANG  AND  S.   M. 
KRASSNER. 

Blood  removed  from  the  body  cavity  of  normal  Phascolosoma  gouldii,  a  sipunclid  worm, 
was  found  to  be  sterile  when  cultured  on  ZoBell's  sea  water  agar  at  room  temperature. 

Several  strains  of  marine  bacteria  were  destroyed  within  twenty-four  hours  when  more 
than  100  million  organisms  were  injected.  An  occasional  infection  was  produced  which 
eventually  killed  the  Phascolosoma.  Blood  removed  from  the  worms  was  incubated  at  room 
temperature  with  varying  concentrations  of  different  bacteria  and  in  a  number  of  individual 
tests  became  sterile  within  six  to  twenty-four  hours.  Consistent  sterility  was  obtained  within 
six  to  twenty-four  hours  when  the  combination  of  bacteria  and  blood  was  kept  at  0°  C.  Various 
control  preparations  kept  these  bacteria  alive  for  days  at  this  temperature.  At  0°  C.  incuba- 
tion activity  was  found  in  both  serum  and  cells.  Destruction  of  1  million  organisms  was 
obtained  with  0.2  cc.  of  whole  blood  within  twenty-four  hours.  Sera  and  bacteria  kept  at 
0°  C.  failed  to  agglutinate. 

Enrichment  studies  on  the  photosynthetic  sulfur  bacteria.     EDWIN  H.  BATTLEY. 

Although  several  genera  of  the  Thiorhodaceae  and  Chlorobacteriaceae  have  been  recog- 
nized, largely  on  the  basis  of  a  morphological  classification,  extensive  studies  of  these  organisms 
have  been  hindered  by  a  lack  of  pure  cultures.  This  lack  may  possibly  exist  because  classical 
methods  of  selectively  isolating  these  bacteria  from  their  natural  habitat  most  frequently  give 
rise  to  organisms  of  the  Chlorobiuin  or  of  the  Chromatium  type.  For  this  reason  it  was  thought 
worth  while  to  search  for  methods  of  selectively  growing  other  types  of  photosynthetic  sulfur 
bacteria.  An  inspection  of  their  marine  habitats  made  it  appear  that  these  bacteria  grew  most 
abundantly  in  the  presence  of  decomposing  plant  or  animal  matter.  This  made  it  seem  that 
classical  enrichment  media,  supplemented  with  vitamins,  trace  elements  and  various  organic 
carbon  donors,  might  select  differently.  Thus,  a  basic  medium  containing  the  usual  amounts 
of  phosphate,  carbonate,  sulfate,  ammonia  nitrogen,  potassium,  magnesium,  calcium  and  iron 
was  used,  plus  2.4  per  cent  sodium  chloride.  Zinc,  boron,  cobalt,  copper  and  manganese  were 
added  as  trace  elements,  and  biotin,  calcium  pantothenate,  inositol,  nicotinamide,  para-amino 
benzoic  acid,  thiamin  hydrochloride  and  riboflavin  as  vitamins.  Specific  media  were  made  by 
adding  to  the  basic  medium  0.4  per  cent  sodium  sulfide  or  1.0  per  cent  sodium  thiosulfate, 
and  0.1  per  cent  sodium  acetate,  0.05  per  cent  peptone  or  0.05  per  cent  yeast  extract.  The 
specific  media  were  adjusted  to  pH  7.0  or  8.0.  Mixtures  were  made  of  the  specific  media 
and  samples  of  marine  mud.  Glass-stoppered  bottles  were  then  filled  with  the  mixtures  and 
placed  at  room  temperature  under  strong  illumination.  Abundant  growth  usually  occurred 
within  three  or  four  days.  After  the  third  transfer  predominant  organisms  were  present  to 

1  Supported  by  a  grant-in-aid  from  the  National  Institutes  of  Health. 


344         PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

the  extent  of  90  per  cent  or  better.     About  half  of  these  organisms  have  not  been  previously 
described. 

A  Bermndian  marine  Vaucheria  at  Cape  Cod.     A.  J.  BERNATOWICZ. 

Vaucheria  nasuta  Taylor  and  Bernatowicz,  heretofore  known  only  from  Bermuda,  is  re- 
ported from  the  salt  marsh  at  Barnstable,  Massachusetts.  With  this  record  the  number  of 
marine  Vaucherias  known  from  northeastern  North  America  is  raised  to  ten,  of  which  seven 
occur  on  Cape  Cod.  Records  of  the  earlier  finds  suggested  that  reproduction  in  local  Vaucherias 
may  be  confined  to  the  winter  months,  but  the  present  material  was  reproductive,  although 
sparsely,  in  August. 

An  inhibitory  extract  of  chick  tissues.     MAXWELL  H.  BRAVERMAN. 

The  imposition  of  extracts  of  adult  tissues  upon  embryonic  systems  has  proved  an  excellent 
tool  for  investigating  the  order  of  tissue  differentiation  and  mode  of  tissue  interaction.  The 
organ  or  organ  part  to  be  used  in  the  test  is  cut  from  a  freshly-killed  animal  and  immediately 
put  into  Tyrode's  solution  (adjusted  to  pH  7.4-7.6)  at  0°  C.  All  subsequent  operations  are 
carried  out  at  this  temperature.  The  tissue  is  homogenized  in  a  tissue  homogenizer  by  hand 
or  in  a  test  tube  fitted  with  a  plastic  plunger  which  is  mechanically  rotated.  The  homogenate 
is  centrifuged  at  5000  X  g  in  a  pre-cooled  centrifuge  and  the  supernatant  filtered  through  a 
Seitz  filter  under  pressure.  The  clear  liquid  is  stored  for  the  t\vo  or  three  hours  between 
preparation  and  utilization  in  injection  bottles  which  hold  10  cc. 

The  bottles  are  heated  immediately  before  use  to  about  35°  C.  by  placing  them  in  small 
water-filled  beakers  on  a  slide  warmer.  Two-tenths  cc.  of  the  liquid  is  removed  into  a 
0.25-cc.  syringe  previously  sterilized  at  160°  C.  for  40  minutes  in  an  autoclave.  The  solution 
is  slowly  injected  beneath  the  blastoderm  of  an  egg  which  has  been  incubating  for  24  hours. 
To  make  the  injection,  a  square  window  is  sawed  in  the  shell  with  an  "Xacto"  No.  34  "razor 
saw"  and  a  small  amount  of  albumin  removed  by  means  of  oral  suction,  into  a  glass  pipette. 
An  attempt  is  made  to  put  the  needle  in  as  closely  parallel  to  the  egg  surface  as  possible,  thus 
liberating  the  fluid  close  to  the  blastoderm.  The  hole  is  sealed  with  Scotch  tape. 

The  eggs,  after  24  hours'  incubation  at  37.5°  C.  are  in  Lillie's  stage  4  or  5.  The  injected 
eggs  are  examined  from  24  hours  to  five  days  after  injection. 

The  explanation  of  the  tu'o-day  physiological  anticipation  of  barometric  pressure 
changes.1     FRANK  A.  BROWN,  JR.  AND  FRANKLIN  H.  BARNWELL. 

It  has  been  reported  that  a  wide  variety  of  animal  and  plant  species  exhibit,  notably  in 
their  5-6-7  PM  metabolic  rate,  a  lead  correlation  with  the  mean  daily  barometric  pressure 
of  the  second  day  thereafter.  This  they  do  even  when  maintained  in  constant  conditions, 
including  pressure.  The  mean  lead  correlations  have  been  reported  to  range  from  r  —  .27 
to  r  =  .84  with  an  average  of  about  0.5.  In  attempting  to  account  for  this  extraordinary 
capacity  of  living  things  it  was  recalled  that  the  6  PM  metabolic  rate  had  been  found  correlated 
with  the  concurrent  2-6  PM  rate  of  barometric  pressure  change.  It  was  now  found  that  the 
2-6  PM  rate  of  pressure  change  itself,  in  Chicago,  during  each  of  the  years  1954  to  1957, 
while  showing  little  or  no  correlation  with  the  mean  pressure  of  the  same  day  (r  —  —  .14), 
was  showing  a  maximum  correlation  (r=:  +  .35)  with  the  pressure  of  the  second  day  there- 
after. The  form  of  the  lag-lead  correlation-relationships  between  these  two  pressure  parame- 
ters was  found  to  resemble  very  closely  the  form  of  the  published  lag-lead  correlation  relation- 
ships between  mean  pressure  and  the  6  PM  metabolic  rate  in  organisms  as  diverse  as  fiddler 
crabs  and  potatoes.  From  this,  it  is  concluded  that  a  direct  response  of  the  organism  to  some 
pervasive  external  force  correlated  with  the  2-6  PM  rate  of  pressure  change  comprises  the 
means  of  the  reported  organismic  anticipation  of  the  pressure  changes. 

1  These  studies  were  aided  by  a  contract  between  the  Office  of  Naval  Research,  Department 
of  Navy,  and  Northwestern  University,  NONR-122803. 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY         345 

The  rhythmic  nature  of  metabolism  in  Ilyanassa  in  constant  conditions.1  FRANK 
A.  BROWN,  JR.,  WILLIAM  J.  BRETT  AND  H.  MARGUERITE  WEBB. 

Hourly  values  of  Da-consumption  of  the  mud  snail,  Ilyanassa,  were  obtained  continuously 
for  33  days  under  conditions  of  constant  temperature,  pressure  and  illumination.  On  no  day 
were  less  than  16  nor  more  than  48  snails  involved.  During  this  study  there  was  found  to  be 
a  highly  significant  solar-day  cycle  (9.7%)  with  a  major  maximum  about  midnight  and  a 
lesser  maximum  about  noon.  There  was  a  lunar-day  cycle  (6.6%)  with  a  broad  maximum 
centered  on  lunar  zenith  and  a  lesser  maximum  at  nadir.  Mean  daily  rates  of  Oa-consumption 
exhibited  a  lunar  monthly  cycle  with  maximum  (first  to  sixth  day  after  full  moon)  being 
45%  greater  than  for  the  minimum  (third  to  eighth  day  after  new  moon)  (p  <  0.001).  Also 
the  5-7  AM  values  of  Oa-consumption  (as  deviations  without  sign  from  the  daily  mean)  cor- 
related with  the  concurrent  daily  2-6  AM  mean  barometric  pressure  change ;  r  =  0.587,  N  —  33, 
t  =  5.0.  There  was  also  a  highly  significant  correlation,  but  of  a  different  character,  between 
the  2-6  PM  mean  pressure  change  and  the  5-7  PM  deviation  in  O2-consumption  from  the  daily 
mean.  There  were  noted  to  be  striking  similarities  between  the  forms  of  the  mean  solar-  and 
lunar-day  cycles,  of  Ilyanassa  and  the  forms  of  the  mean  cycles  for  the  sea-weed,  Fucus,  simi- 
larly obtained  under  constant  conditions. 

Correlation  between  O. -.-Consumption  in  Fucus  in  constant  conditions,  including 
pressure,  and  specific  barometric-pressure  parameters.'1-  FRANK  A.  BROWN, 
JR.,  H.  MARGUERITE  WEBB  AND  WILLIAM  J.  BRETT. 

Oxygen-consumption  was  monitored  for  about  one  lunar  month  during  each  of  the  five 
summers,  1954  through  1958,  in  Fucns  kept  in  constant  conditions  of  temperature,  light,  pres- 
sure, and  humidity.  A  total  of  130  uninterrupted  days  of  data  was  obtained.  Highly  signifi- 
cant solar-day  (amplitude  5.8%;  two  maxima,  4  AM  and  10  AM)  and  lunar-day  (amplitude 
4.6%;  major  maximum  about  zenith  +  6  hours,  major  minimum  about  nadir  +  6  hours)  cycles, 
and  consequent  synodic  monthly  ones  were  found.  Reducing  concurrent  barometric  pressure 
and  Fucus  Oa-consumption  data  for  the  five-year  period  by  averaging  all  data  obtained  on  each 
of  30  corresponding  days  re :  new  moon  and  full  moon,  a  correlation  was  found  between  the 
mean  2-6  AM  rate  of  barometric  pressure  change  (without  sign)  and  the  concurrent  daily 
3-5  AM  percentage  deviation  in  Oa-consumption  (with  sign)  from  the  daily  mean  rate;  r  =  0.67, 
JV  =  30,  t  =  6.5.  There  was  also  a  correlation  between  the  average  2-6  PM  rate  of  barometric 
pressure  change  (with  sign)  and  the  mean  5-7  PM  percentage  deviation  in  Oa-consumption 
(with  sign)  ;  r  =  0.65,  N-  30,  t  —  6.l.  Since  the  barometric  pressure  parameters  are  essen- 
tially random  in  their  day-to-day  fluctuations,  it  is  concluded  that  the  cycle-periods,  and,  in 
part,  cycle-form  as  well,  are  exogenous  in  Fucus. 

The  interrenal  of  the  sting  ray.     K.  A.  BROWNELL  AND  F.  A.  HARTMAN. 

The  interrenal  tissue  of  the  sting  ray  (Dasyatis  centrum)  is  usually  limited  to  a  single 
organ,  shaped  like  a  thick  handled  dumb-bell.  It  lies  posteriorly  between  the  kidneys,  slightly 
embedded  in  the  renal  tissue  on  the  right.  Rarely,  small  isolated  islets  of  interrenal  tissue 
may  be  found  in  the  same  neighborhood.  The  gland  can  be  found  best  by  its  shape  and  loca- 
tion since  its  color  is  so  nearly  that  of  the  kidney,  due  to  the  thick-walled  capsule  which 
covers  the  characteristic  cream-colored  tissue.  When  considered  in  relation  to  body  size,  the 
sting  ray  has  one  of  the  smallest  interrenals  among  the  elasmobranchs.  Fifteen  males  pos- 
sessed glands  0.00057  ±  0.000051  per  cent  of  the  body  while  twenty-two  females  possessed  glands 
0.00070  ±  0.00026  (standard  error)  per  cent  of  the  body. 

For  histological  study  the  capsule  should  be  removed  to  permit  more  rapid  penetration 
of  the  fixative.  The  gland  is  composed  of  rather  small  cells  without  distinct  zonation.  These 
cells  are  arranged  in  clusters  of  various  sizes  and  shapes.  The  nuclei  are  spherical  and 

1  These  studies  were  aided  by  a  contract  between  the  Office  of  Naval  Research,  Department 
of  Navy,  and  Northwestern  University,  NONR-122803. 


346         PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

irregularly   located   in    the    cell.     In    some    specimens,    cells    of   the    peripheral    region    contain 
more  cytoplasm  than  those  more  centrally  located. 

Chromosomes  of  the  estuarine  isopod,  Cyathura  sp.     W.  D.  BURBANCK  AND  MADE- 
LINE P.  BURBANCK. 1 

Specimens  of  Cyathura  sp.  were  collected  for  cytological  study  in  May  and  June  of  1958 
from  Pocasset  River  which  empties  into  Buzzards  Bay  about  fifteen  miles  north  of  Woods 
Hole,  Mass.  These  isopods  are  abundant  along  the  edge  of  the  channel  in  the  upper  reaches 
of  the  marsh  and  were  the  subject  of  an  intensive  ecological  study  from  September,  1957  to 
September,  1958.  To  obtain  gonadal  material,  the  animals  were  beheaded  and  the  contents 
of  the  body  cavity  dissected  out  onto  a  coverslip.  Several  drops  of  Nissenbaum's  (10:2:2:5  of 
mercuric  chloride,  acetic  acid,  formalin  and  tertia^  butyl  alcohol)  followed  by  acetic  alcohol, 
95%  and  100%  alcohols  and  a  colloidin-ether  solution  fixed  the  material  and  affixed  it  to  the 
coverslip.  Staining  was  done  in  warm  Gomori's  haematoxylin.  Female  cyathuras  collected 
in  May  contained  eggs,  but  it  was  difficult  to  retain  the  material  on  the  coverslips,  and  what 
eggs  remained  affixed,  contained  no  discernibly  stained  nuclei.  Males  collected  in  May  and 
June  contained  mature  sperm  and  sperm  in  various  stages  of  development.  In  some  testes 
there  were  prophase  stages  of  what  appeared  to  be  the  first  maturation  division  and  in  the 
vasa  deferentia  of  the  same  animal  were  sperm  with  long  tails.  Usually  each  testis  contained 
several  stages  of  spermatogenesis.  Chromosome  counts  were  made  of  bivalents  at  metaphase 
of  the  first  maturation  division  and  of  metaphase  and  anaphase  chromosomes  of  the  second 
maturation  division.  The  n  number  of  chromosomes  is  5.  The  chromosomes  are  small,  rang- 
ing from  less  than  1  n  at  Telophase  II  to  Metaphase  I  bivalents  about  3  /JL  long.  One  of  the 
five  is  much  smaller  than  the  others. 

Observations  on  the  structure  of  the  cercaria  of  H'unasthla  qnissetensis.     ROBERT 
R.  CARDELL,  JR.  AND  DELBERT  E.  PHILPOTT. 

Cercaria  were  obtained  in  the  free  swimming  form,  fixed  in  1%  osmic  acid,  and  electron 
microscopic  studies  carried  out  on  the  structure  of  the  tail.  Incidental  to  the  study  of  the 
tail,  observations  were  made  on  the  spines,  bacilliform  rods  and  cuticle. 

The  cuticle  of  the  tail  was  found  to  be  one  micron  thick,  possessing  an  outer  single  mem- 
brane with  many  indentations  and  a  double  basement  membrane.  Small  mitochondria  were 
found  scattered  throughout  the  cuticle  but  were  concentrated  near  the  smooth  muscle  layer, 
found  immediately  below  the  basement  membrane.  The  smooth  muscle  appeared  as  concentric 
bands  around  the  tail,  approximately  0.5  micron  in  thickness.  A  layer  of  striated  muscle 
approximately  three  micra  thick  was  found  below  the  smooth  muscle,  and  was  directed 
obliquely  to  the  long  axis  of  the  tail.  A  study  of  the  muscle  did  not  reveal  the  characteristic 
A  and  I  bands  and  distance  between  the  Z  membranes  was  0.8  micron.  The  muscle  tissue 
did  not  appear  to  enter  the  body  of  the  cercaria.  Further  investigation,  however,  is  necessary 
to  substantiate  this  observation.  Mitochondria  were  concentrated  in  a  band  just  below  the 
striated  muscle  whereas  the  nuclei  were  located  in  the  center  of  the  tail. 

The  cuticle  of  the  body  was  found  to  be  four  micra  thick  with  an  outer  membrane  and 
a  basement  membrane.  In  the  cuticle  structure  were  found  many  unidentified  spherical  to  oblong 
osmiophilic  structures.  The  bacilliform  rods  were  0.4  micron  in  diameter,  2.3  micra  in  length, 
with  a  canal  through  the  center  and  concentric  lines  around  the  canal  in  cross  section.  The 
spines,  which  were  embedded  in  the  cuticle,  were  found  to  be  triangular  in  cross  section  with 
an  altitude  of  0.4  micron.  The  base  of  the  spines  rested  above  a  thickened  basement  membrane. 

Regulation  of  flashing  in  the  firefly.     JAMES  CASE  AND  JOHN  BUCK. 

The  debated  question  of  whether  firefly  luminescence  is  controlled  by  the  nervous  system 
was  settled  by  demonstrating  with  Plwturis  vcrsicolor  that  a  characteristic  neural  volley  re- 

1  Supported  by  the  McCandless  Fund  of  Emory  University  and  an  Office  of  Naval 
Research-Marine  Biological  Laboratory  Institutional  Grant. 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY         347 

corded  at  the  terminal  ganglion  of  the  ventral  nerve  cord  precedes  each  spontaneous  flash  by 
60  to  70  msecs.  Upon  electrical  stimulation  the  neuro-photogenic  system  exhibits  facilitation, 
treppe,  tetany,  and  has  strength-duration  relations  similar  to  those  of  arthropod  nerve-muscle. 
Temperature  coefficients  for  flash  latency  in  the  isolated  organ  are  2.7  at  10°  C,  2.2  at  20°  C., 
and  2.0  at  30°  C.  Intact  fireflies  differ  from  decapitated  specimens  in  exhibiting  faster  cord 
transit  rate,  0.66  m/sec.  versus  0.09  m/sec.,  as  well  as  lower  threshold. 

These  and  other  measurements  indicate  that  the  neural  element  of  the  neuro-photogenic 
system  is  similar  to  that  of  the  insect  neuro-muscular  apparatus.  However,  the  behavior  of 
the  effector  element  differs  from  that  of  muscle  in  a  number  of  ways.  Response  latency  of 
photogeny  to  presumably  direct  stimulation  is  9  msec.,  longer  than  in  either  Mnemiopsis  or 
polynoid  photogeny  and  much  longer  than  in  striated  muscle.  In  addition  to  flashing,  the 
organ  seems  able  to  produce  a  long  lasting  glow  which  can  be  enhanced,  or  depressed  under 
certain  conditions,  by  repetitive  stimulation  both  in  the  presence  and  absence  of  flashing. 
Masking  experiments  show  that  the  multiple  flashes  characteristic  of  some  species  may  be 
given  by  as  little  as  five  per  cent  of  the  total  lantern  area  and  hence  are  not  necessarily  due 
to  different  segmental  organs  or  populations  of  photogenic  cells  lighting  in  relays.  However, 
the  organization  of  the  lantern  does  permit,  on  occasion,  the  luminescence  of  different  areas 
of  photogenic  tissue  independently  and  in  varying  sequence. 

A  source  of  the  to.ric  factor(s)  in  scalded  starfish.1  ALFRED  B.  CHAET  AND 
STAFFORD  I.  COHEN. 

The  significance  of  the  toxic  factor  theory  in  heat  death  of  invertebrates  (Phascolosoma 
gouldii  and  Asterias  jorbesi}  has  been  previously  demonstrated  (Chaet,  1951,  1955,  1956).  The 
present  report  deals  with  the  origin  of  the  toxin  released  from  scalded  starfish  which  causes 
autotomy  and  eventual  death  when  injected  into  normal  recipient  Asterias. 

Starfish  were  dissected  into  seven  fractions:  central  discs,  lateral  portion  of  ray,  aboral 
portion  of  ray  (including  tube  feet),  hepatic  caeca,  gonads  and  coelomic  fluid.  These  fractions 
were  then  suspended  in  sea  water  (except  in  the  case  of  coelomic  fluid)  and  heated  in  non- 
toxic  bags  for  1%  minutes  at  76°  C.  Injections  (0.15  ml./gm.)  into  recipient  starfish  showed 
all  fractions,  except  the  coelomic  fluid,  to  contain  a  heat-stable,  dialyzable  toxin  similar  to 
that  obtained  from  in  vivo  scalded  starfish.  It  is  interesting  to  note  that  although  the  cells 
of  the  coelomic  fluid  proved  to  be  the  source  of  the  toxic  factor  in  Phascolosoma  gouldii,  the 
cell-containing  coelomic  fluid  of  starfish  did  not  release  any  toxin  when  heated  in  vitro. 

In  a  search  for  tissue  common  to  all  six  fractions  yielding  the  toxic  factor,  two  tissues 
were  analyzed ;  namely,  nerve  and  epithelial.  Non-toxic  extracts  were  obtained  from  radial 
nerves  which  had  been  heated  in  vitro.  On  the  other  hand,  when  the  layer  of  epithelium  which 
lines  the  perivisceral  cavity  was  isolated  and  heated,  a  toxin  was  in  fact  released.  The  physical 
properties  of  the  "epithelial  toxin,"  as  well  as  its  biological  activity,  have  been  measured.  Like 
the  toxin  found  in  scalded  starfish,  it  is  a  heat-stable,  dialyzable  substance. 

Survival  of  Uca  pugnax  in  sand,  water  and  vegetation  contaminated  with  2,4  di- 
chlorophenoxyacctic  acid.  C.  LLOYD  CLAFF,  FREDERICK  N.  SUDAK  AND 
VALERIE  MOLONEY. 

Fiddler  crabs  (Uca  pugnax}  were  exposed  to  various  concentrations  of  the  commercial 
weed  killer  2,4  dichlorophenoxyacetic  acid  which  was  sprinkled  on  the  sand  and  vegetation 
in  their  confinement  basins.  In  a  series  of  animals  which  were  exposed  continuously  to  con- 
centrations of  10,000,  5000,  2500,  and  1000  p.p.m.  (recommended  spray  concentration),  "2,4-D" 
was  100%  lethal  after  108  hours'  exposure  to  10,000  p.p.m.  (50%  dead  in  72  hours)  and 
5000  p.p.m.  (50%  dead  in  96  hours),  after  10  days  in  2500  p.p.m.  and  14  days  in  1000  p.p.m. 

Another  series  of  animals  were  exposed  to  "2,4-D"  for  12  hours,  rinsed  in  fresh  sea  water, 
and  placed  in  confinement  basins  containing  fresh  sea  water,  sand  and  vegetation.  Fifty  per 
cent  of  the  animals  exposed  to  10,000  p.p.m.  were  dead  in  72  hours  after  they  were  placed  in 

1  This  study  was  supported  by  a  grant  from  the  National  Science  Foundation. 


348         PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

uncontaminated  basins;  80%  were  dead  after  two  weeks.     A  single  12-hour  exposure  to  recom- 
mended spray  concentration  (1000  p.p.m.)  was  lethal  for  20%  of  the  animals  within  two  weeks. 

A  studv  of  riboniicleic  odd  during  the  development  of  Ilyanassa  obsoleta.^     ].  R. 
COLLIER. 

A  micromethod  based  on  the  procedure  of  Ogur  and  Rosen  was  used  for  separation 
of  the  ribonucleic  acid  (RNA)  in  the  Ilyanassa  egg,  and  the  amount  of  RNA  was  determined 
by  spectrophotometry. 

The  fertilized  egg  contains  0.0032  gamma  of  RNA.  The  yolk  platelets  were  separated 
from  the  cytoplasm  by  low  speed  centrifugation  and  all  of  the  RNA  was  recovered  in  the 
cytoplasmic  fraction. 

By  the  end  of  the  third  day  of  development,  at  20°  C,  there  had  occurred  a  three-fold 
increase  in  the  RNA  content  of  the  embryo.  After  this  stage  the  RNA  content  continues 
to  increase  only  slightly.  No  determinations  were  made  on  the  fully  differentiated  veliger. 

The  RNA  content  of  the  first  two  blastomeres  was  determined,  and  the  AB  blastomere 
was  found  to  contain  0.0015  ±  0.0001  gamma  and  the  CD  blastomere  0.0019  ±  0.0001  gamma 
of  RNA.  The  sum  of  the  RNA  found  in  the  two  blastomeres  represents  a  recovery  of  106.2 
per  cent  of  the  RNA  of  the  egg.  The  AB  and  CD  blastomeres  receive  46.8  and  59.3  per  cent, 
respectively,  of  the  RNA  of  the  whole  egg.  The  distribution  of  the  volume  of  the  hyaline 
protoplasm  to  the  first  two  blastomeres  is:  AB,  42.1  per  cent;  CD,  57.8  per  cent.  Thus,  if 
the  volume  of  the  hyaline  protoplasm  is  used  as  a  reference  unit,  these  data  indicate  that  the 
RNA  is  not  concentrated  in  either  of  the  first  two  blastomeres.  Using  the  dipeptidase  activity, 
or  protein  content  of  the  egg  and  blastomeres  as  a  reference  unit,  there  appears  to  be  a  slight 
concentration  of  RNA  in  the  AB  blastomere. 

Some  characterisation  of  the  egg  membrane  lytic  agent  derived  from  spcnn  extracts 
of  Hydroides  lic.\-agonus.~    ARTHUR  L.  COLWIN  AND  LAURA  HUNTER  COLWIN. 

It  has  been  shown  (Colwin  and  Colwin,  1958a)  that  an  extract  prepared  from  frozen- 
thawed  sperm  can  dissolve  the  principal  component  of  the  egg  membrane,  viz.,  the  thick  middle 
layer,  but  fails  to  attack  the  much  thinner  outer  border  layer  and  inner  border  layer.  To 
characterize  the  lytic  agent  the  following  measures  were  taken.  Frozen-thawed  sperm  was 
ground  with  sand  in  sea  water  and  centrifuged  for  90  minutes  at  25,000  X  g  in  the  refrigerated 
Spinco.  The  resultant  supernatant  had  strong  lytic  activity  and  completely  dissolved  the 
thick  middle  layer.  The  supernatant  was  inactivated  if  treated  with  solutions  of  crystalline 
trypsin  or  crystalline  chymotrypsin ;  in  each  case  a  final  concentration  of  0.177%  was  used 
and  the  pH  was  adjusted  to  that  of  sea  water.  In  control  eggs,  treated  with  these  enzyme 
preparations,  the  thick  middle  layer  was  not  affected.  If  the  trypsin  was  first  treated  with 
soy  bean  trypsin  inhibitor  and  then  added  to  the  sperm  supernatant,  there  was  no  loss  of  lytic 
activity.  The  active  lytic  agent  in  the  supernatant  is  non-dialyzable  but  inactivated  when 
boiled.  Saturation  of  the  supernatant  to  various  degrees  with  (NH4)2SO4  gave  the  following 
results:  25%  saturation  produced  a  brownish  green  precipitate  with  lytic  activity.  After 
removal  of  this  precipitate,  additional  solid  (NH4)2SO4  was  added  to  bring  the  supernatant 
to  about  60%  saturation;  this  treatment  produced  a  white  precipitate,  also  with  lytic  activity. 
After  removal  of  this  second  precipitate  further  solid  (NH4):>SO4  was  added  to  complete  the 
saturation  of  the  remaining  supernatant ;  this  produced  a  very  small  amount  of  white  precipitate 
with  little  or  no  lytic  activity  and  a  supernatant  which  was  completely  inactive.  All  these 
precipitates  as  well  as  the  final  supernatant  were  dialyzed  against  sea  water  before  testing. 

From  the  above  observations  it  is  concluded  that  the  lytic  agent  in  the  sperm  extract  is 
probably  a  protein,  presumably  of  an  enzymatic  nature. 

1  Supported  in  part  by  a  grant    (A-1899)    from  the  National   Institutes  of  Health,   U.   S. 
Public  Health  Service. 

2  Supported  by  a  Grant   (RG-4948)   from  the  National  Institutes  of  Health,  U.  S.  Public 
Health  Service. 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY         349 

The  effects  of  certain  enzymes  and  other  substances  on  the  egg  membranes  of 
Hydroidcs  hexagonus.^     LAURA  HUNTER  COLWIN  AND  ARTHUR  L.  COLWIN. 

During  a  study  of  sperm  entry  observations  were  made  of  the  ways  in  which  various 
substances  affected  the  "vitelline  membrane."  Eggs  immersed  in  test  solutions  were  examined 
under  the  light  microscope.  A  sperm  extract  (Colwin  and  Colwin,  1958)  dissolves  the  thick 
middle  layer  of  the  membrane  without  destroying  the  thinner  outer  and  inner  border  layers. 

In  the  concentrations  used,  hyaluronidase  dissolved  the  middle  layer,  appeared  to  separate 
certain  elements  of  the  outer  border  layer  and  may  have  weakend  the  inner  border  layer  in 
some  way.  Trypsin  and  chymotrypsin  had  no  apparent  effect  on  the  middle  and  inner  layers 
but  seemed  to  cause  slight  fraying  of  the  outer  border  layer.  Alpha-amylase  had  no  apparent 
effect  on  any  part  of  the  membrane,  either  when  used  directly,  or  following  previous  treatment 
of  the  eggs  with  trypsin,  chymotrypsin  or  sperm  extract,  followed  by  rinsing  in  sea  water. 

Protamine  sulfate  and  digitonin  did  not  dissolve  any  layer  of  the  membrane.  Sodium 
lauryl  sulfate  may  have  modified  the  outer  border  layer  but  did  not  dissolve  the  middle  or 
inner  layers. 

In  glucose,  glycerol,  urea,  sodium  chloride,  and  distilled  water  the  middle  layer  swelled 
and  disappeared.  If  sea  water  was  added  before  the  dissipation  became  complete,  part  or  all 
of  the  middle  layer  material  reappeared,  sometimes  widely  dispersed.  The  outer  border  layer 
expanded  like  a  balloon  and  usually  broke  and  was  shed ;  in  some  solutions  its  remnant  per- 
sisted, in  others  it  seemed  to  vanish.  The  inner  border  layer  disappeared  in  sodium  chloride 
and  in  glucose,  and  became  weaker  or  thinner  in  some  of  the  other  solutions.  More  detailed 
studies,  probably  by  electron  microscopy,  are  needed  to  clarify  the  changes  in  the  inner  and 
outer  border  layers.  Sea  water  at  pH  ranging  from  5.7  to  8.5  did  not  appear  to  destroy  any 
of  the  three  layers  of  the  membrane. 

Throughout  these  experiments  the  same  results  were  observed  in  fertilized  and  in  unferti- 
lized eggs. 

Membrane  removal  from  the  egg  of  the  annelid,  Hydroides.2     DONALD  P.  COSTELLO. 

During  a  comparative  study  of  the  membranes  of  marine  invertebrate  eggs,  it  was  found 
that  the  eggs  of  Hydroidcs  hcxagonus  could  be  divested  of  their  vitelline  membranes.  If  un- 
fertilized eggs  are  treated  with  successive  changes  of  isosmotic  NaCl  brought  to  a  pH  of  10.5 
by  the  addition  of  Na«COs  (Costello,  1945),  the  membranes  become  sticky,  the  eggs  clump 
together,  the  vitelline  membranes  elevate  (at  first  irregularly  and  asymmetrically),  and,  with 
gentle  agitation,  the  egg  membranes  rupture  or  dissolve  to  permit  the  eggs  to  roll  out.  The 
membranes  themselves  disappear  (dissolve)  except  where  a  large  egg  clump  was  present. 
More  than  one  change  of  the  alkaline  NaCl  is  required  to  eliminate  the  divalent  cations  of 
sea  water,  on  which  the  stability  of  the  membrane  depends.  During  treatment,  the  germinal 
vesicles  break  down  (indicating  parthenogenetic  activation)  and  later  the  polar  bodies  are 
extruded ;  some  of  the  denuded  eggs  cleave.  A  plasma  membrane  is  present  on  the  surface 
of  the  denuded  egg,  and  this  becomes  crenated  on  further  exposure  to  alkaline  NaCl,  just  prior 
to  eventual  cytolysis.  The  action  of  the  alkaline  NaCl  can  be  partially  reversed  in  its  early 
stages,  or  stopped  at  any  stage,  by  returning  the  eggs  to  sea  water.  The  vitelline  membranes 
of  fertilized  eggs  may  be  removed  in  essentially  the  same  manner. 

Hydroides  egg  membranes  do  not  dissolve  in  1%  Duponal  (sodium  lauryl  sulphate)  in 
sea  water  (or  in  NaCl),  as  does  the  Nereis  egg  membrane  (Osterhout,  1950).  Duponal- 
treatment  demonstrates  that  the  vitelline  membrane  consists  of  two  layers,  and  that  there  are 
numerous  weak  spots  (microvilli?),  through  which  materials  from  within  the  egg  may  be 
forced  out. 

Experiments  on  the  development  of  fragments  and  isolated  blastomeres  of  the  Hydroides  egg 
are  now  feasible  and  will  be  undertaken. 

1  Supported  by  a  Grant  (RG-4948)  from  the  National  Institutes  of  Health,  U.'  S.  Public 
Health  Service. 

-Aided  by  a  grant  (RG-5328)  from  the  National  Institutes  of  Health. 


350         PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

The  action  of  substituted  phenols  on  the  conversion  of  glucose-1-C1*  and  glucose- 
6-C11  to  carbon  dioxide  by  the  eggs  of  Arbacia  punctulata.  ROBERT  K.  CRANE, 
A.  K.  KELTCH,  C.  P.  WALTERS  AND  G.  H.  A.  CLOWES. 

In  confirmation  and  extension  of  previous  work,  it  has  been  found  that  the  absolute  and 
relative  rates  of  CO»  formation  from  glucose-l-Cu  and  glucose-6-C14  by  24-hour  embryos  of 
Arbacia  can  be  altered  by  low  concentrations  of  a  series  of  substituted  phenols.  Seven  repre- 
sentative compounds  were  tested  and  it  was  found  that  they  could  be  divided  into  three  groups 
according  to  their  pattern  of  action.  In  the  absence  of  phenol  the  rate  of  CO2  formation  from 
glucose-1-C14  was  2-3  times  the  rate  from  glucose-6-C14.  The  first  group  (2,4-dinitrophenol, 
4,6-dinitro-o-cresol,  2,4-dinitro-4-chlorophenol,  and  2,4-dichlorophenol)  was  characterized  by 
stimulation  of  the  rate  of  COa  formation  from  both  compounds  and  an  enhancement  of  the 
rate  from  glucose-6-C"  relative  to  that  from  glucose-1-C14.  The  rates  from  both  compounds 
were  maximally  stimulated  by  the  same  phenol  concentration  and  these  stimulated  rates  were 
approximately  equal  to  each  other.  The  second  group  (2,4,5-trichlorophenol  and  pentachloro- 
phenol)  also  produced  stimulation  of  COa  formation  from  both  glucose-1-C14  and  glucose-6-C14. 
However,  this  group  did  not  enhance  the  relative  rate  from  glucose-6-C14  to  the  same  extent : 
at  maximal  stimulation,  COs  formation  from  glucose-1-C14  still  exceeded  that  from  glucose-6-C14 
by  50  per  cent  of  the  control  amount.  The  third  pattern  of  action  was  exhibited  by  2,4-dinitro- 
thymol,  which  did  not  stimulate  CO2  formation  at  any  concentration  tested.  On  the  contrary, 
at  a  concentration  of  1.6  X  10~5  M,  dinitrothymol  reduced  COa  production  from  glucose-1-C14 
to  60  per  cent  of  the  control  value  and  that  from  glucose-6-C14  to  less  than  4  per  cent.  The 
first  two  groups  appear  to  inhibit,  although  to  different  degrees,  the  phosphogluconate  pathway 
of  glucose  utilization.  Dinitrothymol,  on  the  other  hand,  appears  to  exert  a  heretofore  unsus- 
pected profound  inhibition  on  the  glycolytic  pathway.  Experiments  on  the  site,  as  well  as 
the  mode  of  action,  of  these  substituted  phenols  are  projected. 

Substrate  induction  of  adenosine  deaminase  activity  in  Arbacia  embryos.     DAVID 

DUBNAU.1 

Enzyme  induction  may  furnish  a  valuable  tool  with  which  to  study  the  synthesis  of  specific 
proteins  in  developing  tissues.  The  induction  of  enzymes  by  their  substrates  has  also  been 
utilized  in  the  formulation  of  a  number  of  hypotheses  concerning  the  mechanism  of  cellular 
differentiation.  A  substrate-induced  increase  of  adenosine  deaminase  activity  has  been  demon- 
strated in  Arbacia  punctulata  embryos  and  is  reported  below.  Sufficient  adenosine  to  provide 
a  final  concentration  of  0.1  mg./cc.  was  added  to  cultures  immediately  after  first  cleavage. 
Enzyme  was  assayed  at  intervals  in  homogenates  by  following  the  disappearance  of  added  sub- 
strate spectrophotometrically.  Specific  activity  was  referred  to  protein.  The  level  of  adenosine 
used  had  slightly  adverse  effects,  resulting  in  the  formation  of  plutei  with  short,  blunted  arms. 
The  control  animals,  raised  in  sea  water  alone,  evidenced  a  gradual  increase  in  enzyme  activity 
as  development  proceeded.  After  20  hours  in  adenosine,  the  specific  activity  of  the  enzyme 
in  the  experimental  embryos  was  10-20%  less  than  that  in  the  controls.  This  was  interpreted 
as  due  to  the  adverse  effect  of  adenosine,  as  noted  above.  Thereafter  a  rise  in  the  level  of 
enzyme  in  the  experimental  embryos  occurred.  By  90-96  hours  after  addition  of  adenosine, 
the  specific  activity  in  the  experimental  embryos  was  from  32%  to  85%  greater  than  that 
in  the  controls.  Induction  was  also  demonstrated  by  measurements  made  forty  hours  after 
the  addition  of  adenosine  to  one-day  plutei.  The  adverse  effect  of  adenosine  can  be  mitigated 
by  the  use  of  lower  inducer  concentrations.  Preliminary  evidence  indicates  that  this  treatment 
results  in  a  more  pronounced  elevation  of  activity  than  was  obtained  with  higher  inducer  levels. 

Permeability  studies  on  Arbacia  punctulata  eggs.  R.  G.  FAUST  AND  A.  K. 
PARPART. 

Volume  changes  of  Arbacia  eggs  were  produced  by  the  addition  of  ethylene  glycol  or 
NaCl  to  sea  water.  These  changes  were  recorded  by  means  of  a  photoelectric  densimeter.  It 

1  Pre-doctoral  fellow  of  the  National  Science  Foundation,  1957-58. 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY         351 

has  been  demonstrated  by  many  other  investigators  that  fertilization  increases  the  permeability 
of  the  egg  membrane.  However,  the  role  played  by  the  hyaline  layer  upon  permeability  of 
fertilized  eggs  has  been  controversial. 

Trypsinized  fertilized  eggs,  which  have  a  distinct  hyaline  layer  but  no  fertilization  mem- 
brane, were  washed  for  two  minutes  with  a  solution  consisting  of  equal  volumes  of  1  M  glu- 
cose and  0.5  M  NaCl.  Thus,  these  fertilized  eggs  had  neither  a  fertilization  membrane  nor 
hyaline  layer.  They  were  returned  to  sea  water  and  tested  in  the  densimeter.  The  permeability 
of  these  eggs  to  water  and  ethylene  glycol  was  the  same  as  that  of  normal  fertilized  eggs 
having  both  fertilization  membranes  and  hyaline  layers.  The  increase  in  permeability  caused 
by  fertilization  is  therefore  independent  of  the  hyaline  layer  or  the  fertilization  membrane. 

Direct  evidence  for  a  distal  retinal  pigment  dark-adapting  hormone  in  Palaemonetes 
vulgaris.^  MILTON  FINGERMAN,  MILDRED  E.  LOWE  AND  BANGALORE  I. 

SUNDARARAJ. 

Heretofore,  there  has  been  no  direct  demonstration  of  dark-adapting  hormone  for  the 
distal  retinal  pigment  of  any  crustacean  kept  under  constant  illumination.  Previous  evidence 
for  a  dark-adapting  hormone  was  based  on  differences  in  rates  of  dark  adaptation  between 
control  prawns  and  those  injected  with  extracts  of  various  organs,  followed  by  transfer  to 
darkness.  In  the  present  investigation  one-eyed  prawns  were  kept  on  a  black  background 
under  a  constant  illumination  of  27  ft.  c.  The  state  of  the  distal  retinal  pigment  was  slightly 
less  than  midway  between  the  fully  light-adapted  and  the  fully  dark-adapted  conditions ;  so 
the  presence  in  extracts  of  a  light-adapting  or  a  dark-adapting  hormone  could  be  demonstrated. 
Boiled  extracts  of  whole  eyestalks  produced  maximal  light  adaptation  followed  in  two  hours 
by  dark  adaptation  that  lasted  about  five  hours.  The  light-adapting  and  the  dark-adapting 
effects  of  extracts  of  the  sinus  gland  equalled  those  of  the  optic  ganglia.  Extracts  of  trito- 
cerebral  commissures  produced  slight  light  and  dark  adaptation.  The  supraesophageal  ganglia 
and  circumesophageal  connectives  without  the  commissures  had  no  dark-adapting  effect. 
With  the  commissure  attached,  the  light-adapting  ability  of  these  tissues  was  decreased,  pre- 
sumably due  to  the  presence  of  the  dark-adapting  hormone  in  the  commissure.  Addition  of 
the  commissure  to  extracts  of  eyestalks  similarly  decreased  their  light-adapting  potency. 

Influence  of  long-term  background  adaptation  on  the  lability  of  chromatophores 
and  the  sources  of  chromatophorotropins  in  Palaemonetes  vulgarise  MILTON 
FINGERMAN,  MURIEL  I.  SANDEEN  AND  MILDRED  E.  LOWE. 

To  study  the  effects  of  long-term  background  adaptation  on  endocrine  sources  and  on 
the  target  organs,  groups  of  Palaemonetes  were  placed  on  black  and  on  white  backgrounds. 
At  intervals  of  2  hours,  1,  2,  4,  6,  8  and  14  days  the  rate  of  migration  of  the  dark  red  pigment 
was  determined  after  moving  the  animals  to  the  opposite  background.  The  rates  of  pigment 
migration  gradually  decreased.  The  midpoint  of  pigment  dispersion,  chromatophore  stage  3, 
was  reached  in  15  minutes  by  1-day  white-adapted  animals  and  not  for  60  minutes  by  14-day 
white-adapted  animals.  Pigment  concentration  was  less  affected.  Chromatophore  stage  2.5 
\vas  achieved  in  15  minutes  by  1-day  black-adapted  animals  and  in  30  minutes  by  8-day  black- 
adapted  animals.  The  nervous  system  source  of  chromatophorotropins  studied  was  the  cir- 
cumesophageal connectives  with  the  tritocerebral  commissure  attached.  Extracts  of  these 
organs  from  animals  kept  on  black  or  on  white  backgrounds  for  2  hours  and  ;for  14  days 
were  assayed  on  one-eyed  animals  for  dark  red  pigment  concentrating  and  dispersing  activity. 
After  14  days  of  background  adaptation  extracts  from  the  animals  on  white  had  much  more 
dispersing  effect,  activity  of  5.7,  than  those  on  black,  activity  of  0.7,  and  less  concentrating 
effect,  activity  of  0.9  compared  to  2.9.  In  animals  adapted  for  only  2  hours  the  dispersing 
activity  of  tissues  from  animals  on  white  was  2.6,  compared  to  2.3  from  animals  on  black. 
The  concentrating  activity  of  tissues  from  animals  on  white  was  1.7  and  from  animals  on 

1  This  investigation  was  supported  by  Grant  No.  B-838  from  the  National  Institutes  of 
Health. 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

black,  2.6.  These  results  offer  evidence  in  support  of  the  hypothesis  that  these  tissues  pro- 
duce hormones,  particularly  red  dispersing  substance,  which  function  in  normal  background 
responses. 

Further  studies  on  the  chromatophorotropins  of  Palaemonetes  vulgaris.1     MILTON 

FlNGERMAN,  BANGALORE  I.  SUNDARARAJ  AND  MURIEL  I.   SANDEEN. 

Two  studies  were  initiated  to  define  the  nature  of  the  antagonistic  chromatophorotropins 
tontrolling  dispersion  and  concentration  of  the  dark  red  pigment  in  Palaemonetes.  Filter 
paper  electrophoresis  of  tissue  extracts  was  performed  at  5-7°  C.  for  two  hours  with  JI//10 
borate  buffer,  pH  7.2  at  500  V.  and  0.1-0.2  mA.  The  eyestalks  contained  an  electronegative 
red  pigment  dispersing  and  an  electropositive  concentrating  substance.  The  supraesophageal 
.ganglia  plus  the  circumesophageal  connectives  contained  an  electropositive  and  an  electronega- 
tive red  pigment  dispersing  substance.  Comparison  of  extracts  of  eyestalks,  fresh  and  boiled, 
revealed  that  the  former  produced  considerable  red  pigment  concentration  and  no  significant 
red  pigment  dispersion  while  the  latter  produced  much  less  pigment  concentration  and  a  sig- 
nificant amount  of  pigment  dispersion.  To  analyze  this  difference  sinus  glands  were  separated 
from  optic  ganglia  and  fresh  and  boiled  extracts  of  each  were  assayed.  Neither  fresh  nor 
boiled  extracts  of  the  sinus  glands  produced  any  pigment  dispersion  although  boiling  decreased 
the  amount  of  pigment  concentration  produced.  Fresh  extracts  of  optic  ganglia  produced 
pigment  concentration  and  dispersion.  Boiled  extracts  produced  an  increased  amount  of  pig- 
ment dispersion.  Boiling  extracts  of  other  parts  of  the  central  nervous  system  likewise  in- 
creased their  pigment  dispersing  potency.  Keeping  fresh  extracts  of  eyestalks  or  other  parts 
of  the  nervous  system  at  room  temperature  for  four  hours  was  about  equivalent  to  boiling 
for  one  minute,  the  pigment  concentrating  activity  decreased  and  the  dispersing  activity 
increased. 

Sodium  and  potassium  exchanges  in  photosensitized  fish   red  cells.     JAMES   W. 
GREEN  AND  THOMAS  A.  BORGESE. 

The  object  of  the  present  study  has  been  to  examine  the  action  of  the  fluorescent  dye 
rose  bengal  on  Na  and  K  exchanges  in  fish  red  cells.  Blood  was  generally  drawn  by  heart 
puncture,  placed  in  isotonic  NaCl  solution  and  defibrinated.  After  centrifugation  the  cells 
were  washed  twice  in  saline  and  refrigerated  until  used,  generally  within  two  days  of  collection. 
Erythrocytes  from  the  elasmobranch  Dasyastis  (sting  ray)  and  the  teleosts  Scomber  (mackerel) 
and  Lophius  (goose  fish)  were  used.  Hemolysis  curves  of  cells  photosensitized  to  2  X  10~6  M 
rose  bengal  by  exposure  to  light  from  a  12  W  fluorescent  bulb  were  determined.  The  times 
to  50%  hemolysis  following  a  two-minute  irradiation  were :  sting  ray  41  minutes,  goose  fish 
30  minutes,  and  mackerel  16  minutes. 

For  the  ion  exchange  experiments,  irradiation  times  of  one  or  two  minutes,  of  1.8% 
erythrocyte  suspensions,  were  used.  The  suspensions  were  reconstituted  and  incubated  at 
room  temperature  in  an  agitation  roller  with  either  Na24  or  K42  and  glucose  saline  for  periods 
up  to  8  hours.  All  erythrocytes  exhibited  an  increase  in  Na24  and  a  decrease  in  K42  when 
photosensitized.  The  most  satisfactory  experiments,  those  with  mackerel  cells,  showed  that 
K42  exchanged,  in  photosensitized  cells,  only  47.8%  as  rapidly  as  the  controls  while  Na24  ex- 
changed 200%  more  rapidly.  In  the  photosensitized  cells  glucose  disappearance  was  50% 
greater  than  the  controls.  It  is  tentatively  concluded  that  the  nucleated  fish  erythrocyte  is 
more  sensitive  than  the  mammalian  red  cell. 

A  crescent-shaped  figure  in  the  liyaline  layer  of  the  Arbacia  egg.     ETHEL  BROWNE 
HARVEY. 

A  crescent-shaped  figure  often  appears  at  the  surface  of  the  Arbacia  punctulata  egg  if 
the  eggs  in  the  "streak"  stage,  13  to  30  minutes  after  fertilization  at  23°  C.,  are  placed  for 
5  minutes  or  longer  in  N/200  NaOH  (in  distilled  water)  ;  this  has  been  found  to  be  the 

1  This  investigation  supported  by  the  National  Institutes  of  Health. 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY         353 

most  favorable  solution.  The  crescent  is  darker  than  the  surrounding  protoplasm  and  is 
sharply  outlined  in  black.  It  is  a  curved  band  with  pointed  ends,  varying  in  width  from 
about  2  ,u  to  about  20  /x;  it  is  sometimes  short  but  may  extend  completely  around  the  cell, 
or  nearly  so;  the  egg  swollen  in  this  hypotonic  solution  measures  about  130  /x  in  diameter, 
the  normal  egg  measures  74  fi.  The  band  tends  to  be  thin  in  earlier  stages.  In  stages  before 
the  crescent  is  formed,  this  is  represented  by  a  somewhat  spherical  mass  of  dark-staining  cyto- 
plasm. Thirty-five  minutes  or  more  after  fertilization,  eggs  similarly  treated  do  not  show  a 
crescent.  The  crescent  occurs  in  eggs  from  which  the  fertilization  membrane  has  been  re- 
moved, but  not  in  eggs  from  which  the  hyaline  layer  has  been  removed  by  calcium-free  sea 
water,  indicating  that  it  lies  in  this  layer;  it  does  not  extend  into  the  interior  of  the  egg.  ^  It 
does  not  appear  with  distilled  water  alone,  without  the  alkali.  Alkalis  other  than  sodium 
can  likewise  be  used:  KOH,  Ca(OH)2,  NH4OH. 

The  crescent  is  formed  in  centrifuged  eggs,  bearing  no  relation  to  the  stratification;  it 
is  also  found  in  the  white  half-eggs,  rather  pale  but  outlined  in  black ;  and  also  in  the  red 
half-eggs,  small  and  quite  dark.  It  occurs  in  parthenogenetic  as  well  as  in  fertilized  eggs. 

Since  the  crescent  occurs  during  the  "streak"  or  "pre-spindle"  stage,  that  is,  after  the 
monaster  and  before  the  amphiaster,  it  seems  probable  that  it  is  connected  with  the  division 
of  the  centrosome,  two  parts  of  which  come  to  lie  one  at  each  pole  of  the  spindle.  How- 
ever, the  position  and  orientation  of  the  crescent  do  not  always  seem  to  correspond  with  that 
of  the  preceding  streak. 

There  is  also,  with  the  same  treatment  with  N/200  NaOH,  a  darkening  of  the  cleavage 
furrow  in  dividing  eggs,  where  the  hyaline  layer  becomes  thickened,  and  later  in  the  layer 
between  the  two  blastomeres.  There  is  no  darkening  of  the  micromeres. 

Many  stains  and  reagents  have  been  tried  in  order  to  determine  the  chemical  nature  of 
the  crescent,  but  nothing  has  been  found  to  stain  the  crescent  differentially  except  that  there 
is  a  slight  greenish  tinge  with  methyl  green,  a  bluish  tinge  with  methylene  blue,  and  it  stains 
pinkish  lavender  with  toluidin  as  does  also  the  hyaline  layer.  Among  other  substances  tried 
were:  Sudan  III,  Millon's,  Schiff's  and  ninhydrin  reagents,  Feulgen  stain,  Janus  green, 
pyronine,  neutral  red,  aceto-carmine,  picro-carmine.  No  dyes  were  found  to  stain  a  figure 
in  the  cytoplasm  such  as  a  crescent  without  the  treatment  with  dilute  alkali.  Nothing  more 
is  seen  with  a  phase  microscope  than  with  a  light  microscope. 

Studies  on  membrane  elevation  in  the  eggs  of  Chaetopterus  and  Nereis.^     CATH- 
ERINE HENLEY. 

It  has  been  suggested  by  Costello  (1958,  Physiol.  Zoo!.}  that  the  waves  of  contraction 
which  are  apparent  in  the  vitelline  membrane  of  the  Chaetopterus  egg,  beginning  about  20 
minutes  after  insemination,  may  be  due  to  the  rhythmic  release  of  colloidal  material.  To  test 
this  hypothesis,  the  effects  have  been  studied  of  several  agents  on  membrane  elevation  in  the 
inseminated  eggs  of  Chaetopterus  and  Nereis. 

When  fertilized  Chaetopterus  eggs  were  cold-treated  in  pre-chilled  filtered  aerated  sea 
water  (1  to  3°  C),  beginning  5  minutes  after  insemination,  for  periods  of  \V-2  to  3  hours,  there 
was  an  asymmetrical  exaggeration  of  membrane  elevation.  This  was  first  apparent  within 
10  minutes  following  the  end  of  cold-treatment,  as  a  localized  surface  crenulation  of  the  mem- 
brane. Gradually,  the  perivitelline  space  increased  in  width,  the  space  being  widest  at  one 
sector  but  also  discernibly  wider  than  normal  for  the  entire  circumference  of  the  egg-membrane 
complex.  In  eggs  which  were  treated  for  longer  periods  (2Vi  to  3  hours),  the  membrane  ele- 
vation eventually  terminated  in  a  bursting  of  the  membrane,  so  that  the  eggs  were  denuded. 

Cold-treated  Chaetopterus  eggs  with  exaggerated  membrane  elevation  were  placed  in  a 
solution  of  gum  arabic  in  sea  water.  The  membranes  promptly  collapsed  back  against  the 
surfaces  of  the  eggs,  indicating  that  the  colloidal  osmotic  pressure  within  the  membrane  (pre- 
sumably the  cause  of  membrane  elevation)  could  be  "neutralized"  by  externally  applied  col- 
loidal osmotic  pressure. 

Fertilized  Nereis  limbata  eggs  cold-treated  for  90  minutes,  beginning  5  minutes  after 
insemination,  showed  no  evidence  of  exaggerated  membrane  elevation. 

1  Aided  by  a  grant  to  Dr.  D.  P.  Costello  from  the  National  Institutes  of  Health,  RG-5328. 


354         PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

Uninseminated  Chaetopterus  eggs  which  were  x-irradiated  with  doses  of  20,000  and  40,000  r 
and  then  fertilized  showed  no  exaggerated  membrane  elevation  during  the  first  hour  after 
treatment,  in  contrast  to  the  results  reported  by  Redfield  and  Bright  (1921)  for  irradiated 
.Nereis  eggs. 

Mercaptoethanol  and  Tctrahyincna,     GEORGE  G.  HOLZ,  JR. 

The  effects  of  2-mercaptoethanol  on  morphogenesis,  nuclear  division  and  cleavage  have 
been  tested  on  Tetrahymena  pyrtformis  (MTL,  VI)  whose  division  was  synchronized  by  tem- 
perature changes  (5  alternate  half-hour  periods  at  43°  and  35°  C.).  Sixty  to  80%  were 
blocked  at  43°  in  the  anarchic  field  stage  of  stomatogenesis  and  the  anaphase  stage  of  micro- 
nuclear  division.  Normally  the  first  synchronous  division  occurred  55  minutes  after  the  last 
43°  exposure,  and  during  the  final  10  minutes  stomatogenesis  and  nuclear  division  resumed  and 
cytoplasmic  cleavage  began.  Mercaptoethanol  (0.0004—0.005  M)  introduced  immediately  after 
the  last  43°  period  delayed  division.  Higher  concentrations  prevented  it;  the  ciliates  remained 
at  the  stage  characteristic  of  "heat  block." 

Application  of  the  thiol  (0.025  M)  during  early  cleavage  retarded  but  did  not  prevent 
fission.  Macronuclear  division  did  not  occur,  micronuclear  division  was  normal,  and  one 
daughter  was  amacronucleate. 

Introduction  of  mercaptoethanol  at  intervals  after  the  first  division  showed  that  the  second 
synchronous  division  could  be  delayed  or  prevented,  depending  upon  the  concentration  used. 
When  it  was  added  (0.025  M)  10-20  minutes  before  cleavage,  the  ciliates  were  blocked  at 
stages  of  morphogenesis  and  micronuclear  division  characteristic  of  the  time  of  addition. 
Ciliates  treated  earlier  never  began  stomatogenesis  or  micronuclear  division.  There  was  no 
accumulation  of  a  characteristic  blocked  stage  like  that  produced  by  cyclic  heat  treatments. 

Starved  ciliates  introduced  to  nutrient  medium  containing  mercaptoethanol  (0.0025-0.025  M ) 
failed  to  reproduce  in  6  hours  and  grew  only  to  the  extent  shown  by  controls.  They  showed 
a  nuclear  constitution  and  infra-ciliature  characteristic  of  the  period  between  divisions. 

The  above  results  demonstrate  the  lack  of  a  sharp  specificity  of  action  of  mercaptoethanol 
on  a  particular  stage  of  morphogenesis  or  micronuclear  division,  and  a  possible  inhibitory  action 
on  cell  syntheses  necessary  for  growth. 

The  action  of  pcntahalophcnols  on  oxygen  consumption  and  cell  division  and  on 
the  glucose-6-phosphate  dehydrogenase  of  the  eggs  of  Arbacia  punctulata.  A.  K. 
KELTCH,  H.  H.  HIATT,  C.  P.  WALTERS  AND  G.  H.  A.  CLOWES. 

In  extension  of  previous  work,  the  compounds  pentachlorophenol  and  pentabromophenol 
were  tested  at  graded  concentrations  for  their  possible  influence  on  oxygen  consumption 
and  cell  division  of  fertilized  Arbacia  eggs  and  on  the  glucose-6-phosphate  dehydrogenase 
activity  of  extracts  of  unfertilized  eggs.  The  influence  on  cell  division  and  oxygen  consump- 
tion was  similar  to  that  previously  observed  with  a  large  series  of  nitro-  and  halophenols. 
Oxygen  consumption  was  stimulated  by  both  pentahalophenols.  As  the  concentration  of  phenol 
was  increased,  the  stimulation  increased  in  degree,  reached  a  maximal  value,  and  decreased. 
Cell  division,  on  the  other  hand,  was  reversibly  inhibited.  At  a  concentration  of  10~G  M 
pentachloro-  or  pentabromophenol,  cell  division  was  inhibited  50  per  cent  and  oxygen  consump- 
tion was  stimulated  about  80  per  cent.  Glucose-6-phosphate  dehydrogenase  activity  was  in- 
hibited by  these  compounds.  Fifty  per  cent  inhibition  was  obtained  with  9  X  10"6  M  penta- 
chlorophenol and  10"5  M  pentabromophenol. 

Dehydrogenase  activity  in  developmental  stages  of  Spisula  as  measured  with  a 
tetrasolium  salt.  EVELYN  KIVY-ROSENBERG,  KAREN  STEEL  KAGEY  AND  JOSEPH 
CASCARANO. 

A  quantitative  estimate  of  endogenous  dehydrogenase  activity  during  developmental  stages 
in  Asterias  had  been  sought  earlier  (Kivy-Rosenberg  and  Zweifach,  1956,  B'wl.  Bull.,  Ill) 
using  two  tetrazolium  salts  as  indicators — -TTC  and  NT — but  the  toxicity  of  these  salts  made 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY         355 

such  studies  not  feasible.  Since  then  investigations  on  specific  substrate-dependent  dehydro- 
genase  activity  have  been  in  progress. 

A  comparative  study  was  undertaken  of  Spisula  but  endogenous  activity  again  was  not 
feasible  since  INT,  which  is  a  more  active  acceptor  than  TTC  or  NT,  is  highly  insoluble 
in  sea  water.  Attention  was  turned  once  again  to  studies  of  specific  substrate-dependent  de- 
hydrogenase  activity,  using  several  stages  in  development  including  uninseminated  and  insemi- 
nated eggs.  Samples  of  both  fresh  and  frozen  eggs  were  incubated  at  37-38°  C.  for  one  hour 
in  a  medium  containing  one  of  a  series  of  substrates  including  succinate,  and  with  DPN  as 
cofactor,  alpha-glycerophosphate,  glucose,  glutamate.  malate,  lactate,  beta-hydroxybutyrate, 
ethanol.  The  formazan  was  extracted  and  amounts  of  reduced  tetrazolium  determined  photo- 
metrically. This  activity  was  expressed  as  micrograms  of  formazan  per  milligram  of  dried 
tissue. 

As  in  Asterias,  malate-dependent  dehydrogenase  activity  was  the  greatest  and  alpha- 
glycerophosphate  dehydrogenase  activity  less  but  still  second.  The  other  dehydrogenases  all 
showed  some,  though  limited  activity.  Quantitatively,  Spisula  shows  more  uniformity  than 
Asterias.  With  respect  to  metabolic  changes  during  development,  limited  observations  indicate 
that  both  malate  and  aplha-glycerophosphate  dehydrogenases  become  less  active  following 
insemination. 

In  Spisula  the  quantity  of  eggs  is  quite  limited  and  so  microchemical  determinations  were 
made  to  parallel  macrochemical  ones.  Homogenates  of  both  fresh  and  frozen  uninseminated 
and  inseminated  eggs,  first  cleavage,  and  early  larvae  were  incubated  in  a  similar  manner. 
The  substrate-dependent  dehydrogenase  activity  was  expressed  as  micrograms  of  formazan 
per  milligram  of  protein.  With  this  method,  the  malate  and  alpha-glycerophosphate-dependent 
dehydrogenase  activity  ranked  as  it  had  in  the  whole  egg.  There  is,  however,  a  discrepancy 
seen  between  reactions  of  the  organized  cell  and  disorganized  material  of  the  cell  which  will 
be  further  investigated.  In  the  homogenized  state  there  is  a  rise  in  malate-dependent  dehy- 
drogenase activity  following  insemination,  rather  than  a  fall.  This  is,  in  general,  true  of 
alpha-glycerophosphate  dependent  activity  also. 

Changes  in  the  levels  of  triphosphopyridine  nucleotidc  in  the  eggs  of  Arbacia  punc- 
tulata  subsequent  to  fertilization:  Presence  of  pyridine  nueleotide  transhydro- 
genase  and  diphosphopyridinc  nueleotide  kinase.  STEPHEN  M.  KRANE  AND 
ROBERT  K.  CRANE. 

Glucose  utilization  in  the  eggs  of  Arbacia  punctitlata  is  predominantly  via  pathways  re- 
quiring triphosphopyridine  nueleotide.  The  amount  of  glucose  metabolized  by  these  pathways 
has  been  shown  to  increase  strikingly  in  the  24-hour  embryos  compared  to  the  unfertilized 
eggs.  To  determine  some  of  the  factors  controlling  the  rate  of  carbohydrate  utilization,  there- 
fore, the  steady-state  concentrations  of  oxidized  (TPN)  and  reduced  (TPXH)  triphospho- 
pyridine nueleotide  were  measured  fluorometrically.  TPN  was  assayed  on  neutralized  tri- 
chloracetic  acid  extracts  using  TPN-specific  isocitric  dehydrogenase ;  TPNH  on  neutralized 
sodium  carbonate  extracts  using  TPNH-specific  glutathione  reductase.  Recovery  of  added 
TPN  was  100  per  cent.  Recovery  of  added  TPNH  varied  from  33  to  56  per  cent  and  the 
use  of  an  internal  standard  was  required.  TPN  levels  in  unfertilized  eggs  and  24-hour 
embryos  were  11  and  14  millimicromoles  per  gram  wet  weight,  respectively,  values  which  are 
close  to  the  lower  limit  of  sensitivity  of  the  method  used.  In  contrast,  TPNH  levels  in  four 
experiments  on  unfertilized  eggs  averaged  19  millimicromoles  per  gram  (range  7-32)  and 
in  24-hour  embryos  67  millimicromoles  per  gram  (range  57-90).  Eggs  one  hour  after  fertiliza- 
tion (two  assays)  contained  57  and  61  millimicromoles  of  TPNH  per  gram.  Since  the  con- 
centrations of  TPN  were  low  compared  to  TPNH,  pyridine  nueleotide  transhydrogenase  activity 
was  assayed  fluorometrically  using  the  3-acetylpyridine  analog  of  DPN  as  electron  acceptor. 
In  homogenates  at  27°  C.  rates  as  high  as  14  micromoles  per  hour  of  TPNH  converted  to 
TPN  per  gram  of  eggs  were  found.  Activity  of  DPN  kinase  was  also  found  but  the  presence 
of  inhibitors  in  the  crude  homogenate  prevented  the  determination  of  absolute  rates  for  this 
enzyme.  Further  work  will  be  needed  to  establish  unequivocally  the  very  striking  increase 
in  TPNH  level  and  the  apparent  variation  in  transhydrogenase  activity  incident  to  fertilization 
and  development  of  the  eggs.  Similar  assays  on  Arbacia  sperm  are  projected. 


356         PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

The  biological  and  chemical  mechanisms  of  protein  utilisation  by  Hydra.     HOWARD 
M.  LENHOFF. 

By  inducing  a  feeding  response  in  Hydra  with  reduced  glutathione  it  was  possible  to 
make  them  ingest  tissue  containing  protein  labeled  with  radioactive  sulfur.  The  fate  of  the 
ingested  protein  was  studied  by  chemical  fractionation  and  radioautography.  The  results 
demonstrated  that:  (1)  The  protein  was  not  digested  in  the  gastrovascular  cavity,  but  rather 
the  tissue  was  broken  down  into  small  particles ;  no  significant  hydrolysis  of  the  protein  to 
polypeptides  or  amino  acids  occurred  extracellularly.  (2)  About  80-90  per  cent  of  the  food 
particles  were  phagocytized  by  the  gastrodermal  cells  within  5  hours  after  ingestion  of  the 
food  into  the  gastrovascular  cavity.  (3)  only  the  gastrodermal  cells  in  the  upper  two-thirds 
of  the  body  tube  were  active  in  engulfing  most  of  the  food ;  a  very  slight  amount  of  food 
particle  engulfment  occurred  in  the  lower  parts  of  the  tentacles.  (4)  The  initial  products 
of  intracellular  protein  digestion  were  alcohol-soluble  proteins  or  polypeptides.  The  presence 
and  function  of  alcohol-soluble  proteins  in  animal  tissues  has  hitherto  never  been  described. 
Kinetic  experiments  demonstrated  that  these  unusual  proteins  supplied  precursor  material-  for 
the  synthesis  of  alcohol-insoluble  proteins.  Thus,  the  alcohol-soluble  proteins  formed  by  Hydra 
seem  to  act  in  a  similar  capacity  as  do  the  alcohol-soluble  storage  proteins  of  plants,  the  prola- 
mines.  (5)  During  asexual  reproduction  about  15-25  per  cent  of  the  parent's  radioactivity 
was  distributed  to  each  bud,  depending  upon  the  conditions  of  growth.  (6)  Twenty-five  per 
cent  of  the  radioactivity  was  excreted  into  the  environment  during  five  days  of  starvation. 
(7)  The  Hydra  egested  its  solid  wastes  by  inflating  itself  with  water  after  most  of  the  food 
particles  were  engulfed.  When  this  water  was  released,  most  of  the  solid  wastes  were 
flushed  out. 

Experimental  induction  of  cleavage  fnrrou's  in  the  Arbacia  egg.1     DOUGLAS  MARS- 
LAND  AND  WALTER  AUCLAIR. 

As  previously  reported,  premature  furrows,  which  always  cleave  the  egg  at  right  angles 
to  the  centrifugal  axis,  can  be  induced  at  various  times  up  to  35  minutes  ahead  of  the  normal 
cleavage  schedule.  The  induced  furrows  appear  in  a  high  percentage  of  the  eggs,  starting 
about  3  minutes  following  a  4-minute  period  of  centrifugation  at  high  pressure  (8-10,000 
lbs./in.2)  and  high  force  (41,000  Xg). 

The  treatment  always  ruptures  the  nuclear  membranes,  liberating  Feulgen-positive  material 
which  comes  to  lie  in  or  near  the  mitochondrial  zone.  Less  drastic  treatments  may  break 
the  nuclei,  however,  without  inducing  the  reaction.  In  fact,  the  furrowing  reaction  has  only 
been  observed  when  certain  cytoplasmic  vacuoles  are  also  broken.  These  vacuoles  are  some- 
what smaller  than  the  echinochrome  vesicles  and  each  vacuole  contains  from  1-4  granules 
which  stain  metachromatically  (red)  with  toluidine  blue.  Eggs  receiving  a  non-inducing 
centrifugation  show  hundreds  of  these  vacuoles  in  the  hyaline  and  mitochondrial  zones,  but 
after  an  inducing  treatment,  the  eggs  show  only  a  diffuse  non-granular  metachromatic  coloring 
in  the  hyaline  zone,  especially  near  the  oil  cap  and  in  the  neighboring  perivitelline  space. 
Numerous  metachromatic  vacuoles  are  also  observed  in  close  association  with  the  peripheral 
parts  of  the  asters  in  non-induced  centrifuged  eggs ;  and  during  telophase  some  of  these 
vacuoles  are  seen  to  move  peripherally  into  the  cell  cortex. 

The  data  suggest  that  the  metachromatic  granulated  vacuoles  play  some  role  in  the 
induction  of  the  furrowing  reaction.  It  is  suggested  that  induction  may  depend  upon  the 
liberation  of  material  from  the  vacuoles  after  they  have  reached  the  cell  cortex.  However, 
material  from  the  nucleus  may  also  be  involved,  since  furrow-inducing  treatments  always 
rupture  the  nuclear  as  well  as  the  vacuolar  membranes. 

Graded  and  all-or-none  electrical  acuity  in  insect  muscle  fibers.     F.  V.  McCANN, 
R.  WERMAN  AND  H.  GRUNDFEST. 

In  muscle  fibers  of  Romalea  microptera  modifications  were  produced  in  the  electrically 
excitable  responses  by  applying  divalent  alkali  earth  cations.  The  effects  were  analyzed  with 

1  Work  supported  by  grant  C-807  from  the  National  Cancer  Institute,  U.S.P.H.S. 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY         357 

intracellular  stimulations  and  recording.  Confirming  earlier  work,  normal  electrically  ex- 
citable responses  of  the  muscle  fibers,  whether  evoked  by  neural  stimuli  or  by  intracellular 
depolarizations,  are  graded.  They  arise  with  vanishingly  brief  latency,  are  small  and  decre- 
mentally  propagated.  Maximal  directly  evoked  electrical  responses  produce  only  local  visually 
observable  contractions.  When  10%  of  the  external  Na+  is  replaced  with  Ba++,  spontaneous 
repetitive  firing  occurs  in  the  fast  axon  and  in  the  muscle  fibers.  Hyperpolarization  of  the 
latter  stops  their  repetitive  discharge.  Frequently,  however,  no  postsynaptic  potentials  are 
then  disclosed.  Brief  direct  stimuli  also  induce  repetitive  firing,  without  neural  activity. 
Therefore,  Ba++  can  initiate  repetitive  activity  in  the  muscle  fibers  by  direct  action.  Membrane 
conductance  is  usually  decreased  in  the  presence  of  Ba++,  the  time  constant  and  space  constant 
both  increasing  while  the  resting  potential  is  unchanged.  The  threshold  for  electrical  stimula- 
tion decreases  markedly.  Probably  as  a  result  of  these  several  factors,  the  electrically  excitable 
activity  changes  from  graded  to  all-or-none  responsiveness.  Simultaneous  recording  with 
several  microelectrodes  shows  that  this  change  occurs  only  in  some  regions  of  the  fiber. 
Spikes  therefore  may  arise  at  a  site  some  distance  from  an  intracellular  stimulating  electrode, 
and  sometimes  at  several  sites  independently,  then  propagating  into  other  regions  that  are 
gradedly  responsive.  The  duration  of  the  response  is  greatly  increased.  Higher  concentra- 
tions of  Ba++  further  increase  spike  height  and  markedly  prolong  the  responses  which  then 
resemble  spikes  of  cardiac  muscle  and  may  last  up  to  30  seconds.  Brief  repolarization  by  an 
inward  current  pulse  abolishes  the  spike.  The  current  required  to  do  this  depends  on  the 
membrane  potential  when  the  pulse  is  applied.  The  threshold  is  lowered  and  the  response 
amplitude  and  duration  are  increased  by  high  concentrations  of  Ca++,  but  the  magnitudes  of 
these  changes  are  relatively  small  with  this  ion.  On  the  other  hand,  Sr++  appears  to  be  almost 
as  effective  as  is  Ba++  in  converting  the  gradedly  responsive,  electrically  excitable  membrane 
into  the  regenerative  variety  which  produces  all-or-none  spikes. 

Hyperglyccinia  and  islet  damage  after  intracardiac  injection  of  allo.van  in  toadfish. 
P.  F.  NACE,  J.  E.  SCHUH,  L.  R.  MURRELL  AND  A.  D.  DINGLE. 

Histological  and  blood  sugar  studies  of  toadfish  injected  with  alloxan  by  gill  arch,  sub- 
cutaneous, intraperitoneal  and  intramuscular  routes  have  shown  very  variable  responses  to 
doses  of  400  to  800  mg./kg.  A  series  of  approximately  200  experimental  and  200  control 
fish  has  shown  a  very  consistent  response  to  the  intracardiac  injection  of  10%  iced  aqueous 
alloxan.  For  an  unnumbered  lot  of  Eastman  Kodak  alloxan,  a  dose  of  700  mg./kg.  was 
effective  in  almost  all  cases,  with  very  low  mortality.  Other  lots  tested  have  required  higher 
or  lower  dose  levels. 

Marked  hyperglycemia  was  observed  three  hours  after  treatment.  This  persisted  for 
about  two  days.  From  2  to  6  days  after  treatment,  a  gradual  decline  to  normal  levels  was 
found.  Principal  islets  of  animals  sacrificed  in  the  first  two  days  after  treatment  exhibited 
extensive  beta  cell  damage,  with  nuclear  pycnosis  and  cytoplasmic  degeneration.  Islets  fixed 
6  days  after  treatment  appear  nearly  normal,  with  small  necrotic  foci. 

The  course  of  blood  sugar  and  cellular  changes  in  this  animal,  Opsanus,  differs  markedly 
from  that  seen  in  the  catfish,  Ictalwus,  in  which  both  blood  sugar  elevation  and  cell  damage 
persist  without  apparent  trend  toward  recovery. 

This  work  was  supported  by  Research  Grant  A-1129,  National  Institute  of  Arthritis  and 
Metabolic  Diseases,  U.S.P.H.S. 

The  effect  of  bacterial  cndoto.vins  and  biogenic  amines  on  the  pliagocytic  behavior 
of  endothelial  elements  in  the  frog,  Rana  pipiens.  ARNOLD  L.  NAGLER  AND 
BENJAMIN  W.  ZWEIFACH. 

Although  bacterial  endotoxins  are  not  lethal  in  the  frog  (Rana  pipiens)  in  any  dose, 
extracts  of  E.  coli  (500  /xg/40  gm.)  administered  intravenously  facilitated  the  clearance  (as 
measured  photometrically)  of  a  carbon-gelatin  suspension  from  the  bloodstream;  values  for 
K  (phagocytic  index)  were  0.132  at  45  minutes  (26  animals),  in  contrast  to  control  values 
of  0.045  in  30  animals  (a  300%  increase).  This  stimulating  action  was  no  longer  evident 
after  two  hours. 


358         PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

In  search  for  mediators  of  this  effect,  frogs  were  pre-treated  with  48/80  and  polymyxin  B, 
agents  known  to  release  histamine,  5-hydroxytryptamine  (serotonin),  norepinephrine  and 
heparin.  Both  drugs  counteracted  the  stimulating  action  of  endotoxins  on  RES  clearance. 
Various  biologic  amines  were  injected  i.v. — epinephrine,  norepinephrine,  serotonin  and  histamine. 
Epinephrine  was  the  only  agent  with  a  stimulating  effect  on  the  RES.  As  anticipated,  an 
adrenergic  blocker,  dibenzyline,  abolished  the  endotoxin  effect  on  the  RES.  Dibenzyline  did 
not  counteract  the  stimulating  action  of  epinephrine,  suggesting  that  this  effect  was  not  due 
to  its  pressor  properties.  When  equivalent  amounts  of  serotonin  were  mixed  with  epinephrine, 
there  was  no  stimulation  of  RES  function.  Passive  transfer  of  blood,  removed  after  the  frogs 
had  received  polymyxin  B  or  48/80,  indicates  the  presence  of  an  inhibitory  agent,  1.2  ml.  of 
heparinized  blood  serving  to  counteract  the  usual  stimulating  action  of  endotoxin  and  epinephrine 
on  the  RES.  Histologic  inspection  reveals  that  normally  the  uptake  of  carbon  is  restricted 
to  the  liver  and  spleen.  Increased  phagocytic  function,  induced  by  endotoxin  or  by  epinephrine, 
is  associated  with  extensive  deposits  of  carbon  along  the  peritubular  capillaries  and  glomerular 
loops.  Agents  which  prevented  stimulation  of  the  RES,  prevented  uptake  of  carbon  by  the 
kidney  vessels. 

The  data  suggest  that  in  the  frog  reno-portal  system,  endotoxins  result  in  a  more  rapid 
clearance  of  particulate  materials  from  the  bloodstream  through  an  effect  primarily  on  vessels 
of  the  kidney  mediated  by  the  release  of  epinephrine. 

Changes  in  behavior  of  the  cell  ivall  and  cytoplasm  due  to  injuries  in  Nitella  fle.rilis. 

W.  J.  V.  OSTERHOUT. 

(I)  When  an  uninjured  cell  of  Nitella  flexilis  was  placed  for  a  few  minutes  in  0.01  per 
cent  cresyl  blue  solution  at  pH  9  the  dye  in  molecular  form  penetrated  rapidly  into  the  cell 
and  dissociated  in  the  acid  sap.     When  this  cell  was  transferred  to  a  buffer  solution  at  pH  9 
containing  no  dye,  little  or  no  dye  escaped  from  the  cell.     The  cell  wall  remained  pale  violet, 
and  the  vacuole  appeared  deep  blue. 

(II)  A  cell  was  bent  until  a  protoplasmic  mass  was  formed  as  a  result  of  the  injury.     This 
cell  was  placed  in  the  dye  solution  at  pH  9  as  in   (I).     The  dye  penetrated  rapidly  into  the 
uninjured  parts  of  the  cell  and  collected  in  the  vacuole  while  the  injured  spot  appeared  less 
colored  until  the  dye  became  uniformly  distributed  in  the  vacuole.     When  this  cell  was  trans- 
ferred to  a  buffer  solution  at  pH  9  as  in  (I)  the  dye  came  out  from  the  injured  spot  but  not 
elsewhere.     The    injured   spot   became   more    stained   while    the    rest   of   the    cell    became    less 
stained. 

(III)  If  an  uninjured  cell  was  placed  for  a  few  minutes  in  0.05  per  cent  dye  at  pH  5.5 
the  cell  wall  became  purple  but  no  dye  was  found  in  the  vacuole.     When  this  cell  was  trans- 
ferred to  a  buffer  solution  at  pH  9  the  dye  from  the  cell  wall  rapidly  penetrated  the  cell  until 
the  cell  wall  became  pale  violet  and  the  vacuole  deep  blue. 

(IV)  When  a  cell  was  bent  as  in   (II)   and  was  placed  in  the  dye  solution  at  pH  5.5  as 
in    (III)    the   cell   wall   was   deeply   stained   after   a   few   minutes   except   at   the   injured    spot 
which  was  much  less  stained.     There  was  no  dye  in  the  vacuole.     When  this  cell  was  trans- 
ferred to  a  buffer  solution  at  pH  9  as  in   (III)   the  dye  rapidly  penetrated  the  uninjured  parts 
of  the  cell  and  collected  in  the  vacuole  but  the  injured  spot  remained  much  less  stained. 

These  results  indicate  that  the  injuries  alter  the  cell  wall  and  the  cytoplasm. 

Changes  in  permeability  to  an  acid  dye  due  to  protoplasmic  lesions  in  Nitella  flexilis. 
W.  J.  V.  OSTERHOUT. 

(I)  Single  internodal  cells  of  Nitella  flexilis  were  employed. 

(II)  Buffer  solution  at  pH  5.5  was  made  up  in  0.02  M  phosphate  buffer  mixture.     Buffer 
solution  at  pH  8  was  made  up  in  0.02  M  phosphate  or  borate  buffer  mixture.     The  concentration 
of  the  dye  solution  was  0.05   per  cent  unless  otherwise  stated.     Staining  of  the   protoplasmic 
mass  was  determined  after  two  minutes'  exposure  to  the  dye  solution.     The   exit  of  the  dye 
was   determined   after   five   minutes   in   a   buffer   solution   containing   no   dye   unless   otherwise 
stated. 

(III)  The  dye  did  not  readily  enter  the  cells  unless  they  were  injured. 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY  359 

(IV)  The  cell  was  bent  until  a  protoplasmic  mass  was  formed  as  a  result  of  the  lesion. 
This  was  rapidly  stained  in  the  dye  solution  at  pH  5.5.     The  same  result  was  obtained  with 
the  dye  in  distilled  water. 

(V)  If  a  cell  was  placed  in  the  dye  solution  at  pH  8  the  dye  did  not  stain  the  protoplasmic 
mass  rapidly.     There  was  very  little  staining  after  two  minutes'  exposure. 

(VI)  If  a  cell  was  immersed  for  five  minutes  in  the  buffer  solution  at  pH  8  and  then 
transferred  to  the  dye  solution  at  pH  5.5  there  was  a  rapid  staining  of  the  protoplasmic  mass. 

(VII)  The  dye  came  out  from  the  protoplasmic  mass  into  distilled  water,  into  a  buffer 
solution  at  pH  8  or  at  5.5,  all  containing  no  dye  and  also  into  the  buffer  solution  at  pH  8 
containing  0.05  per  cent  dye. 

(VIII)  Since  the  color  of  the  dye  at  pH  8  became  paler  after  a  few  minutes  higher  con- 
centrations of  the  dye  up  to  0.5  per  cent  were  used  and  the  same  results  were  obtained. 

These  results  indicate  that  the  permeability  to  the  dye  is  altered  at  the  point  of  bending 
so  that  the  protoplasmic  mass  can  take  up  an  acid  dye  rapidly  when  the  rest  of  the  cell  re- 
mains unstained.  The  staining  of  the  cell  wall  is  too  slight  to  play  an  important  role  here. 

Inhibitory  effect  of  electrolytes  on  the  penetration  of  organic  molecules  into  Nitella 
fle.vilis.     W.  J.  V.  OSTERHOUT. 

(I)  A  basic  dye,  azure   B,  penetrated  into  the  cells  of  Nitella  fle.vilis  more   rapidly  at 
higher  pH  values  in  which  the  dye  was  largely  in  molecular  form.     The  dye  dissociated  in 
the  acid  sap  and  accumulated  since  it  could  not  come  out  of  the  cell  rapidly  in  this  form. 

(II)  The  control  cells  were  kept  in  solution  .A  formerly  described  by  the  author.     These 
cells  were  transferred  to  0.005  per  cent  dye  solution  at  pH  8  (in  0.007  M  borate  buffer  mixture) 
at  22°   Centigrade,  and  after  one  minute  there  was  0.008  per  cent  dye  in  the  vacuole.     This 
was  taken  as  a  standard  with  which  other  experiments  were  compared. 

(III)  Previous  exposure  to  distilled  water  for  one  hour  did  not  alter  the  rate  of  pene- 
tration of  the  dye  as  compared  with  the  standard. 

(IV)  Previous  exposure  to  0.001  M  CaCU  solution  for  five  minutes  did  not  alter  the  rate 
of  penetration  of  the  dye  as  compared  with  the   standard.     The   same  results  were   obtained 
when  similar  experiments  were  made  with  MgCU,  MgSOi,  and  LaCla.     All  pH  values  were 
adjusted. 

(V)  Previous  exposure  to  0.005  M  KC1  solution  for  five  minutes  brought  about  45  per 
cent  decrease  in  the  rate  of  penetration  of  the  dye  as  compared  with  the  standard.     The  same 
results  were  obtained  when  similar  experiments  were  made  with  KNOs  and  KsSOi. 

(VI)  This  inhibitory  effect  of  the  salts  with  monovalent  cations  was  completely  abolished 
when  the  cells  were  subsequently  exposed  for  five  minutes  to  0.001  M  salts  with  bivalent  and 
trivalent  cations   (under  IV). 

(VII)  The  concentration  of  the  halides  in  the  control  sap  was  0.12  M.     This  remained 
unchanged  when  the  cells  were  exposed  to  the  salt  solutions  mentioned  under  (IV)  and  (V)  for 
one-half  hour. 

These  results  confirm  those  obtained  by  Marian  Irwin  in  experiments  on  the  inhibitory 
effects  of  sodium  salts  on  the  penetration  of  cresyl  blue  and  subsequent  abolition  by  salts  of 
bivalent  and  trivalent  cations. 

Rate  of  recovery  of  centrifitgally-deformed  mast  cells  as  a  function  of  age  in  the  rat. 
JACQUES  PADAWER/  DOUGLAS  MARSLAND  ~  AND  WALTER  AUCLAIR. 

Rat  peritoneal  fluid,  containing  numerous  free-floating  mast  cells,  was  centrifuged  at 
41,000  X  g  and  12,000  lbs./in.2  pressure  long  enough  to  deform  appreciably  most  of  the  cells. 
By  this  procedure,  pressure-induced  cytoplasmic  solation  allowed  the  normally  spherical  mast 
cells  to  elongate.  Rates  of  return  to  spherical  shape  were  then  measured  both  on  a  popula- 
tion and  on  an  individual  cell  basis.  Two  age  groups  were  investigated,  7-9-week  and 
11-12-month  old  rats.  In  the  young  adult  animals,  population  recovery  was  much  faster  than 

1  Supported  by  grant  from  the  American  Heart  Association. 

2  Supported  by  grant  C-807  from  National  Cancer  Institute. 


360         PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

in  the  older  rats.  From  a  time-lapse  photographic  study  of  individual  cells,  this  difference 
was  shown  to  result  from  an  appreciable  lag  prior  to  the  inception  of  rounding  in  many  of 
the  cells  from  the  older  rats,  as  well  as  from  morphological  readjustments  involving  transi- 
tions from  one  type  of  deformation  to  another,  a  process  not  commonly  observed  with  cells 
from  the  younger  rats.  Shortening  of  deformed  mast  cells  was  found  to  proceed  as  a  definite 
function  of  time.  A  few  non-spherical  mast  cells  normally  occurred  in  the  peritoneal  fluid 
of  the  older  rats  and  these,  too,  eventually  rounded  up  in  vitro.  From  the  data,  it  appears 
that  the  differences  observed  between  the  two  age  groups  studied  are  ascribable  to  all  the 
cells  of  the  respective  populations. 

A  novel  method  for  correcting  astigmatism  in  electron  microscopes.     DELBERT  E. 
PHILPOTT. 

Most  recently  produced  electron  microscopes  are  capable  of  resolution  of  approximately 
ten  Angstrom  units.  However,  this  resolution  lasts  for  a  very  short  period  of  time  and  then 
slowly  declines.  This  is  due  to  contamination  of  the  objective  aperture  which  is  usually  nec- 
essary to  provide  adequate  contrast  in  the  final  image.  Externally  compensatable  microscopes 
make  it  possible  to  visually  correct  most  of  the  astigmatism  while  viewing  the  defect  with 
some  suitable  specimen.  The  final  correction  must  be  done  on  photographic  plates.  The 
purpose  of  the  following  method  is  to  provide  visual  correction  down  to  the  limit  of  resolution. 

Two  sets  of  coils,  one  above  the  other,  when  placed  on  opposite  sides  of  an  electron  beam 
can  be  made  to  wobble  the  beam  when  it  is  out  of  focus  if  alternating  current  is  fed  to  these 
coils.  The  disadvantage  of  the  above  is  that  any  astigmatism  in  any  direction  except  ninety 
degrees  to  the  beam  wobbler  will  cause  the  focus  to  be  off  proportionately  to  the  degree  of 
astigmatism.  By  placing  another  set  of  coils  ninety  degrees  to  the  first  set,  and  making  the 
unit  mechanically  rotatable,  one  set  can  be  put  in  the  direction  of  astigmatism  and  the  other 
perpendicular  to  the  beam  (where  no  astigmatism  exists).  The  microscope  will  now  focus 
properly  with  one  set  of  coils  and  overfocus  with  the  second  set.  External  compensation 
can  now  be  applied  until  the  beam  wobbling  in  the  direction  of  astigmatism  ceases.  Since 
the  beam  wobbling  increases  the  ability  to  see  the  astigmatism,  visual  correction  can  be 
carried  out  without  the  need  to  resort  to  photographs.  This  saves  time  and  correction  carried 
out  during  operation  is  not  subject  to  mechanical  or  electrical  change  before  the  pictures  are 
taken. 

Conduction  in  Phascolosoma  fusiform  muscle.     CHARLES  L.  RALPH  AND  C.  LADD 
PROSSER. 

The  fusiform  muscle  of  Golfingia  (Phascolosoma)  gouldi,  a  non-striated  retractor,  is  a 
thread-like  structure  along  the  mesentery  of  the  ascending  intestine.  Its  fibers  are  approxi- 
mately 4  ^  in  diameter,  1  mm.  in  length  and  have  a  helical  configuration  when  contracted. 
Electrical  stimulation  of  the  muscle  evoked  a  twitch-like  contraction  lasting  8-10  seconds. 
Frequently,  repetitive  contractions,  occurring  at  regular  intervals  and  continuing  for  several 
minutes,  followed  a  single  stimulation.  A  quick  stretch  of  1-2  mm.  produced  a  twitch  essen- 
tially like  that  evoked  by  electrical  stimulation.  These  responses  were  graded  in  tension 
and  latency  according  to  the  amount  of  stretch  applied.  Tetracaine,  d-tubocurarine,  and  high 
calcium  concentrations  all  modified  the  twitch  by  slowing  the  rate  of  tension  development 
and  extending  its  duration  4-5  times  the  normal  length.  Acetylcholine  stimulated  the  muscle, 
but  physostigmine  did  not  potentiate  its  effects.  Although  there  is  a  nerve  extending  the 
length  of  the  muscle  there  is  no  evidence  that  it  mediates  excitation.  Tetracaine  (10~4)  failed 
to  block  conduction.  Degenerated  muscle  segments  showing  no  nerve  fibers  when  examined 
with  methylene  blue  still  responded  to  stretch  and  electrical  stimulation.  High  amplification 
oscilloscope  recordings  from  external  electrodes  failed  to  show  nerve  spikes  preceding  the 
muscle  action  potential.  The  latter  were  propagated  at  about  1  cm./sec.,  characteristic  of 
muscle  conduction.  When  a  portion  of  the  muscle  was  prevented  from  moving,  as  by  wrap- 
ping the  middle  part  several  times  around  a  glass  rod,  there  was  no  activity  beyond  the  fixed 
point.  Several  methods  of  suspending  the  muscle  demonstrated  that  unless  the  stimulated 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY         361 

muscle  was  allowed  to  develop  tension  and  produce  stretch  in  a  non-excited  region,  there  was 
no  propagation  of  excitation.  It  is  concluded  that  in  the  fusiform  muscle  conduction  is 
effected  by  means  of  intercellular  stretch. 

A  photosensitive  pigment  from  the  dorsal  skin  and  eyespots  of  the  starfish,  Asterias 
forbesi.1     MORRIS  ROCKSTEIN,  JANICE  COHEN  AND  SANFORD  A.  HAUSMAN. 

Improvement  in  extraction  procedures,  consisting  chiefly  of  an  increase  to  five  of  the 
number  of  acid  buffer  "wash  extractions,"  resulted  in  the  elimination  of  an  overlying  violet 
pigment  and  a  resultant  "rosy  peach"  to  the  2%  aqueous  digitonin  extract  of  the  dorsal  skin. 
Despite  this  visible  change  in  color,  the  X  max  of  this  pigment  remained  unchanged  at  495  m/* ; 
the  minimum  absorbance  was  shifted  somewhat  to  about  385  m/a.  However,  the  lowering  of 
the  Dmln/Dmax  from  values  of  as  high  as  0.7-0.8  (cf.  Biol.  Bull,  113:  353-354,  1957)  to  as 
low  as  0.45  indicated  as  improved  purity  of  the  extracted  photosensitive  pigment.  The  dif- 
ference spectrum  was,  as  expected,  essentially  identical  with  those  obtained  for  exposures  to 
lower  wave-lengths  (340  to  550  m/u).  However,  the  "reverse  bleaching"  effects  of  short- 
term  exposures  to  longer  wave-lengths  (600  to  700  rmx),  with  an  identically  reciprocal  dif- 
ference spectrum,  reported  earlier,  could  be  reversed  by  increasing  the  exposure  time  at 
such  wave-lengths  to  4.5  hours.  It  is  thus  apparent  that  at  longer  wave-lengths  the  photo- 
sensitive pigment  of  the  starfish  is  capable  of  regeneration,  provided  that  such  exposure  is 
limited  (cf.  Hubbard,  R,  and  St.  George,  R.  C.  C,  /.  Gen.  Physiol,  41:  501,  1958).  Heating 
in  boiling  water  bath  for  five  minutes  shifted  the  peak  absorbance  to  455  m/u.  with  a  visible 
color  change  to  a  yellow-brown  peach  hue  accompanied  by  the  deposition  of  a  whitish  pre- 
cipitate. A  Biuret  test  of  this  precipitate  was  positive.  A  Biuret  test  of  an  unheated  digitonin 
extract  of  250  pigment  spots  was  also  positive,  indicating  at  least  10  mg.  of  protein  per  250 
eyespots.  It  appears  as  though  the  photosensitive  pigment  in  the  skin  and  eyespots  of  A. 
forbesi  does  not  involve  the  splitting  of  a  retinene-like  compound  and  a  protein  in  the  presence 
of  effective  light  intensities.  Indeed,  the  splitting  off  of  a  protein  moiety  is  accomplished 
only  with  extreme  denaturation.  A  Carr-Price  test  on  a  chloroform  extract  of  methanol- 
treated  pigment  from  non-dark-adapted  eyespots  gave  a  positive  result. 

Artificial  hybridization  between  two  species  of  Menidia    (silverside  fishes}.     IRA 

RUBINOFF  AND  EVELYN   SHAW. 

M.  menidia  and  M.  bcryllina  are  sympatric  species  in  the  Cape  Cod  area.  They  occupy 
similar  ecological  niches  and  are  frequently  taken  together  in  a  seine  haul.  Moreover,  the 
gonads  of  both  species  are  ripe  at  the  same  time  during  June  and  early  July,  suggesting  that 
these  fish  may  be  capable  of  interbreeding.  However,  no  hybrids  have  been  found  among 
the  many  specimens  collected  in  the  field.  To  determine  whether  or  not  these  fish  can  hybridize, 
reciprocal  crosses  were  made  in  the  laboratory  with  16  adult  M.  menidia  and  51  adult  M. 
bcryllina.  In  the  classification  of  these  species,  the  main  distinguishing  character  is  the  number 
of  rays  in  the  anal  fin;  adult  specimens  of  M.  menidia  have  22-25  rays  (mean,  23.3),  those 
of  M.  beryllina  have  14-17  rays  (mean,  15.2).  Since  these  ranges  in  the  adults  do  not 
overlap,  this  character  was  analyzed  in  the  Fi  generation. 

Good  yields  of  fertilized  eggs  were  obtained  from  both  reciprocal  crosses.  All  groups 
began  hatching  9  days  after  fertilization,  and  in  most  specimens  the  rays  could  be  counted 
by  the  24th  day  after  hatching.  The  average  numbers  of  fin  rays  of  both  hybrid  crosses 
fall  between  those  of  the  parents.  The  mean  numbers  and  ranges  of  the  hybrid  and  control 
groups  were  as  follows;  M.  menidia  $  X  M.  bcryllina  d,  20.8  (19-23);  M.  beryllina  ?  X  M. 
mcnidia  <3,  18.6  (17-20);  M.  menidia^  XM.  menidia  J,  22.8  (22-25);  M.  beryllina^  X  M. 
beryllina  J,  15.8  (14-17). 

The  fact  that  these  forms  can  hybridize  in  the  laboratory  raises  questions  about  the  natural 
isolating  mechanisms  that  tend  to  keep  them  taxonomically  discrete. 

1  This  research  was  supported  in  part  by  a  grant  from  the  National  Science  Foundation 
(NSF  G-3517). 


362         PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

The  effect  of  ^'-irradiation  of  the  early  fish  embryo.     ROBERTS  RUGH  AND  ERIKA 
GRUPP. 

The  poikilothermic  eggs  of  Fundulus  (fish)  are  radio-resistant  when  compared  with 
eggs  of  homoiothermic  forms.  There  is  a  progressive  decrease  in  the  sensitivity  with  early 
developmental  changes,  the  pre-cleavage  stage  being  the  most  sensitive  and  the  post-gastrula- 
tion  stage  the  most  resistant.  The  relative  values  were  500  r  to  10,000  r. 

This  study  was  designed  to  determine  whether  the  nervous,  muscular,  or  circulatory  sys- 
tems might  be  specifically  affected  following  x-irradiation  of  pre-differentiation  stages.  It 
was  found  that  exposure  prior  to  gastrulation,  even  at  the  2-4-cell  stage,  could  affect  neural 
development  without  comparable  effects  on  the  muscular  or  circulatory  systems.  Fish  embryos 
were  produced  without  heads,  without  eyes,  and  with  various  degrees  of  deletion  of  anterior 
neural  elements.  In  these  same  embryos,  which  survived  well  but  did  not  hatch,  there  was 
normal  but  slightly  retarded  pulsation  of  the  heart  (sometimes  even  without  corpuscles),  and 
muscular  movements  of  the  caudal  end.  Thus  it  appears  that  failure  of  development  of  the 
nervous  system  may  be  caused  some  time  before  the  onset  of  neural  differentiation  while  the 
circulatory  and  muscular  systems  are  not  comparably  affected.  The  effect  appears  to  be 
largely  cephalic  since  there  is  response  of  caudal  structures  to  tactile  stimulation. 

Ionic  regulation  in  a  spider  crab.     RICHARD  C.  SANBORN. 

Animals  respond  to  ionic  and  osmotic  changes  in  their  environment  by  regulating  the 
ionic  and  osmotic  concentration  of  their  body  fluid  or  by  allowing  the  body  fluid  to  reflect 
external  changes  and  adjusting  the  tissues  to  such  variations.  Examination  of  several  organs 
of  an  animal  the  osmotic  concentration  of  whose  blood  varies  with  changing  external  con- 
centration, Libinia  cmarginata,  shows  that  they  are  surrounded  by  a  tough,  multicellular  con- 
nective tissue  sheath.  I  have  tested  the  ability  of  the  sheath  surrounding  the  supraeoesophageal 
and  thoracic  ganglia  of  this  animal  to  regulate  the  osmotic  and  ionic  composition  of  the  tissues 
within.  Isolated  ganglia  with  the  cut  ends  of  the  nerves  ligatured  were  treated  with  varied 
concentrations  of  some  twenty  compounds ;  changes  in  weight  and  in  internal  concentration 
of  relevant  compounds  and  ions  were  measured  by  appropriate  techniques.  The  tissues,  when 
surrounded  by  an  intact  sheath,  undergo  net  gains  or  losses  of  water,  glycerol,  and  the  following 
ions :  bicarbonate,  chloride,  magnesium,  potassium,  sodium  and  probably  calcium.  In  parallel 
experiments  no  net  gain  or  loss  of  glucose,  sucrose,  sulfate,  or  lithium  is  observed  at  external 
concentrations  varying  from  100  to  1600  milliosmoles  per  liter.  When  the  integrity  of  the 
sheath  is  interrupted,  the  final  concentration  and  the  rate  of  penetration  of  the  ions  and  com- 
pounds are  markedly  changed. 

Preliminary  measurements  with  a  micro-electrode  indicate  that  the  potential  between  sea 
water  and  the  extracellular  space  inside  the  sheath  is  about  30  mv.,  while  the  neurons  within 
the  sheath  have  resting  potentials  of  60  to  80  mv.  When  soaked  in  sea  water,  the  ensheathed 
ganglia  lose  potassium  and  take  up  sodium  and  chloride.  Similar  behavior  was  described  for 
leg  muscle  fibers  of  this  species  by  Stephenson  (1955,  Biol.  Bull.).  On  the  basis  of  these 
experiments  it  seems  that  the  adjustment  of  Libinia  to  environmental  changes  in  salt  concen- 
tration is  carried  out,  in  large  measure,  by  the  connective  tissue  sheath  rather  than  by  the 
cells  or  by  osmoregulatory  organs.  Similar  mechanisms  have  been  shown  to  operate  in 
another  arthropod  group,  the  insects. 

Aided  by  a  grant  from  the  National  Science  Foundation. 

Water  relations  of  the  Spisula  egg.     VICTOR  SCHECHTER. 

The  phase  of  the  work  here  reported  has  been  directed  chiefly  toward  the  effect  of  water, 
introduced  under  hypotonic  conditions  in  the  medium,  upon  nuclear  dynamics. 

1.  Cleavage  furrows  continue  to  appear  after  nuclear  activity  has  been  inhibited  by  means 
of   water   narcosis.     It   is   a   significant   implication   that   the    cortical    phenomena   of   cleavage 
are  not  rigidly  locked  to  the  mitotic  figure  but  are,  to  some  extent  at  least,  autonomous. 

2.  The   germinal  vesicle   is   less   sensitive   to   water   than   are   the   pronuclei.     The   critical 
point  for   inhibition   of  maturation   does   not   occur   until   a   dilution   containing   only  40%    sea 
water,  while  male  and  female  pronuclei  cease  their  interaction  in  60%  sea  water. 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY         363 

3.  There  is  a  slight  but  definite  difference  in  the  relative  sensitivity  of  the  micromere  and 
the  macromere  of  the  first  cleavage,  resulting  in  a  transient  3-celled  stage,  as  one  blastomere 
precedes  the  other  in  cleavage. 

4.  Recovery  time  of  the  egg  as  a  whole  from  water   narcosis   is   of  lesser   or   of  equal 
duration  to  exposure  time  down  to  a  dilution  of  50%  sea  water.     Beyond  this  point,  recovery 
time  exceeds  exposure  time,  thus  defining  the  limits  of  injury  to  the  protoplasmic  complex. 

5.  There  is  proof  that  the  plasma  membrane  at  the  surface  of  the  egg,  like  the  nuclear 
membrane  itself,  is  impermeable  to  the  osmotic  constituents  of  the  nucleus. 

Urethan  inhibition  of-  cleavage  in  the  CJiaetopterus  egg  and  its  antagonism  by 
various  substances.  HERBERT  SCHUEL. 

When  eggs  of  the  marine  worm  Chaetopterus  pergamentaceus  are  placed  in  a  \%  solu- 
tion of  urethan  (ethyl  carbamate)  in  sea  water  5  minutes  after  insemination,  cleavage  is  in- 
hibited and  the  characteristic  increase  in  cytoplasmic  viscosity,  the  "mitotic  gelation,"  does 
not  take  place.  Ordinarily  when  eggs  are  allowed  to  remain  in  \%  urethan  (at  21°  C.) 
very  few  if  any  eggs  begin  to  divide  until  about  4  hours  after  insemination.  The  percentage 
of  eggs  found  to  have  divided  under  these  conditions  during  this  period  ranges  from  0% 
to  6%.  The  addition  of  small  amounts  of  proteolytic  enzymes,  of  isotonic  calcium  chloride, 
or  of  certain  basic  substances  makes  it  possible  for  more  of  the  eggs  to  cleave  while  still  in 
the  urethan  solution.  Thus  at  two  hours  after  insemination,  from  10%  to  30%  of  such  eggs 
have  already  divided.  The  following  substances  were  found  to  be  effective:  0.1%  to  0.0001% 
trypsin,  0.1%  to  0.0001%  chymotrypsin,  0.037  M  to  0.007  M  calcium  chloride,  0.01%  to  0.00001% 
histamine,  0.01%  to  0.0001%  protamine,  1.0%  to  0.0001  %  arginine,  and  1.0%  to  0.0001%  lysine. 
In  no  case,  however,  is  there  more  than  a  temporary  recovery,  because  the  eggs  are  able  to 
divide  only  a  few  times  and  do  not  develop  into  larvae. 

Additional  evidence  for  somatic  reduction  in  the  metamorphosis  of  the  ileum  of 
mosquitoes  by  the  use  of  tritiated  thymidine.1  JOSEPH  E.  SCHUH,  S.  J.  AND 
GEORGE  CARANASOS. 

During  the  larval  life  of  the  mosquito,  the  epithelium  of  the  ileum  grows  by  increase  in 
size  of  cell  rather  than  by  cell  multiplication.  Berger  (1938)  described  the  origin  and  fate 
of  these  large  multiple  complexes  containing  96,  48,  24,  12  and  the  diploid  number  of  6  chromo- 
somes. The  larval  epithelium  does  not  undergo  histolysis  but  these  large  cells  undergo  several 
somatic  reduction  divisions  during  metamorphosis,  giving  rise  to  the  numerous  small  cells 
of  the  adult  hind  gut.  Tritiated  thymidine  gave  promise  of  being  a  very  suitable  tool  for  a 
further  study  of  this  unique  phenomenon. 

Larvae  of  Acdcs  acgypti  were  exposed  to  varying  amounts  of  tritiated  thymidine  in  their 
culture  medium  in  various  stages  of  larval  development  and  for  different  lengths  of  time. 
Newly  hatched  larvae,  late  first  instar,  second,  and  third  and  fourth  instar  larvae  were 
treated  for  12,  24  and  48  hours  with  25,  50  and  100  pC  of  tritium  per  10  ml.  of  culture 
medium.  Pupal  hindguts  were  dissected  in  insect  Ringer  solution  and  aceto-orcein  smears 
prepared.  Cover-slips  were  removed  by  the  dry  ice  method  and  radioautographs  prepared 
by  placing  Kodak  Ltd.  AR  10  Radioautographic  Stripping  Film  over  the  specimen.  The  film 
was  exposed  for  from  5  to  10  days  and  developed  according  to  the  method  of  Taylor  (1956). 

Preliminary  results  indicate  that  the  later  the  treatment  is  begun  the  fewer  "hot"  nuclei 
are  found  in  the  metamorphosing  and  metamorphosed  hind  gut.  The  hind  guts  of  pupae  de- 
veloped from  larvae  treated  in  the  third  and  fourth  instar  for  12  and  24  hours  contained  both 
"hot"  and  "cold"  multiple  complexes,  side  by  side.  These  were  in  the  late  prophase  stage 
and  were  coming  up  for  their  first  division.  The  hind  guts  of  pupae  from  larvae  treated 
shortly  after  hatching  and  exposed  to  the  tritium  until  pupation  or  late  fourth  instar  showed 
the  majority  of  the  cells  of  the  metamorphosed  hind  gut  to  be  "hot." 

Affinity  of  tissues  in  reconstituting  tunicates.     SISTER  FLORENCE  MARIE  SCOTT. 

Tunicates  provide  a  rich  source  of  material  for  the  study  of  tissue  affinity.  The  organs 
are  simple,  consisting  of  a  single  layer  of  cells  and  basement  membrane ;  histological  charac- 

1  Supported  by  a  grant  from  the  U.  S.  Atomic  Energy  Commission. 


364         PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

teristics  are  easily  discernible ;  tissues  are  versatile  in  their  capacity  to  regenerate  and  recon- 
stitute. The  entire  animal,  moreover,  can  be  used  in  a  variety  of  experiments  constructed  to 
investigate  the  behavior  of  whole,  though  simple,  systems  in  their  ability  to  recognize  their 
related  structures  and  to  re-establish  former  associations.  Zooids  of  Amaroecium  constel- 
latum,  twelve  hours  after  attachment,  when  adult  organization  has  differentiated  but  feeding 
activities  have  not  yet  commenced,  are  divided  into  three  parts  to  insure  separation  of  organs 
and  are  immediately  dissociated  into  floating  masses  of  tissue  within  the  tunic.  The  experi- 
mental animals  are  implanted  in  tunics  of  older  evacuated  individuals  which  provide  attach- 
ment and  facilitate  their  return  to  running  sea  water  for  optimal  conditions  of  development. 
The  tissues  begin  promptly  to  reconstitute  themselves.  They  retain  their  histological  charac- 
teristics throughout  the  period  of  recovery  from  maceration.  Necrotic  cells  are  extruded 
into  the  host  tunic  space.  Parts  of  organs  aggregate  together  and  recover  their  former  organic 
status.  After  twelve  or  twenty-four  hours  of  recovery,  the  parts  of  systems  re-assemble  into 
their  original  relationships.  They  accomplish  this  reunion  by  a  process  of  directed  growth, 
cells  from  the  anterior  end  of  the  oesophagus  growing  to  meet  corresponding  masses  of  cells 
from  the  oesophageal  funnel  at  the  base  of  the  branchial  basket.  If  there  are  masses  of 
caudal  elements  or  cellular  detritus  in  their  paths,  they  move  around  the  intervening  masses 
to  effect  the  union.  In  cases  where  fragments  of  the  organs  are  too  widely  separated  to  be- 
come re-integrated  into  their  original  positions  in  a  system,  the  fragments  reconstitute  them- 
selves and  regenerate  anterior  and  posterior  sections  and,  then,  attach  themselves  to  their 
parent  structure  in  the  proper  linear  axis.  There  may  be,  therefore,  a  smaller  pharynx  at- 
tached to  the  main  pharynx  or  a  hernia-like  segment  attached  to  the  intestine. 

Inhibiting  action  of  a  triphenylethanol  derivative  on  the  development  of  eggs  of 
Arbacia  punctulata  and  on  the  fertilising  capacity  of  the  sperm.1  SHELDON  J. 
SEGAL  AND  ALBERT  TYLER. 

The  anti-fertility  action  in  rats  of  l-(/>-2-diethylaminoethoxy-phenyl)-l-phenyl-2-/>-ani- 
sylethanol  (referred  to  as  MER-25)  has  been  attributed  to  interference  with  pre-implantation 
zygotic  development.  It  has  been  proposed  that  MER-25  may  have  a  direct  detrimental  effect 
on  the  zygote  or  may  influence  it  indirectly,  by  altering  the  oviducal  environment.  Tests  of 
possible  direct  action  were  carried  out  on  the  gametes  and  developing  zygotes  of  the  sea 
urchin  since  this  avoids  the  complicating  conditions  of  internal  fertilization  and  viviparity. 
A  concentration  of  2  X  10~6%  of  MER-2S,  added  to  the  sea  water  medium  5  minutes  after 
fertilization,  blocks  first  cleavage  of  over  50%  of  treated  eggs.  Those  which  initiate  develop- 
ment do  not  progress  beyond  the  blastula  stage.  Higher  concentrations  of  the  compound 
(2X10"*%)  prevent  the  normal  onset  of  development  in  virtually  all  the  newly-fertilized 
eggs.  At  lower  doses  (2  X  10~7%)  the  compound's  inhibitory  activity  is  also  manifest,  although 
a  small  percentage  of  the  zygotes  (%  the  expected  number)  may  progress  through  gastrulation 
and  develop  into  normal  plutei.  The  blocking  effect  of  MER-25  on  fertilized  eggs  may  be 
reversed  by  washing  after  exposure  to  even  highly  active  concentrations.  When  unfertilized 
eggs  are  treated  with  the  above-mentioned  concentrations  of  MER-25  a  normal  number  remain 
fertilizable  but  developmental  arrest,  as  noted  previously,  occurs.  Washing  unfertilized  eggs 
removes  the  inhibiting  effect.  The  effectiveness  of  MER-25  is  not  enhanced  significantly  by 
prior  trypsin  treatment  of  the  unfertilized  eggs.  The  compound  also  inhibits  both  motility  and 
fertilizing  capacity  of  spermatozoa.  Ninety  to  ninety-nine  per  cent  of  the  spermatozoa  in  a 
1%  sperm  suspension  become  immotile  within  3  minutes  in  a  2  X  10~4%  solution;  the  suspen- 
sion is  unable  to  fertilize  normal  eggs. 

Striicture-activity-relationships  concerning  the  inhibitory  activity  of  synthetic  estro- 
gens and  some  triphenylethanol  derivatives  on  developing  eggs  of  Arbacia  punc- 
tulata.1  SHELDON  J.  SEGAL  AND  ALBERT  TYLER. 

Establishment  of  the  direct  inhibiting  activity  of  l-(/'-2-diethylaminoethoxyphenyl) -1- 
phenyl-2-/>-anisylethanol  (referred  to  as  MER-25)  on  Arbacia  egg  development  has  led  to 
an  investigation  of  a  related  group  of  triphenylethanol  derivatives  and  of  several  synthetic 

1  Supported  by  the  Population  Council  and  by  research  grant  (C-2302)  from  the  National 
Cancer  Institute,  U.S.P.H.S. 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY         365 

estrogens  with  structural  similarities.  MER-25,  at  a  concentration  of  2  X  10~5%  does  not 
impair  fertilizability  of  eggs,  but  subsequent  cleavage  is  prevented  or  proceeds  abnormally. 
Those  eggs  that  divide  are  generally  blocked  in  an  abnormal  blastula  stage  and  remain  intact 
for  36-48  hours  before  disintegrating.  If  treatment  is  initiated  at  stages  up  to  the  just- 
hatching  blastula,  development  is  blocked  before  gastrulation,  the  embryos  again  remaining 
intact  and  motile  for  a  considerable  time.  Post-gastrulation  stages  are  not  visibly  affected 
by  treatment  with  MER-25.  At  the  same  concentration,  the  synthetic  estrogens,  stilbestrol 
and  hexestrol,  are  cytotoxic  to  all  embryos,  pre-  or  post-gastrulation.  When  administered 
to  unfertilized  eggs,  these  compounds  prevent  subsequent  fertilization.  Unfertilized  eggs, 
newly-fertilized  eggs  and  developing  zygotes  (including  well-formed  plutei)  become  non- 
viable  and  cytolyze  within  several  hours  after  exposure  to  these  compounds.  This  appears 
to  be  a  general  cytotoxic  effect,  distinct  from  the  blockage  action  exhibited  by  MER-25. 
The  same  concentration  of  a  third  synthetic  estrogen,  tri-(/>-anisyl)-chloroethylene  (TACE) 
has  no  inhibitory  action  on  Arbacia  development.  The  unsaturated  parent  compound,  tri- 
phenylethylene,  is  likewise  ineffective.  Several  derivatives  of  the  latter,  with  side-chains 
bearing  structural  similarities  to  the  diethylaminoethoxyphenyl  group  and  with  the  1 -carbon 
hydroxyl  substitution  of  MER-25,  exhibit  the  blockage  activity.  The  data  suggest  that  the 
anti-zygotic  activity  of  MER-25  is  not  related  to  its  general  structural  similarity  to  some 
synthetic  estrogens  nor  does  the  activity  derive  from  the  triphenylethylene  moiety  which  pro- 
vides the  basic  configuration  for  the  compounds  in  this  series. 

A  study  of  current  orientation  as  a  stimulus  to  schooling  behavior  in  Menidia. 
EVELYN  SHAW. 

Most  fish,  including  Menidia,  show  a  positive  rheotaxis  when  placed  in  a  moderate  current 
flow.  This  response  is  found  as  early  as  hatching.  Newly  hatched  Mcnidia  immediately 
orient  upstream  and  maintain  a  constant  swimming  speed  within  the  current.  It  seemed 
possible  that  orientation  into  a  current  might  be  an  effective  stimulus  for  the  development 
of  the  parallel  pattern  of  swimming  found  among  schooling  fish.  Orientation  into  the  current 
could,  for  instance,  accustom  pre-schooling  fish  to  seeing  their  species  mates  in  certain  visual 
patterns  which  would  influence  the  fish  in  their  mutual  response  in  such  a  way  that  this 
familiar  visual  pattern  would  be  maintained.  To  evaluate  the  influence  of  current  flow  on 
the  development  of  schooling,  fish  were  reared  to  a  length  of  15  mm.  in  bowls  of  still  water. 
Under  these  conditions,  schooling  developed  at  the  same  age  and  with  the  same  characteristic 
patterns  found  among  fishes  reared  in  a  moderate  current  flow. 

A   study   of  visual   attraction   as   a   stimulus   to   schooling    behavior  in   Menidia. 
EVELYN  SHAW. 

Visual  attraction  as  a  primary  stimulus  for  schooling  has  been  reviewed  by  Morrow  and 
Atz.  In  order  to  determine  whether  visual  attraction  is  an  important  stimulus  in  the  develop- 
ment of  schooling  in  Menidia,  experiments  have  been  carried  out  by  a  technique  which  was 
described  last  year.  This  technique  consists  in  presenting  a  freely-swimming  fish  to  a  fish  of  the 
same  size  enclosed  in  a  narrow  glass  tube.  The  studies  of  last  year  on  early  schooling  fish 
(about  12  mm.  in  length)  have  been  extended  to  include  fish  of  lengths  varying  from  5  to  16  mm. 
It  was  found  that  fish  of  5,  6  or  7  mm.  in  length  did  not  approach  the  enclosed  fish.  Fifty 
per  cent  of  fish  of  8  to  14  mm.  in  length  approached  and  adopted  a  parallel  orientation  for 
brief  periods  (2  to  3  seconds).  Eighty  per  cent  of  fish  of  15-16  mm.  in  length  oriented 
and  swam  parallel  to  the  enclosed  fish.  This  orientation  was  maintained  for  periods  of  time 
up  to  one  minute  during  which  the  direction  of  swimming  changed  as  many  as  seven  times. 
In  all  cases,  the  freely-swimming  fish  did  not  respond  to  the  enclosed  fish  immediately  after 
presentation  of  the  tube.  The  latency  of  response  in  fish  of  8-14  mm.  in  length  averaged 
3%  minutes  and  in  fish  of  15-16  mm.,  50  seconds.  Contrary  to  our  observations  with  this 
technique,  it  should  be  noted  that  freely-swimming  fish  of  5-7  mm.  in  length  approach  one 
another.  They  do  not,  however,  orient  with  one  another.  Two  of  the  possible  explanations 
for  this  difference  in  behavior  are  (1)  approach  by  fish,  5-7  mm.  in  length,  is  not  relevant 
to  schooling  behavior,  (2)  that  fish  of  this  age  require  a  response  from  a  species  mate  which 
cannot  be  given  by  a  fish  which  is  restricted  within  a  glass  tube. 


366         PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

The  occurrence  of  amicronucleate  tetrahymenae  as  facultative  parasites  in  embryos 
of  the  catfish  Ameiurus*     CARL  CASKEY  SPEIDEL. 

Ciliated  protozoan  parasites  were  found  by  P.  B.  Armstrong  in  a  few  embryos  of  the 
catfish  Ameinrus  collected  from  a  fresh  water  pond  in  Falmouth,  Massachusetts.  When 
examined  by  us  the  living  embryos  were  in  the  yolk-sac  stage  and  the  parasites  were  present 
especially,  though  not  exclusively,  in  the  developing  central  nervous  system.  Superficial  ob- 
servation at  once  indicated  that  the  parasites  were  much  like  the  tetrahymenae  previously 
reported  by  us  as  facultative  parasites  in  tadpoles  of  the  toad  Bufo,  now  classed  as  Tetrahymena 
corlissi.  An  important  difference,  however,  was  the  absence  of  a  micronucleus  in  the  catfish 
parasites.  The  organisms  were  observed  further  in  cultures  in  which  the  food  consisted  of 
excised  pieces  of  tadpole  tissues.  Their  details  of  structure  as  well  as  their  methods  of 
movement,  feeding,  reproduction,  and  invasion  of  tissues  seemed  quite  like  those  of  T.  corlissi. 
Furthermore,  like  T.  corlissi  the  catfish  parasites  were  able  to  invade  the  tissues  of  living 
tadpoles  rendered  vulnerable  by  experimental  lesions  or  by  x-ray  irradiation. 

Thus,  the  catfish  parasites  appear  to  be  a  naturally  occurring  strain  of  amicronucleate 
tetrahymenae  possibly  to  be  classified  as  T.  corlissi.  Final  determination  of  the  species, 
however,  must  await  a  study  of  silver-stained  specimens. 

Radiation-induced  variations  in  the  micronucleus  of  Tetrahymena  corlissi.1     CARL 
CASKEY  SPEIDEL. 

As  previously  reported  for  two  strains  of  Tetrahymena  corlissi  the  micronucleus,  a  center 
rich  in  deoxyribonucleic  acid,  was  eliminated  by  repeated  severe  x-ray  treatments.  A  like 
result  has  now  been  obtained  with  a  third  strain  which  was  found  invading  the  tissues  of 
tadpoles  of  Pscudacris  which  had  been  subjected  to  ultraviolet  radiation.  The  tadpoles  and 
the  tetrahymenae  of  this  strain  were  collected  in  a  swamp  near  Charlottesville,  Virginia. 

Further  studies  have  made  it  clear  that  marked  micronuclear  variations  could  be  induced 
by  single  x-ray  treatments  of  from  400-700  kr.  Although  a  single  dose  of  500  kr  killed  the 
great  majority  of  irradiated  tetrahymenae,  a  few  survived  and  multiplied.  These  gave  rise 
to  a  progeny  made  up  of  individuals  that  differed  with  respect  to  micronuclear  content. 
Such  descendants  were  kept  under  observation  for  a  year  for  each  of  two  strains.  The 
descendants  of  one  strain  (Woods  Hole  Strain  W)  exhibited  the  following  micronuclear 
variations:  a  few  had  no  micronucleus;  a  majority  had  a  single  micronucleus  of  reduced 
size  (some,  however,  of  normal  size)  ;  some  had  2  micronuclei ;  and  a  few  had  3,  4,  or  5 
micronuclei.  A  clone  culture  derived  from  a  single  descendant  gave  rise  to  individuals  with 
similar  micronuclear  variations.  This  result  seemed  to  indicate  that  there  had  been  a  profound 
disturbance  in  the  normal  mechanism  by  which  during  binary  fission  each  daughter  cell 
received  one  micronucleus.  The  descendants  of  another  strain  (Thompson's  Charlottesville 
Strain  Th-X)  after  a  dose  of  500  kr  were  almost  entirely  without  a  micronucleus.  A  clone 
culture  derived  from  one  of  these  was  made  up  of  amicronucleate  individuals  only. 

Micronuclear  variations  like  those  listed  above  were  also  induced  in  Tetrahymena  corlissi 
by  repeated  ultraviolet  radiation.  This  type  of  radiation,  however,  was  much  less  effective 
than  x-radiation  in  producing  amicronucleate  individuals. 

Conduction  in  dogfisli  spiral-valve  retractor  and  Phascolosoma  proboscis  retractor 
muscles.     W.  W.  STEINBERGER  AND  C.  LADD  PROSSER. 

The  retractor  of  the  spiral-valve  is  a  sheet  of  non-striated  muscle  arranged  in  fasciculi 
with  individual  fibers  3  /x  in  diameter  and  0.1  mm.  long.  Simultaneous  recordings  of  mechan- 
ical contractions  and  external  action  potentials  show  in  response  to  electrical  stimuli  a  burst 
of  spike-like  action  potentials  preceding  sustained  contractions  lasting  several  minutes.  During 
the  maintained  tension  electrical  activity  decreased  (sometimes  ceasing)  only  to  increase  (or 
reappear)  with  the  onset  of  relaxation.  The  action  potentials  and  the  corresponding  tension 
waves  then  decrease  and  cease.  A  quick  short  mechanical  stretch  is  an  adequate  stimulus 

1  This  investigation  was  supported  by  a  research  grant  (PHS  RG-4326  C)  from  the 
National  Institutes  of  Health,  Public  Health  Service. 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY         367 

giving  a  similar  response  pattern.  Action  potential  propagation  is  about  2  cm./sec.  Methylene 
blue  stain  indicated  the  presence  of  nerve  fibers  but  no  evidence  of  nerve  impulses  prior  to 
muscle  response  were  seen  when  recording  externally  with  high  amplification.  Resting  poten- 
tials as  high  as  70  mV.  were  recorded  with  microelectrodes.  Intracellular  action  potentials 
did  not  overshoot  and  are  spike-like  with  a  duration  of  approximately  0.2  second.  The  spiral- 
valve  retractor  muscle  is  similar  to  mammalian  visceral  muscle  in  that  there  is  interfiber  con- 
duction independent  of  nerves  and  shows  graded  cellular  potentials.  When  a  region  of  the 
retractor  was  mechanically  fixed  propagation  of  action  potentials  occurred  through  the  me- 
chanical block  showing  conduction  in  the  absence  of  mechanical  pull. 

Previous  evidence  showed  that  unlike  vertebrate  smooth  muscles  the  Phascolosoma  pro- 
boscis retractor  contracts  entirely  by  nervous  control.  The  muscle  action  potential  shows 
fast  and  slow  components  conducted  in  different  nerve  fibers.  Microelectrode  records  showed 
that  many  muscle  fibers  give  both  fast  and  slow  potentials,  while  some  give  only  one  type. 
Many  of  the  individual  muscle  fibers  have,  therefore,  double  innervation.  Intracellular  poten- 
tials are  low  (40  mV.)  probably  due  to  the  microelectrode  not  sealing  in  the  fibers  (5  /*). 
Quick  stretch  failed  to  stimulate  the  proboscis  retractor. 

Enzymatic  inactivation  of  cJiromatophorotropic  principles  from   the  fiddler  crab, 
Uca.     G.  C.  STEPHENS  AND  J.  P.  GREEN. 

Extracts  of  the  green  gland  of  Uca  retained  their  chromatophorotropic  activity  for  at 
least  forty-eight  hours  provided  the  extract  was  boiled  for  ten  minutes.  By  contrast,  unboiled 
kidney  extract  was  inactive  twelve  to  twenty-four  hours  after  preparation.  This  suggested 
that  the  green  gland  might  possess  the  capacity  to  inactivate  chromatophorotropins  from  other 
sources  in  the  organism  such  as  the  sinus  gland,  thoracic  cord  and  brain  and  circumesophageal 
connectives. 

In  order  to  test  this  possibility,  extracts  of  these  organs  as  well  as  of  the  green  gland 
were  prepared  at  suitable  concentrations  and  boiled  for  ten  minutes.  Unboiled  extracts  of 
hepatopancreas,  green  gland,  skeletal  muscle  and  heart  were  prepared.  Each  unboiled  extract 
was  tested  for  its  capacity  to  inactivate  the  chromatophorotropins  from  the  four  sources  men- 
tioned. Equal  volumes  of  unboiled  extract  and  each  of  the  boiled  extracts  were  mixed,  strepto- 
mycin added,  the  mixture  allowed  to  stand  at  room  temperature  for  twelve  to  twenty-four 
hours,  and  then  injected  into  previously  prepared  destalked  assay  animals.  The  extracts  of 
skeletal  muscle  and  heart  were  inactive.  The  previously  reported  presence  of  an  inactivating 
material  in  the  hepatopancreas  was  confirmed.  In  addition,  inactivation  of  chromatophoro- 
tropic principles  by  unboiled  kidney  extract  was  observed. 

Activity  of  trypsin  and  papain  was  also  tested  on  boiled  extracts  of  endocrine  sources. 
Both  proved  effective  in  destroying  chromatophorotropic  activity  though  trypsin  was  effective 
only  at  very  high  concentrations  (7%). 

Hepatopancreas  extracts  are  capable  of  digestion  of  gelatin  but  extracts  of  the  green  gland 
are  not. 

The  sensitivity  of  these  chromatophorotropins  to  trypsin  and  papain,  together  with  their 
stability  to  boiling,  suggests  they  may  be  polypeptide  in  nature.  These  observations  further 
suggest  that  inactivation  of  extracts  by  the  green  gland  may  be  more  specific  in  character 
while  inactivation  by  hepatopancreas  may  be  a  consequence  of  its  general  proteolytic  capacity. 

Chromatophorotropic  principles  of  the  green  gland  of  the  fiddler  crab,  Uca.     G.  C. 
STEPHENS,  B.  GUTTMAN  AND  J.  P.  GREEN. 

Chromatophorotropic  activity  of  the  green  glands  of  the  fiddler  crab,  Uca  pugilator,  was 
measured  by  the  following  technique.  Male  donor  and  assay  animals  were  prepared  by  re- 
moving the  eyestalks,  so  that  the  melanophores  became  punctate  and  the  guanophores  dispersed. 
Green  glands  were  removed  from  these  animals  and  ground  in  sea  water  to  make  an  extract 
with  the  concentration  of  10  glands  per  0.5  cc.  of  water.  Five-hundredths  cc.  of  this  extract 
was  then  injected  into  each  assay  animal.  The  chromatophores  were  staged  at  intervals  of 
Vz,  1,  lJ/2  and  2  hours  after  injection  and  their  total  excursion  from  the  initial  condition  was 
used  as  a  measure  of  activity  of  the  extract. 


368         PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

Green  glands  of  donor  animals  produced  strong  black  expansion  and  white  contraction ; 
there  is  also  some  indication  of  a  red  concentrating  element.  As  a  control,  various  concentra- 
tions of  ammonium  chloride  and  urea  in  sea  water  were  injected;  these  produced  no  chromato- 
phore  changes.  Green  gland  extract  shaken  with  Permutit  to  remove  ammonia  retained 
chromatophorotropic  activity.  A  dilution  curve  was  obtained  showing  chromatophorotropic 
activity  as  a  function  of  extract  concentrations. 

An  attempt  was  made  to  modify  the  chromatophorotropic  activity  of  green  gland  extracts 
by  injecting  extracts  of  sinus  gland  or  thoracic  cord  into  donor  animals  and  permitting  them 
to  respond  before  removal  of  their  glands.  The  majority  of  such  experiments  produced  a 
highly  significant  decrease  in  chromatophorotropic  activity  of  the  subsequently  assayed  green 
glands  when  compared  with  those  of  donors  which  did  not  receive  extracts.  However,  other 
experiments  failed  to  exhibit  such  a  difference,  particularly  when  the  green  gland  activity 
of  the  uninjected  donors  was  initially  low.  Some  additional  explanation  is  required  to  account 
for  the  variability  encountered  in  the  green  gland  extracts  of  both  destalked  and  normal 
donor  animals. 

Studies  on  the  effect  of  population  size  on  the  diurnal  inclanophore  rhythm  of  the 
fiddler  crab,  Uca.  _  G.  C.  STEPHENS,  J.  P.  GREEN,  B.  GUTTMAN  AND  R.  A. 

SCHINSKE. 

Thirty-six  Uca  pugnax  were  isolated  in  individual  glass  jars  and  placed  in  a  darkroom 
along  with  two  pans  of  25  animals.  The  melanophores  of  these  animals  were  staged  for 
forty-eight-  to  sixty-hour  periods  six  times,  providing  a  discontinuous  record  of  the  melano- 
phore  rhythm  over  a  period  of  two  months. 

No  maintained  drift  was  evident  in  the  time  relations  of  the  cycle  in  either  group  although 
departures  as  large  as  four  hours  from  the  originally  established  rhythm  were  observed.  The 
previously  reported  decline  in  the  amplitude  of  the  rhythm  was  observed  as  an  increasingly 
obvious  effect.  The  over-all  stability  of  such  isolated  animals  with  respect  to  time  of  dispersion 
of  their  chromatophores  is  indicated  by  the  fact  that  at  the  the  end  of  two  months  of  isola- 
tion, only  four  of  the  32  animals  surviving  had  lapsed  totally  out  of  phase.  The  form  and 
timing  of  the  cycle  of  individual  animals  varied  considerably  from  one  day  to  the  next,  with 
differences  as  great  as  eight  hours  being  observed  in  time  of  dispersion  and  contraction. 

Numerous  observations  were  made  in  an  effort  to  determine  whether  this  effect  on  ampli- 
tude was  mediated  through  the  release  of  some  metabolite  or  other  chemical  agent.  Isolated  ani- 
mals were  subjected  to  periodic  changes  of  the  surrounding  medium  using  extracts  of  endo- 
crine glands,  green  glands  and  water  recovered  from  pans  containing  groups  of  animals.  The 
amplitude  of  the  rhythm  which  they  exhibited  was  then  compared  with  that  of  suitable  controls. 
Grouped  animals  were  placed  in  running  sea  water  and  compared  with  controls  at  the  same 
temperature  maintained  in  standing  water.  Finally,  isolated  animals  maintained  in  a  common 
water  supply  were  compared  with  totally  isolated  animals.  Evidence  for  any  chemical  media- 
tion of  the  amplitude  effect  was  occasional  and  dubious  so  that  one  must  conclude  on  negative 
grounds  that  physical  contact  between  individuals  is  necessary  and  sufficient  for  maintenance 
of  normal  amplitude  of  the  diurnal  melanophore  rhythm. 

Relation  of  halogen  position  to  physiological  properties  of  mono-,  di-,  and  tridiloro- 
phenoxyacetic  acid.  FREDERICK  N.  SUDAK,  C.  LLOYD  CLAFF  AND  ALAN 
GREENBERG. 

Previous  experiments  in  our  laboratory  have  shown  that  2,4  dichlorophenoxyacetic  acid 
(300  mg./kg.)  rendered  rats  poikilothermic  in  relation  to  changes  in  ambient  temperature. 
Animals  treated  with  this  compound  were  unable  to  increase  their  metabolism  or  maintain 
body  temperature  in  a  cold  environment  (7°  C.).  Conversely  these  animals  could  not  lose 
body  heat  nor  control  their  metabolism  when  exposed  to  warm  (35°  C.)  air.  The  effect  of 
2,4,5  trichlorophenoxyacetic  acid  (300  mg./kg.)  was  similar  to  that  of  "2,4-D"  on  metabolic 
and  body  temperature  responses  to  changes  in  ambient  temperature.  Phenoxyacetic  acid,  ortho-, 
and  parachlorophenoxyacetic  acid  (300  mg./kg.)  were  without  effect. 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY         369 

A   bacteria-free  inhibitor  of  regeneration  in  Tubularia.     KENYON  S.  TWEEDELL. 

Inhibition  of  regeneration  in  Tubularia  can  be  produced  by  bacteria-free  filtrates  of  the 
culture  medium.  The  inhibitor  was  obtained  from  adult  hydranths  removed  from  the  colony 
with  portions  of  the  cut  stems  attached.  Fully  mature  individuals  were  avoided.  The  animals 
were  washed  in  5  washes  of  sterile  sea  water  and  placed  in  bacterial  filtered  sea  water  in  a 
ratio  of  3  animals/ml.  The  inhibitor  was  collected  in  a  modified  twin-spout  Ehrlenmeyer  flask. 
Rotation  of  the  animals  was  accomplished  by  an  aerator  leading  inside  to  the  flask  bottom. 
The  temperature  was  maintained  at  15°  C.  by  means  of  a  refrigerated  bath  to  suppress  bac- 
terial activity.  Every  hour  the  collecting  fluid  was  bled  off  through  a  side  arm  and  imme- 
diately returned  to  the  flask  through  a  pressure-operated  Seitz  bacterial  filter  (type  EK  filter). 
Potential  growth  of  any  bacterial  population  was  thus  prevented.  Harvest  of  the  inhibitor 
water  was  taken  at  12  or  18  hours.  The  resulting  fluid  was  clear  and  odorless  with  a  lower 
bacterial  count  than  that  of  normal  sea  water. 

The  inhibitor  water  was  applied  to  freshly  amputated  stems  kept  in  a  water  bath  at  15°  C. 
After  70  hours,  all  control  stems  had  regenerated.  The  stems  in  12-hour  inhibitor  filtrates 
never  emerged.  The  inhibitor  often  affected  differentiation  but  did  not  restrict  internal  circu- 
lation or  movement  of  the  coenosarc.  Qften,  small  bulbs  of  coenosarc  moved  beyond  the  cut 
ends  of  the  stems  or  occasional  abortive  pinch  stages  (constriction  without  differentiation) 
were  encountered.  Complete  cessation  of  activity  was  obtained  with  the  18-hour  filtrates. 

Stems  treated  with  either  12-  or  18-hour  bacterial  filtrated  inhibitor  and  kept  at  21°  C. 
regenerated  at  a  greater  rate  but  gave  identical  results. 

Production  of  S^-labclled  fertilizin  in  eggs  of  Arbacia  pnnctulata.^  ALBERT  TYLER 
AND  RALPH  R.  HATHAWAY. 

The  fertilizins  of  eggs  of  sea  urchins  are  glycoproteins  containing  considerable  sulfate 
which  is  probably  ester-linked.  It  seemed  likely,  then,  that  the  administration  of  S35  in  the 
form  of  inorganic  sulfate  during  the  period  of  ripening  of  oocytes  would  lead  to  its  incorpora- 
tion in  the  fertilizin.  This  expectation  has  been  realized. 

Ripe  females  were  induced  to  shed  their  eggs  by  means  of  KCl-injection.  They  were 
then  each  given  a  series  of  three,  alternate-day,  50-microcurie  injections  of  an  S^-labelled 
sulfate  solution.  The  animals  were  kept  in  non-circulating  sea  water  (six  per  3  liters),  which 
was  changed  before  each  injection.  The  day  after  the  last  injection  of  the  series  the  animals 
were  placed  in  running  sea  water  overnight.  The  eggs  were  then  collected,  by  the  usual 
KCl-injection  procedure,  in  the  cold,  and  washed  several  times  with  cold  sea  water.  The 
washings  showed  no  appreciable  content  of  S35.  The  fertilizin  was  then  dissolved  off  the 
eggs,  by  lowering  the  pH  of  the  suspension  to  about  3.5  to  4,  and  found  to  be  highly  radio- 
active. A  sample  of  fertilizin  (ca.  15  mg.)  from  about  1  ml.  (packed  volume)  of  eggs  gave 
counts  (gas-flow  type  counter)  of  about  60,000  per  minute,  corresponding  roughly  to  an 
activity  of  0.003  microcurie  per  mg.  fertilizin,  about  one-fourth  of  which  is  sulfate.  Four 
series  of  injections  were  run  with  12  to  18  animals,  most  of  which  were  used  continuously 
and  produced  fair  yields  (ca.  1  to  4  ml.)  of  eggs  after  each  series,  with  approximately  the 
above  mentioned  content  of  S85  in  their  fertilizin. 

The  influence  of  protoporphyrin-nitroresorcinol  and  other  phenols  on  .\--radiation 
sensitivity  of  Parauicciuin  caudatum.  RALPH  WiCHTERMAN,2  HARVEY  SOLO- 
MON AND  FRANK  H.  J.  FIGGE.S 

We  previously  reported  that  when  phenol  was  added  to  clonal  cultures  of  paramecia  before 
irradiation,  the  LD/50  dose  fell  from  340  kr  to  less  than  18  kr.  A  trinitro-resorcin  proto- 

1  Supported  by  a  research  grant  (C-2302)  from  the  National  Cancer  Institute,  U.  S.  Pub- 
lic Health  Service  and  by  AEC  Contract  AT  (30-1) -1343  to  the  Marine  Biological  Laboratory. 

-  Part  of  a  project  aided  by  a  contract  between  the  Office  of  Naval  Research,  and  Temple 
University  (NR  104-475)  and  the  Committee  on  Research,  Temple  Univ. 

3  Supported  by  the  American  Cancer  Society  (Maryland  Division),  Grant.  No.  CY3580 
(Cl)  from  the  Cancer  Division  of  USPHS,  and  the  Anna  Fuller  Fund. 


370         PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

porphyrin  complex  was  prepared  to  determine  its  effect  on  radiation  sensitivity  of  Paramccium 
caudatum  and  later  to  test  the  tendency  of  the  complex  to  accumulate  in  tumors.  At  the  same 
time,  trinitroresorcinol  was  tested  in  addition  to  a  series  of  other  tri-,  di-,  and  mono-hydric 
phenols.  The  porphyrin-resorcinol  compound  increased  the  radiation  sensitivity  of  paramecia. 

Paramecia  from  clear  lettuce  medium  were  placed  in  phenol  solutions  ranging  from  1  to 
10,000  to  1  in  50,000  and  then  exposed  to  50  kr  of  x-irradiation  in  closed  one-mi.  Lucite 
chambers  completely  free  of  air  pockets.  Immediately  after  irradiation,  the  paramecia  were 
exposed  to  the  air.  The  organisms  died  within  one-half  to  two  hours  (depending  upon  the 
concentration  and  potency  of  the  chemical)  in  chambers  containing  phenol-,  ortho-,  meta-,  and 
para-nitrophenol,  resorcinol,  catechol,  and  phlorglucinol.  Neither  the  irradiated  controls,  nor 
the  controls  treated  with  the  above  chemicals  were  affected.  Non-irradiated  hydroquinone  and 
pyrogallol  solutions  exposed  to  air  were  so  toxic  that  paramecia  did  not  survive  a  1  to  500,000 
concentration. 

These  experiments  indicate  that  the  phenol  in  the  culture  medium  is  probably  converted 
into  hydroquinone,  resorcinol,  catechol,  pyrogallol,  and  similar  partially  oxidized  phenols  which 
result  from  reaction  with  the  peroxides  formed  during  the  irradiation.  The  fact  that  the 
paramecia  irradiated  in  phenol  solutions  do  not  die  until  the  solution  is  exposed  to  air  indicates 
that  these  slightly  toxic  irradiation  products  require  autoxidation  in  air  to  exhibit  maximum 
toxicity. 

Effect  of  temperature  on  circulation  in  Cistenides.     CHARLES  G.  WILBER. 

The  trumpet  worm,  Cistenides,  has  a  major  pulsating  blood  vessel  which  is  clearly  visible 
through  the  body  wall  of  the  living  animal.  Observations  of  the  rate  of  pulsation  of  the 
vessel  can  be  made  readily  by  examining  the  animal  under  good  illumination  after  it  is  removed 
from  its  cone-shaped  tube.  Approximately  50  of  these  worms  were  exposed  to  aerated  sea 
water  at  various  temperatures  above  20°  C.  The  rate  of  pulsation  of  the  blood  vessel  was 
observed  under  low  magnification  (10X)  and  recorded.  The  average  rate  over  a  30-minute 
period  at  a  given  temperature  was  calculated  and  plotted.  If  the  logarithm  of  the  rate  in 
beats  per  minute  is  plotted  against  the  water  temperature  in  "  C.,  a  good  straight  line  is 
obtained  between  20°  C.  and  31°  C.  Between  31°  C.  and  35°  C.  the  slope  of  the  line  is  de- 
creased. Above  35°  C.  the  animal  shows  obvious  distress  and  the  rate  of  pulsation  of  the 
blood  vessel  becomes  very  irregular.  The  response  of  the  heart  to  temperatures  below  20°  C. 
is  now  under  study  but  adequate  data  are  not  yet  available  for  report.  Qi0  value  between 
20°  C.  and  30°  C.  is  3;  between  31°  C.  and  35°  C.,  approximately  2.  There  is  some  indica- 
tion that  lysergic  acid  diethylamide,  yohimbine  and  other  drugs  influence  the  rate  of  pulsation 
of  the  vessel  in  question.  Such  drug  studies  are  now  in  progress. 

Partial  support  of  this  work  came  from  a  National  Science  Foundation  Grant. 

The  morphology   of   the   copepod   Congericola  pallida  from   the  gills   of   Conger 
vulgaris  taken  at  Woods  Hole.     CHARLES  H.  WILLEY. 

Congericola  pallida  Van  Beneden  1854  is  found  infrequently  along  the  European  coast 
but  has  not  been  hitherto  reported  from  American  waters.  One  large  conger  among  eleven 
examined  harbored  about  fifty  mature  female  specimens  of  the  copepod.  No  males  were 
encountered.  A  few  scattered  accounts  of  the  European  forms  have  been  published,  but  as 
pointed  out  by  Wilson  in  a  monograph  on  the  Dichelesthiidae,  a  dearth  of  information  exists 
on  the  morphology  of  the  species.  The  thoracic  appendages  are  of  importance  taxonomically 
and  have  been  differently  described  by  different  investigators.  Examination  of  living  and 
preserved  material  reveals  that  there  are  four  pairs  and  all  are  biramose.  The  endopodites 
and  exopodites  of  the  first  pair  are  two- jointed  and  those  of  the  second  and  third  pairs  are 
three-jointed.  The  rami  of  the  fourth  pair  are  larger  and  consist  of  but  a  single  segment. 
Differences  reported  in  the  width  of  certain  thoracic  segments  are  explained  by  variation  in 
the  degree  of  antero-posterior  contraction  of  the  body.  Longitudinally  contracted  individuals 
exhibit  a  third  thoracic  segment  a  little  narrower  than  the  second  while  in  fully  extended 
specimens  they  are  essentially  the  same  width.  The  structures  of  the  internal  organs,  studied 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY         371 

from  whole  mounts  and  sections,  show  no  essential  differences  when  compared  with  other 
members  of  the  Family  Dichelesthiidae.  It  is  concluded  that  the  present  material  is  speci- 
fically identical  with  the  European  form. 

Carbon  monoxide  in  the  float  of  Physalia.     JONATHAN  B.  WITTENBERG. 

At  the  turn  of  the  century  Schloesing  and  Richard  found  the  gases  contained  in  the  float 
of  the  portuguese-man-of-war,  Physalia,  to  be  essentially  similar  to  air  in  their  content  of 
argon,  nitrogen  and  oxygen.  In  addition  to  these  we  have  now  found  a  fourth  component, 
carbon  monoxide.  Individuals  collected  at  Woods  Hole  contained  in  the  float  gases  amounts 
of  carbon  monoxide  varying  from  traces  to  eight  per  cent  of  the  total  gas.  The  majority  of 
individuals  contained  from  one  to  five  per  cent. 

Carbon  monoxide  was  determined  volumetrically  by  reaction  with  a  solution  of  cuprous 
chloride  in  ammonium  chloride,  and  was  identified  by  a  characteristic  color  reaction  catalyzed 
by  palladium  chloride,  and  by  the  characteristic  carboxyhemoglobin  spectrum  exhibited  by  blood 
equilibrated  with  the  float  gases.  Carbon  monoxide  accounted  for  all  of  the  combustible  gas 
present. 

Although  carbon  monoxide  is  known  to  occur  in  the  air  spaces  of  some  marine  algae,  to 
the  author's  knowledge  the  present  finding  represents  the  second  discovered  occurrence  of 
carbon  monoxide  in  animal  tissues  (the  first  being  its  origin  during  the  degradation  of 
hemoglobin). 

Glucagon  and  blood  glucose  in  Lophius  piscatorius.1     PAUL  A.  WRIGHT. 

Since  the  realization  that  bullfrogs  had  very  low  blood  glucose  values,  or  in  28%  of  the 
cases  glucose  absence,  we  have  been  interested  to  know  whether  any  other  vertebrate  was 
also  anomalous  in  this  respect.  As  tested  by  the  same  modified  Nelson  procedure  used  for 
the  frog,  marine  fishes  showed  average  glucose  values  as  follows  (number  of  animals  in 
parentheses)  :  Mustelus  cams,  87  mg.  %  (8),  Raia  diaphanes,  78  mg.  %  (4),  Dasyatis  centroura, 
54  mg.  %  (7),  Scrranus  atrarius,  57  mg.  %  (8),  Prionolus  strigatus,  66  mg.  %  (6),  and 
Tautoga  onitis,  59  mg.  %  (12).  Lophius  piscatorius  (13),  in  contrast,  gave  an  average  value 
of  only  7.6  mg.  %,  with  a  high  of  15  mg.  %  and  two  animals  with  glucose  absence.  Intra- 
arterial  administration  of  crystalline  glucagon  (10  /xg./kg.  and  100  ^g./kg.)  failed  to  induce 
any  detectable  hyperglycemia  within  40  minutes  after  injection.  Failure  to  respond  to  glucagon 
puts  Lophius  in  a  category  with  the  salamander,  Taricha  torosa,  as  reported  by  Miller  and 
Wurster.  Bullfrogs,  on  the  other  hand,  develop  hyperglycemia  rapidly  in  response  to  glucagon 
injection. 

Extracts  of  the  principal  islet  of  Lophius,  prepared  according  to  the  method  of  Sutherland 
and  de  Duve,  induced  a  consistent  hyperglycemia  (40  mg.  %  in  40  minutes)  after  intra- 
arterial  injection  in  bullfrogs.  Such  extracts  were  still  potent  after  incubation  with  0.1% 
cysteine  at  38°  C.  for  4  hours,  thus  destroying  any  possible  epinephrine  or  insulin  contamination, 
and  are  therefore  presumed  to  contain  glucagon. 


LALOR  FELLOWSHIP  REPORTS 

The  osmotic  behavior  of  marine  oocytc  nuclei.     CLIFFORD  V.  HARDING. 

Osmotic  properties  of  nuclei  have  been  reported  for  intact  amphibian  and  marine  oocytes 
and  for  isolated  amphibian  germinal  vesicles.  Unlike  amphibian  germinal  vesicles,  however, 
starfish  oocyte  nuclei  do  not  swell  upon  isolation  into  simple  salt  solutions  or  glass-distilled 
water.  On  the  other  hand,  isolated  germinal  vesicles  of  starfish  and  Spisula,  as  well  as  those 
of  Hydroidcs  (Ashton),  do  change  volume  with  change  in  colloid  osmotic  pressure  of  the 

1  Supported  by  grants  from  the  U.  S.  Public  Health  Service,  A-1280  (C)  and  the  Horace 
H.  Rackham  Fund,  University  of  Michigan. 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

surrounding  medium.  For  example,  they  showed  a  decrease  in  diameter  when  placed  in  solu- 
tions of  polyvinylpyrrolidone  (PVP,  mol.  weight,  40,000)  of  5%  or  greater,  made  up  either 
in  0.53  M  KC1  or  glass-distilled  water.  This  is  similar  to  the  results  obtained  previously 
with  amphibian  oocytes.  It  is  possible  to  demonstrate  this  also  in  starfish  nuclei  which 
remain  within  cytolyzed  oocytes.  Oocytes  which  had  been  cytolyzed  showed  no  increase  in 
nuclear  diameter.  They  did,  in  fact,  show  a  decrease  (Feldherr).  However,  when  these 
cytolyzed  cells  were  placed  in  5%  PVP  in  glass-distilled  water,  there  was  a  rapid  shriveling 
of  the  nuclei.  In  some  cases,  there  was  a  return  to  spherical  shape  of  the  nuclei  within  30 
minutes,  indicating  the  possibility  that  the  PVP  may  be  penetrating  these  nuclei.  Similar 
experiments  were  carried  out  in  which  the  cytolyzed  oocytes  were  transferred  to  0.53  M  KC1 
before  being  placed  in  the  PVP,  which  was  also  made  up  in  0.53  M  KC1.  These  experiments 
were  complicated  by  the  fact  that  the  cytolyzed  cells  themselves  decreased  markedly  in 
volume  when  placed  in  the  KC1-PVP  solution,  obscuring  any  changes  in  the  nuclei.  The 
differences  observed  between  amphibian  and  starfish  nuclei  may  represent  inherent  differences 
in  the  properties  of  the  nuclei,  or,  perhaps,  differences  in  the  extent  of  injury  sustained  by  the 
nuclei  as  a  result  of  the  process  of  isolation. 

Uptake  of  tritium-labeled  thymidine  by  Arbacia  eggs  and  embryos.     CLIFFORD  V. 
HARDING  AND  WALTER  L.  HUGHES. 

Investigations  were  carried  out  to  measure  the  uptake  of  tritium-labeled  thymidine  into 
Arbacia  punctulata  eggs  and  embryos,  and  to  determine  if  this  uptake  resulted  in  any  inhibi- 
tion of  development.  Unfertilized  eggs  and  embryos  at  various  stages  of  development  (early 
cleavage  through  gastrulation)  were  incubated  for  different  periods  of  time  in  low  concen- 
trations of  tritium-labeled  thymidine  (3-8  microcuries  per  culture;  culture  volumes  varied 
from  20  to  150  ml.).  Samples  of  eggs,  embryos  and  supernatant  culture  media  were  preserved 
in  absolute  alcohol.  The  samples  were  washed  further  with  absolute  alcohol  or  1  per  cent 
NaCl,  dried,  digested  in  H2SO4 — HNOs,  and  counted  in  a  liquid  scintillation  counter.  An 
internal  standard  was  used  to  correct  for  quenching.  The  samples  of  embryos  consistently 
showed  an  uptake  of  tritium  into  the  insoluble  phase,  and  they  showed  increased  counts  with 
increased  time  of  incubation.  The  activities  varied  from  2  to  20  disintegrations  per  minute 
per  egg,  depending  primarily  on  the  time  of  incubation.  Uptake  occurred  as  early  as  106 
minutes  after  fertilization,  which,  at  20°  C.  is  just  after  the  second  cleavage.  Unfertilized 
eggs,  on  the  other  hand,  showed  no  concentration  of  tritium  activity.  It  is  suggestive  that 
the  activity  determined  in  the  developing  embryos  is  truly  built  into  DNA;  however,  it  would 
be  important  to  isolate  and  determine  the  specific  activity  of  the  DNA  in  order  to  establish 
this.  There  was  no  noticeable  inhibition  of  development  in  the  tritiated  eggs.  This  is  per- 
haps not  surprising  in  view  of  the  comparatively  large  amount  of  tritium  necessary  to  inhibit 
the  multiplication  of  cells  in  tissue  culture  (Painter,  Drew  and  Hughes,  1958).  It  would  be 
of  interest  to  extend  the  studies  on  Arbacia,  using  higher  concentrations  of  tritium-labeled 
thymidine,  and  determining  the  distribution  of  insoluble  activity  within  the  egg  by  radio- 
autography. 

Active  transport  of  oxygen.     JONATHAN  B.  WITTENBERG. 

Two  structures  participate  in  the  transport  of  oxygen  gas  from  the  circulating  blood  into 
the  swimbladder  of  fishes.  These  are  the  gas  gland  and  a  vascular  counter-current  exchanger, 
the  rete  mirabile,  supplying  blood  to  that  gland.  We  were  emboldened  by  Ruud's  description 
of  certain  antarctic  fishes,  totally  lacking  hemoglobin,  to  attempt  experimentally  to  reduce  to 
low  levels  the  circulating  oxyhemoglobin  of  locally  available  fishes,  and  in  that  way  study 
the  function  of  the  gas  gland  uncomplicated  by  the  action  of  the  rete. 

Toadfish  (Opsanus  tail)  were  made  to  breathe  gas  mixtures  containing  varying  amounts 
of  carbon  monoxide  (0.2  to  35  per  cent  carbon  monoxide,  50  per  cent  oxygen,  balance  nitrogen), 
which  especially  in  the  higher  ranges  should  be  sufficient  to  convert  essentially  all  of  the  blood 
hemoglobin  to  carboxyhemoglobin.  These  animals  continued  to  secrete  oxygen  into  the 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY          373 

swimbladder,  thus  proving  that  the  gas  gland  cells  are  capable  of  transporting  oxygen  from 
the  blood  plasma  into  the  swimbladder.  In  addition  to  oxygen  the  secreted  gas  contained  a 
substantial  concentration  of  carbon  monoxide.  Since  animals  breathing  0.2  and  5  per  cent 
carbon  monoxide  secreted  gas  mixtures  containing  10  and  30  per  cent  carbon  monoxide,  re- 
spectively, carbon  monoxide  transport  must  also  be  considered  to  be  an  active  process. 

From  the  compositions  of  the  gas  mixtures  breathed  and  secreted,  the  relative  affinity  of 
the  glandular  oxygen  transporting  system  for  oxygen  and  for  carbon  monoxide  may  be  ap- 
proximated. Over  the  entire  range  of  carbon  monoxide  concentrations  studied,  the  affinity 
for  carbon  monoxide  was  found  to  be  3  to  8  times  as  great  as  the  affinity  for  oxygen. 

It  is  reasonable  to  assume  that  an  iron-heme  protein  is  implicated  in  the  active  transport 
of  oxygen. 


Vol.  115,  No.  3  December,  1958 

THE 

BIOLOGICAL  BULLETIN 

PUBLISHED  BY  THE   MARINE  BIOLOGICAL  LABORATORY 


SOME  ASPECTS  OF  REPRODUCTIVE  BIOLOGY  IN  THE 

FRESH-WATER  TRICLAD  TURBELLARIAN, 

CURA  FOREMANII * 

JOHN  MAXWELL  ANDERSON  AND  JEANNE  CAROL  JOHANN 
Department  of  Zoology,  Cornell   University,  Ithaca,  Nciv   York 

Apparently  alone  among  the  fresh-water  triclad  turbellarians  for  which  the 
details  of  reproduction  are  known,  Cura  foremanii  is  capable  of  sexual  reproduction 
under  conditions  which  prevent  the  occurrence  of  copulation  between  two  indi- 
viduals. This  was  established  for  a  Rhode  Island  strain  of  this  species  by  Ander- 
son (1952a),  who  isolated  individuals  on  the  day  of  hatching  and  found  that  upon 
reaching  sexual  maturity  each  worm  deposited  numerous  cocoons,  a  large  majority 
of  which  were  usually  fertile  and  produced  normal  juveniles.  Furthermore,  it  was 
established  that  reproduction  could  continue  in  this  way  for  long  periods  without 
apparent  detriment  to  the  viability  or  reproductive  capacity  of  the  strain.  In 
one  series  of  observations,  terminated  after  20  months,  a  pedigreed  line  of  worms 
produced  9  successive  generations,  each  generation  represented  by  one  individual 
isolated  as  soon  as  it  had  hatched  and  maintained  in  isolation  until  its  first  off- 
spring appeared  (Anderson,  1952b  and  subsequent  unpublished  observations). 
As  these  worms  were  apparently  capable  of  maintaining  reproductive  activities 
indefinitely  without  opportunity  for  copulation,  and  as  we  had  never  observed 
copulation  in  mass  or  paired  cultures  of  Cura,  it  was  suggested  that  copulation 
between  individuals  might  not  be  a  normal  prelude  to  sexual  reproduction  in  this 
species.  Kenk  (1935)  has  reported  that  Cura  foremanii  lacks  a  true  copulatory 
bursa,  an  organ  invariably  present  in  copulating  species ;  in  Cura  it  is  represented 
only  by  its  stalk,  which  connects  with  one  of  the  medial  branches  of  the  intestine 
and  forms  a  genito-intestinal  canal.  These  facts  support  the  suspicion  that  Cura 
must  reproduce  without  copulation. 

In  the  initial  report  of  the  unique  reproductive  habits  of  Cura  foremanii,  it  was 
impossible  to  state  whether  reproduction  in  isolated  individuals  involved  fertiliza- 
tion of  eggs  by  spermatozoa  from  the  same  worm  or  parthenogenetic  development 
of  unfertilized  eggs.  Neither  parthenogenesis  nor  self-fertilization  had  been  re- 
ported in  other  fresh-water  triclads  by  previous  authors,  and  no  direct  evidence 
favoring  either  possibility  was  obtained  from  our  observations. 

1  Referred  to  in  previous  publications  as  Curtisia  foremanii;  best  taxonomic  account  is  that 
of  Kenk  (1935).  The  name  Curtisia  (given  this  worm  in  1916  by  von  Graff,  in  honor  of 
Prof.  W.  C.  Curtis)  is  preoccupied;  Strand  (1942)  has  proposed  the  substitution  of  Cura 
for  Curtisia. 

375 


376          JOHN  MAXWELL  ANDERSON  AND  JEANNE  CAROL  JOHANN 

The  apparently  simple  question  of  the  nature  of  the  reproductive  process  in 
this  species  has  proved  surprisingly  difficult  to  answer  unequivocally.  Evidence 
bearing  on  the  central  problem  has  been  sought  by  attacking  several  subsidiary 
questions,  such  as  the  following : 

1  )  Is  sexual  reproduction  in  isolation  characteristic  of  the  species  as  a  whole, 
or  is  it  limited  to  the  stock  with  which  the  original  work  was  done? 

2 )  In  sexually  mature  and  reproductive  individuals,   reared   in   isolation,  are 
spermatozoa  present   in  the  seminal   receptacles    (the  anterior   extremities   of  the 
ovovitelline  ducts,  adjacent  to  the  ovaries),  as  they  were  reported  to  be  by  Curtis 
(1900)   and  by   Stevens    (1904)    in  animals  taken  from  the  wild  and  from  mass 
cultures  ? 

3)  If  spermatozoa  are  found  here  in  isolated  individuals,  when  do  they  first 
arrive  at  the  seminal  receptacles?     Is  there  any  correspondence  between  the  time 
of  first  appearance  of  spermatozoa  at  the  ovaries  and  the  time  of  deposition  of 
first  cocoons  by  isolated  worms  just  attaining  sexual  maturity?     Or  can  an  indi- 
vidual   deposit    fertile    cocoons    before    spermatozoa    first    appear    in    its    seminal 
receptacles  ? 

4)  Do  eggs  deposited  in  cocoons  by  isolated  individuals  show  any  evidence  of 
having  been  penetrated  by  spermatozoa  ? 

5)  Do  spermatozoa  ever  appear  in  the  bursa  stalk  (genito-intestinal  canal)   of 
mature  worms  taken  from  mass  culture^  ?     That  is,  is  there  evidence  that  what 
remains  of  the  copulatory  bursa  is  used  in  copulation  in  mass  cultures?     If  sperma- 
tozoa are  found  here,  how  do  worms  from  mass  cultures  compare  in  this  respect 
with  mature  and  reproductive  individuals  reared  in  isolation? 

This  paper  reports  the  results  of  investigations  designed  to  provide  answers 
to  these  and  related  questions  and  discusses  the  relationship  between  this  infor- 
mation and  the  major  problem  of  the  nature  of  the  reproductive  process  in  Cnra 
foremanii. 

MATERIALS  AND  METHODS 

The  worms  on  which  these  observations  have  been  made  constitute  several 
stocks  of  diverse  geographical  origins.  One  stock  represents  descendants  of  the 
specimens  collected  in  1950  from  a  stream  near  Geneva,  Rhode  Island,  in  which 
reproduction  in  isolation  was  originally  described.  Another  group  of  worms  origi- 
nated with  several  specimens  collected  in  Cascadilla  Creek,  near  Ithaca,  New  York. 
A  third  stock  consists  of  offspring  of  several  mature  specimens  collected  from  a 
stream  in  the  Adirondacks,  near  Warrensburg,  New  York,  by  Neal  1\.  Foster. 
The  fourth  strain  was  purchased  from  a  biological  supply  house,  having  been  col- 
lected in  Powder  Mill  Park,  near  Rochester,  New  York. 

The  methods  used  in  maintaining  these  stocks  are  essentially  those  described 
earlier  (Anderson,  1952a),  with  some  modifications.  All  worms  have  for  the 
past  few  years  been  kept  in  a  constant-temperature  room  under  continuous  illumi- 
nation. Temperatures  have  varied  over  long  periods  between  21°  and  22.5°  C. : 
illumination  is  provided  by  two  40-watt  fluorescent  tubes  about  four  feet  above 
the  table  on  which  the  cultures  are  kept.  Specimens  have  been  maintained  both 
in  mass  cultures  and  in  isolation.  Each  of  the  isolated  worms  is  kept  in  about 


REPRODUCTION  IN  CURA  FOREMANII  377 

50  ml.  of  water  in  a  4-ounce  jar.  Small  mass  cultures  (6  to  12  individuals)  are 
kept  in  larger  amounts  of  water  in  identical  jars ;  larger  mass  cultures  of  several 
dozen  individuals  occupy  wide-mouth  jars  containing  approximately  200  ml.  of 
water.  All  culture  jars  are  tightly  closed  with  metal  screw  caps. 

The  sources  and  types  of  water  used  for  the  cultures  have  been  subject  to 
considerable  variation  through  the  past  several  years.  When  the  Rhode  Island 
strain  was  first  transferred  to  Ithaca,  and  for  some  years  subsequently,  these  worms 
and  the  locally-collected  stocks  were  kept  in  water  obtained  from  Cascadilla  Creek 
at  the  Cornell  University  fish  hatchery.  In  1957  all  stocks  were  changed  to  a 
"modified  tap  water"  suggested  by  Loomis  and  Lenhoff  (1956),  prepared  by  adding 
50  mg./L  each  of  disodium  ethylenediaminetetraacetate  and  CaCl.2  to  tap  water. 
For  the  past  few  months  we  have  used  unmodified  Ithaca  tap  water  without  ap- 
parent unfavorable  effects  on  the  cultures. 

All  worms  are  fed  regularly,  once  each  week.  Isolated  worms  are  given  bits 
of  fresh  mouse-liver,  while  the  mass  cultures  are  fed  liver  and  such  other  organs 
as  heart,  spleen,  and  kidneys  of  freshly-killed  mice.  Food  is  allowed  to  remain  in 
the  jars  for  at  least  several  hours  and  sometimes  overnight,  after  which  the  rem- 
nants are  removed  and  the  water  changed. 

The  worms  are  examined  carefully  at  one-  or  two-day  intervals.  For  each 
of  the  isolated  worms,  records  are  kept  of  the  dates  of  deposition  and  hatching  of 
all  cocoons,  and  of  the  number  of  young  produced.  Juvenile  worms  are  removed 
from  these  cultures  as  soon  as  possible  after  hatching ;  they  are  either  transferred 
to  mass  cultures  or  set  up  individually  in  isolation  to  maintain  and  extend  selected 
pedigreed  stocks.  Following  the  scheme  previously  described  (Anderson,  1952a), 
each  such  isolated  juvenile  receives  a  code  designation  denoting  its  generation  and 
its  ancestry. 

For  the  specific  purposes  of  the  present  investigation  it  was  necessary  to  prepare 
serial  sections  of  mature  wrorms  from  both  mass  and  isolation  cultures,  as  well  as 
of  a  series  of  developing  juveniles  sacrificed  at  weekly  intervals  from  the  time  of 
hatching  to  the  attainment  of  sexual  maturity.  Specimens  were  anesthetized  by 
placing  them  in  a  solution  of  MS-222,-  1:1500  by  weight,  as  suggested  by  Manner 
(1957)  ;  when  relaxed,  the  worms  were  flooded  with  either  Zenker-acetic  or 
Kelly's  fluid.  They  were  kept  extended  and  moderately  flattened  during  fixation 
by  compressing  them  gently  under  a  coverslip.  Following  standard  procedures, 
the  specimens  were  imbedded  in  Tissuemat ;  serial  sections  were  prepared  at  7  ^, 
mounted,  and  stained  in  Harris'  hematoxylin.  Several  freshly  deposited  cocoons 
were  also  fixed,  imbedded,  sectioned,  and  similarly  stained,  to  provide  material 
for  determination  of  the  nuclear  condition  of  the  eggs  they  contained. 

OBSERVATIONS  AND  DISCUSSION 

The  ability  of  Cura  forcnianii  to  carry  on  active  sexual  reproduction  in  isola- 
tion is  not  limited  to  the  Rhode  Island  strain  in  which  it  was  originally  described. 
The  other  three  stocks  with  which  we  have  worked,  collected  from  various  locali- 
ties in  central  and  northern  New  York,  have  all  proved  capable  of  the  same  repro- 

-  A  supply  of  this  reagent  was  generously  provided  by  Dr.  Perry  W.  Gilbert.  In  our 
experience,  exposure  of  planarians  to  MS-222  at  this  concentration  almost  invariably  induces 
aversion  of  the  proboscis,  and  the  animal  relaxes  in  this  condition. 


JOHN  MAXWELL  ANDERSON  AND  JEANNE  CAROL  JOHANN 

ductive  behavior.  It  therefore  seems  reasonable  to  conclude  that  this  kind  of 
reproduction,  whatever  the  details  of  the  process  may  be,  is  a  general  characteristic 
of  the  species. 

In  all  mature  specimens  of  which  sections  have  been  examined,  masses  of 
'spermatozoa  have  been  found  in  the  seminal  receptacles.  Figure  1  is  presented  as 
typical.  This  section  is  from  an  adult  worm  killed  in  the  act  of  releasing  a  finished 
cocoon  from  its  genital  atrium.  As  the  figure  shows,  the  ovaries  contain  de- 
veloping eggs  in  various  stages  of  maturity,  and  the  seminal  receptacles  are  occu- 
pied by  masses  of  spermatozoa.  It  is  to  be  emphasized  that  in  view  of  the 
anatomical  relationships  of  the  reproductive  systems  in  fresh-water  triclads,  these 
spermatozoa  could  have  reached  the  seminal  receptacles  only  by  moving  anteriorly 
in  the  ovovitelline  ducts  from  the  genital  atrium  (see  Curtis,  1900,  and  Kenk,  1935, 
for  anatomical  details  of  the  reproductive  system  of  Cum).  Furthermore,  since 
this  individual  had  been  maintained  in  isolation  since  the  day  it  hatched,  these 
spermatozoa  could  have  originated  only  in  its  own  testes  and  must  have  been 
emitted  through  its  penis ;  spermatozoa  are  never  found  wandering  about  in  the 
mesenchyme  but  are  always  restricted  to  the  genital  ducts. 

All  our  observations  indicate  convincingly  that  in  fully  mature,  actively  repro- 
ducing specimens  of  Cur  a  the  seminal  receptacles  are  invariably  packed  with 
spermatozoa ;  in  isolated  individuals  these  can  have  come  only  from  the  testes  of 
the  same  animal.  It  is  of  interest  to  determine  whether  in  young  worms,  just 
reaching  sexual  maturity,  cocoons  can  be  deposited  before  spermatozoa  are  pro- 
duced and  reach  the  seminal  receptacles,  or  whether  spermatozoa  are  already  present 
in  the  seminal  receptacles  at  the  time  of  deposition  of  the  first  cocoon.  Table  I 
lists  the  ages  at  which  28  individuals  of  diverse  origins,  reared  in  isolation  under 
constant  conditions  of  temperature,  light,  and  feeding,  deposited  their  first  cocoons. 
There  is  considerable  variation,  not  apparently  correlated  with  strain  differences 
or  any  other  obvious  factors ;  the  mean  age  at  sexual  maturity  for  this  group  of 
worms  is  57.6  ±2.5  days.  Among  these  specimens,  as  among  large  numbers  of 
other  isolated  individuals  for  which  records  have  been  kept,  40  days  is  the  minimum 
age  at  sexual  maturity  ;  in  all  our  experience  with  these  worms,  no  isolated  indi- 
vidual has  ever  deposited  its  first  cocoon  before  about  six  weeks  from  its  day  of 
hatching. 

The  problem  now  becomes  one  of  determining  whether  there  is  any  correspond- 
ence between  this  earliest  first-cocoon  record  and  the  time  of  arrival  of  spermatozoa 
in  the  seminal  receptacles.  Our  study  of  this  aspect  of  the  problem  involved 
the  preparation  and  study  of  sections  of  a  series  of  individuals  of  known  ages,  at 
weekly  intervals  from  one  week  to  seven  weeks  after  the  day  of  emergence  from 
the  cocoon.  This  afforded  an  opportunity  for  a  general  study  of  the  development 
of  both  male  and  female  systems,  but  since  the  details  of  such  a  study  are  not 
germane  to  the  present  problem  only  a  summary  of  the  principal  events  will  be 
given.  Several  individuals  of  each  age  were  studied,  and  there  was  some  varia- 
tion in  the  stage  of  development  reached  by  different  animals  of  the  same  age. 
Those  showing  the  most  rapid  rate  of  development  will  be  described,  as  they 
probably  represent  the  group  that  would  have  shown  earlier-than-average  deposi- 
tion of  first  cocoons.  At  two  ivecks,  early  ovaries  and  testes  are  recognizable, 
containing  chiefly  gonia  and  early  gametocytes.  At  three  weeks,  the  gonads  are 


REPRODUCTION  IN  CURA  FOREMANII 


379 


PLATE  I 

All  figures  are  photomicrographs  of  sections  cut  at  7  /x  and  stained  in  Harris'  hematoxylin. 
The  scales  represent  10  microns;  that  given  for  Figure  3  indicates  magnification  for  Figures 
1  and  4  also. 

FIGURE  1.  Portion  of  ovary  and  seminal  receptacle  of  a  mature  individual,  reared  in  iso- 
lation, fixed  at  the  moment  of  deposition  of  a  finished  cocoon.  At  upper  right,  developing  eggs 
in  various  stages  ;  at  lower  left,  mass  of  spermatozoa  in  seminal  receptacle. 

FIGURE  2.  Fertilized  egg  in  a  cocoon,  surrounded  by  yolk  cells.  Second  polar  body  (pb) 
has  been  emitted  ;  note  vesicular  female  pronucleus  and  darkly-stained  sperm  nucleus. 

FIGURE  3.  Same  section  as  that  shown  in  Figure  2,  at  a  slightly  different  focus,  showing 
chromosomes  in  polar  body  (  pb ) . 

FIGURE  4.  Portion  of  ovary  and  seminal  receptacle  of  an  individual  reared  in  isolation 
for  five  weeks  frcm  the  day  of  hatching.  Note  developing  eggs  in  ovary  (upper  right)  and 
mass  of  spermatozoa  already  present  in  seminal  receptacle  (lower  left)  in  this  animal  which 
is  just  approaching  sexual  maturity. 


380 


JOHN  MAXWELL  ANDERSON  AND  JEANNE  CAROL  JOHANN 


TABLE  I 
Time  required  for  attainment  of  sexual  maturity  at  22.5°  C. 


Worm 

Date  hatched 

Date  of  1st  cocoon 

Days  to  1st  cocoon 

V9-la-la 

1/8 

2/17 

40 

T3-9a 

1/7 

2/17 

41 

V4-la 

10/9 

11/19 

42 

V4-lb 

10/9 

11/19 

42 

*AD-9 

10/24 

12/10 

47 

*AD-5 

10/24 

12/11 

48 

V9-la 

11/8 

12/26 

49 

T6-8a 

1/8 

2/28 

51 

*AD-8 

10/24 

12/14 

51 

fW2-lb 

2/11 

4/4 

52 

TlO-lla 

12/28 

2/18 

52 

P8-la 

11/4 

12/26 

52 

V9-12a 

12/20 

2/11 

53 

T10-lla-3a 

3/20 

5/12 

53 

*AD-3-2b 

12/9 

1/31 

53 

VIO-la 

11/13 

1/8 

56 

TlO-la 

11/1 

12/28 

57 

*AD-9-3a 

1/7 

3/3 

57 

t\V2-la 

2/11 

4/10 

58 

V6-la 

11/1 

12/30 

59 

*AD-3-2a 

12/9 

2/7 

60 

P6-la-lla 

3/31 

5/31 

61 

V4-lb-la 

11/29 

2/7 

71 

V2-la 

11/11 

1/22 

72 

VIO-lOa 

12/20 

3/11 

81 

TlO-la-la 

1/8 

4/2 

84 

P6-la 

11/4 

1/29 

86 

V4-la-7a 

1/2 

3/29 

86 

*  Adirondack*  strain. 

f  Rochester  strain. 

Unmarked  individuals  are  of  Rhode  Island  or  Ithaca  strains. 

larger  and  contain  more  advanced  stages  in  gametogenesis ;  in  addition,  the  earliest 
signs  of  development  of  the  posterior  genital  complex  are  found  at  this  age.  At 
four  weeks,  large  oocytes  are  present  in  the  ovaries,  and  the  testes  contain  what 
appear  to  be  well-developed  spermatozoa.  The  genital  atrium  and  associated 
structures  are  in  an  advanced  state  of  development,  but  there  are  no  signs  that 
the  ovovitelline  ducts  and  the  ductus  deferentes  are  complete.  At  five  weeks, 
the  most  advanced  animal  studied  showed  fully  developed  ovaries  containing  eggs 
well  on  the  way  to  maturity  ;  the  testes  were  packed  with  mature  spermatozoa ;  the 
genital  complex  posterior  to  the  pharynx  was  completely  formed,  and  sperm  ducts 
and  oviducts  were  present.  Most  significantly,  at  the  age  of  35  days  from  hatch- 
ing, this  individual's  seminal  receptacles  were  filled  with  spermatozoa.  A  portion 
of  a  section  of  this  worm,  showing  some  of  these  features,  is  presented  as  Figure  4. 
All  individuals  studied  at  si.v  and  seven  weeks  were  in  similarly  advanced  stages 
of  sexual  maturity,  although  none  had  deposited  its  first  cocoon  before  fixation ; 
in  all,  the  seminal  receptacles  were  filled  with  spermatozoa.  The  results  of  these 


REPRODUCTION   IX  CURA  FOREMANII  381 

studies  are  consistent  with  the  conclusion  that  spermatozoa  are  always  present  in 
the  seminal  receptacles  before  the  individual  deposits  its  first  cocoon,  and  thus  that 
all  eggs  leaving  the  ovaries,  even  those  first  produced  by  an  individual  just  reach- 
ing sexual  maturity,  must  pass  through  a  cloud  of  spermatozoa  before  reaching 
the  genital  atrium  for  inclusion  in  a  cocoon. 

Stevens  (1^04).  in  her  studies  of  reproduction  and  early  development  in  wild 
and  mass-cultured  specimens  of  Cura  jorciuanii  (referred  to  in  her  paper  as 
Planana  simplissima,  following  Curtis'  identification),  reports  that  the  first  matura- 
tion division  of  oogenesis  takes  place  within  the  ovary,  and  that  the  secondary 
oocytes  released  into  the  seminal  receptacles  are  then  penetrated  by  the  sperma- 
tozoa. The  second  maturation  division  occurs  as  the  cocoon  is  being  deposited, 
or  very  shortly  thereafter,  the  sperm  nucleus  remaining  quiescent  until  after  the 
emission  of  the  second  polar  body.  AYe  have  now  determined  that  in  isolated 
individuals,  just  as  in  the  cases  reported  by  Stevens,  spermatozoa  are  always 
present  in  the  seminal  receptacles  after  the  attainment  of  sexual  maturity.  The 
problem  remaining  is  to  determine  whether  in  these  isolated  individuals  the  sec- 
ondary oocytes  passing  through  the  seminal  receptacles  are  penetrated  by  sperma- 
tozoa, or  whether  they  remain  unfertilized  and  develop  parthenogenetically. 

For  information  on  this  point  we  have  turned  to  a  study  of  the  contents  of 
freshly  deposited  cocoons.  Each  cocoon  contains  four  to  six  eggs,  extremely  diffi- 
cult to  locate  among  the  myriads  of  yolk  cells  with  which  they  are  surrounded. 
\Ye  were  unsuccessful  in  our  attempts  to  identify  eggs  in  squash  preparations  of 
fresh  cocoons  but  were  able  to  locate  several,  in  various  stages  of  development,  in 
serial  sections.  One  of  these  eggs  is  shown  in  Figures  2  and  3  ;  these  are  photo- 
graphs of  the  same  section,  taken  at  slightly  different  levels  of  focus,  showing  the 
condition  of  the  egg  shortly  after  the  formation  of  the  second  polar  body.  This 
cell  contains,  in  addition  to  the  vesicular  female  pronucleus,  a  densely  staining  mass 
which  clearly  represents  the  quiescent  sperm  nucleus  (compare  with  Stevens' 
figures,  1904).  Needless  to  say,  a  section  such  as  this  is  exceptional;  but  there 
is  no  reason  for  believing  that  the  condition  it  reveals  is  anything  but  typical.  On 
the  basis  of  such  evidence  we  are  led  to  conclude  that  the  eggs  of  isolated  indi- 
viduals are  iertilized,  as  they  pass  through  the  seminal  receptacles,  by  spermatozoa 
from  the  testes  of  the  same  animal. 

Although  we  may  be  confident  that  self-fertilization  is  the  rule  in  isolated 
individuals,  we  have  as  yet  no  conclusive  evidence  as  to  whether  reproduction  in 
mass  cultures  involves  copulation.  Kenk  (1935),  reporting  the  absence  of  a 
copulatory  bursa  in  Cum,  nevertheless  assumed  (on  what  grounds  we  do  not  know) 
that  copulation  occurs:  "In  copulation,  the  relatively  small  penis  is  inserted  into 
the  posterior  part  of  the  bursa  stalk  'vagina'  of  the  co-copulant"  (p.  82).  In  our 
own  investigations,  three  individuals  taken  from  mass  cultures  were  fixed,  sec- 
tioned, and  stained,  and  their  bursal  stalks  were  carefully  searched  for  the  presence 
of  spermatozoa.  In  only  one  of  these  were  traces  of  spermatozoa  found,  but 
spermatozoa  were  found  also  in  the  bursal  stalks  of  worms  reared  in  isolation, 
in  about  the  same  proportion  of  cases.  This  is  not  a  reliable  kind  of  evidence, 
for  two  reasons.  If  copulation  does  occur  between  individuals,  we  do  not  know 
how  long  spermatozoa  remain  in  the  bursal  stalk  "vagina"  before  migrating  into 
the  seminal  receptacles ;  those  animals  from  mass  cultures  in  which  the  stalks 


382  JOHN  MAXWELL  ANDERSON  AND  JEANNE  CAROL  JOHANN 

were  empty  might  simply  represent  cases  in  which  this  migration  had  been  com- 
pleted. Furthermore,  there  is  no  certainty  that  spermatozoa  might  not  be  de- 
posited in  the  bursal  stalk  by  the  penis  of  the  same  individual,  or  might  wander 
into  it  seeking  the  openings  of  the  ovovitelline  ducts ;  in  the  isolated  worms,  sperma- 
tozoa must  have  reached  the  stalk  in  some  such  manner.  Since  the  stalk  forms 
a  genito-intestinal  connection,  functioning,  as  Kenk  suggests,  to  carry  off  "super- 
fluous" sperm  to  the  intestine  for  digestion,  the  spermatozoa  found  in  the  stalks 
of  our  isolated  worms  may  simply  be  on  their  way  to  this  fate. 

The  evidence  bearing  on  the  question  of  copulation  in  these  worms  is  indirect 
and  inadequate,  but  it  seems  a  significant  fact  that  the  only  species  in  which 
sexual  reproduction  is  known  to  occur  in  the  absence  of  copulation  is  also  a 
species  in  which  the  copulatory  bursa  has  been  lost  (or  never  developed  at  all) 
and  is  represented  only  by  a  genito-intestinal  canal.  To  settle  the  question  we 
need,  simply,  to  observe  two  individuals  in  the  act  of  copulation ;  failing  this,  we 
need  a  fertile  individual  showing  some  distinct  heritable  peculiarity  of  color  or 
form  which  could  be  followed  through  breeding  experiments.  In  our  seven  years' 
experience  with  this  species  we  have  observed  neither  copulation  nor  a  distinctively- 
marked  individual ;  this  is  an  extremely  uniform  species.  Exposure  of  large  num- 
bers of  the  worms  to  x-radiation  might  produce  a  mutation  which  could  serve  as 
a  genetic  marker.  Such  evidence  as  we  now  have,  however,  indicates  the  possi- 
bility, if  not  the  probability,  that  Cur  a  foremanii,  which  in  isolation  can  substitute 
self-fertilization  for  copulation,  may  in  fact  have  eliminated  copulation  as  a  normal 
feature  of  its  reproductive  processes. 

SUMMARY  AND  CONCLUSIONS 

A  careful  re-examination  of  the  details  of  sexual  reproduction  in  isolated  indi- 
viduals of  the  triclad  species  Cura  foremanii  has  been  carried  out,  designed  par- 
ticularly to  determine  whether  this  reproduction  involves  self-fertilization  or  simply 
the  parthenogenetic  development  of  unfertilized  eggs.  The  following  conclusions 
have  been  reached : 

1  )  Without  apparent  detriment  to  the  species,  sexual  reproduction  can  con- 
tinue for  many  generations,  and  evidently  indefinitely,  under  conditions  of  isola- 
tion which  prevent  the  occurrence  of  copulation  between  individuals.  This  is  true 
of  four  different  stocks  of  the  species,  of  diverse  geographical  origin. 

2)  All    individuals    examined    during   active    reproduction    showed    masses    of 
spermatozoa  occupying  the  seminal  receptacles  through  which  the  eggs  must  pass 
in  moving  from  the  ovaries  to  the  genital  atrium. 

3)  Studies  of  the  development  of  the  reproductive  systems  in  immature  iso- 
lated worms  of  known  ages  reveal  that  spermatozoa  are  mature  and  have  already 
migrated  to  the  seminal  receptacles  as  early  as  35   days  after  the  emergence  of 
the  individual  from  its  cocoon. 

4)  Under  constant  conditions,  isolated  individuals  deposit  their  first  cocoons 
some  time  between  the  40th  and  the  86th  day  after  emergence ;  thus,  the  eggs  in 
even  these  first  cocoons  have  always  been  exposed  to  spermatozoa  in  the  seminal 
receptacles. 

5)  During  this  exposure  to   spermatozoa,   the   eggs  are  penetrated   by  them; 
eggs  have  been  observed  in  freshly  deposited  cocoons  showing  second  polar  bodies 


REPRODUCTION  IN  CURA  FOREMANII  383 

and  vesicular  female  pronuclei,  as  well  as  distinctly  staining  sperm  nuclei.  Devel- 
opment of  these  self-fertilized  eggs  is  presumably  normal. 

6)  It  is  thus  clear  that  sexual  reproduction  in  isolated  individuals  of  Cura 
foremanii  involves  self-fertilization  and  not  parthenogenesis. 

One  tantalizing  question  remains  unanswered  :  whether  copulation  ever  occurs 
in  Cura,  even  among  individuals  in  mass  cultures.  This  species  lacks  a  copulatory 
bursa,  possessing  only  the  bursa  stalk  which  serves  as  a  genito-intestinal  canal. 
Copulation  has  not  been  observed  during  our  experience  with  this  species,  and 
clearly  from  the  standpoint  of  normal  reproduction  and  development  the  process 
is  completely  superfluous.  Breeding  experiments  with  marked  individuals  might 
settle  the  question ;  until  these  can  be  devised,  it  seems  probable  that  Cura  foremanii 
may  have  dispensed  with  copulation  as  a  feature  of  its  sexual  reproductive 
processes. 

LITERATURE  CITED 

ANDERSON,  J.  M.,  1952a.     Sexual  reproduction  without  cross-copulation  in  the  fresh-water  tri- 

clad  turbellarian,  Curtisia  foremanii.     Biol.  Bull.,  102:   1-8. 
ANDERSON,  J.  M.,  1952b.     A  further  report  on  sexual  reproduction  without  cross-copulation  in 

the  freshwater  triclad  turbellarian,  Curtisia  foremanii.     Anat.  Rcc.,  113:  601. 
CURTIS,  W.   C,   1900.     On   the   reproductive   system   of   Planaria   simplissima,   a   new   species. 

Zool.  Jahrb.,  Abt.  f.  Anat..  13:  447-466. 
VON  GRAFF,  L.,  1916.     Tricladida.     In:  H.  G.  Bronn   (ed.),  Klassen  mid  Oninnngen  des  Thier- 

i-eichs.  Ed.  IV,  Abt.  Ic,  Teil  II. 

KENK,  R.,  1935.     Studies  on  Virginian  triclads.     /.  Elisha  Mitchell  Sci.  Soc.,  51  :  79-126. 
LOOMIS,  W.  F.,  AND  H.  M.  LENHOFF,  1956.     Growth  and  sexual  differentiation  of  Hydra  in 

mass  cultures.     /.  E.vp.  Zool.,  132  :  555-573. 

MANNER,  H.  W.,  1957.     Anesthetize  those  planaria!     Turto.v  Ncit's.  35:    134. 
STEVENS,  N.  M.,  1904.     On  the  germ  cells  and  the  embryology  of  Planaria  simplissima.     Proc. 

Acad.  Nat.  Sci.,  Philadelphia,  56:  208-220. 
STRAND,  E.,  1942.     Miscellanea  nomenclatoria  zoologica  et  palaeontologia,  X.     Folia  Zoologica 

et  Hydrobiologica,  11:  386-402. 


FORM-STABILITY  OF  CILIATES  IN  RELATION  TO 
PRESSURE  AND  TEMPERATURE1 

WALTER  AUCLAIR  AND  DOUGLAS  MARSLAND 

Department  of  Biology,  Nciv  York   University.  Washington  Square  College,  New   York   City, 
and  tlie  Marine  Biological  Laboratory,  ]]roods  Hole,  Mass. 

The  first  use  of  hydrostatic  pressure  as  an  experimental  parameter  in  biological 
research  is  credited  to  Regnard  (1884a,  1884b,  1884c)  and  Certes  (1884),  work- 
ing independently.  Both  of  these  early  workers  were  impressed  by  the  variety 
of  living  forms  that  had  been  recovered  by  the  deep-sea  dredging  expedition  of 
the  Talisman  in  1882-1883.  Regnard,  particularly,  became  interested  in  the  effects 
of  pressure  per  sc.  He  eliminated  changes  in  the  gaseous  equilibria  by  applying 
the  pressure  directly  to  the  aqueous  medium  and  he  studied  the  effects  of  pressures 
ranging  up  to  1000  atmospheres  on  a  wide  variety  of  small  aquatic  organisms.  In 
the  present  connection,  he  observed  that  various  ciliates,  crowded  in  stagnant 
water  and  subjected  to  pressures  of  600  to  1000  atmospheres  for  10  minutes,  be- 
came immobile  and  distended,  and  that  ciliary  movement  stopped.  ;Within  two 
hours  after  decompression  many  of  the  organisms  seemed  to  have  recovered 
completely. 

Ebbecke  (1935,  1936)  described  the  effect  of  pressure  on  paramecia.  Expo- 
sure to  pressures  of  500  atmospheres  for  periods  of  from  10  to  30  minutes  mainly 
effected  a  change  in  the  shape  of  the  ciliates,  i.e..  their  bodies  became  more  sphe- 
roidal. At  pressures  of  from  800  to  1000  atmospheres  for  periods  extending  from 
5  to  30  minutes,  the  organisms  became  spheroidal  and  many  underwent  cytolysis. 
The  rounding  effects  were  reversible  after  a  recovery  period  spanning  several  days. 
At  2000  atmospheres  there  was  a  drastic  rounding  of  the  cells,  followed  by  com- 
plete cytolysis  of  all  the  organisms. 

Hodapp  and  Luyet  (1947)  studied  the  mechanism  of  death  of  paramecia  sub- 
jected to  high  hydrostatic  pressure.  They  obtained  a  typical  sigmoid  curve  of 
the  percentage  of  paramecia  killed  by  pressures  varied  systematically  from  500-1200 
atmospheres,  each  pressure  being  maintained  for  two  minutes.  At  950  atmospheres 
about  50  per  cent  of  the  cells  were  killed.  Hodapp  and  Luyet  also  varied  the 
time,  the  temperature,  the  rate  at  which  the  pressure  was  increased  and  decreased, 
and  the  age  of  the  cultures,  and  found  that  the  total  compression  time  and  the 
culture  age  were  most  important  in  relation  to  lethality.  Temperature  (between 
10°-22°  C.)  and  the  rate  of  pressure  increase  were  reported  to  have  little  or  no 
effect  upon  lethality.  In  this  connection.  ho\vever,  it  should  be  realized  that 
Hodapp  and  Luyet  did  not  employ  a  windowed  pressure  chamber  and  could  not 
observe  the  effects  until  after  the  organisms  were  removed  from  the  chamber. 
Consequently,  the  compression  and  decompression  effects  could  not  be  differentiated. 

In  1934,  D.  E.  S.  Brown,  studying  frog  muscle,  first  recognized  the  interrela- 

1  Work  supported  by  Grant  C807  (c  1-10)   from  the  National  Cancer  Institute,  U.S.P.H.S. 

384 


FORM-STABILITY  OF  CILIATES  385 

tionship  between  pressure  and  temperature  in  biological  systems,  and  an  apprecia- 
tion of  this  important  relationship  did  much  to  clarify  subsequent  investigations 
in  the  field.  Further  insight,  particularly  with  reference  to  the  problem  of  how 
pressure  induces  the  solation  of  protoplasmic  gels,  was  provided  by  a  classification 
of  gelational  phenomena,  published  by  Freundlich  in  1937.  Experiments  by  Hey- 
man  (1935,  1936)  had  shown  that  certain  gel  systems,  represented  by  methyl  cel- 
lulose, behave  oppositely  to  gelatin.  The  methyl  cellulose  type  of  system  displays 
a  volume  increment  (+  AF)  upon  gelation;  and  Freundlich  deduced  that  such 
gelations  must  be  endergonic  in  nature.  Then  Marsland  and  Brown  (1942)  studied 
the  sol-gel  equilibria  of  myosin,  methyl  cellulose,  and  gelatin  as  affected  by  hydro- 
static pressure  and  temperature.  These  experiments  showed  that  myosin  (in 
vitro),  methyl  cellulose,  and  protoplasmic  gels,  generally,  must  be  placed  in  a 
common  class  of  system  because  all  undergo  solation  as  a  result  of  compression 
and  cooling.  Gelatin,  on  the  other  hand,  gelates  more  firmly  with  cooling  and 
compression  and  must  represent  a  different  class  of  system.  These  workers  also 
emphasized  the  speed  with  which  protoplasmic  sol-gel  equilibria  may  be  shifted, 
particularly  in  fresh  myosin  preparations,  and  they  postulated  the  intervention  of 
an  enzyme  system  (an  ATP-ase  complex)  which  likewise  is  sensitive  to  pressure 
and  temperature. 

The  studies  of  Marsland  (1950)  demonstrated  that  the  cortical  plasmagel  of 
dividing  sea  urchin  eggs  reacts  to  pressure-temperature  treatments  as  do  other 
intracellular  gels.  Landau  ct  al.  (1954),  performing  similar  experiments  on  the 
plasmagel  system  of  Amoeba  protens,  came  to  the  same  conclusion,  namely,  that  the 
gel  system  is  weakened  by  higher  pressures  and  lower  temperatures,  within  the 
physiological  range. 

It  is  apparent,  therefore,  that  pressure-temperature  conditions  may  effect  pro- 
found changes  in  cell  structure  and  that  such  changes  are  determined,  at  least 
partly,  by  pressure-temperature  effects  upon  the  gelational  state  of  the  protoplasm. 
The  purpose  of  the  present  work,  accordingly,  is  to  study  the  form-stability  of  two 
representative  ciliates,  Blepharisma  and  Paramecium,  under  systematically  varied 
conditions  of  pressure  and  temperature.  Generally,  it  has  been  supposed  that  the 
characteristic  morphology  and  structural  integrity  of  these  organisms  is  maintained, 
not  only  by  a  tough,  flexible  surface  pellicle,  but  also  by  a  peripheral  gelated  layer 
of  cytoplasm,  often  referred  to  as  "ectoplasm."  In  the  previous  pressure  studies 
on  Paramecium,  the  organisms  were  not  observed  clearly  during  the  compression 
period  so  that  compression  effects  could  not  be  distinguished  from  the  effects  of 
decompression;  and  in  the  previous  studies  on  Blepharisma  (Hirshfield  ct  al.,  1957) 
no  extensive  variation  in  temperature  was  employed. 

MATERIALS  AND  METHODS 

The  ciliates,  Blepharisma  nadiilans  and  Paramecium  caudatum,  provided 
excellent  material  for  this  study  because  of  their  elongate  shape  which  tends  to 
become  spheroidal  when  structural  instability  develops. 

The  original  Blepharisma  culture  was  obtained  from  Dr.  H.  I.  Hirshfield;  and 
the  paramecia  were  derived  from  a  mass  culture  maintained  for  many  years  at  New 
York  University.  Both  species  were  cultured  in  a  lettuce-Pseudomonas  oral  is 


386  WALTER   AUCLAIR  AND  DOUGLAS  MARSLAND 

medium  according  to  the  method  of  Hirshfield  (personal  communication).  The 
Blepharisma  were  maintained  in  an  incubator  at  a  temperature  of  20°  C,  whereas 
the  paramecia  were  kept  at  room  temperature  (25°— 27°  C.). 

Periodically,  single  organisms  were  isolated  and  placed  in  the  separate  spots 
of  a  12-spot  Klein  agglutination  slide  for  five  days.  The  contents  of  each  depres- 
sion were  then  transferred  to  a  test-tube  of  lettuce  medium  which  had  been  inoculated 
three  days  previously  with  Pseudomonas  oralis.  Such  Blepharisma  clones  were 
used  for  experimental  purposes  only  from  the  seventh  to  the  eleventh  day  after 
isolation.  These  gave  fairly  consistent  data,  but  organisms  from  older  cultures 
displayed  a  marked  increase  in  pressure  sensitivity.  Paramecium  cultures  were 
more  stable  and  gave  fairly  consistent  results  over  a  three-week  period. 

The  constant-temperature  housing,  used  with  the  pressure  apparatus,  has  been 
described  by  Marsland  (1950).  The  apparatus  provides  for  a  rapid  build-up  and 
release  of  pressure,  and  for  constant  microscopic  observation  while  the  organisms 
are  under  pressure.  In  the  pressure  bomb  the  protozoa  were  kept  in  viewr  by 
confining  them  within  a  small  plastic  chamber  (6.5  mm.  diameter  and  2  mm.  depth) 
which  was  closed  above  and  below  by  glass  coverslips  held  in  position  by  Lubriseal 
films. 

Ten  to  thirty  ciliates  were  placed  in  the  chamber  for  each  experiment,  and  the 
remaining  volume  (approximately  85  ml.)  of  the  pressure  chamber  was  filled  with 
Brandwein  solution.  The  duration  of  exposure  to  any  given  pressure  was  fifteen 
minutes.  The  pressures  ranged  from  7000  to  11,000  psi.  and  the  temperatures 
employed  were  12°,  15°,  20°,  and  25°  C.  The  organisms  were  counted  several 
times  during  the  progress  of  each  experiment,  and  then  at  the  end  of  the  15-minute 
period  the  percentage  of  cytolyzed  individuals  was  determined.  At  least  60 
Blepharisma  in  a  total  of  four  or  more  experiments  were  used  at  each  pressure  and 
temperature,  at  least  in  all  critical  ranges. 

RESULTS 

Blepharisma:  pressure-temperature  effects  on  form-stability 

Blepharisma  from  old  mixed  cultures  were  very  sensitive  to  pressure.  In  such 
cultures  (at  20°  C.)  4000  psi.  (lbs./in.-)  usually  was  sufficient  to  cause  a  rounding 
up  of  all  the  specimens  and  subsequently  cytolysis  occurred  in  over  75  per  cent. 
Also,  aging  cloned  cultures  showed  a  steady  decrease  in  resistance  to  pressure.  In 
fact,  during  the  fourth  or  fifth  week  after  cloning,  pressures  of  about  4000  psi. 
became  sufficient  to  cause  breakdown  of  the  organisms,  as  was  the  case  with  the 
mixed  cultures.  Occasionally,  young  cloned  cultures  were  found  which  displayed 
a  similar  super-sensitivity  to  the  pressure-temperature  conditions.  Perhaps  such 
clones  were  derived  from  a  weak  or  aberrant  individual.  In  any  event  they  were 
not  used  for  further  experimentation. 

Blepharisma  from  typical  young  cloned  cultures  showed  little  or  no  tendency 
to  become  rounded,  regardless  of  the  experimental  temperature,  until  the  pressure 
exceeded  7000  psi.  Moreover,  there  was  virtually  no  cytolysis  within  the  15-minute 
experimental  period.  At  higher  pressures,  however,  a  number  of  the  specimens 
first  became  rounded  and  then  cytolyzed.  Furthermore,  the  temperature  of  the 


FORM-STABILITY  OF  CILIATES 


387 


TABLE  I 

Percentage  of  cytolysis  in  Blepharisina  after  15 -minute  exposure  to  various  pressure-temperature 

conditions.     These  are  the  results  of  the  individual  experiments.     In  each  experiment  the 

number  of  lysed  specimens  is  given  in  relation  to  the  total  number  treated 


T°  C. 

psi. 

8000 

0000 

10,000 

11,000 

12 

10-20 

12-15 

17-18  =  100% 

— 

13-25 

15-20 

8   15 

27  35  =  77% 

31-60  =  52', 

IS 

4-20 

6-10 

9-10 

15-15  =  100% 

3-12 

7-13 

23-25 

9-23 

10-20 

11-12 

7-15 

10-15 

13-15 

8-1  > 

20-22 

23-70  =  33% 

O         1  v" 

11-17 

76-84=91% 

52-88  =  59% 

20 

5-25 

7-15 

6-10 

14-15 

3-15 

5-15 

9-10 

8-8 

2-10 

6-18 

14-15 

15-15 

3-10 

8-20 

9-10 

4-13 

5-15 

13-15 

37-38=100% 

2-10 

4   11 

3-10 

4-11 

51-60  =  85% 

22-93  =  24% 

39-105=37% 

25 

3-20 

3-10 

5-6 

15-15  =  100'  t 

2-26 

4-11 

7-8 

2-16 

4-14 

17-20 

2-22 

4-10 

10-12 

3-10 

13-17 

9-82  =  1  1  % 

x_7        1  \J 

6-15 

9-12 

M    1  A 

24-70  =  34% 

i  \j    i\t 

74-91=81', 

experiment  had  a  distinct  influence  upon  the  percentage  of  suseptibility,  as  is  shown 
in  Table  I. 

The  character  of  the  rounding  and  of  the  subsequent  cytolysis  varied  somewhat 
in  relation  to  the  intensity  of  the  pressure  treatment  and  to  the  experimental 
temperature.  However,  under  critical  conditions — which  may  be  defined  as  any 
pressure-temperature  combination  which  yields  just  50  per  cent  cytolysis  in  15 
minutes — the  reactions  were  generally  similar.  Thus  it  is  possible  to  describe  the 
variations  which  occurred  under  sub-critical,  critical,  and  super-critical  conditions 
which,  respectively,  yielded  more  and  more  cytolysis  within  the  experimental  time. 


388  WALTER   AUCLAIR  AND  DOUGLAS  MARSLAND 

The  rounding  and  cytolysis  reactions  under  slightly  sub-critical  conditions 
(9000  psi./25°  C.)  are  shown  in  Figure  1.  Under  such  conditions,  generally 
speaking,  the  shortening  seldom  exceeded  25  per  cent  of  the  original  length ;  the 
number  of  rounded  specimens  increased  only  gradually  during  the  experimental 
period ;  and  the  tapered  anterior  end  of  the  organism  tended  to  retain  a  fairly  close 
semblance  of  its  original  architecture.  Generally,  motility  was  absent  or  at  least 
drastically  retarded  in  the  rounded  specimens. 

Lysis,  as  was  the  case  under  all  conditions  studied,  occurred  only  subsequent  to 
the  rounding  reaction.  Under  sub-critical  conditions  the  time  of  the  lysis  was 
distributed  quite  evenly  throughout  the  test  period.  In  each  specimen,  however,  the 
lysis  was  sudden,  sometimes  being  initiated  in  the  tapered  anterior  end  (Fig.  1.  C), 
and  sometimes  in  the  swollen  posterior  half,  near  the  contractile  vacuole.  It 
appeared  to  involve  a  sudden  rupturing  of  the  cell  surface  and  a  disruption  of  the 
cytoplasm  into  a  number  of  rounded  free-floating  pieces  (Fig.  1,  D).  Occasionally, 
some  of  these  protoplasmic  fragments  become  motile  after  pressure  was  released. 


B  C 


FIGURE  1.  Blepharisma :  rounding  and  cytolysis  reactions  under  sub-critical  conditions 
(9000  psi./25°  C.).  A  and  B:  Gradual  shortening  of  specimen;  successive  exposures  taken 
4  and  7  minutes  after  pressure  build-up.  C :  Sudden  cytolysis,  exposure  30  seconds  after  B. 
D :  Rounding  up  of  cytoplasmic  remnants,  one  minute  after  cytolysis.  After  decompression 
some  of  these  fragments  may  become  motile.  Photographs  retouched. 

Under  distinctly  super-critical  conditions,  rounding  and  lysis  developed  rapidly 
and  most  of  the  susceptible  specimens  had  reacted  within  the  first  five  minutes. 
The  shortening  of  specimens  was  distinctly  greater,  although  often  there  was  some 
persistence  of  the  tapered  anterior  end  up  to  the  moment  of  lysis.  The  lysis  was 
more  complete ;  the  protoplasmic  fragments  were  smaller ;  these  fragments  showed 
less  tendency  to  round  up,  and  they  did  not  develop  motility  subsequent  to 
decompression. 

Under  intermediate  conditions,  in  and  around  the  critical  range,  the  rounding 
and  cytolysis  reactions  were  intermediate  in  character. 

Blepharisma:  decompression  effects 

The  sudden  release  of  pressure,  under  critical  or  nearly  critical  conditions,  gave 
rise,  within  two  minutes,  to  an  abrupt,  further  shortening  of  all  the  non-cytolyzed 
specimens,  accompanied  by  a  momentary  stoppage  of  any  persisting  ciliary  activity. 


FORM-STABILITY  OF  CILIATES  389 

This  shortening  (Fig.  2)  was  more  abrupt  than  the  pressure-induced  rounding. 
Immediately  after  shortening,  a  few  specimens  displayed  sudden  lysis,  but  this 
decompression  lysis  did  not  involve  more  than  five  per  cent  of  the  animals.  In 
fact,  most  of  the  specimens  regained  their  motility  within  some  ten  minutes,  and 
after  30  to  200  minutes  they  presented  a  fairly  normal  form  and  appearance. 

Blepharisma :  pressure-temperature  parameters  of  cytolvsis 

As  may  be  seen  in  Table  I,  the  percentage  of  cytolysis  obtained  at  any  given 
pressure  represents  a  temperature-dependent  value.  The  sensitivity  to  pressure 
cytolysis  increases  very  definitely  with  decreasing  temperature  within  the  experi- 
mental range  (25°-12°  C).  This  is  shown  more  clearly  when  the  data  are  plotted, 
as  in  Figure  3.  Conversely,  the  resistance  to  pressure  cytolysis  increases  with 
increasing  temperature,  as  is  shown  in  Figure  4.  There  it  may  be  seen  that  the 


V 

S 


B 


FIGURE  2.  Blepharisma  :  shortening  under  pressure  (slightly  sub-critical  conditions,  i.e., 
9000  psi./25°  C.)  followed  by  rapid  shortening  after  decompression.  A:  Exposure  13  minutes 
after  pressure  build-up.  B  :  One  minute  after  decompression.  C  :  Two  minutes  later.  D  :  An- 
other two  minutes  later.  Photographs  retouched. 

pressure  which  is  just  adequate  to  induce  cytolysis  in  50  per  cent  of  the  treated 
specimens  increases  regularly  as  the  temperature  increases,  within  the  given  range. 

Blepharisma  :  pressure-centrifuge  experiments 

Quantitative  measurements  of  the  solational  effects  of  pressure  are  difficult  to 
obtain  with  Blepharisma.  The  pressure-centrifuge  method,  which  has  been  used 
widely  for  other  cells  (Marsland,  1956),  is  not  very  suitable.  The  orientation  of 
the  specimens  in  the  centrifugal  field  shows  considerable  variation,  and  the  instability 
of  form  and  cellular  integrity  at  higher  pressures  gives  further  difficulty. 

It  was  possible,  however,  to  obtain  qualitative  data  which  showed  unequivocally 
that  pressure  does  induce  solational  changes  in  the  cytoplasm  of  Blepharisma. 
Many  of  the  specimens  centrifuged  for  one  minute  at  5000  X  gravity  at  3000  psi. 
showed  a  distinct  clearing  of  the  centripetal  half  of  the  cell  —  by  virtue  of  the 
centrifugal  displacement  of  food  vacuoles  and  other  granular  bodies  —  to  a  degree 
that  was  never  found  in  control  specimens,  centrifuged  simultaneously  at  atmospheric 
pressure. 


390 


WALTER  AUCLAIR  AND  DOUGLAS  MARSLAND 


Paramecium:  comparative  observations 

Generally  speaking,  the  pressure-temperature  effects  on  Paramecium  and 
Blepharisma  were  similar.  However,  there  were  two  important  differences :  1 ) 
Paramecium  was  distinctly  more  sensitive  to  pressure  lysis,  and  2)  decompression 
lysis,  which  was  almost  negligible  in  Blepharisma,  became  very  significant  in 
Paramecium. 

For  Paramecium,  the  critical  pressure  for  50  per  cent  lysis  was  2000-3000  psi. 
lower  than  for  Blepharisma,  at  each  of  the  two  temperatures  (20°  and  25°  C.) 
which  were  studied.  Under  such  critical  conditions  (e.g.,  7000  psi./20°  C.)  the 
animals  shortened  moderately  and  displayed  gradually  diminishing,  distinctly  ir- 
regular locomotion,  which  ceased  only  if  and  when  cytolysis  occurred.  Most  of 
the  cytolysis  occurred  during  the  last  5  minutes  of  the  15-minute  compression  period. 
Moreover,  two  somewhat  different  types  of  lysis  were  observed  with  roughly  equal 
frequency.  One  type  seemed  to  involve  a  detachment  of  the  pellicle,  with  the 
formation  of  one  or  more  large  hyaline  blisters  which  later  broke,  liberating  the 
deeper  granular  cytoplasm  (Fig.  5).  The  other  type,  in  contrast,  seemed  to 


lOOr 


0 


FIGURE  "3 

8  9  10  II 

PRESSURE-  1000  LBS.  /  SQ.  IN. 


FIGURE  3.     Blepharisma  :  percentage  of  cytolysis  as  a  function  of  pressure,  at  four  temperatures. 
Cytolyzed  cells  were  counted  exactly  15  minutes  subsequent  to  pressure  build-up. 


FORM-STABILITY  OF  CILIATES 


391 


CO 


CO 
CD 


8    9 

O 

I 
UJ 

QC 

ID 

00       o 

co    8 

UJ 

o: 
o. 


FIGURE-4 


10    12          15  20  25 

TEMPERATURE     °C 

FIGURE  4.  Blepharisma:  critical  pressure  yielding  50  per  cent  cytolysis  after  15-minute 
exposure,  plotted  as  a  function  of  temperature.  The  data  of  this  figure  are  derived  from 
Figure  3. 


represent  a  more  generalized  breakdown  of  the  cell  surface  in  either  the  anterior 
or  posterior  half  of  the  animal,  with  a  less  abrupt  scattering  of  the  granular  cyto- 
plasm (Fig,  6).  With  Paramecium,  moreover,  regardless  of  the  conditions  or 
type  of  cytolysis,  there  was  very  little  tendency  for  the  cytoplasmic  remnants  to 
round  up,  or  to  wall  themselves  off  from  the  surrounding  medium. 

The  decompression  lysis  under  critical  conditions  usually  involved  more  than 
half  of  the  surviving  specimens,  particularly  when  the  decompression  was  rapid 
(within  one  second).  Sudden  decompression  was  followed  within  about  two 
minutes  by  an  abrupt  further  shortening  of  all  surviving  specimens,  followed 
immediately  by  a  generalized  cytolysis  of  the  majority.  Specimens  that  escaped 
cytolysis,  on  the  other  hand,  gradually  regained  normal  form  and  motility  within 
2—3  hours. 

Under  super-critical  compression  (8000  psi./20°  C.),  the  degree  of  rounding 
was  greater ;  and  most  of  the  lysis,  which  involved  more  than  60  per  cent  of  the 
specimens,  occurred  during  the  first  ten  minutes  of  the  compression  period.  Then, 
following  rapid  decompression,  all  surviving  specimens  shortened  still  more  and 
quickly  underwent  cytolysis. 


392  WALTER   AUCLAIR  AND  DOUGLAS  MARSLAND 


A  B  C  D 


FIGURE  5.  Paramecium :  one  type  of  pressure  cytolysis  (8000  psi./25°  C.).  A  and  B: 
Shortened  specimen,  photographed  9  and  10  minutes  after  compression.  C  :  Two  minutes  later, 
cytolysis  starting.  D :  30  seconds  later,  showing  hyaline  blisters  which  are  about  to  break. 
Photographs  retouched. 

Distinctly  sub-critical  conditions  (5000  psi./20°  C.)  produced  no  clearly  defined 
effects  ;  but  at  6000  psi.  a  slight  degree  of  shortening  was  noted,  although  locomotion 
appeared  to  continue  in  normal  manner.  Abrupt  decompression,  under  these 
conditions,  produced  a  further  sudden  shortening  of  the  specimens,  but  there  was 
no  cytolysis  and  usually  the  animals  regained  their  normal  form  within  one  hour. 

DISCUSSION 

The  problem  of  how  pressure  exerts  its  effects  upon  cellular  systems  has  been 
approached  from  several  angles.  Regnard  (1891)  interpreted  his  results  in  terms 
of  an  imbibition  of  water  by  the  ciliated  cells.  However,  since  no  volume  increase 
can  be  found  in  pressurized  cells,  this  hypothesis  has  not  been  pursued.  Hodapp 
and  Luyet  (1947)  suggested  a  disturbance  of  the  permeability  mechanism  and  an 
injury  of  the  neuromotor  apparatus  as  the  main  factors  involved  in  pressure  lysis. 
However,  as  stated  previously,  they  were  unable  to  observe  the  organisms  during 
the  pressure  period.  As  to  their  findings  that  temperature  and  the  rate  of  applica- 
tion and  release  of  pressure  had  no  effect  on  lethality,  at  the  high  pressures  they 
employed,  it  is  probable  that  the  decompression  effects  were  very  drastic  and 
negated  these  variables. 


A  B 


FIGURE  6.  Paramecium:  another  more  generalized  type  of  pressure  lysis  (9000  psi./25°  C.). 
A:  Shortened  intact  specimen  5  minutes  after  pressure  build-up.  B:  5  minutes  later,  sudden 
cytolysis  initially  involving  all  of  the  anterior  half  of  the  specimen.  Photographs  retouched. 


FORM-STABILITY  OF  CILIATES  393 

Pressure-temperature  effects  on  cell  jonn 

A  more  fruitful  approach,  perhaps,  is  to  interpret  the  observed  effects  on  the  cell 
form  and  integrity  of  Blepharisma  and  Paramecium  in  terms  of  the  now  well 
established  action  of  pressure  and  temperature  upon  intracellular  gel  structures.  To 
some  extent  this  approach  has  been  adopted  by  Ebbecke  (1936),  who  had,  however, 
very  little  experimental  evidence.  Moreover,  Ebbecke  postulated  that  pressure 
exerts  its  effect  upon  the  gel  system  indirectly  via  an  action  upon  cell  metabolism, 
rather  than  directly  and  indirectly,  as  proposed  by  Marsland  and  Brown  (1942). 

It  now  seems  reasonable  to  assume  that  the  cortical  cytoplasm  of  ciliates, 
immediately  subjacent  to  the  pellicle,  is  firmly  gelated  and  that  this  plasmagel  layer 
plays  a  significant  role  in  helping  to  maintain  the  unique  form  of  the  particular 
species.  Also,  it  seems  possible  that  the  plasmagel  layer  of  the  ciliate  may  possess 
contractile  properties  which  can  be  instrumental  in  producing  changes  of  form  and 


orientation  during  normal  locomotion. 


Experimental  evidence  in  regard  to  the  foregoing  question  is  not  very  extensive, 
however.  A  plasmagel  structure  is  indicated,  to  be  sure,  by  the  fact  that  the 
peripheral  layer  of  cytoplasm,  in  which  the  trichocysts  are  lodged,  does  not  become 
involved  in  the  protoplasmic  streaming  when  cyclosis  occurs.  Also,  it  is  frequently 
observed,  not  only  in  the  pressure-temperature  experiments,  but  also  when  Para- 
mecium and  other  ciliates  are  exposed  to  toxic  substances  or  merely  flattened  under 
a  coverslip,  that  the  pellicle  may  peel  away  from  the  subjacent  cytoplasm  and  form  a 
hyaline  blister  of  large  or  smaller  size.  When  this  happens  the  granular  cytoplasm, 
from  which  the  pellicle  has  become  detached,  may  persist,  retaining  its  stability  for 
a  minute  or  two.  Then  it  disintegrates  and  pours  forth  its  granular  components 
into  the  hyaline  fluid  which  fills  the  blister. 

All  the  evidence  of  the  present  experiments  indicates  that  the  shortening  and 
rounding  of  the  cells  induced  by  suitably  high  pressure  and  modified  by  temperature 
are  mediated  by  a  solation  of  the  plasmagel  layer.  Qualitatively,  the  susceptibility 
of  this  gel  to  pressure  solation  is  established  by  the  pressure-centrifuge  experiments 
and  quantitatively  the  pressure-temperature  parameters  of  this  gel  system  are  very 
similar  to  those  which  have  been  established  in  various  other  protoplasmic  gels 
(Marsland,  1956).  Apparently  a  shortening  and  rounding  of  these  elongate 
ciliated  cells  occur,  under  the  agency  of  tensional  forces  in  the  cell  surface,  when- 
ever the  subjacent  plasmagel  structure  is  weakened  below7  a  certain  critical  resistance 
level. 

Pressure-tempera  tit  re  lysis 

Pressure  lysis,  apparently,  is  always  preceded  by  a  rounding  of  the  cells ;  and, 
generally  speaking,  the  more  drastic  the  rounding  the  greater  is  the  lysis  tendency. 
It  seems  likely,  therefore,  that  cell  form  and  cell  integrity  may  be  determined  In- 
similar  underlying  factors. 

A  firmly  maintained  plasmagel  structure  would  serve,  most  probably,  not  only 
to  stabilize  the  total  form  of  the  cell,  but  also  to  preserve  the  orientation  and  spatial 
configuration  of  many  of  the  microscopic  and  submicroscopic  constituents  of  cell 
structure.  Moreover,  if  the  solation  is  drastic  enough  to  allow  for  a  rounding  of 
the  cell,  the  rounding  itself  tends  to  disturb  and  disorient  the  configuration  of  the 


394  WALTER  AUCLAIR  AND  DOUGLAS  MARSLAND 

protoplasmic  constituents.  Cytolysis,  perhaps,  may  involve  a  detachment  of  the 
pellicle  from  the  subjacent  plasmagel,  with  a  concomitant  disarrangement  of  the 
ciliary  origins  and  trichocysts,  or  it  may  involve  some  other  type  of  disorientation. 
In  any  event,  it  seems  to  occur  whenever  drastic  solation  occurs.  Thus  it  is  not 
surprising  to  note  that  preliminary  dosages  of  UV-irradiation,  utilizing  wave-lengths 
which  have  a  primary  effect  upon  the  proteins  of  the  peripheral  cytoplasm,  predispose 
Blepharisma  to  pressure  cytolysis,  presumably  as  a  result  of  a  weakening  effect 
upon  the  plasmagel  structure  (Hirshfield  et  al.,  1957). 

Decompression  lysis 

This  phenomenon,  which  was  particularly  conspicuous  in  Paramecium,  can  be 
interpreted,  perhaps,  in  terms  of  a  rapid  post-pressure  reconstruction  and  contrac- 
tion of  the  plasmagel  system.  A  similar  phenomenon,  in  fact,  has  been  described 
for  Amoeba  by  Landau,  Zimmerman  and  Marsland  (1954).  Many  studies  have 
shown  that  pressure  solation  is  rapidly  reversible  upon  decompression  and  the 
Amoeba  study  indicates  that  the  newly  reconstituted  gel  system  tends  to  contract 
sharply,  presumably  as  a  result  of  an  accumulation  of  metabolites  which  are  not 
fully  utilized  during  the  pressure  period  (Landau  ct  al.,  1954).  In  any  event,  both 
Blepharisma  and  Paramecium  always  showed  an  abrupt  contraction  about  two 
minutes  after  release  from  any  extensive  critical  or  super-critical  pressure- 
temperature  treatment  and,  particularly  in  the  case  of  Paramecium,  this  abrupt 
contraction  was  very  frequently  accompanied  by  cytolysis.  Precisely  why  cytolysis 
should  occur  under  these  circumstances  is  problematical.  It  may  be  supposed, 
however,  that  such  a  contraction  would  tend  to  disrupt  the  surface  architecture  of 
the  cell,  especially  if  it  occurs  before  a  proper  stabilization  of  the  cell  structure  has 
occurred.  Furthermore,  these  observations  indicate  that  the  peripheral  gelated 
cytoplasm  of  the  ciliate  displays  a  potential  contractility,  and  that  this  layer  may 
play  a  role  in  effecting  changes  of  form  and  orientation,  during  locomotion,  and  in 
performing  the  work  of  cell  division. 

Metabolic  relationships 

The  increasing  susceptibility  of  older  cloned  cultures  in  regard  to  pressure- 
temperature  cytolysis  raises  some  interesting  questions.  A  continued  source  of 
metabolic  energy  appears  to  be  necessary  for  the  maintenance  of  protoplasmic  gel 
structures  (see  Marsland,  1956)  ;  but  why  should  such  structures  tend  to  be  weaker 
in  aging  clones?  Lettre  (1952)  has  suggested  that  cell  form  and  stability  may 
be  dependent  upon  the  level  of  ATP  reserve  in  the  cell,  but  why  should  this  tend 
to  diminish  with  age?  ATP-sensitive  proteins,  capable  of  forming  potentially 
contractile  gel  systems,  seem  to  be  present  in  various  relatively  unspecialized 
cells — in  the  slime  mold  (Loewy,  1952  and  Ts'o  et  al.,  1956),  in  sea  urchin  eggs 
(Mirsky,  1936),  in  fibroblasts  and  other  tissue  cells  (Weber,  1955  and  Hoffman- 
Berling,  1954),  and  in  Amoeba  (Landau  et  al.,  1954).  At  present,  however,  it  is 
entirely  problematical  as  to  whether  age-changes  in  the  gel  structure  result  from 
changes  in  metabolism,  changes  in  the  constituent  proteins  or,  at  least  partly,  from 
other  unknown  changes. 


FORM-STABILITY  OF  CILIATES  395 

SUMMARY 

1.  Two  ciliates,  Blepharisma  undulans  and  Paramecium  caudatum,  were  studied 
with  reference  to  form  stability  and  integrity  (resistance  to  cytolysis)  under  varying 
conditions   of  hydrostatic  pressure    (up   to    10,000   lbs./in.2)    and   of  temperature 
(12°-25°  C.). 

2.  At  lower  pressures  the  specimens  retained  their  elongate  form,  but  at  higher 
levels,  depending  on  temperature,  species,  and  age  of  the  cloned  cultures,  the  cells 
gradually  become  shorter  and  more  rounded.     Following  this  form  change,  cytolysis 
occurred  in  a  varying  percentage  of  the  specimens.     Older  cloned  cultures  showed 
a  greater  and  more  variable  susceptibility  to  the  pressure-temperature  effects,  so 
that  selected  younger  cultures  were  used  for  the  quantitative  evaluations. 

3.  For   Blepharisma,  the  critical  pressure,   which  gave    50  per  cent   cytolysis 
within  a  15-minute  compression  period,  displayed  a  distinct  temperature  dependence, 
being  8000  psi.  at  12°  C.,  8700  at  15°  C.,  9200  at  20°  C..  and  9300  at  25°  C. 
Paramecium,  in  contrast,  showed  a  distinctly  greater  sensitivity,  the  critical  pressure 
for  50  per  cent  cytolysis  at  20°  C.  being  some  2000  psi.  lower  than  for  Blepharisma. 

4.  Rapid  decompression,  following  any  critical  or  super-critical  pressure  treat- 
ment,  produced   an   abrupt    further    shortening    (contraction)    of    the    specimens, 
accompanied  by  a  cytolysis  of  some  of  the  previously  resistant  individuals.     For 
Blepharisma,  decompression  cytolysis  involved  only  about  5  per  cent  of  the  animals. 
Paramecium,  however,  was  much  more  sensitive  and  virtually  100  per  cent  became 
involved. 

5.  An  interpretation  of  these  changes  in  cell  form  and  integrity  is  given  in 
terms  of  pressure-temperature  effects  upon  protoplasmic  gel  structure,  particularly 
with  reference  to  the  solation  of  the  peripheral  plasmagel  layer  of  the  cytoplasm. 

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MIRSKY,  A.  E.,   1936.     Protein  coagulation  as  a  result  of  fertilization.     Science,  84:   333-334. 

REGNARD,  P.,  1884a.  Note  sur  les  conditions  de  la  vie  dans  les  profondeurs  de  la  mer.  (".  R. 
Soc.  Biol.,  36:  164-168. 

REGNARD,  P.,  1884b.  Note  relative  a  1'action  des  hautes  pressions  sur  quelques  phenomenes 
vitaux  ( mouvement  des  cils  vibratiles,  fermentation).  C.  R.  Soc.  Biol..  36:  187-188. 

REGNARD,  P.,  1884c.  Recherches  experimentales  sur  1'influence  des  tres  hautes  pressions  sur 
les  organismes  vivants.  C.  R.  Acad.  Sci..  98:  745-747. 

REGNARD,  P.,  1891.     La  vie  dans  les  eaux.     C".  R.  Assoc.  Francaise;  20th  session,   (1)  :  393-429. 

Ts'o,  P.  O.  P.,  L.  EGGMAN  AND  J.  YIXOGRAD,  1956.  The  isolation  of  myxomyosin,  an  ATP- 
sensitive  protein  from  the  plasmodium  of  a  myxomycete.  /.  Gcu.  Physiol.,  39:  801-812. 

WEBER,  H.  H.,  1955.  Adenosine  triphosphate  and  motility  of  living  systems.  The  Harvey 
Lectures  (1953-1954);  Academic  Press,  New  York. 


OBSERVATIONS  ON  THE  SYMBIOSIS  OF  THE  SEA  ANEMONE 

STOICHACTIS  AND  THE  POMACENTRID  FISH, 

AMPHIPRION  PERCULA1 

DEMOREST  DAVENPORT  AND  KENNETH  S.  NORRIS 

The  I'nii'crsity  of  California,  Santa  Barbara,  Goleta,  California,  and 
Marineland  of  the  Pacific,  Palos  I'crdes.  Califo 


urina 


The  partnership  between  certain  tropical  damselfishes  and  sea  anemones  has 
excited  the  interest  of  students  of  natural  history  for  almost  a  century.  The  most 
significant  investigations  of  the  symbiosis  have  been  those  of  Sluiter  (1888),  Ver- 
wey  (1930)  and  Gohar  (1948),  who  have  given  us  some  knowledge  of  the  ecology 
and  behavioral  characteristics  of  the  animals.  In  1947  Gudger  reviewed  all  the 
observations  that  had  been  made  up  to  that  time,  and  in  1950  Baerends  first  specu- 
lated about  the  possible  role  of  releasers  in  the  maintenance  of  the  association. 

However,  this  symbiosis,  like  many  others,  still  poses  many  unanswered  ques- 
tions. The  physiological  and  behavioral  mechanisms  which  maintain  the  animals 
in  partnership  have  not  been  investigated  with  present-day  techniques. 

It  has  not  been  clear  whether  the  fish  responds  to  chemical,  tactile  or  visual 
stimuli  from  the  host,  nor  whether  the  behavior  of  the  anemone  is  affected  by 
stimuli  from  the  fish.  The  mechanism  whereby  the  fish  is  protected  from  the 
nematocysts  of  the  host  has  been  a  mystery.  In  spite  of  the  fact  that  it  is  gen- 
erally supposed  that  nematocysts  are  not  under  nervous  control  but  that  they  fire 
off  independently  upon  adequate  stimulation,  several  investigators  have  speculated 
that  in  such  partnerships  the  presence  of  the  fish  in  some  way  causes  the  coelen- 
terate  host  to  put  its  nematocysts  "out  of  action"  (Baerends,  1957,  p.  262).  The 
question  remains  whether  the  fish  simply  fails  to  provide  adequate  stimuli  to  dis- 
charge the  nematocysts,  or  whether  a  factor  is  produced  by  the  fish  which  markedly 
raises  the  threshold  of  discharge  of  the  nematocysts  and  thus  affords  protection. 
Finally,  it  remains  to  be  determined  whether  or  not  the  fish  is  immune  to  the 
poison  of  the  nematocysts. 

Recently  at  Marineland  of  the  Pacific  it  became  possible  to  investigate  the 
partnership  between  Ainphiprion  pcrcula  (Lacepede)  and  the  giant  anemone 
Stoichactis  (Fig.  1 ).  We  directed  our  attention  primarily  to  the  physiological  and 
behavioral  mechanisms  involved  in  the  protection  of  the  fish  against  the  nemato- 
cysts of  its  host  and  in  the  course  of  the  work  were  able  to  re-examine  and  re- 
establish some  of  the  observations  of  Vervey  and  Gohar. 

1  Contribution  No.  6,  Marineland  of  the  Pacific  Biological  Laboratory.  This  work  was 
carried  out  under  the  contract  of  the  senior  author  with  the  Office  of  Naval  Research.  We 
wish  to  express  our  appreciation  to  Marineland  of  the  Pacific  for  its  hospitality  and  facilities, 
and  to  Dr.  Cadet  Hand  for  a  tentative  identification  of  our  anemone. 

397 


398 


DEMOREST  DAVENPORT  AND  KENNETH  S.  NORRIS 


FIGURE  1.     The  anemone  Stoichactis  and  two  partner  Amphiprion  percula.     Photographed 
in  the  exhibition  aquarium  at  Marineland  of  the  Pacific.     Approximately  X  %. 


MATERIAL  AND  METHODS 

Experimental  fish  were  obtained  on  the  reefs  near  Nasugbu,  Batangas  Province, 
Luzon,  in  the  Philippine  Islands  by  commercial  collectors.  Our  single  specimen 
of  the  host  anemone  was  taken  at  the  same  locality.  Verwey  (1930)  describes  an 
anemone,  probably  identical  with  ours,  from  Batavia  Bay,  Java  (Anemone  1, 
Plate  XV,  Fig.  2)  which  he  says  is  colonized  in  nature  by  Amphiprion  percula 
alone.2  Ours  was  provided  by  the  collectors  specifically  as  the  host  of  A.  percula. 
At  this  writing  it  is  still  alive  at  Marineland.  We  believe  it  to  be  Stoichactis 
kenti  (Haddon  and  Shackleton)  although  precise  identification  will  not  be  pos- 
sible until  examination  of  the  internal  anatomy  can  be  made  after  the  animal  is 
preserved. 

The  anemone  was  received  at  Marineland  in  January  of  1957,  and  our  experi- 
ments were  started  on  September  3,  1957.  Thus  the  animal  was  acclimated  to 
Marineland  sea  water  for  a  period  of  somewhat  over  8  months.  During  the  period 
of  our  observations  it  was  maintained  at  25°  C.  in  a  60-gallon  "photographic" 
redwood  aquarium,  which  was  so  constructed  that  a  sheet  of  glass  could  be  in- 
serted to  isolate  fishes  from  the  anemon3  when  desired. 

We  received  a  total  of  thirteen  specimens  of  Amphiprion  percula.     The  previous 

2  In  the  aquarium  at  Batavia  this  anemone  \vas  readily  occupied  by  Amphiprion  akallopisus 
and  A.  polynemus,  in  addition  to  A.  percula. 


AMPHIPRION  AND  STOICHACTIS  399 

history  of  these  fish  is  totally  unknown  to  us.  Probably  some  or  all  of  the  animals 
were  collected  from  anemones.  However,  according  to  Dr.  Jose  Montilla  of  the 
Division  of  Marine  Fisheries  in  Manila,  this  species  of  Amphiprion  does  not  always 
live  in  association  with  anemones  in  the  Philippines ;  hence  some  of  our  experi- 
mental fish  may  have  been  free-living.  Also,  it  is  known  that  A.  percula  lives 
in  association  with  at  least  two  species  of  anemones  (Verwey,  1930).  Therefore 
the  host  habit  of  any  that  may  have  been  commensal  is  also  unknown  to  us. 

Prior  to  the  experiments,  two  fish,  A  and  B,  were  kept  in  partnership  with 
the  anemone  for  several  weeks.  These  gave  us  controls  which  we  knew  were 
"acclimated"  to  the  anemone.  Nine  other  fish  (C  to  K)  had  been  isolated  from 
any  possible  sensory  contact  with  an  anemone  host  for  a  period  of  not  less  than 
six  weeks.  In  the  following  experiments  these  are  spoken  of  as  "unacclimated"  fish. 

Two  other  A.  percula  (L  and  M)  which  had  occasionally  been  put  in  with 
the  anemone  for  exhibition  purposes  prior  to  our  experiments  were  also  used. 
One  of  these  (L)  was  the  largest  animal  in  our  sample,  measuring  65  mm.  standard 
length.  Fish  M,  a  small  animal,  was  sacrificed  in  a  physiological  experiment. 

The  age  and  sex  of  our  Amphiprion  pcrcula  were  not  determined. 

A  single  adult  specimen  (56  mm.  standard  length)  of  Amphiprion  frenatus 
Brevoort  was  available  for  specificity  studies.  This  fish  had  lived  in  the  exhibition 
aquarium  with  the  anemone  for  the  eight  months  prior  to  our  experiments  but  was 
never  observed  to  enter  it. 

The  most  careful  precautions  were  taken  to  maintain  all  glassware,  forceps, 
scissors,  dip  nets,  and  other  tools  free  from  contamination  with  organic  materials, 
because  of  the  well-known  sensitivity  of  nematocysts  to  such  substances.  All  items 
employed  in  the  manipulation  of  fishes  or  isolated  anemone  tentacles  were  scrubbed 
with  detergent,  washed  in  distilled  water,  dipped  in  ether-alcohol  and  allowed  to 
dry  without  contact.  Fish  to  be  sacrificed  were  dissected  with  clean  instruments 
in  clean  Petri  dishes.  Whenever  possible,  experimental  fish  were  not  handled 
at  all  but  were  trapped  in  the  aquaria  with  clean  500-cc.  beakers.  When  it  was 
necessary  to  use  a  nylon  dip  net,  the  net  was  first  boiled  and  rinsed. 

Experiments  on  the  discharge  of  nematocysts  from  isolated  tentacles  were  con- 
ducted in  clean  watch  glasses.  A  new  tentacle  was  prepared  for  each  test.  Ten- 
tacles were  isolated  by  clipping  them  off  at  the  base  with  clean,  fine-tipped  scissors. 
They  were  stimulated  mechanically  with  a  clean  glass  rod  drawn  to  a  fine  point, 
and  electrically  stimulated  with  a  platinum  wire-glass  electrode  drawn  to  a  fine 
capillary  point.  The  electrode  was  connected  in  a  circuit  with  a  standard  induc- 
torium,  key,  and  a  6-volt  dry  cell.  A  small  piece  of  aluminum  foil  dipped  into 
the  sea  water  in  the  watch  glass  served  as  the  other  electrode.  Between  each 
experiment,  the  watch  glass,  the  glass  rods,  the  platinum  wire,  and  the  capillary 
tube  were  washed  with  ether-alcohol ;  the  capillary  tube  was  refilled  with  clean 
sea  water,  and  the  aluminum  electrode  was  replaced. 

In  certain  experiments,  one-cm,  cubes  of  plastic  sponge  were  used.  These 
were  cut  from  the  center  of  a  new  commercial  sponge  by  use  of  a  clean  single-edge 
razor  blade. 

OBSERVATIONS  ON  THE  "PROCESS  OF  ACCLIMATION" 

It  has  been  observed  (Gohar,  1948)  that  the  acclimation  of  an  Amphiprion  to 
an  anemone  may  take  a  considerable  length  of  time.  The  details  of  this  acclima- 


400  DEMOREST  DAVENPORT  AND  KENNETH  S.  NORRIS 

tion  remain  virtually  unknown.  We  felt  that  careful  observation  of  this  process 
might  give  us  insight  into  the  mechanism  which  protects  the  fish  from  nematocyst 
discharge. 

Accordingly,  a  series  of  nine  experiments  were  performed  in  which  we  intro- 
duced individual  unacclimated  Amphiprion  f>crcnla  into  the  observation  tank  with 
the  anemone.  These  tests  revealed  a  fairly  stereotyped  series  of  events  which 
terminated  in  the  acclimation  of  each  new  fish  to  the  anemone.  The  results  of 
these  experiments  are  summarized  below. 

An  unacclimated  fish  introduced  into  the  tank  a  foot  or  so  away  from  the  anem- 
one, usually  approached  the  anemone  within  a  few  minutes  and  began  to  swim 
under  the  disk,  around  the  column,  and  occasionally  over  the  top  of  the  disk  a 
centimeter  or  more  away  from  the  tentacles.  Such  fish  spent  most  of  their  time 
under  the  disk  at  this  stage  and  sometimes  were  seen  nibbling  at  the  column  of 
the  anemone.  Most  fish  seemed  to  "recognize"  the  anemone  within  a  few  minutes 
and  swam  toward  it.  However,  in  two  tests,  two  fish  failed  to  react  noticeably 
to  the  anemone  for  20  and  27  minutes,  respectively.  In  both  cases  another  fish 
was  introduced  directly  onto  the  disk  of  the  anemone  where  it  shortly  took  up 
residence.  In  both  tests  the  unreactive  fish  then  came  rather  quickly  toward  the 
anemone,  apparently  in  response  to  the  fish  already  in  occupancy,  and  began  the 
characteristic  acclimation  process. 

As  the  process  proceeded,  passage  over  the  disk  became  more  and  more  fre- 
quent and  the  "acclimating"  fish  moved  closer  and  closer  to  the  tentacles.  Swim- 
ming was  accomplished  by  a  distinctive  series  of  slow  vertical  undulations,  in 
which  the  tail  was  usually  held  a  little  lower  than  the  rest  of  the  body.  Eventually, 
on  one  of  these  trips  over  the  disk,  the  fish  would  touch  a  tentacle  or  two,  usually 
with  the  ventral  edge  of  its  anal  fin  or  the  lower  margin  of  its  caudal  fin.  Com- 
monly this  resulted  in  a  moderate  adherence  of  the  tentacle  to  the  fin  and  contrac- 
tion of  the  tentacle.  The  fish  then  jerked  itself  free  with  a  violent  flexure  of  its 
body  and  usually  raced  off  the  disk.  Xot  all  newly  introduced  Amphiprion  caused 
clinging  upon  their  first  contact  with  tentacles,  but  it  was  the  general  rule.  How- 
ever, this  adherence  failed  to  deter  the  fish,  which  nearly  always  returned  imme- 
diately to  the  anemone,  either  under  the  disk  or  over  the  tentacles.  In  our  ex- 
periments the  time  from  initial  introduction  until  the  first  physical  contact  between 
fish  and  anemone  varied  from  less  than  1  minute  to  65  minutes. 

After  this  initial  contact  the  fish  typically  came  closer  and  closer  to  the  tentacles, 
touching  them  with  increasing  regularity.  The  reaction  to  the  clinging  of  tentacles 
became  less  and  less  violent  until  a  sudden  flexure  of  the  animal's  body  was  the 
only  reaction  given  by  the  fish.  Mouthing  or  nipping  of  tentacles  was  often 
observed  in  this  and  later  stages. 

The  clinging  and  contraction  of  tentacles  upon  contact  with  the  fish  gradually 
became  less  until  it  ceased  altogether.  At  the  same  time  the  fish  began  to  swim 
deeper  among  the  tentacles,  using  the  same  slow  undulating  movements  as  when  it 
had  cruised  above  the  disk. 

Once  the  fish  was  swimming  in  fairly  constant  contact  with  the  tentacles  of 
the  anemone,  a  very  striking  change  in  its  behavior  occurred.  The  general  speed 
of  swimming  suddenly  increased  until  the  Amphiprion  was  dashing  back  and  forth 
over  the  disk  of  the  anemone,  flailing  unreactive  tentacles  aside  with  violent  move- 


AMPHIPRION  AND  STOICHACTIS  401 

ments  of  its  body.  Often  the  fish  raced  beneath  the  anemone  and  appeared  in  one 
of  the  folds  of  the  disk  margin,  its  head  completely  ringed  in  tentacles.  The  fish 
frequently  maintained  this  vantage  point  for  a  few  seconds,  holding  position  with 
rapid  alternate  fanning  movements  of  its  pectoral  fins,  after  which  it  might  dash  onto 
the  disk  again  for  another  foray  among  the  tentacles.  The  powerful  swimming 
typical  of  this  stage  of  the  acclimation  process  was  accomplished  by  rapid  and  strong 
lateral  body  flexures.  The  impression  given  by  the  swimming  behavior  of  the  fish 
after  final  acclimation  was  that  the  fish  was  "bathing"  its  entire  skin  surface  among 
the  tentacles. 

At  this  point  we  considered  the  fish  to  be  fully  acclimated  to  the  anemone,  since 
no  further  clinging  or  tentacle  contraction  appeared.  The  time  required  for 
complete  acclimation  varied  from  about  one  minute  to  nearly  three  hours,  with  an 
average  time  of  one  hour. 

If  a  fully  acclimated  fish  was  removed  from  the  anemone  and  its  fins  or  body 
carefully  scraped  with  a  scalpel,  and  then  returned  to  the  anemone,  the  scraped  areas 
caused  both  clinging  and  tentacle  contraction.  However,  fish  treated  in  this  manner 
did  not  then  begin  the  acclimation  process  anew  but  stayed  among  the  tentacles  until 
clinging  waned  and  disappeared.  These  fish  gave  evidence  of  discomfort  from  the 
clinging  tentacles  by  jerking  themselves  free.  They  did  not,  however,  rush  off  the 
disk.  It  would  seem  that  treating  the  fish  in  this  way  partially  broke  down  their 
protection. 

Acclimation  involves  development  of  visual  recognition  of  the  anemone  by  the 
fish.  This  was  demonstrated  by  removing  fully  acclimated  fish  from  the  anemone 
and  placing  them  in  a  compartment  of  the  observation  tank  separated  from  the 
anemone  by  a  heavy  glass  sheet.  Incoming  water  was  introduced  into  the  isolation 
compartment,  flowed  over  and  around  the  partition,  and  was  discharged  from  the 
compartment  containing  the  anemone  to  prevent  chemical  gradients  from  occurring 
which  could  guide  the  fish.  In  every  case  acclimated  fish  oriented  strongly  toward 
the  anemone  which  they  could  see  through  the  glass,  by  gathering  at  the  glass 
nearest  it  and  swimming  up  and  down  with  their  heads  directed  toward  their  host. 

The  behavior  of  an  Amphiprion  which  has  been  resident  for  a  time  in  an  anemone 
is  somewhat  different  from  that  of  a  newly  acclimated  animal.  The  general  level 
of  activity  becomes  lower  though  such  a  fish  normally  moves  much  more  rapidly 
than  an  unacclimated  fish.  After  acclimation  of  the  fish  is  complete  the  anemone 
tends  to  become  a  strongly  defended  territory.  Acclimated  fishes  often  refuse  to 
leave  the  anemone's  folds  even  if  it  is  lifted  from  the  water. 

EXPERIMENTS  ON   PROTECTION  AGAI-NST  THE  HOST 

In  these  experiments  we  wished  to  determine  initially  whether  the  presence  of 
the  fish  close  to  but  not  in  contact  with  the  surface  of  the  anemone  had  any  observable 
effect  on  the  anemone. 

Experiment  No.  1 .  A  V-j-inch  I.D.  plastic  tube  was  cleaned  with  alcohol-ether. 
A  small  A.  percula  was  slipped  into  the  tube  and  shaken  down  it  until  it  protruded 
slightly  from  the  end.  When  the  fish  was  held  as  close  as  %  mm.  from  the 
tentacles,  they  showed  no  reaction  whatever.  A  similar  test  with  a  control  Fundulns 
parvipinnis  gave  identical  results.  Contact  of  a  single  tentacle  with  the  Fundulus 
resulted  in  immediate  massive  discharge  and  clinging. 


402  DEMOREST  DAVENPORT  AND  KENNETH  S.  NORRIS 

No  interaction  at  a  distance  between  the  partners  or  between  prey  and  anemone 
could  be  observed. 

Next,  in  the  hope  that  we  might  be  able  to  identify  and  localize  the  mechanism 
of  protection,  we  designed  the  following  experiments  in  which  direct  stimulation 
of  the  anemone  was  employed. 

Experiment  No.  2.  As  a  control,  we  investigated  the  reaction  of  the  anemone  to 
stimulation  with  a  clean,  flame-polished  glass  rod.  In  a  number  of  repeated  tests 
we  saw  that  such  stimulation  caused  "clumping"  of  the  tentacles,  marked  adherence 
to  the  rod  (discharge  of  nematocysts),  retraction  of  the  tentacles,  and  retraction  of 
the  lobe  of  the  disk  in  the  vicinity  of  the  point  of  stimulation.  Far  greater 
mechanical  stimulation  and  agitation  of  tentacles  and  disk  by  Amphiprion  produce 
no  noticeable  response  from  the  anemone. 

Experiment  No.  3.  We  trapped  an  Amphiprion  in  a  beaker  and  held  it  by  the 
lower  jaw  in  the  tips  of  a  pair  of  fine-tipped  forceps.  Twice  we  drew  it  forcibly 
across  the  disk  of  the  anemone,  bringing  it  into  violent  contact  with  the  tentacles. 
There  was  no  discernible  reaction  from  the  anemone.  The  fish  when  released 
immediately  entered  the  tentacles  in  a  normal  manner  and  "bathed"  itself  among 
them. 

An  adult  Fnnditlits  parvipinnis  was  brought  into  contact  with  the  anemone  and 
was  seized  in  the  characteristic  way,  involving  widespread  adhesion,  tentacle  con- 
traction, and  infolding  of  the  disk. 

An  Amphiprion  percnla,  trapped  in  a  beaker  and  held  with  forceps  by  the  jaw. 
was  brought  into  contact  with  a  large  specimen  of  the  eastern  Pacific  anemone, 
Anthopleura  xanthogrammica.  There  was  immediate  widespread  clinging  so  that 
the  fish  had  to  be  pulled  forcibly  from  the  anemone. 

Experiment  No.  4.  An  Amphiprion  was  sacrificed,  and  we  cut  a  cross-sectional 
piece  of  flesh,  including  skin,  from  it  with  a  carefully  cleaned  scalpel.  We  made  a 
similar  preparation  from  Fundulus.  The  two  preparations  were  placed  next  to  each 
other  on  the  disk  of  the  anemone.  The  flesh  from  Amphiprion  was  slowly  worked 
to  the  edge  of  the  disk  and  cast  off,  while  the  Fundulus  meat  was  enveloped  and 
ingested.  The  experiment  was  later  repeated  with  similar  results. 

Experiment  No.  5.  We  caught  an  Amphiprion,  placed  it  in  a  clean  Petri  dish 
and  killed  it  by  severing  the  head.  We  then  dissected  off  a  strip  of  skin,  taking  the 
greatest  care  to  prevent  contact  of  both  surfaces  of  the  strip  with  other  skin  surfaces. 
We  brought  this  piece  of  freshly-removed  skin  into  contact,  on  its  outer  surface, 
with  several  tentacles  of  the  anemone.  No  clinging  occurred  except  for  slight 
adherence  at  the  edge  of  the  piece  of  skin.  When  the  skin  was  brought  into  contact 
on  its  inner  surface,  the  tentacles  immediately  clung  strongly  to  it. 

This  experiment  was  repeated  twice  with  identical  results.  Strips  of  skin  from 
the  same  fish  were  used. 

Experiment  No.  6.  We  heated  the  two  pieces  of  skin  used  in  the  preceding 
experiment  to  90°  C.  for  ten  minutes  in  sea  water  in  separate  clean  test  tubes.  The 
preparations  were  cooled.  When  we  brought  the  outside  surface  of  these  heat- 
treated  pieces  into  contact  with  tentacles,  clinging  immediately  occurred. 

Experiment  No.  7 .  A  V^-cm.  cube  of  muscle  without  skin  was  cut  from  the 
caudal  peduncle  of  the  Amphiprion  percnla,  taking  great  care  not  to  bring  it  in 
contact  with  skin  surface.  It  was  placed  on  the  disk  of  the  Stoichactis.  and  was 


AMPHIPRION  AND  STOICHACTIS  403 

immediately  seized.  The  tentacles  clumped  around  the  piece  and  infolding  of  the 
disk  margin  occurred.  The  anemone's  response  differed  in  no  discernible  way 
from  its  response  to  Fundulus  meat. 

Experiment  No.  8.     Four  cubes  were  cut  from  a  commercial  plastic  sponge. 

In  the  following  tests,  the  "clinging  reaction"  of  a  small  group  of  tentacles  was 
tested.  When  the  plastic  cube  was  brought  in  contact  with  the  tentacles,  the 
reaction  was  classified  arbitrarily  from  0  (no  clinging)  to  +  +  +  +  (very  strong 
adhesion).  In  each  test  a  different  group  of  tentacles  was  selected.  The  time 
required  for  release  of  the  cube  was  noted. 

a.  A   clean   control   cube:   tentacles   retracted;   clinging   0-+ ;   time   of   release 
<  1  second.     This  control  was  repeated  several  times  with  identical  results. 

b.  A  similar  cube  of  which  all  surfaces  had  been  rubbed  over  the  skin  of  Am- 
phiprion percula:  results  identical  with  the  control.     This  test  was  repeated 
several  times  with  similar  results. 

c.  A  cube  rubbed  over  the  skin  of  Ampkiprion  frcnatits:  clinging  ++  ;  retrac- 
tion of  tentacles,  release  time  20  seconds. 

d.  A  cube  rubbed  over  the  skin  of  an  adult  Garibaldi,  Hypsypops  nibicunda 
(an  eastern  Pacific  pomacentrid  fish)  :  clinging   +  +  +  +  ,   released  after  2 
minutes  45  seconds. 

e.  A  cube  rubbed  over  the  skin  of  Funditlns  parvipinnis:  clinging  +  +  +  +  ,  re- 
leased after  3  minutes  45  seconds. 

Experiment  No.  9.  Four  new  cubes  were  cut.  Two  of  these  were  rubbed 
over  Amphiprion  percula.  One  clean  cube  and  one  mucus-covered  cube  were 
heated  to  100°  C.  for  ten  minutes  in  a  dry  oven  and  cooled. 

a.  The  clean  control  cube:  tentacles  retracted;  clinging  0-  +  ,  time  of  release 

;  1   second.     Heavy  pressure  caused  sufficient  clinging  to   hold   the  cube 
for  as  long  as  4  seconds. 

b.  A  clean  cube,  heat  treated:  results  identical  with  control. 

c.  A   mucus-covered,   unheatecl   cube :   tentacular   retraction ;   clinging   0,   even 
under  strong  pressure ;  time  of  release,  immediate. 

d.  A  mucus-covered,  heat-treated  cube:  identical  with  control  (a). 
Identical  results  were  obtained  in  a  second  series  of  tests.     In  this  experiment 

we  see  that  stronger  mechanical  stimulation  than  was  used  in  Experiment  8  in- 
duced clinging  of  brief  duration  in  a  control  sponge.  If  there  was  a  coating  of 
Amphiprion  percula  mucus  on  the  sponge,  clinging  could  not  be  induced  even 
with  strong  pressure.  But  if  the  coating  of  Amphiprion  mucus  was  heat-treated, 
its  protective  effect  was  obliterated. 

The  effect  of  heat  was  also  shown  in  Experiment  6. 

Experiment  No.  10.  On  May  14,  1958,  while  the  anemone  was  located  in  a 
display  tank,  two  large  groups  of  eggs  were  found  attached  to  the  rock  occupied 
by  the  anemone.  One  patch  was  being  guarded  by  an  adult  goby  Bathygobius 
soporator  ( Cuvier  and  Valenciennes)  and  the  other  patch,  which  was  attached  in 
a  crevice  directly  beneath  the  anemone,  was  guarded  by  two  adult  Amphiprion 
percula,  which  had  been  allowed  to  become  resident  in  the  anemone.  The  eggs  of 
both  species  were  tested  for  protection  against  the  nematocysts  of  the  anemone. 

When  the  intact  egg  of  the  goby  was  touched  against  a  tentacle,  clinging 
occurred,  and  the  tentacle  bent  into  a  clump  with  four  or  five  other  tentacles.  No 


404  DEMOREST  DAVENPORT  AND  KENNETH  S.  NORRIS 

movements  of  the  disk  were  noted.  When  eggs  were  released  in  the  water  over 
the  anemone  and  allowed  to  drift  onto  the  tentacles  the  same  effects  were  produced. 
When  these  tests  were  repeated  using  Amphiprion  eggs,  the  following  results 
were  obtained.  Even  when  an  egg  was  pressed  against  a  tentacle  with  sufficient 
pressure  to  bend  the  tentacle  no  clinging  resulted.  Eggs  dropped  onto  the  disk- 
through  the  water  caused  no  reaction.  Quite  evidently  Amphiprion  eggs  are  as 
effectively  protected  as  the  adult. 

EXPERIMENTS  ON  THE  DISCHARGE  OF  NEMATOCYSTS 
FROM  ISOLATED  TENTACLES 

Our  next  experiments  were  designed  to  determine  whether  Amphiprion  mucus 
raised  the  threshold  of  nematocyst  discharge.  Isolated  tentacles  were  stimulated 
mechanically  or  electrically  while  being  observed  through  a  dissecting  microscope. 

Mechanical  stimulation.  Pantin  (1942)  showed  that  direct  mechanical  stimula- 
tion of  the  isolated  tentacle  of  Anemonia  sulcata  with  a  clean  glass  bead  failed  to 
cause  discharge.  Experiment  No.  2  showed  that  stimulation  of  the  /;/  situ  tentacle 
tip  of  Stoichactis  with  a  smooth  flame-polished  glass  rod  results  in  clinging.  Isolated 
tentacles  of  Stoichactis  appear  to  be  more  sensitive  to  mechanical  discharge  than 
those  of  Anemonia.  Even  when  the  greatest  care  was  taken  in  transferring  a 
tentacle  to  a  clean  watch  glass  in  clean  sea  water  for  isolation  and  stimulation,  its  tip 
very  frequently  stuck  to  the  bottom  of  the  glass  for  a  few  seconds. 

In  our  preparations  mechanical  stimulation  was  effected  by  fine  glass  rods  or 
by  using  the  tip  of  the  capillary  tube  of  the  glass  electrode.  Variation  in  the 
sensitivity  of  tentacles,  the  ease  with  which  nematocysts  could  be  mechanically  dis- 
charged and  our  inability  to  deliver  mechanical  stimuli  of  precisely  controlled 
intensity  made  it  difficult  to  obtain  a  truly  quantitative  picture  of  threshold  changes 
and  intensity  of  discharge. 

Our  observations  on  the  results  of  mechanical  stimulation  by  the  capillary  tube 
of  the  electrode  may  lie  summarized  as  follows  (discharge  classified  arbitrarily 
from  0  to  +  +  +  +): 

a.  Stimulation    witli    the   clean    capillar\    tube:     An    initial    stimulation    flight 
touching)  at  the  tip  of  the  tentacle  typically  produced  a  moderate  discharge  (  +  +  ). 
Similar  stimulation  halfway  between  the  tip  of  the  tentacle  and  its  cut  base  results 
in  a  lighter  discharge  ( + ) .     Repeated  mechanical  stimulation  at  both  points  results 
in  progressively  less  discharge.     Reduction  of  the  discharge  is  not  due  to  exhaustion 
of  the  nematocyst  supply,   as  subsequent  electrical   stimulation   produces   massive 
discharge  at  the  same  points. 

b.  Stimulation  with  the  tip  of  the  capillary  tube  covered  zvith  a  pad  of  Fundulus 
mucus  (control}  :     Initial  light  mechanical  stimulation  at  the  tip  produced  massive 
discharge  (  +  +  +  +  )  and  clinging  to  the  mucus  pad. 

c.  Stimulation   with    (lie   tip   of   the   capillary    tube   covered   with    mucus  from 
Amphiprion   percula:     Light   stimulation  of  both  tip  and   middle   of  the  tentacle 
produced  no  discharge  (0).     If  the  tentacle  was  held  in  place  by  a  clean  glass  rod 
and  stimulated  at  another  point  by  the  mucus-covered  capillary  tube  so  forcibly  as 
to  deform  the  tentacle,  the  discharge  of  a  few  isolated  nematocysts  occurred  but 
no  clinging  resulted.     The  effect  of  the  pad  of  Amphiprion  mucus  appeared  to  be 


AMPHIPRION  AND  STOICHACTIS  405 

limited  to  the  area  in  contact  with  the  mucus,  for  if  an  uncovered  portion  of  the  glass 
tube  came  in  contact  with  the  tentacle,  discharge  would  occur  at  this  point  hut  not 
at  points  protected  by  the  mucus  pad. 

d.  Stimulation   with    the   capillary   tube   covered  u'illi    a    pad   of   mucus  from 
Amphiprion  frenatus:     Light  touch  at  the  tentacle  tip— »+  +  .     A  touch  at  the 
side  of  the  tentacle  —»  0.     This  test  did  not  appear  to  be  significantly  different  in 
results  from  control  (a). 

e.  Stimulation  with   the  capillary  tube  covered  tvith   mucus  from    the   base   of 
Stoichactis:  Not  significantly  different  from  control  (a). 

Electrical  stimulation.  Stimulation  by  faradic  current  was  produced  according 
to  the  standard  method  described  above.  The  single  excised  tentacle,  in  clean  sea 
water,  was  first  tested  for  mechanically-induced  discharge  by  light  contact  with  the 
electrode  at  a  point  halfway  between  its  tip  and  base.  The  tentacle  was  then  given 
a  series  of  three-second  bursts  of  faradic  stimulation  at  the  same  point,  starting  with 
the  inductorium  at  its  lowest  setting  (12). 

Table  I  shows  the  threshold  and  intensity  of  discharge  under  different  conditions 
in  a  series  of  tentacle  preparations.  Intensity  of  discharge  was  arbitrarily  classified 
from  0  to  +  +  +  +  .  The  sensitivity  of  the  control  series  in  which  mucus  was 
absent  varied  widely.  It  appears  that  this  variation  in  sensitivity  reflects  variation 
in  the  threshold  of  the  different  preparations,  since  after  the  initial  mechanically- 
induced  discharge,  stimulation  by  pressing  the  electrode  against  the  side  of  the 
tentacle  elicited  no  further  discharge  in  the  four  preparations.  When  a  pad  of 
Fundulus  mucus  was  placed  over  the  tip  of  the  electrode,  the  very  lightest  mechanical 
contact  of  the  electrode  tip  with  the  side  of  the  tentacle  elicited  some  discharge  ( + ) . 
Hence,  if  the  threshold  of  electrically-induced  discharge  had  been  lowered  by  the 
mucus,  it  could  not  be  discerned.  Intensity  of  discharge  at  high  levels  of  electrical 
stimulation  did  not  appear  to  differ  from  the  controls.  When  pads  of  mucus  from 
Amphiprion  percula,  A.  frenatus  and  the  anemone  itself  were  used,  results  did  not 
differ  significantly  from  the  controls. 

It  is  quite  clear  that  the  presence  of  mucus  from  the  partner  fish  did  not  raise 
the  threshold  of  electrically-induced  discharge  of  nematocysts.  It  is  also  interesting 
to  note  that  maximum  discharge  in  all  cases  was  elicited  within  a  narrow  range  of 
inductorium  setting  (4-5). 

DISCUSSION 

The  above  investigations  were  principally  directed  toward  understanding  the 
physiological  and  behavioral  mechanisms  which  maintain  the  animals  in  partnership 
and  which  protect  the  fish  from  the  nematocysts  of  its  host. 

The  reactions  of  unacclimated  fish  to  the  anemone  were  described  in  detail. 
These  reactions  differed  considerably  within  our  sample,  but  it  must  be  remembered 
that  the  history  of  our  nine  fish  was  unknown  to  us.  Some  may  have  been  free- 
living  and  some  commensal  with  other  species  of  anemone.  However,  the  entire 
sample  ultimately  became  acclimated  to  the  Stoichactis.  There  is  unquestionably 
a  bond  which  attracts  A.  percula  to  this  anemone  and  keeps  the  fish  in  it,  once  the 
acclimation  process  is  completed.  This  process  was  first  observed  by  Gohar  (1948) 
who  says  (p.  39)  :  "Fish  of  the  commensal  species  may  develop  partnership  with 
such  anemones  as  Discosomum  giganteum  by  cautiously  approaching  it.  The 


406 


DEMOREST  DAVENPORT  AND  KENNETH  S.  NORRIS 


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AMPHIPRION  AND  STOICHACTIS  407 

association  is  completed  in  one  to  a  few  days."  Once  the  association  is  completed 
the  bond  is  stonger ;  acclimated  fish  rarely  wander  away  from  the  anemones,  while 
unacclimated  ones  may  wander  all  over  the  aquarium.  In  the  course  of  the 
acclimation  process  we  observed  the  "cautiousness"  noted  by  Gohar.  A  fish 
touches  the  tentacles,  often  sticks  at  first  and  flees.  But  it  keeps  coming  back, 
making  more  and  more  contact  until  no  tentacular  clinging  occurs.  We  have  not 
as  yet  identified  those  signs  which  attract  the  fish,  beyond  confirming  Verwey's 
observation  (1930)  that  the  fish  respond  to  visual  cues.  Since  our  anemone  was 
kept  in  still  water,  part  of  the  visual  cue  to  an  unacclimated  fish  may  have  been 
absent,  since  in  nature  the  Stoichactis  must  be  in  almost  constant  motion  in  its 
shallow  water  habitat.  We  have  not  as  yet  investigated  the  possibility  that  specific 
chemical  releasers  from  the  anemone  may  be  an  important  part  of  the  bond.  Tactile 
stimuli  may  also  be  important,  for  the  fish  appears  to  "seek"  contact  with  the 
anemone  during  the  acclimation  process.  The  process  of  acclimation  may  be 
recognized  by  the  action  of  the  fish  of  bringing  more  and  more  of  its  body  in 
contact  with  the  host.  The  strength  and  effectiveness  of  stimuli  from  the  anemone 
certainly  affect  the  rate  of  attainment  of  the  ultimate  equilibrium  between  the 
partners,  which  is  the  consummation  of  the  acclimation  process. 

The  behavior  of  the  anemone  in  relation  to  the  fish  was  also  carefully  observed. 
Some  writers  have  claimed  that  the  commensals,  even  without  contact,  affect  the 
behavior  of  the  tentacles.  Crespigny  (1869)  said  (p.  10)  :"  .  .a  Premnas  now 
passes  over  the  anemone  and  immediately  the  tentacula  become  erect  and  diverge, 
while  their  extremities  become  clubby.  .  .  ."  Herre  (1936),  working  with  the 
symbionts  used  in  our  investigation,  says  (p.  167)  :  "But  when  an  Amphiprion 
darted  in  among  the  beautiful  but  dangerous  tentacles,  they  curled  away  from  the 
intrepid  invader."  We  have  never  observed  any  such  action  at  a  distance,  in  spite 
of  efforts  to  elicit  some  response  by  bringing  an  acclimated  Amphiprion,  held  by  its 
lower  jaw  or  immobilized  in  a  plastic  tube,  within  a  fraction  of  a  millimeter  of  the 
host.  In  the  former  test,  water  currents  from  the  fish's  pectoral  fins  gently  waved 
the  anemone's  tentacles,  but  no  such  response  appeared  as  that  described  above. 
Even  when  an  acclimated  Amphiprion  was  dragged  across  the  disk,  no  response 
occurred  which  was  not  attributable  to  mechanical  disturbance ;  we  suspect  that  the 
observations  of  Crespigny  and  Herre  were  merely  the  result  of  water  currents. 

Gohar  implies  that  the  fish  in  some  way  affect  the  nerve  net  of  the  anemone 
when  he  speaks  of  the  activity  of  the  fishes  appearing  (p.  38)  "as  if  they  were 
.  .  .  sympathetically  caressing"  closed  anemones  so  that  they  opened.  This  observa- 
tion was  also  made  by  Verwey  (1930).  Gudger  feels  that  such  behavior  involves 
a  certain  "gentle  massage."  This  activity  may  be  effective  in  bringing  the  anemone 
back  to  its  expanded  state,  and  if  so,  then  the  fish  is  affecting  the  neuromotor 
apparatus  of  the  host.  But  the  purposive  implication  is  unwarranted,  since  the 
behavior  of  the  fish  is  probably  not  very  different  from  that  when  the  anemone  is 
already  expanded  and  may  be  under  the  control  of  the  same  stimuli  as  those  eliciting 
typical  "acclimating  activity." 

We  have  produced  other  evidence  that  the  presence  of  the  fish  may  affect  the 
neuromotor  apparatus  of  the  host,  for  contact  by  an  unacclimated  fish  may  result 
in  localized  retraction  of  tentacles,  and,  if  stimulation  is  particularly  strong,  a  slight 
infolding  of  the  disk  may  occur.  This  reaction  is  similar  to  that  elicited  by  contact 


408        DEMOREST  DAVENPORT  AND  KENNETH  S.  NORRIS 

with  prey,  but  is  not  as  intense,  involving  briefer  tentacular  retraction  and  a  weaker, 
more  localized  infolding  of  the  disk.  The  frequently  violent  activity  of  the 
acclimated  fish  has  no  apparent  effect  on  the  anemone  whatever.  It  is  as  though 
a  physiological  barrier  had  been  set  up  during  the  acclimation  process.  We  believe 
that  the  weak  response  of  the  anemone  to  an  unacclimated  fish  indicates  that  the  fish 
has  not  yet  reached  a  state  in  which  it  fails  to  stimulate  the  host  and  that  low 
intensity  stimuli  of  the  same  nature  as  those  received  from  prey  are  "getting 
through."  It  seems  apparent  that  in  the  acclimation  process,  repeated  contact  with 
the  anemone  is  necessary  for  the  establishment  of  both  the  physiological  protective 
barrier  and  the  "bond  of  association"  between  the  animals.  We  do  not  know  the 
exact  nature  of  the  barrier.  If  one  places  a  skinless  piece  of  Amphiprion  meat  on 
the  disk  it  is  consumed,  while  a  piece  with  skin  attached  is  rejected  and  ultimately 
falls  off  the  disk.  Furthermore,  if  a  piece  of  Amphiprion  meat  with  skin  and  a  piece 
of  Fundulus  meat  with  skin  are  placed  side  by  side  on  the  disk  the  former  is  rejected 
and  the  latter  consumed.  It  appears  as  though  the  anemone  "discriminated" 
between  them.  From  this  we  are  forced  to  conclude  that  a  factor  is  present  in  the 
skin  which  affects  the  stimulus-response  chain  in  the  anemone.  But  the  factor  may 
do  this  indirectly  by  preventing  nematocyst  discharge,  if,  for  instance,  the  normal 
feeding  reactions  depend  upon  the  reception  of  information  from  receptors  in  the 
tentacles  which  are  sensitive  to  bursts  of  nematocyst  discharge,  or  to  substances 
released  from  prey  that  has  been  "stung." 

We  are  persuaded  that  the  protection  of  the  fish  against  its  host's  nematocysts 
does  not  involve  a  simple  inability  on  the  part  of  the  fish  to  give  adequate  stimuli 
for  discharge.  We  have  shown  that  (1)  the  strongest  stimulation  of  an  isolated 
tentacle  by  a  glass  rod  covered  with  Amphiprion  mucus  results  in  little  or  no 
discharge  whereas  like  stimulation  by  a  clean  rod  causes  a  burst  of  nematocysts ; 
(2)  when  an  Amphiprion  is  dragged  across  the  disk  of  the  host  no  discharge  or 
adherence  occurs;  (3)  the  inner  surface  of  a  piece  of  Amphiprion  skin  sticks 
immediately  to  the  tentacles,  while  the  outer  surface  does  not;  (4)  heat-treatment 
of  the  skin  abolishes  the  protection;  (5)  a  sponge  covered  with  Amphiprion  mucus 
will  not  stick  to  the  tentacles,  even  when  firmly  pressed  against  them,  while  a  clean 
sponge  will;  (6)  heat-treatment  of  a  mucus-covered  sponge  destroys  the  protection; 
(7)  Amphiprion  is  immediately  seized  by  another  anemone  (Anthopleura).  All 
these  observations  argue  for  the  existence  of  a  heat-labile  factor  present  on  the 
outer  surface  of  the  skin  of  Amphiprion,  which  raises  the  threshold  of  discharge  of 
nematocysts  in  the  host  Stoichactis. 

What  is  the  function  of  the  behavioral  process  we  call  acclimation  ?  We  believe 
that  this  process,  which  other  workers  have  suggested  serves  to  change  the  condition 
of  the  anemone,  is  more  probably  a  mechanism  which  changes  the  condition  of  the 
fish  as  the  result  of  repeated  contact  between  it  and  the  anemone.  It  remains  to 
be  determined  whether  the  fish  has  immunity  to  the  nematocyst  contents  and 
whether  acclimation  has  any  relation  to  the  maintenance  of  this  immunity.  There 
is  a  possibility,  though  we  cannot  offer  conclusive  proof,  that  acclimation  may  be 
related  to  changes  in  the  mucus  coat  of  the  fish.  Frequently,  prior  to  complete 
acclimation,  the  protection  of  a  fish  is  not  perfect.  Perhaps  increasing  contact  with 
the  anemone  induces  a  greater  general  secretion  of  mucus  or,  specifically,  more  of 
the  active  principle  in  the  mucus.  Rough  handling  of  the  fish  renders  it  susceptible 


AMPHIPRION  AND  STOICHACTIS  409 

to  localized  stinging,  which  may  result  from  "breaks"in  the  protective  mucus  coat. 
During  the  early  stages  of  acclimation  the  fin  tips  are  the  sites  of  nearly  all  localized 
clinging.  These  edges  are  precisely  the  areas  which  are  first  brought  carefully  into 
contact  with  the  anemone.  Similar  clinging  occurs  at  the  site  of  a  wound  in  a 
damaged  fish,  but  after  a  short  time,  if  the  wound  is  not  great,  clinging  no  longer 
occurs.  This  may  indicate  the  spread  of  mucus  over  the  wound,  renewing  the 
integrity  of  the  protective  coat.  It  would  seem  that  the  characteristic  fluttering 
movements  of  Amphiprion  when  on  the  disk  of  the  anemone  would  be  particularly 
effective  in  spreading  mucus  over  the  various  sharp  fin  edges. 

Clearly  there  remain  many  unsolved  problems.  It  appears  that  the  protective 
principle  in  the  mucus  coat  takes  its  effect  locally,  is  fast-acting  and  specific.  It 
would  be  of  interest  to  determine  its  rate  of  decay  and  to  find  out  whether,  after 
cessation  of  contact  between  a  tentacle  and  Amphiprion  mucus,  there  is  some  effect, 
however  brief,  on  the  threshold  of  nematocyst  discharge.  One  would  wish  to  know 
a  good  deal  more  about  the  chemical  nature  of  the  principle  and  whether  it  is  present 
in  other  fishes  such  as  Nomeus,  the  commensal  of  the  Portuguese  Man-of-War, 
Physalia. 

SUMMARY 

1.  The  behavioral  process  is  described  whereby  the  fish  Amphiprion  percula, 
after  long  isolation  from  the  anemone  Stoichactis,  effects  its  association  with  the 
host. 

2.  This  process  appears  to  involve  a  gradual  acclimation  to  the  host,  brought 
about  by  increasing  contact  with  the  host's  tentacles.     This  appears  to  effect  the 
establishment  of  both  the  "bond"  and  the  physiological  protective  barrier  between 
the  animals. 

3.  Evidence  is  presented  that  an  active  principle  is  present  in  the  mucus  secreted 
on  the  outer  surface  of  the  integument  of  Amphiprion  which  raises  the  threshold  of 
mechanically-induced  discharge  of  the  host's  nematocysts.     This  factor  does  not 
affect  the  threshold  of  electrically-induced  discharge.     It  is  fast-acting,  specific  in 
its  effect  and  heat-labile.     It  is  not  present  in  the  muscle  of  the  fish. 

4.  After  contact  between  the  host  and  an  acclimated   commensal  no  feeding 
reactions  can  be  observed  in  the  anemone  such  as  occur  when  similar  contact  is 
made  between  Stoichactis  and  prey  fish  or  between  other  anemones  and  Amphiprion. 
It  is  possible  that  this  "inhibition"  of  the  anemone  may  be  the  result  of  a  direct  effect 
on  the  nervous  system  by  the  active  principle.     However,   it   would   seem   more 
probable  that  this  absence  of  feeding  reactions  even  on  violent  contact  may  depend 
upon  the  fact  that  nematocysts  are  not  discharged.     Perhaps  stimuli  from  receptors 
in  the  tentacles  sensitive  to  nematocyst  discharge  or  to  substances  from  "stung" 
prey  are  necessary  for  the  initiation  of  feeding  reactions. 

LITERATURE  CITED 

BAERENDS,   G.   P.,   1950.     Specializations   in  organs   and  movements   with   a   releasing   function. 

Symposium  Soc.  E.vp.  Bio!.,  4:  337-360. 
BAERENDS,   G.    P.,    1957.     The   ethological   analysis   of   fish   behavior.     /;;:    The    Physiology   of 

Fishes,  M.  E.   Brown,   Edit.     New  York,   Academic   Press,   Inc. 
DE  CRESPIGNY,  C.  C,  1869.     Notes  on  the  friendship  existing  between  the  Malacopterygian  fish 

Premnas  biacitlcatus  and  the  Actinia  crassiconiis.     Proc.  Zool.  Soc.  London:  248-249. 


410        DEMOREST  DAVENPORT  AND  KENNETH  S.  NORRIS 

GOHAR,  H.  W.  F.,  1948.     Commensalism  between  fish  and  anemone.     Publ.  Mar.  Sta.  Ghardaqa, 

6:  35-44. 
GUDGER,  E.  W.,   1947.     Pomacentrid  fishes  symbiotic  with  giant  sea  anemones   in   Indo-pacific 

waters.     /.  Asiat.  Soc.  Beng.,  12:  53-76. 
HERRE,  A.  W.  C.  T.,  1936.     Some  habits  of  Amphiprion  in  relation  to  sea  anemones.     Copeia, 

3:  167-168. 

PANTIN,  C.  F.  A.,  1942.     The  excitation  of  nematocysts.     /.  £.r/>.  Bio!..  19:  294-310. 
SLUITER,  C.  P.,  1888.     Ein  mergwiirdiger  Fall  von  Mutualismus.     Zool.  Ans.,  11:  240-243. 
VERWEY,  J.,  1930.     Coral  reef  studies,  I.     The  symbiosis  between  damselfishes  and  sea  anemones 

in  Batavia  Bay.     Trcubia,  12  :  305-366. 


A  COMPARISON  OF  THE  EFFECTS  OF  GOITROGENS  ON 

THYROID  ACTIVITY  IN  TRITURUS  VIRIDESCENS 

AND  DESMOGNATHUS  FUSCUS  1 

JAMES  NORMAN  DENT  -  AND  W.  GARDNER  LYNN  3 

Biology  Division,   Oak  Ridge  National  Laboratory,*   Oak   Ridge,   Tennessee,   Department   of 

Biology,  University  of  Virginia,  Charlottesville,  Virginia,  and  Department  of  Biology, 

Catholic    University   of  America,    Washington,   D.    C. 

Salamanders  of  the  genus  Triturus  have  been  used  widely  in  studies  of  the 
histology,  cytology,  seasonal  variation,  and  responses  of  the  endocrine  glands. 
Relatively  few  investigations  of  these  matters  have  been  made  with  members  of 
other  urodele  genera.  It  would  be  interesting  to  know  to  what  extent  findings 
based  on  study  of  ah  exclusively  aquatic  newt  are  applicable  to  urodeles  that  are 
terrestrial  in  habit.  There  is  some  indication  that  the  thyroid  of  Triturus  may  differ 
significantly  in  at  least  one  respect  from  that  of  the  terrestrial  salamander, 
Desmognathus,  namely,  in  the  histological  changes  elicited  by  treatment  with  the 
goitrogenic  drug,  thiourea.  Adams  (1946)  found  that  a  high  dosage  and  a  long 
period  of  treatment  are  required  to  bring  about  hypertrophy  and  hyperplasia  of 
the  thyroid  gland  of  Triturus  mridescens  whereas  Fisher  (1953)  and  Wheeler 
(1953)  reported  that  the  thyroid  of  Desrnognathus  fuscus  responds  much  more 
readily  and  typically  to  this  goitrogen.  Since,  however,  the  doses,  environmental 
conditions,  and  length  of  treatment  differed  considerably  in  these  studies,  it  is  not 
possible  to  conclude  with  any  assurance  that  Triturus  is  refractory  to  the  effects 
of  thiourea. 

In  our  experiments  a  comparison  is  made  of  the  responses  of  these  two  sala- 
manders to  treatment  with  two  different  goitrogens,  thiourea  and  potassium  per- 
chlorate.  The  effects  of  these  goitrogens  on  radioiodine  uptake  by  the  thyroid 
and  on  the  histology  of  the  gland  were  the  basis  for  the  comparison. 

MATERIALS  AND  METHODS 

Specimens  of  Triturus  (Dicinyctylus)  viridcscens  viridcscens  (Rafinesque) 
were  collected  on  October  20,  1955,  from  a  pond  near  Monterey,  Virginia. 
Specimens  of  Desmognathus  fuscus  fuscus  (Rafinesque)  were  taken  from  beneath 
stones  at  the  edge  of  a  small  stream  on  a  thickly  wooded  hillside  near  Oliver 
Springs,  Tennessee,  over  the  period  June  23-July  23,  1956.  All  animals  were 
maintained  in  the  laboratory  for  a  minimum  of  two  weeks  before  being  subjected 
to  experimental  treatment.  Both  before  and  during  treatment  they  were  fed  every 
other  day,  the  Triturus  with  ground  lean  beef  fortified  with  cod  liver  oil  and  calcium 
phosphate,  and  the  Desmognathus  with  live  meal-worm  larvae.  Throughout  the 

1  Supported  in  part  by  AEC  Contract   No.   AT- (40-1) -2000. 

2  University  of  Virginia. 

3  The  Catholic  University  of  America. 

4  Operated  by  Union  Carbide  Corporation  for  the  U.  S.  Atomic  Energy  Commission. 

411 


412  JAMES  NORMAN    DENT  AND  W.  GARDNER  LYNN 

experimental  period  the  specimens  were  kept  in  a  constant-temperature  room  at 
19.0°  ±  1.0°  C. 

Goitrogens  were  injected  into  the  body  cavity  on  alternate  days.  Some  animals 
were  given  injections  of  0.1  ml.  of  a  l.O'/o  aqueous  solution  of  thiourea ;  others 
received  injections  of  0.1  ml.  of  a  0.2%  aqueous  solution  of  potassium  perchlorate. 
Controls  were  given  injections  of  0.1  ml.  of  distilled  water.  The  injections  were 
made  with  a  27-gauge  needle  introduced  into  the  body  cavity  through  the  muscles 
at  the  base  of  the  hind  leg.  We  chose  this  site  to  prevent  the  loss  of  fluid  that 
sometimes  occurs  when  injections  are  made  directly  through  the  abdominal  wall. 
Successive  injections  were  always  given  on  alternate  sides. 

The  uptake  and  turnover  of  radioiodine  in  the  thyroids  of  experimental  and 
control  animals  were  observed  by  the  following  method  :  The  animal  to  be  studied 
was  injected  intraperitoneally  with  0.1  ml.  of  10 //  Holtfreter's  solution  containing 
50.0  /Ac/ml,  of  I  131.  At  fixed  intervals  over  a  2^ -day  period  thereafter  the  animal 
was  anesthetized  in  an  aqueous  solution  of  tricaine  methane  sulfonate  ( 1  part  in 
1000)  and  the  radioactivity  of  the  thyroid  and  heart  regions  was  measured  by  a 
scintillation  counter  consisting  of  a  1.5-inch  Nal  crystal  cemented  to  the  window 
of  an  RCA  type-5819  photomultiplier  tube  and  a  conventional  amplifier  and  binary 
sealer.  The  crystal  and  photomultiplier  were  mounted  in  a  lead  cylinder  5.2  cm. 
thick  with  a  collimating  slit  measuring  4.0  by  12.0  mm.  The  ventrum  of  the 
anesthetized  animal  was  apposed  to  the  lead  cylinder  with  the  region  to  be  counted 
directly  over  the  slit. 

The  procedure  just  described  was  evolved  from  a  series  of  preliminary  experi- 
ments carried  out  to  ascertain  the  optimal  dosage  of  I  1:!1  for  reliable  counts,  the 
time  required  for  maximum  uptake  by  the  thyroid,  the  extent  of  individual  variation 
in  uptake  among  control  animals,  and  the  possible  effects  of  frequent  anesthetization 
on  the  uptake.  In  one  preliminary  experiment,  counts  were  made  at  eight  successive 
levels  on  the  anterior-posterior  axes  of  Ii:u-injected  animals  from  the  snout  to  the 
base  of  the  tail,  giving  a  profile  of  radioactivity.  Relatively  high  counts  were 
obtained  in  the  region  of  the  thyroid  and  in  the  abdomen.  The  heart  region  was 
selected  as  representative  of  the  tissues  in  general  (other  than  the  gut)  for  com- 
parison with  the  thyroid  region.  The  counting  on  these  two  regions  was  done  by 
centering  the  thyroid  or  the  heart,  as  located  with  relation  to  external  anatomical 
characters,  over  the  collimating  slit.  During  the  experiment  the  mean  background 
count  was  1.68  counts  per  second.  It  did  not  vary  significantly  over  the  60-hour 
counting  period. 

At  the  conclusion  of  the  measurements  of  radioactivity  the  animals  were  killed. 
The  lower  jaws,  containing  the  thyroid  glands,  were  fixed  in  a  solution  containing 
equal  parts  of  Bouin's  solution  and  ethylene  glycol  monethyl  ether.  Sectioned  thy- 
roids were  stained  with  Harris'  hematoxylin  and  Ponceau  de  zylidine-orange  IT 
(Gray,  1952)  for  histological  study. 

The  results  to  be  described  here  were  based  on  information  obtained  from  the 
study  of  72  salamanders  divided  into  two  series,  one  made  up  of  animals  that 
received  injections  for  30  days  and  one  of  animals  that  had  46  days  of  treatment. 
Each  series  consisted  of  six  groups  of  six  animals  each ;  one  group  of  control,  one  of 
thiourea-treated,  and  one  of  perchlorate-treated  specimens  for  each  of  the  two 
species. 


EFFECTS  OF  GOITROGENS  IN  SALAMANDERS  413 

RESULTS 

1.  Effects  of  thionrca  and  perchlorate  treatment  upon  the  histology  of  the  thyroid 

The  thyroid  glands  of  the  control  animals  of  the  30-  and  46-day  series  did  not 
differ  significantly  and  will  he  described  together.  The  thyroids  of  ten  Desmog- 
nathus appeared  to  have  moderate  secretory  activity.  The  follicles  were  relatively 
large,  the  colloid  was  homogeneous,  and  there  was  a  moderate  number  of  chronio- 
phobe  droplets.  The  epithelium  was  cuboidal  to  low  columnar.  There  was  some 
but  not  a  great  deal  of  individual  variation  among  the  ten  specimens.  The  extremes 
of  variation  in  epithelial  height,  follicle  size,  and  vacuolization  of  the  colloid  are 
shown  in  the  photomicrographs  (Figs.  1  and  2).  The  thyroids  of  ten  Tritnnts 
controls  presented  a  definite  contrast  with  those  just  described.  The  follicles  were 
larger,  the  epithelium  much  more  flattened,  and  chromophobe  droplets  were  either 
entirely  absent  or  quite  sparse.  There  was  less  individual  variation  in  this  group 
than  in  the  Desmognathus  controls  and  a  single  photomicrograph  will  suffice  to 
illustrate  the  entire  set  (Fig.  3). 

Examination  of  five  pairs  of  thyroids  of  Desmognathus  given  treatment  \vith 
thiourea  for  30  days  (15  injections)  revealed  a  definite  response  indicated  by  a 
marked  increase  in  height  of  the  follicular  epithelium,  a  folding  of  the  follicle  walls, 
a  reduction  in  the  amount  of  colloid  present,  and  an  increase  in  the  number  of 
chromophobe  droplets.  The  thyroids  of  animals  given  this  treatment  for  46  days  (23 
injections)  showed  little  difference  from  those  just  described  except  for  a  further 
reduction  in  the  amount  of  colloid.  A  typical  example  is  shown  in  Figure  4.  The 
thyroids  of  Tritnnts  given  thiourea  for  30  days  or  for  46  days  were  almost  precisely 
like  those  of  controls.  The  only  definite  change  is  an  indication  of  hyperemia  in 
the  glands,  the  sections  showing  enlarged  capillaries  and  many  more  blood  cor- 
puscles than  were  seen  in  controls  (Fig.  5). 

Potassium  perchlorate  treatment  produced  a  still  greater  effect  than  thiourea 
treatment  in  Desmognathus.  Even  after  30  days,  intrafollicular  colloid  was  almost 
entirely  lacking  and  the  increase  in  epithelial  height  was  quite  striking  (Fig.  6). 
Thyroids  of  animals  treated  wTith  perchlorate  for  46  days  showed  no  significant 
further  change,  perhaps  indicating  that  a  maximal  response  had  already  been 
elicited.  In  Trititrus,  perchlorate  was  no  more  effective  than  thiourea  in  causing 
histological  changes  in  the  thyroid.  In  fact,  there  was  not  even  an  indication  of 
hyperemia  in  the  glands  of  the  perchlorate-treated  specimens ;  the  glands  were 
indistinguishable  from  those  of  controls  (Fig.  7). 

2.  Effect  of  goitrogcns  on  uptake  and  release  of  7131 

Table  I  gives  the  compiled  results  of  measurements  of  radioactivity  in  thyroids 
of  the  animals  of  the  46-day  series  in  comparison  with  radioactivity  in  their  heart 
regions  during  a  60-hour  period  after  injection  with  I  131.  As  has  been  pointed 
out,  the  measurements  made  in  the  heart  region  are  presumed  to  be  representative 
of  the  non-thyroidal  tissues  in  general,  except  the  gut,  where  iodine  was  being 
concentrated  and  eliminated.  The  measurements  were  corrected  for  the  physical 
decay  of  I131  that  followed  the  injection,  and  an  analysis  of  variance  was  made  of 
the  means  of  the  corrected  counts. 


414 


JAAIES  NORMAN  DENT  AND  W.  GARDNER  LYNN 


^"L/  ;  "^kaJ**  •*  , 

%        %*^        **- 


'    - 


FIGURES  1-7. 


EFFECTS  OF  GOITROGENS  IN  SALAMANDERS  415 

The  least  significant  difference  beween  any  pair  of  means  in  Table  I  was  found 
to  be  2.63  at  the  5%  level.  The  interactions  among  these  individual  means  are 
apparently  of  considerable  physiological  significance  and  will  be  interpreted  in  the 
following  section  of  this  paper.  It  is  well  to  note  also  that  the  differences  between 
the  over-all  mean  counts  for  the  two  species,  for  the  two  types  of  treatment  and 
control  maneuvers,  for  the  two  locations  of  counting,  and  for  the  six  periods  of 
counting  were  all  highly  significant  in  the  statistical  sense,  p  being  less  than  0.1% 
in  each  instance. 

DISCUSSION 
7.  General  comment  and  histological  findings 

The  histology  of  the  thyroid  gland  of  Tri turns  viridescens  and  the  seasonal 
changes  it  undergoes  have  been  described  by  Morgan  and  Fales  (1942)  who  found 
that  the  thyroid  is  moderately  active  in  early  winter,  gradually  increases  in  activity 
during  mid- winter  and  spring  (up  to  the  breeding  season)  but  has  low  activity 
during  the  summer  months.  Our  Triturus  controls  had  thyroids  that  agreed 
closely  with  their  description  of  the  summer  thyroid  characterized  by  flattened 
epithelial  cells  and  abundant  homogeneous  colloid.  No  comparable  study  of 
seasonal  varation  is  available  for  Desmognathus.  Our  findings  showed,  however, 
that  there  was  a  definite  difference  in  the  histology  of  the  thyroid  in  these  two 
salamanders  at  the  same  season  and  under  the  same  temperature  conditions, 
Desmognathus  showing  histological  indications  of  a  much  higher  level  of  activity 
than  Triturus. 

The  experimental  use  of  thiourea  as  a  goitrogenic  agent  is  now  well  known. 
Although  the  precise  details  of  its  mode  of  action  are  still  not  completely  understood, 
it  is  generally  accepted  that  it  does  not  interfere  with  the  ability  of  the  thyroid  gland 
to  concentrate  iodide  but  does  inhibit  its  ability  to  utilize  iodide  for  hormone 
synthesis  (Pitt-Rivers,  1950;  Roche  and  Michel,  1955).  As  a  result  of  this 
inhibition,  the  level  of  thyroid  hormone  in  the  blood  of  animals  treated  with  thiourea 
falls,  causing  increased  production  of  thyroid-stimulating  hormone  (TSH)  by  the 
pituitary.  In  turn,  the  elevation  of  the  TSH  level  induces  hypertrophy,  hyper- 
plasia,  and  hyperemia  in  the  thyroid  and  the  release  of  its  intrafollicular  colloid. 
The  "goitrogenic"  effects  of  thiourea  thus  result  from  the  pituitary  stimulation 
rather  than  from  the  direct  action  of  the  drug  itself.  In  our  experiments,  the  30- 
or  46-day  treatments  of  Desmognathus  with  thiourea  brought  about  all  these 
structural  changes  in  the  thyroid.  Triturus,  similarly  treated,  showed  no  response 

FIGURES  1  AND  2.  Sections  of  thyroid  glands  from  control  specimens  of  Desmognathus 
killed  in  July.  Histological  evidence  of  moderate  secretory  activity  ranges  from  the  condition 
shown  in  Figure  1  to  that  shown  in  Figure  2. 

FIGURE  3.  Thyroids  of  Triturus  killed  in  July  gave  a  uniform  appearance  of  inactivity 
as  illustrated  in  this  section. 

FIGURE  4.  Section  of  thyroid  gland  from  Desmognathus  treated  with  thiourea.  Marked 
secretory  activity  is  indicated. 

FIGURE  5.     Thyroid  section  from  Triturus  treated  with  thiourea. 

FIGURES  6  AND  7.  Thyroid  sections  from  Desmognathus  (6)  and  Triturus  (7)  treated 
with  potassium  perchlorate.  Note  marked  response  shown  in  Desmognathus  and  lack  of 
response  in  Triturus. 


416  JAMES  NORMAN   DENT  AND  W.  GARDNER  LYNN 

aside  from  an  increase  in  the  vascularity  of  the  gland.  This  lack  of  response  in 
Triturus  agrees  with  the  findings  of  Adams  (1946)  who  immersed  specimens  of 
Triturus  viridescens  in  thiourea  solutions  that  were  increased  in  strength  over  a 
period  of  42  days  to  0.528%  and  then  allowed  the  animals  to  remain  in  this  con- 
centration for  44  days  longer.  Such  animals  showed  no  change  in  thyroid  histology 
as  compared  with  controls.  Adams  found  that  animals  treated  similarly  with 
solutions  of  twice  this  strength  did  show  an  increase  in  epithelial  height  and  a 
reduction  in  colloid,  indicating  that  a  sufficiently  high  dosage  may  produce  a 
response.  Since,  however,  only  two  of  the  ten  animals  so  treated  survived  to  the 
end  of  the  experiment,  it  seems  clear  that  this  concentration  is  quite  toxic  and  the 
changes  in  thyroid  structure  may  be  related  to  this  toxicity.  Fisher  ( 1953)  reported 
histological  changes  in  the  thyroid  of  Dcsmognathus  after  thiourea  administration 
that  are  in  complete  accord  with  our  findings.  His  animals  were  treated  by 
immersion  in  a  0.05%  solution  of  thiourea  and,  although  this  is  only  one-tenth  as 
strong  as  the  weakest  solution  used  by  Adams,  it  caused  hyperplasia,  loss  of  colloid, 
and  hyperemia — all  of  which  became  apparent  after  only  two  weeks  and  had  in- 
creased markedly  by  six  weeks.  Although  Fisher's  experiments  were  performed 
in  January  and  February  and  the  season  at  which  Adams'  work  was  done  is  not 
given,  the  results  of  these  two  studies  seemed  to  indicate,  as  did  our  findings,  that 
the  structure  of  the  thyroid  of  Triturus  was  relatively  unaffected  by  treatment  with 
thiourea  whereas  that  of  Desmognathus  was  markedly  altered. 

The  effects  of  perchlorate  have  apparently  not  been  investigated  previously  for 
either  of  these  animals.  Studies  on  mammals  indicate  that  the  action  of  perchlorate 
is  quite  unlike  that  of  thiourea,  for  it  interferes  with  the  process  of  iodide  concen- 
tration (Wyngaarden,  Wright  and  Ways,  1952).  The  thyroids  of  animals  given 
effective  doses  of  this  drug  are  unable  to  accumulate  iodine  from  the  plasma.  This, 
of  course,  prevents  synthesis  of  thyroid  hormone  (TH)  and,  just  as  in  thiourea 
treatment,  the  resultant  lowering  of  the  TH  level  and  rise  in  TSH  level  cause  the 
histological  changes  in  the  thyroid  that  are  usually  associated  with  a  high  rate  of 
secretory  activity.  In  our  experiments  the  histological  results  of  perchlorate 
treatment  precisely  paralleled  those  obtained  with  thiourea.  The  Dcsmognathus 
thyroid  was  strikingly  affected  but  that  of  Triturus  remained  unchanged. 

Desmognathus,  then,  after  treatment  with  either  of  these  two  goitrogenic  drugs, 
gives  indication  of  the  increased  TSH  production,  which  would  be  expected  on 
the  basis  of  the  work  with  mammals.  Triturus  shows  little  or  no  sign  of  any 
increased  TSH  level.  The  lack  of  response  in  Triturus  could  be  explained  in  at 
least  two  different  ways.  One  possibility  is  that  this  salamander  is  refractory  to  the 
drugs  so  that,  at  the  dosage  level  used,  thiourea  and  perchlorate  both  failed  to 
inhibit  hormone  synthesis.  If  this  were  the  case,  the  treatment  would  have  caused 
no  lowering  of  the  TH  level  in  the  blood ;  therefore,  no  stimulation  of  the  pituitary 
to  increased  TSH  production  would  have  occurred.  The  other  possibility  is  that 
the  drugs  were  effective  in  preventing  hormone  synthesis  but  that  the  pituitary 
failed  to  respond  in  the  usual  way  to  the  TH  decrease.  Then  no  increase  in  TSH 
would  occur  and  none  of  the  effects  of  this  hormone  on  the  thyroid  would  be 
observed.  The  results  of  the  study  of  radioiodine  uptake  will  serve  to  indicate 
which  of  these  alternatives  is  correct. 


EFFECTS  OF  GOITROGENS  IN  SALAMANDERS 


417 


2.  Iodine  uptake  in  control  animals 

It  was  observed  (Table  I)  that  in  the  Desmognathus  controls  the  counts  in  the 
thyroid  region  were  not  significantly  higher  than  those  in  the  heart  region  at  6 
hours  after  injection  of  I131.  By  12  hours,  however,  they  had  risen  to  a  high 
level.  They  then  declined  steadily,  reaching  about  one-half  the  peak  level  by 
2.5  days,  but  remained  significantly  higher  than  the  counts  in  the  heart  region 
throughout  this  period.  Triturus  controls,  on  the  other  hand,  showed  neither  an 
initial  difference  in  counts  between  thyroid  and  heart  nor  a  steady  decline  in  the 
counts  of  the  thyroid  region  during  the  2.5-day  period,  although  the  counts  at 
the  heart  level  declined  steadily  as  I131  was  eliminated  from  the  body,  with  the 
result  that  the  thyroid  counts  were  significantly  higher  than  the  heart  region  counts 
from  24  hours  on.  Comparison  of  the  thyroid  counts  with  heart  counts  showed 
that  radioiodine  was  taken  up  actively,  reached  a  high  level,  and  was  rapidly  re- 
leased by  the  thyroid  of  Desmognathus  whereas  in  Triturus  the  gland  accumulated 
I131  more  sluggishly  and  in  lesser  amounts  and  the  turnover  (during  2.5  days)  was 
negligible.  These  results  are  in  accord  with  the  histological  findings,  which  indi- 
cate a  greater  physiological  activity  of  the  thyroid  in  untreated  Desmognathus  than 
in  untreated  Triturus. 

3.  Iodine  uptake  in  thiourea-treated  animals 

Table  I  shows  that  Desmognathus  treated  with  thiourea  had  a  high  initial  up- 
take of  I131  by  the  thyroid  (falling  in  the  same  range  as  the  peak  for  the  controls) 
and  then  a  rapid  loss ;  the  differences  between  thyroid  and  heart  regions  were  there- 


TABLE  I 

Radioactivity  (corrected  for  physical  decay)  in  counts  per  second  uf  specimens  of  Triturus  viridescens 

and  Desmognathus  fuscus  at  the  indicated  periods  of  time  after  injection  with  5  /j.c.  of  7131  after 

46  days  of  treatment  with  goitrogens.     Each  number  represents  the  mean  of  counts  made  on 

six  specimens.      The  least  significant  difference  (at  the  5%  level)  calculated 

for  these  means  is  2.63 


Time  after 

Control  animals 

Animals  treated  with 
thiourea 

Animals  treated  with 
KC1O4 

SD&CICS 

I131  inj. 

hours 

Region  of 

Region  of 

Region  of 

Region  of 

Region  of 

Region  of 

thyroid 

heart 

thyroid 

heart 

thyroid 

heart 

Triturus 

6 

10.30 

10.94 

9.64 

9.96 

10.25 

11.68 

viridescens 

12 

11.84 

9.42 

9.90 

9.41 

8.77 

9.85 

24 

11.70 

8.59 

8.90 

8.80 

5.96 

6.22 

36 

11.57 

7.76 

8.86 

8.56 

5.04 

5.03 

48 

11.14 

7.47 

8.79 

8.06 

3.92 

4.06 

60 

11.11 

7.89 

9.10 

8.11 

3.53 

3.95 

Desmognathus 

6 

8.68 

6.80 

10.22 

6.84 

6.40 

6.95 

fuscus 

12 

12.13 

5.66 

8.42 

6.23 

5.58 

6.05 

24 

10.98 

4.56 

6.39 

4.13 

4.67 

4.43 

36 

10.94 

3.78 

4.51 

3.58 

3.91 

3.80 

48 

8.73 

3.64 

4.15 

3.16 

3.14 

3.33 

60 

6.86 

3.35 

3.71 

3.09 

2.84 

3.03 

418  JAMES  NORMAN   DENT  AND  W.  GARDNER  LYNN 

fore  quite  significant  at  6  hours  after  injection  of  I131  but  not  significant  thereafter. 
In  Triturus,  similarly  treated,  the  iodine  content  of  the  thyroid  was  different  from 
that  of  the  controls  only  in  the  24-  and  36-hour  counts  and  the  thyroid  region 
showed  no  count  significantly  higher  than  that  of  the  heart  region  at  any  time. 

These  results  may  be  interpreted  as  follows.  It  is  clear  from  the  histological 
study  that  the  thiourea  treatment  caused  a  hypertrophy  of  the  thyroid  in  Dcsmogna- 
thus  and  one  may  assume  that  this  is  accompanied  by  a  high  affinity  for  iodine. 
This  would  account  for  the  initial  high  uptake  of  I131.  Since,  however,  the  gland 
was  unable  to  bind  iodine,  because  of  the  treatment  with  thiourea,  and  also  since 
the  TSH  level  in  the  blood  was  presumably  high,  the  I131  that  had  been  taken  up 
was  very  rapidly  lost.  In  Tritnnis  the  thyroid  did  not  hypertrophy  and  therefore 
would  not  be  expected  to  have  a  high  affinity  for  iodine.  Indeed,  it  takes  up  I1£ 
and  retains  it  to  about  the  same  degree  as  the  controls.  Not  being  able  to  bind 
I131  the  thyroid  releases  it,  but  since  the  TSH  level  is  apparently  unaffected  by 
treatment  with  thiourea  in  Tritnnis,  the  release  of  I131  from  the  thyroid  is  much 
slower  than  in  Desmognathus. 

4.  Iodine  uptake  in  animals  treated  to/7/;  potassium  pcrchlorate 

Treatment  with  perchlorate  produced  the  same  effect  on  radioiodine  uptake  in 
both  salamanders.  Counts  in  the  thyroid  region  did  not  differ  from  those  in  the 
heart  region  at  any  time.  Indeed,  the  counts  over  the  thyroid  were,  in  general, 
slightly  lower  than  those  over  the  heart,  probably  because  of  the  difference  in  the 
volume  of  circulating  blood  in  the  two  regions.  In  any  case  it  seems  clear  that 
perchlorate  was  equally  effective  in  these  two  animals  in  preventing  any  accumula- 
tion of  I131  by  the  thyroid. 

In  perchlorate-treated  Triturus,  the  counts  in  the  heart  region  declined  more 
rapidly  and  reached  a  significantly  lower  level  than  in  the  control  or  thiourea- 
treated  Triturus.  This  indicates  that  elimination  of  I131  from  the  body  goes  on 
rapidly  when  iodide  uptake  by  the  thyroid  is  blocked  but  is  in  some  way  delayed 
when  the  thyroid  is  actively  concentrating  iodide.  This  delay  occurs  regardless 
of  whether  the  iodine  taken  up  by  the  thyroid  is  ultimately  bound  to  protein.  It 
is  well  known  that,  so  long  as  there  is  no  interference  with  the  thyroid's  ability  to 
concentrate  iodide,  the  gland  acts  as  an  iodine  reservoir,  continually  taking  up 
iodine  from  the  plasma  and  passing  it  back  at  a  slow  but  steady  rate  so  that  the 
decline  in  iodine  content  of  the  plasma  caused  by  elimination  from  the  body  is 
partially  compensated  by  the  exchange  of  iodine  with  the  thyroid.  In  perchlorate- 
treated  animals,  where  no  such  mechanism  is  in  operation,  the  rate  of  decline  in 
the  iodine  content  of  the  plasma  would  depend  solely  upon  the  rate  of  iodine 
excretion.  It  is  noteworthy  that  the  rate  of  decline  in  the  heart-region  counts 
in  Desmognathus  is  essentially  the  same  in  all  three  groups.  The  failure  of  the 
thyroid  to  show  any  iodine  reservoir  effect  in  this  animal,  even  when  its  iodide- 
concentrating  ability  is  unimpaired,  is  probably  to  be  ascribed  to  the  high  activity 
of  the  gland.  In  thiourea-treated  Desmognathus,  although  the  initial  uptake  of 
radioiodine  was  high,  I131  was  very  rapidly  passed  out  to  the  plasma  and,  since 
none  was  bound,  the  decline  in  uptake  in  both  thyroid  and  plasma  was  rapid, 
paralleling  that  of  perchlorate-treated  Desmognathus,  where  no  iodide-concentration 
occurred  at  all.  It  may  be  assumed  that  in  control  Desmognathus  any  unbound 


EFFECTS  OF  GOITROGENS  IN  SALAMANDERS  419 

iodine  is  also  rapidly  exchanged  with  the  plasma,  and  the  counts  in  the  heart  region 
again  fall  rapidly.  Undoubtedly,  the  counts  in  the  thyroid  region  remain  signifi- 
cantly high  in  the  controls  because  in  these  animals,  in  contrast  with  the  thiourea- 
treated  specimens,  a  part  of  the  iodine  is  bound  to  protein  and  is  thus  unavailable 
for  ready  passage  into  the  plasma. 

5.  Possible  adaptive  significance  of  findings 

The  results  of  the  study  of  radioiodine  uptake  indicate  that  the  differences  in 
histological  response  of  the  thyroid  in  Triturus  and  in  Desmognathus  should  not 
be  attributed  to  a  difference  in  the  direct  effects  of  the  goitrogens  on  thyroid 
functioning  in  the  two.  Perchlorate  treatment  definitely  prevented  thyroidal 
build-up  of  I131  in  both,  and  there  is  good  reason  to  conclude  that  thiourea  treat- 
ment interfered  with  binding  of  I131  in  both.  It  would  seem,  therefore,  that  the 
lack  of  histological  change  in  the  Triturus  thyroid,  as  well  as  its  normally  inactive 
state,  must  be  attributed  to  some  unusual  condition  in  the  pituitary,  involving  a 
low  level  of  TSH  production.  Such  an  explanation  has  been  suggested  to  account 
for  what  is  apparently  a  similar  situation  in  the  goldfish  (Carassius  anratus). 
The  goldfish  thyroid  exhibits  a  histological  appearance  of  very  low  activity  through- 
out the  year  and  at  no  season  (Fortune,  1955)  undergoes  hypertrophy  after  ad- 
ministration of  thiourea  (or  other  goitrogens).  It  does,  however,  respond  very 
strongly  to  injected  TSH  both  histologically  (Gorbman,  1940)  and  physiologically 
(Berg  and  Gorbman,  1954).  Fortune  (1956)  suggests  that  the  inactive  thyroid 
of  the  goldfish  (possibly  acquired  through  many  generations  of  artificial  selection) 
may  be  an  important  adaptation  permitting  its  tolerance  of  an  exceptionally  wide 
range  of  temperatures  (0°  to  41°  C.)  and  cites  the  rise  in  thermal  death  point 
from  23°  C.  to  33°  C.  in  the  teleost  Phoxinus  laevis  after  treatment  with  thiourea 
as  supporting  evidence  for  his  suggestion. 

This  hypothesis  may  also  apply  to  Triturus  viridescens.  Although  no  specific 
data  are  available  concerning  the  temperature  range  of  this  species,  it  is  undoubt- 
edly quite  wide.  The  animal  lives  in  pools  and  ponds  in  both  wooded  and  open 
situations  and  has  a  geographical  range  from  southeastern  Canada  to  Georgia  and 
Alabama.  It  is  certainly  able  to  survive  successfully  in  ice-covered  water,  and 
the  water  temperature  in  unshaded  ponds  in  mid-summer  must  frequently  rise 
above  30°  C.  Desmognathus,  on  the  other  hand,  lives  in  moist  situations  usually 
in  close  proximity  to  small  streams  in  deep  woods.  In  our  experience  it  does  not 
survive  in  the  laboratory  at  temperatures  above  25°  C.  and  does  best  below  20°  C. 
Several  rather  obvious  devices  for  testing  this  theory  come  to  mind.  One,  namely, 
observation  of  the  effect  of  TSH  administration  on  iodine  metabolism  in  Triturus, 
is  currently  being  employed  by  us. 

The  authors  wish  to  acknowledge  the  assistance  of  Dr.  A.  W.  Kimball  of  the 
Oak  Ridge  National  Laboratory  Mathematics  Panel  in  the  statistical  analysis. 

SUMMARY 

1.  Specimens  of  Triturus  viridescens  and  Desmognathus  fuse  us  were  injected 
on  alternate  days  with  0.1  ml.  of  1.0%  thiourea.  Others  were  injected  on  alternate 


420         JAMES  NORMAN  DENT  AND  W.  GARDNER  LYNN 

days  with  0.1  ml.  of  0.2%  potassium  perchlorate.  Histological  study  was  made 
of  the  thyroid  glands  of  both  experimental  and  control  animals  after  30  days  and 
46  days  of  treatment.  Measurements  of  uptake  and  turnover  of  injected  I131  were 
made  on  the  animals  treated  for  46  days. 

2.  Evidence  was  obtained  from  the  histological  observations  and  from  the  use 
of  radioiodine  to  show  that  although  the  thyroids  of  control  specimens  of  Desmogna- 
thus were  physiologically  active,  those  of  Tritunts  controls  were  rather  inactive. 

3.  Both   thiourea   and   potassium   perchlorate    inhibited    thyroidal    function    in 
Desmognathus,  as  evidenced  by  both  histological  changes  and  changes  in  radio- 
iodine  uptake. 

4.  In  Triturus,  thiourea  brought  about  only  a  slight  hyperemia  and  potassium 
perchlorate  produced  no  detected  histological  change  in  the  thyroid.     Radiological 
measurements  after  the  injection  of  I131,  however,  indicated  that  the  same  physio- 
logical responses  taking  place  in  Desmognathus  also  occurred  in  Triturus  but  at 
a  lower  level  of  thyroidal  function. 

5.  Measurements  of  radioactivity  in  the  heart  region  demonstrated  that  iodine 
was  readily  excreted  from  all  the  specimens  of  Desmognathus  and  from  the  indi- 
viduals of  Triturus  treated  with  potassium  perchlorate.     Elimination  of  iodine  was 
relatively  slow  in  the  other  specimens  of  Triturus. 

LITERATURE  CITED 

ADAMS,  ANN  ELIZABETH,  1946.     The  effects  of  thiourea  on  the  thyroids  of  Triturus  viridesccns. 

Anat.  Rcc.,  94:  532. 
BERG,  OLGA  A.,  AND  A.  GORBMAN,  1954.     Normal  and  altered  thyroidal  function  in  domesticated 

goldfish  Carassius  auratus.     Proc.  Soc.  Exp.  Biol.  Mcd.,  86:  156-159. 
FISHER,   J.,    1953.     Thyroidal   response   to   goitrogens    by   Desmognathus   fuscus    (Rafinesque) 

using  radioactive   iodine   as   indicator.     The  Catholic    Univ.   of  America   Studies,  27 : 

1-25. 
FORTUNE,  P.  Y.,  1955.       Comparative  studies  of  the  thyroid  function  in  teleosts  of  tropical  and 

temperate  habitats.     /.  E.vp.  Biol.,  32:  504-513. 

FORTUNE,  P.  Y.,  1956.     An  inactive  thyroid  gland  in   Carassius  auratus.     Nature,  178:   98. 
GORBMAN,   A.,    1940.     Suitability   of  the   common   goldfish   for   assay   of   thyrotropic   hormone. 

Proc.  Soc.  Exp.  Biol.  Mcd.,  45 :  772-773. 

GRAY,  P.,  1952.     Handbook  of  Basic  Microtechnique.     Blakiston  Company,  Philadelphia. 
MORGAN,  ANN  H.,  AND  CATHERINE  H.  FALES,  1942.     Seasonal  conditions  and  effects  of  low 

temperature    in    the    thyroid    glands    of    amphibians.     I.     Adult    Triturus    viridescens. 

J.  Morphol,  71 :  357-389. 
PITT-RIVERS,  ROSALIND,  1950.     Mode  of  action  of  antithyroid  compounds.     Physiol.  Rev.,  30 : 

194-205. 
ROCHE,  J.,  AND  R.  MICHEL,  1955.     Nature,  biosynthesis  and  metabolism  of  thyroid  hormones. 

Physiol.  Rev.,  35 :  583-610. 
WHEELER,  A.  J.,  1953.     Temporal  variations  in  histological  appearance  of  thyroid  and  pituitary 

of  salamanders  treated  with  thyroid  inhibitors.     Biol.  Bull.,  104 :  250-262. 
WYNGAARDEN,  J.  B.,  B.  M.  WRIGHT  AND  P.  WAYS,  1952.     The  effect  of  certain  anions  upon 

the  accumulation   and   retention   of   iodide   by   the   thyroid   gland.     Endocrinology,   50 : 

537-539. 


RESPONSE  OF  THE  MALE  REPRODUCTIVE  SYSTEM  OF  LIZARDS 
(ANOLIS  CAROLINENSIS)  TO  UNNATURAL  DAY-LENGTHS 

IN  DIFFERENT  SEASONS 

WADE  FOX  AND   HERBERT  C.  DESSAUER 

Departments  of  Anatomy  and  Biochemistry,  Louisiana  State  University  School  of  Medicine, 

Nezv  Orleans  12,  La, 

In  this  study  we  have  examined  the  response  of  the  male  reproductive  system 
of  the  green  anole,  Anolis  carolincnsis,  to  unnatural  day-lengths  at  different  seasons 
of  the  year.  Evidence  of  the  photoperiodic  control  of  reproduction  in  reptiles  has 
been  slowly  accumulating.  Burger  (1937)  found  that  artificially  increased  day- 
length  stimulated  a  new  spermatogenic  cycle  in  the  red-eared  turtle,  Pseudemys 
scrip ta  elegans.  Clausen  and  Poris  (1937)  reported  unseasonal  gonadal  hyper- 
trophy and  spermatogenesis  in  green  anoles  exposed  daily  to  18  hours  of  light. 
Bartholomew  (1950,  1953)  described  similar  gonadal  recrudescence  in  the  desert 
night  lizard,  Xantusia  vigilis,  maintained  at  16-hour  day-lengths. 

The  above  investigators  have  been  primarily  interested  in  the  condition  of  the 
gonads.  Descriptions  of  accessory  sex  organs  have  not  been  detailed  enough  to 
allow  for  a  positive  statement  that  reptiles  can  be  brought  into  full  breeding  condi- 
tion by  artificially  increasing  day-length.  In  the  experiments  to  be  described 
we  have  examined  both  gonads  and  certain  accessory  sex  organs  to  obtain  a  more 
complete  measure  of  breeding  condition.  Further,  the  influence  which  the  condi- 
tion of  the  reproductive  system  at  the  beginning  of  an  experiment  has  upon  its 
response  to  day-length  alterations  has  not  been  thoroughly  examined  in  all  seasons. 
We  have  studied  the  effect  of  short  days  prior  to  and  during  the  breeding  season, 
as  well  as  the  effect  of  long  days  during  and  following  the  breeding  season.  To 
our  knowledge  there  has  been  no  previous  effort  to  determine  whether  a  seasonal 
"refractory  period,"  characteristic  of  passerine  birds  (for  review  see  Hammond, 
1954),  is  present  in  reptiles  or  if  a  period  of  exposure  to  short  days  is  necessary 
before  a  new  reproductive  cycle  can  be  initiated  (Miller,  1954). 

METHODS 

Animals  were  collected  as  needed  over  a  period  of  three  years  in  the  vicinity 
of  New  Orleans.  Adult  animals  (61  to  71  mm.  snout-vent  length),  that  had  prob- 
ably been  through  a  previous  reproductive  period,  were  separated  from  immatures 
or  sub-adults  (51  to  60  mm.).  The  latter  had  not  been  through  a  previous  com- 
plete reproductive  hypertrophy  but  were  due  to  become  sexually  mature  in  the 
next  normal  breeding  season  (Fox,  1958). 

Methods  of  animal  care  have  been  reported  previously  (Fox  and  Dessauer, 
1957).  Cage  temperatures  were  maintained  at  28  ±  2°  C.  Artificial  lighting  was 
supplied  by  means  of  daylight  fluorescent  lamps  mounted  above  the  cages.  These 
were  regulated  by  automatic  time  switches  to  supply  18L  (hours  of  light  per 

421 


422  WADE  FOX  AND  HERBERT  C.  DESSAUER 

24-hour  period),   16L,   14L,  9L  or  6L.     Animals  exposed  to  natural  day-length 
were  placed  in  front  of  a  north-facing  window. 

Experiments  were  conducted  for  approximately  60  days  unless  otherwise  stated. 
At  the  end  of  each  experiment  lizards  were  killed  with  ether,  and  their  fat  bodies, 
liver  and  left  testis  weighed.  Loss  of  body  weight  during  the  course  of  an  experi- 
ment, and  liver  and  fat  body  weights  at  autopsy  were  useful  in  estimating  the  de- 
gree of  starvation  of  unresponsive  lizards.  The  right  testis  with  attached  epi- 
didymis,  the  ductus  deferens,  and  the  right  kidney  were  fixed  in  Bouin's  solution, 
embedded  in  paraffin,  sectioned  at  10  micra,  and  stained  with  Harris'  hematoxylin 
and  eosin.  The  state  of  spermatogenesis  and  the  cytology  of  interstitial  cells  and 
accessory  sex  organs  were  studied  microscopically.  Measurements  of  the  diam- 
eter of  seminiferous  tubules,  the  diameters  and  epithelial  heights  of  the  ductus 
epididymidis,  ductus  deferens,  and  the  sexual  segment  of  the  kidney  were  made 
with  a  calibrated  ocular  micrometer.  All  mensural  data  were  plotted  as  histo- 
grams and  subjected  to  the  "t  test"  for  possible  significance.  The  size  of  each 
sample,  mortality  and  number  of  starving  animals  (fat  bodies  less  than  1%  of  body 
weight)  are  presented  in  Table  II.  Column  "N"  in  Table  II  is  the  number  used 
in  calculating  standard  deviations  and  "t"  values  for  determining  the  level  of  sig- 
nificance of  the  data  when  it  seemed  justifiable  to  eliminate  the  measurements  on 
starving  animals.  Differences  are  accepted  as  significant  at  the  5%  level  and 
highly  significant  at  the  1  %  level  of  probability. 

In  reporting  the  results,  frequent  reference  is  made  to  arbitrarily  delimited 
stages  of  the  normal  reproductive  cycle  of  wild  male  Anolis.  These  stages  are 
characterized  briefly  in  Table  I ;  detailed  descriptions  are  presented  by  Fox  (1958). 

Although  considerable  effort  was  made  to  locate  and  measure  interstitial  cells 
of  the  testes,  it  was  felt  that  these  data  were  unsatisfactory  for  valid  statistical 
analyses.  Data  on  the  sexual  segment  of  the  kidney,  however,  afford  an  index  to 
androgenic  activity  (Reynolds,  1943).  The  sexual  segment  of  Anolis,  like  that  of 
Sceloporus  (Forbes,  1941),  includes  the  entire  set  of  uriniferous  collecting  ducts 
and  ureter  (Fox,  1958).  Measurements  were  taken  in  the  distal,  middle,  and 
proximal  regions  of  the  collecting  ducts,  but  only  those  of  the  distal  end  are 
recorded  in  Table  II. 

RESULTS 

Experiment  1.     September-November:  Adult  lizards  exposed  to  18L  and  natural 
day-length 

At  the  beginning  of  the  experiment  adults  had  just  completed  a  breeding 
season  and  the  gonads  and  accessory  sex  organs  were  in  their  most  atrophic  state 

TABLE  I 
Arbitrary  stages  in  the  normal  reproductive  cycle  of  Anolis 

Spermatogenesis  Accessory  sex  organs 

Stage  I       Dividing  spermatogonia  Atrophic 

Stage  II     Primary  spermatocytes  predominate  Slight  hypertrophy 

Stage  III   Maximimi  development;  large  numbers  Near  maximum  hypertrophy 

of  spermatocytes  and  spermatids 

Stage  IV   Large  numbers  of  spermatids;  Maximum  hypertrophy 

spermatocytes  reduced 

Stage  V     Only  spermatids  numerous  Partial  atrophy- 
Stage  VI    Expulsion  of  all  active  cells  Atrophic 


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WADE  FOX  AND  HERBERT  C.  DESSAUER 


TESTIS    WEIGHT 


mg 
50- 
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Sept.-Nov. 
Adults 


Sept-Nov. 
Immotures 


Oct. -Dec. 
Immotures 


Nov.  -Jon 
Adults 


Dec. -Feb. 
Adults 


Jon-Mor 
Immotures 


Moy-July 
Adults 


July-Sept 
Adults 


Exp.  NO 


Aug  -Oct. 
Adults 

10 


FIGURE  1.  Testis  weights  demonstrating  the  seasonal  gradient  in  testicular  response  to 
exposure  to  long  photoperiods.  The  terminal  horizontal  bars  indicate  the  range  in  sample 
weights,  the  middle  horizontal  bar  indicates  the  sample  mean  and  the  solid  rectangle  represents 
the  standard  deviation  plotted  on  both  sides  of  the  mean.  N.  D.  =  exposure  to  natural  day- 
lengths  ;  L  =  hours  of  artificial  light  per  24  hours. 


(end  of  Stage  VI).  By  the  end  of  the  experiment,  animals  in  the  wild  had 
initiated  spermatogonial  mitoses  but  their  accessory  sex  organs  were  still  atrophic 
(Stage  I).  The  18L  sample  underwent  a  highly  significant  increase  in  testis 
weight  (Fig.  1)  although  two  apparently  healthy  animals  did  not  respond  to  the 
light  treatment.  Control  animals  retained  small  gonads  that  averaged  significantly 
less  in  weight  than  those  of  wild  animals  in  November  (Fig.  2).  Seminiferous 
tubule  diameters  were  greater  and  spermatogenesis  more  advanced  in  the  experi- 
mental sample  (Table  II).  Whereas  none  of  the  control  animals  advanced  further 
than  spermatogenic  Stage  I,  four  of  the  experimentals  were  classified  as  early 
Stage  III.  The  total  cell  number,  however,  was  about  half  that  present  in  a 
normal  Stage  III  gonad. 

The  accessory  sex  organs  were  partially  stimulated  by  exposure  to  18-hour 
day-lengths  at  this  season  (Table  II).  The  ductus  epididymidis  underwent  a  very 
significant  increase  in  diameter  and  epithelial  height,  in  half  of  the  sample  the 
cells  contained  secretion  granules  (Stages  II  and  III)  ;  in  six  animals  sperm 
were  present  in  the  lumen  (beginning  of  Stage  III).  Secretion  granules  and 
sperm  were  lacking  in  all  the  controls  (Stage  I).  Neither  the  average  of  epithelial 
height  nor  diameter  of  the  ductus  deferens  significantly  increased  in  the  18L  sample 
(Table  II).  In  three  animals,  however,  sperm  had  entered  this  organ  and  it  was 
significantly  enlarged,  convoluted  and  contained  secretion  granules  (Stage  III). 
The  ductus  deferens  of  the  controls  remained  atrophic  (viz.,  empty,  not  convoluted, 
and  with  a  pseudostratified  agranular  epithelium). 

Measurements  of  the  sexual  segment  (Table  II)  did  not  reveal  significant 
differences  between  the  means  of  the  experimental  and  control  samples.  Four 
experimental  animals,  however,  showed  slight  hypertrophy  and  the  presence  of 
eosinophilic  granules  at  the  tips  of  the  epithelial  cells  of  the  ureter  and  the  distal 


DAY-LENGTH  AND  MALE  ANOLIS  CYCLES  427 

ends  of  the  collecting  tubules  (Stage  II).     No  hypertrophy  or  secretion  occurred 
among  the  controls. 

Experiment   2.     September-November:    Immature   lizards   exposed   to   18L   and 
natural  day-length 

Although  the  reproductive  organs  of  these  young  animals  had  achieved  a  partial 
maturity  during  the  previous  summer,  they  were  completely  atrophic  at  the  begin- 
ning of  the  experiment.  Wild  animals  in  this  age  group  were  at  minimal  reproduc- 
tive development  at  the  close  of  the  experiment  (Stage  I). 

Immature  lizards  on  18L  underwent  a  highly  significant  gonadal  response.  Tes- 
tis  weights  were  markedly  greater  (P  <  1%)  than  the  controls  (Fig.  1).  Four 
were  more  responsive  to  the  light  treatment  than  adults  during  the  same  period. 
Spermatogenesis  (Table  II)  almost  reached  peak  activity  (Stage  III)  in  a  few 
individuals,  whereas  none  of  the  controls  progressed  beyond  Stage  I. 

Of  the  accessory  sex  organs  studied,  only  the  ductus  epididymidis  proved  to  be 
significantly  hypertrophied  (P  < ;  1%)  by  the  long  light  treatment.  Over  half  the 
animals  showed  secretory  granules  and  one-third  had  sperm  in  the  epididymis.  In 
all  controls  the  epididymis  was  atrophic  and  without  sperm.  Three  experimental 
animals  showed  enlargement  of  the  ductus  deferens  (Stage  II).  They  had  a  few 
sperm  in  the  lumen,  the  epithelium  was  taller  and  the  cytoplasm  granular.  The 
ductus  deferentia  of  the  controls  were  atrophic  (Stage  I). 

Five  experimental  animals  showed  a  slight  hypertrophy  of  the  sexual  segment  of 
the  kidney  with  secretion  granules  at  the  tips  of  epithelial  cells  near  the  ureter 
(Stage  II).  The  sexual  segment  of  a  few  controls  underwent  a  minor  hypertrophy 
but  no  signs  of  secretion  were  present. 

Experiment  3.     October-December:  Immature  lizards  exposed  to  18L,  16L  and 
9L 

At  the  beginning  of  this  experiment  the  animals  were  in  a  stage  of  minimal 
development.  The  healthy  animals  on  16L  and  18L  showed  a  highly  significant 
increase  in  testis  weight  over  those  kept  at  9L  (Fig.  1).  Animals  that  lost 
weight  did  not  respond  to  the  light  treatment.  Many  kept  on  the  long  days  had  very 
large  seminiferous  tubules  and  had  progressed  into  Stages  III  and  IV  of  sper- 
matogenic  development  (Table  II),  without  achieving,  however,  the  high  volume 
of  sperm  production  characteristic  of  adults  during  the  breeding  season.  Nearly 
half  of  the  9L  animals  attained  the  spermatogenic  development  of  adult  wild  animals 
for  December  (Stage  II),  the  others  remained  in  Stage  I. 

Both  samples  on  18L  and  16L  showed  highly  significant  or  significant  hyper- 
trophy of  the  various  accessory  sex  organs  when  compared  to  those  on  9L.  The 
differences  between  the  18L  and  16L  samples  were  not  statistically  significant.  The 
epididymis  of  the  animals  on  long  days  contained  many  sperm.  The  cytoplasm  of 
the  ductus  epididymis  was  granular  and  the  cell  height  and  diameter  much  enlarged 
(Stages  II  and  III).  The  epididymes  of  controls  were  all  in  Stage  I.  Significant 
numbers  of  animals  on  long  day  treatment  had  sperm  in  the  ductus  deferens.  In 
these  individuals  the  diameter  of  the  ductus  was  increased,  the  epithelium  hyper- 
trophied and  the  cytoplasm  granular  (Stage  III).  A  few  animals  remained  in 
Stage  I  or  II. 


428  WADE  FOX  AND  HERBERT  C.  DESSAUER 

The  sexual  segment  underwent  a  significant  hypertrophy  on  the  18L  (P  =  1%) 
and  16L  (P  —  5%)  regimes.  Secretion  occurred  in  only  five  animals  on  18L  and 
one  on  16L.  In  these  animals  the  epithelium  of  the  ureter  and  the  distal  end  of  the 
collecting  tubules  exhibited  eosinophilic  granules  in  the  apical  %-%  of  the  cells 
(Stage  II).  The  9L  series  showed  only  slight  hypertrophy,  and  no  signs  of  secre- 
tion (Stage  I). 

Experiment  4.     November- January:   Adult  lizards  exposed  to   18L   and   natural 
day-length 

During  November  the  reproductive  system  of  Anolis  is  in  Stage  I.  Lizards  on 
18  hours  of  day-length  underwent  marked  hypertrophy  of  the  testis  (P  <  1%) 
compared  to  controls  on  natural  day-length  (Fig.  1).  The  testes  of  the  controls 
enlarged  also,  but  the  sample  mean  did  not  differ  significantly  from  that  of  wild 
animals  killed  in  January  (Fig.  2). 

Most  of  the  experimental  animals  reached  the  peak  of  seminiferous  tubule  diam- 
eter and  spermatogenic  development  (Stage  III).  A  few  passed  the  peak  and 
advanced  to  Stage  IV.  Four  control  animals  progressed  to  early  Stage  III  but 
these  were  far  from  attaining  peak  development.  The  remainder  of  the  controls 
were  in  spermatogenic  Stage  II. 

The  accessory  sex  organs  of  the  experimentals  were  greatly  hypertrophied 
(P  <  .01;  Table  II).  In  most  of  the  sample  the  ductus  epididymidis  was  filled 
with  sperm  and  the  epithelial  cytoplasm  completely  filled  with  eosinophilic  granules 
(Stages  III  and  IV).  Four  controls  had  a  secretory  epididymis  with  a  few  sperm 
in  the  ductus  (Stage  II).  The  ductus  deferentia  of  the  18L  sample  were  very 
convoluted,  filled  with  sperm  and  highly  secretory  (Stages  III  and  IV).  Those 
of  the  control  sample  were  largely  atrophic  and  empty  (Stages  I  and  II). 

The  sexual  segment  of  the  experimentals  was  hypertrophied  (Table  II)  and 
highly  secretory  at  its  distal  end  in  all  animals  (Stage  II)  and  in  its  middle  portion 
in  half  the  sample  (Stage  III).  Control  animals  showed  no  signs  of  secretion 
although  wild  animals  at  this  time  show  slight  activity  (Stage  II). 

Experiment  5.     December-February:  Adult  lizards  exposed  to  13L,  16L  and  9L 

During  this  period  the  reproductive  system  of  wild  animals  progresses  through 
Stage  II  with  some  individuals  reaching  early  Stage  III.  The  distribution  and 
sample  means  of  testis  weights  were  very  similar  for  animals  exposed  to  9L  and 
16L  (Fig.  1).  Testes  averaged  smaller  in  the  18L  sample.  There  were  no 
significant  differences  in  seminiferous  tubule  diameters  (Table  II).  All  healthy 
animals  in  the  16L  and  18L  series  reached  peak  spermatogenic  development  (Stage 
III),  and  two-thirds  passed  on  to  Stage  IV.  Animals  in  the  9L  sample  were 
largely  in  Stage  III,  but  a  few  merited  assignment  to  Stage  IV.  When  subjected 
to  the  Chi-square  test,  the  16L  and  18L  samples  proved  to  be  very  significantly 
advanced  beyond  the  9L  sample. 

In  the  16L  sample,  but  not  in  the  18L  sample,  the  epithelial  cells  of  the  ductus 
epididymidis  were  significantly  taller  than  those  of  the  9L  sample.  Diameters  of 
both  the  ductus  epididymidis  and  ductus  deferens  were  similar  in  the  9L  and  16L 
samples,  but  averaged  significantly  smaller  in  the  18L  sample.  All  were  secretory 


DAY-LENGTH  AND  MALE  ANOLIS  CYCLES  429 

and  contained  sperm.  The  cluctus  deferens  appeared  to  be  more  highly  convoluted 
in  the  16L  sample  than  in  the  9L  sample,  indicating  greater  sperm  storage.  Those 
of  the  18L  animals  were  less  convoluted  and  contained  considerably  fewer  sperm 
than  either  of  the  two  other  samples. 

The  sexual  segment  was  hypertrophied  in  both  the  9L  and  16L  samples.  In 
the  16L  sample  Stage  III  was  reached  whereas  in  the  9L  sample  the  sexual  segment 
remained  in  Stage  II.  The  distal  ends  of  the  collecting  tubules  were  hypertrophied 
and  secretory  in  both  samples.  The  middle  portions  of  the  collecting  tubules  were 
secretory  in  thirteen  animals  on  16L  but  in  only  one  on  9L.  All  sexual  segments 
of  the  18L  sample  were  atrophic  except  that  of  one  animal  in  which  the  distal  end 
was  secretory. 

Data  on  testes  weights  were  available  for  a  series  of  captive  animals  exposed 
to  natural  day-length  during  this  period.  The  mean  testis  weight  was  comparable 
to  that  found  in  the  9L  and  16L  series  (Fig.  1). 

Experiment  6.     January-March:    Immature  lizards   exposed   to   18L   and  9L 

At  the  beginning  of  this  experiment  smaller  immature  male  lizards  were  in  Stage 
I  and  larger  ones  in  Stage  II.  At  the  end  of  this  experiment  the  mean  testis  weight 
of  the  9L  sample  was  greater  (Fig.  1).  although  not  significantly  so,  than  the  18L 
sample.  However,  animals  on  18L  were  far  advanced  beyond  those  on  9L  in  respect 
to  spermatogenesis  (Table  II).  Most  of  them  appeared  to  have  passed  the  peak  of 
gonad  development  (Stage  IV). 

The  cluctus  epididymidis  was  secretory  and  filled  with  sperm  in  both  samples. 
Epithelial  height  of  the  18L  sample  (Table  II)  was  significantly  greater.  The 
ductus  deferens  was  secretory,  convoluted  and  filled  with  sperm  in  most  animals 
of  both  samples.  Those  of  the  18L  sample  had  a  significantly  greater  diameter, 
appeared  to  be  more  convoluted  and  to  contain  more  sperm.  In  three  animals  of 
the  9L  sample  the  ductus  deferens  was  pseudostratified,  non-secretory  and  contained 
very  few  or  no  sperm. 

The  sexual  segment  was  very  significantly  hypertrophied  in  the  18L  sample. 
In  over  half  of  this  sample  it  was  secretory  in  the  ureter  and  distal  ends  of  the 
collecting  tubules,  and  in  %  it  was  secretory  through  the  middle  portion  (Stage 
III).  Most  sexual  segments  in  the  9L  sample  remained  in  Stage  I ;  in  two  animals 
they  were  secretory  through  the  middle  segment  (Stage  III)  and  in  one  secretion 
occurred  in  the  ureter  and  distal  end  only  (Stage  II). 

Regardless  of  the  length  of  light  exposure,  most  animals  that  underwent  marked 
testicular  development  and  hypertrophy  of  the  accessory  sex  organs  were  over 
55  mm.  snout-vent  length  at  the  start  of  the  experiment  and  grew  to  over  60  mm. 
during  the  two  months  period. 

Miscellaneous  winter  experiments 

Data  available  on  a  few  animals  maintained  for  longer  than  the  standard  60-day 
period  are  pertinent  to  this  study.  Eight  immature  lizards  kept  at  9L  from 
December  10  to  April  4  underwent  considerable  hypertrophy  of  the  reproductive 
system  despite  the  short  day-length.  Testes  ranged  from  8  to  33  mg.  with  a 
mean  of  22  mg.  Spermatogenesis  reached  Stages  III  and  IV  in  the  larger  gonads. 


430  WADE  FOX  AND  HERBERT  C.  DESSAUER 

These  same  individuals  had  sperm  in  the  ductus  epididymidis  and  ductus  deferens, 
both  of  which  were  hypertrophied  and  secretory.  The  sexual  segment  of  two 
larger  and  faster  growing  individuals  was  secretory  through  the  middle  portion 
(Stage  III).  Those  lizards  that  reached  60  mm.  snout- vent  length  had  the  largest 
gonads  and  the  most  hypertrophied  accessory  sex  organs.  In  those  less  than  60 
mm.  the  extent  of  testicular  hypertrophy  was  closely  correlated  with  body  growth. 
Four  animals  of  the  same  size  group  survived  18L  exposure  during  the  above 
period.  The  range  in  testicular  size  (  3  to  33  mg. )  was  similar  to  that  in  the  group 
exposed  to  9L  but  the  gonads  averaged  considerably  smaller  (16.5  mg.).  Only  two 
possessed  large  gonads  with  spermatogenic  activity  equivalent  to  Stage  IV.  One  of 
the  latter  animals,  that  had  grown  more  than  the  rest,  had  hypertrophied  accessory- 
sex  organs  (Stage  III),  the  other  had  atrophic  accessory  sex  organs  and  a  spermatic 
granuloma  of  the  epididymis.  The  remaining  two  animals  were  in  spermatogenic 
Stage  VI.  The  epididymis  and  ductus  deferens  were  either  empty  or  contained 
cellular  debris.  None  of  the  accessory  sex  organs  was  secretory  (Stage  V). 
Four  adult  lizards  on  9L  from  January  9  to  April  4  were  in  spermatogenic  Stage 
IV  and  the  accessory  sex  organs  approached  Stage  III.  Similarly,  ten  adults  on  9L 
from  February  3  to  April  4  were  in  spermatogenic  Stages  III  and  IV  and  their 
accessory  sex  organs  in  Stage  III. 

Experiment  7.     April-June:  Adult  lizards  exposed  to  6L  and  natural  day-length 

On  April  30,  26  adult  male  lizards  were  placed  on  a  regime  of  6L.  Five  of 
these  animals  were  sacrificed  on  June  3  and  compared  to  a  series  of  24  control 
animals  maintained  on  natural  day-length.  Testis  weight  was  very  significantly  less 
in  the  6L  sample  (22  to  33  mg;  29.5  ±  3.1  mg.)1  than  in  the  controls  (24  to  52 
mg. ;  37.0  ±  6.5  mg.),  but  was  larger  than  that  of  two  samples  of  wild  animals 
taken  in  June  (Fig.  2).  Spermatogenesis  in  the  6L  animals  was  in  late  Stage  IV 
typical  of  wild  animals  in  July,  whereas  the  controls  were  in  spermatogenic  Stages 
III  and  early  IV.  The  accessory  sex  organs  of  the  6L  sample  were  secretory  and 
were  not  significantly  different  from  those  of  the  controls. 

Nine  additional  animals  from  the  6L  sample  were  sacrificed  on  June  21  and 
compared  with  a  sample  of  nine  newly  captured  animals.  Testis  weights  were 
very  significantly  less  (4  to  22  mg. ;  14.9  ±  5.5  mg.)  than  those  of  wild  animals 
(16  to  33  mg. ;  23.7  ±  5.6  mg.).  Eight  of  the  6L  animals  were  in  spermatogenic 
Stage  V  (typical  of  August  animals)  and  one  in  Stage  VI.  All  wild  animals  were 
in  spermatogenic  Stage  IV.  The  epididymis  and  ductus  deferens  were  significantly 
reduced  in  size  and  in  secretory  activity  in  a  few  experimental  animals.  All 
experimental  animals  showed  significant  reduction  in  epithelial  height  of  the  sexual 
segment,  but  a  few  retained  secretory  activity  in  the  distal  end  of  the  tubules. 

An  attempt  was  made  to  determine  whether  the  precocious  termination  of  the 
sexual  cycle,  which  was  brought  about  by  exposure  to  short  days,  resulted  in  a 
temporary  gonadal  refractoriness  to  stimulation  by  long  day-lengths.  Six  animals 
which  had  been  maintained  on  6L  from  April  30  to  June  20,  were  exposed  to  18L 
from  June  21  to  September  18.  Three  responded  markedly  and  three  did  not. 
Testis  weights  (3  to  17  mg. ;  10.8  ±  7.2  mg.)  were  significantly  heavier  than  those 
of  a  sample  of  eight  controls  maintained  on  natural  day-length  from  June  4  to 

1  Range;  mean  and  standard  deviation. 


DAY-LENGTH  AND  MALE  ANOLIS  CYCLES  431 

September  18  (2  to  6  mg. ;  3.5  ±  0.6  mg.)  and  a  sample  of  nine  wild  animals  killed 
September  22  (1.5  to  4  mg. ;  2.3  ±  0.7  mg.).  Controls  and  wild  animals  were  at 
the  end  of  the  annual  spermatogenic  cycle  ( Stage  VI )  and  all  accessory  sex  organs 
were  atrophic.  Of  the  six  experimental  animals  two  were  in  spermatogenic  Stage 
VI,  one  advanced  to  Stage  I,  and  three  advanced  to  Stage  II.  The  latter  three 
individuals  exhibited  a  precocious  spermiogenesis  and  the  epididymis  was  hyper- 
trophied  and  contained  sperm.  The  ductus  deferens  and  sexual  segment  were 
secretory  (Stage  II)  in  only  a  single  specimen  in  which  sperm  had  reached  the 
ductus  deferens.  The  two  unresponsive  animals  were  seriously  starved. 

Experiment  8.     May-July:  Adult  lizards  exposed  to  18L,  natural  day-length  and 
9L 

At  the  initiation  of  this  experiment  the  reproductive  system  of  newly  captured 
animals  was  near  peak  development  (Stage  III).  During  July  wild  lizards  undergo 
a  regression  of  spermatogenesis  (Stage  IV),  but  the  accessory  sex  organs  are  still 
near  maximum  development.  The  two  experimental  samples  for  this  period  had 
a  relatively  high  mortality  due  to  starvation  (Table  II).  Spermatogenesis  was 
exhausted  (Stage  VI)  in  the  9L  sample  (Fig.  1)  and  the  accessory  sex  organs 
were  atrophic  (Table  II).  Considerable  variation  existed  in  the  18L  sample.  Its 
average  testis  weight  (Fig.  1)  was  very  significantly  less  than  that  of  controls 
exposed  to  natural  daylight,  but  it  was  nearly  equal  to  the  average  for  wild  animals 
in  July.  Only  one  individual  was  markedly  stimulated  by  the  excessive  light 
treatment.  Its  reproductive  organs  were  at  peak  activity  and  comparable  to  those 
of  an  animal  in  April  or  May.  Except  for  a  slight  increase  in  the  number  of 
primary  spermatocytes,  the  gonads  of  three  animals  were  comparable  to  those  of 
control  or  wild  animals  in  July ;  the  sexual  segment  of  the  kidney,  however,  was 
less  hypertrophied.  The  two  remaining  lizards  had  smaller  gonads  and  an  atypical 
spermatogenic  pattern.  The  germinal  elements  closest  to  the  lumina  of  the 
seminiferous  tubules  were  all  advanced  spermatids  (Stage  V)  whereas  the  outer 
layers  revealed  a  proliferation  of  spermatogonia  and  primary  spermatocytes  (Stage 
I).  In  one  of  these  animals  the  ductus  deferens  and  ductus  epididymidis  were 
moderately  hypertrophied  and  contained  sperm,  in  the  other  these  organs  were 
atrophic.  The  sexual  segment  was  inactive  in  both.  Since  these  animals  gained 
weight,  the  poor  response  to  light  did  not  appear  to  be  due  to  starvation. 

Experiment  9.     July-September:  Adult  lizards  exposed  to  18L  and  natural  day- 
length 

The  sample  means  of  the  experimental  animals  were  very  significantly  greater 
for  all  measurements  taken  (Fig.  1  and  Table  II).  The  testes  of  the  controls  were 
lacking  in  spermatogenic  activity  (Stage  VI).  The  testes  of  the  experimentals 
varied  from  5  to  34  mg.  and  were  judged  to  be  in  spermatogenic  Stages  I-IV. 

Control  and  wild  animals  sacrificed  in  September  have  atrophic  accessory  sex 
organs.  Among  animals  maintained  on  18L,  the  ductus  epididymidis  and  ductus 
deferens  were  highly  convoluted,  filled  with  sperm  and  secretory  in  twelve ;  nearly 
empty  and  much  reduced  in  three ;  empty  and  atrophic  in  two.  The  sexual  segment 
of  the  kidney  was  hypertrophied  and  secretory  in  the  ureter  and  distal  ends  of  the 


432  WADE  FOX  AND  HERBERT  C.  DESSAUER 

ducts  in  six,  and  non-secretory  in  the  others.  The  animals  that  were  least  respon- 
sive to  the  light  treatment  possessed  fat  bodies  weighing  around  100  mg.  (5%  of 
body  weight).  This  represented  half  or  less  the  weight  of  fat  bodies  of  animals 
that  yielded  a  good  response. 

Experiment  10.     August-October:  Adult  lizards  exposed  to  18L,  14L,  and  natural 
day-length 

This  experiment  was  initiated  at  a  time  when  rapid  involution  was  occurring  in 
all  reproductive  organs.  By  the  end  of  the  experiment  both  wild  animals  and 
controls  had  completed  a  reorganization  of  these  organs  and  were  ready  to  initiate 
a  new  cycle.  The  testes  of  most  animals  in  both  experimental  groups  were 
markedly  (P  <  1%)  enlarged  (Fig.  1).  Both  the  maximum  and  average  re- 
sponses of  the  14L  sample  were  considerably  less  than  those  of  the  18L  sample 
in  respect  to  testis  weight  and  spermatogenic  development  (Table  II).  On  the 
basis  of  a  highly  convoluted  and  sperm-packed  ductus  deferens,  four  lizards  on  18L 
were  classified  in  spermatogenic  Stage  IV.  The  fat  bodies  of  the  unresponsive 
lizards  on  18L  weighed  half  as  much  as  those  that  responded  well.  Most  of  the 
unresponsive  animals  on  14L  had  very  large  fat  bodies. 

Hypertrophy  of  the  accessory  sex  organs  of  the  18L  sample  tested  to  be  very 
highly  significant  except  for  the  sexual  segment  of  the  kidney  for  which  P  —  .02. 
Only  the  four  animals  classified  in  spermatogenic  Stage  IV  showed  secretion  in 
the  middle  and  distal  portions  of  the  collecting  ducts.  The  hypertrophy  of  the 
accessory  sex  organs  of  the  14L  sample  proved  to  be  significant,  although  no  secre- 
tion was  found  in  the  sexual  segment  of  the  kidney. 

DISCUSSION 

Degree  of  response  of  Anolis  to  artificially  lengthened  davs.  In  the  fall,  when 
gonads  are  smallest,  a  60-day  exposure  to  18L  was  sufficient  to  produce  numerous 
spermatozoa  in  the  larger  testes  and  a  precocious,  but  limited,  spermiogenesis  in  the 
smaller  ones.  Although  the  size  of  the  gonads  was  only  about  half  that  of  animals 
during  the  breeding  season,  the  relative  increases  were  the  greatest  of  the  entire 
series.  Assuming  that  testes  weights  were  equivalent  to  those  of  \vild  anoles  at  the 
beginning  of  the  experiments,  gonadal  weights  of  both  adults  and  immatures  under- 
went at  least  a  six-fold  increase  (Experiments  1  and  2).  The  closer  to  the  breeding- 
period  (April  through  August)  an  experiment  was  started,  the  more  complete  was 
gonadal  response.  The  relative  increase  in  weight,  however,  was  proportionately 
less. 

A  similar  seasonal  gradient  in  responsiveness  was  observed  in  the  accessory 
sex  organs.  The  sexual  segment  is  a  more  reliable  index  of  this  gradient  than  are 
the  epididymis  and  vas  deferens  in  which  the  apparent  activity  varies  with  sperm 
content.  Exposure  to  18L  for  60  days  starting  in  September  or  October  (Experi- 
ments 1  to  3)  did  not  bring  the  sexual  segment  of  adult  or  immature  lizards  into 
a  secretory  condition  typical  of  the  breeding  period.  However,  in  Experiment  4, 
started  November  14,  half  of  the  adults  developed  secretory  sexual  segments  typical 
of  April  breeding  animals.  Progressively  greater  responses  were  obtained  in  each 
succeeding  experiment  through  the  winter  (Table  II). 


DAY-LENGTH  AND  MALE  ANOLIS  CYCLES  433 

Our  data  on  the  completeness  of  the  testicular  response  correspond  reasonably 
well  with  the  results  of  others  working  with  reptiles  and  birds  (for  references  see 
reviews  by  Hammond,  1954  and  Farner,  1955).  Only  rarely  do  experimental, 
photoperiodic-stimulated  gonads  achieve  the  size  that  is  typical  of  the  particular 
species  during  the  breeding  season;  further,  the  largest  gonads  are  invariably 
induced  just  prior  to  the  natural  time  for  hypertrophy.  The  data  of  Vaugien 
(1955)  on  testis  size  and  bill  color  of  immature  English  sparrows  best  illustrate 
the  seasonal  gradient  of  response. 

Several  factors  have  been  suggested  to  account  for  the  seasonal  variation  in  the 
response  of  the  reproductive  system.  Vaugien  (1955)  considered  the  increasing 
responses  which  he  observed  to  be  due,  in  part,  to  the  increasing  age  of  the  immature 
sparrows.  In  immature  anoles  the  degree  of  response  appears  to  be  correlated 
with  growth  rate  as  well  as  age  (as  determined  by  size).  Vaugien  also  demonstrated 
that  in  immature  English  sparrows  the  longer  the  exposure  to  short  days  prior  to 
capture,  the  greater  the  testicular  response  to  long  day-lengths.  Exposure  to  a  period 
of  short  days  in  the  fall  is  apparently  essential  for  spermatogenesis  in  a  number  of 
species  of  birds.  In  adult  anoles,  exposure  to  long  day-lengths  during  or  following 
the  involution  of  the  testes  (Experiments  9,  10,  and  1)  elicited  a  response  without 
prior  exposure  to  short  photoperiods. 

It  is  generally  accepted  that  the  annual  variation  of  the  reproductive  systems  of 
wild  vertebrates  is  primarily  determined  by  the  cyclic  nature  of  pituitary  secretions. 
However,  Van  Oordt  (1956)  demonstrated  a  seasonal  difference  in  the  sensitivity 
of  spermatogonia  to  pituitary  gonadotropins  in  the  frog,  Rana  tcinporaria.  On  the 
basis  of  thyroxin  injections,  Vaugien  (1955)  postulated  that  during  short  days  the 
testes  of  immature  English  sparrows  become  increasingly  susceptible  to  stimulation. 
It  is  possible,  however,  to  explain  adequately  the  data  on  Anolls  without  assuming 
a  seasonal  change  in  sensitivity  on  the  part  of  the  reproductive  system.  The 
gradient  of  response  of  the  testes  can  be  accounted  for  by  the  number  of  sper- 
matogonia present  at  the  beginning  of  each  experiment,  there  being  fewest  in 
September  and  progressively  more  during  the  winter.  The  gradient  observed  in 
the  accessory  sex  organs  appears  to  depend  both  upon  the  state  of  their  respective 
cells  and  the  state  of  the  interstitial  cells  at  the  beginning  of  each  experiment.  In 
September  the  reproductive  organs  are  atrophic  and  secretory  interstital  cells  are 
virtually  absent  (Fox,  1958).  At  this  time,  a  long  period  of  stimulus  appears 
necessary  since  the  interstitial  cells  must  be  brought  into  activity  before  their 
secretions  can  secondarily  stimulate  the  accessory  sex  organs.  During  later  months 
hypertrophied  interstitial  cells  are  increasingly  more  abundant.  These  probably 
immediately  release  androgenic  hormone  which,  in  turn,  would  bring  about  pro- 
gressively greater  enlargements  of  the  already  partially  hypertrophied  accessory 


sex  organs. 


The  above  explanation  could  account  for  the  marked  response  of  some  individ- 
uals in  Experiments  9  and  10.  Although  the  reproductive  organs  and  interstitial 
cells  of  these  animals  were  declining  at  the  start  of  the  experiments,  they  were  not 
atrophic  and  could  respond  rapidly  to  a  new  stimulus.  Conversely,  the  results 
of  Experiment  7,  in  which  lizards  hastened  into  an  early  atrophy  of  the  reproductive 
organs  by  exposure  to  6L  responded  only  mildly  to  exposure  to  18L,  could  be 
explained  by  the  degree  of  atrophy  at  the  time  the  long  day  stimulus  was  applied. 


434  WADE  FOX  AND  HERBERT  C.  DESSAUER 

On  the  basis  of  data  obtained  by  exposing  normal  and  pinealectomized  anoles  to 
normal  and  long  days,  Clausen  and  Poris  (1937)  have  suggested  that  the  pineal  eye 
inhibits  the  testicular  cycle.  It  is  interesting  to  compare  data  from  our  Experiment 
4  with  their  data  since  the  two  experiments  were  conducted  at  approximately  the 
same  time  of  the  year.  The  testes  weights  of  our  animals  on  either  long  or  short 
days  during  this  period  appear  to  be  nearly  identical  to  those  of  their  pinealectomized 
animals  on  similar  light  regimes.  Of  the  two  samples  they  maintained  on  normal 
day-lengths  the  unoperated  controls  averaged  0.8  g.  less  in  weight  at  the  beginning 
of  the  experiment.  The  average  weight  of  this  sample  (3.92  g.)  suggests  that  it 
was  composed  largely  of  sub-adult  animals.  Since  testis  weight  in  Anolis  has  been 
shown  to  regress  significantly  with  body  size  (Fox,  1958),  the  differences  between 
the  two  samples  could  be  due  to  inequalities  in  sampling.  Similarly,  in  their  two 
samples  kept  on  a  long  day  program,  the  unoperated  animals  averaged  0.7  g.  lighter 
than  the  operated.  However,  even  without  this  consideration  it  is  doubtful  whether 
the  minor  difference  of  1.5  mg.  between  the  mean  testis  weights  would  prove  to 
be  statistically  significant.  In  our  opinion,  the  data  of  Clausen  and  Poris  (1937) 
do  not  afford  adequate  proof  that  the  pineal  eye  acts  as  an  inhibitor  of  the  male 
reproductive  system  in  Anolis. 

Studies  on  Anolis  and  other  species  emphasize  the  importance  of  examining 
reproductive  structures  other  than  the  testes  or  sperm-storing  organs  when  deter- 
mining the  breeding  status  of  an  animal.  The  presence  of  viable  sperm  may  not 
always  be  a  reliable  indicator  as  to  whether  or  not  an  individual  is  in  full  breeding 
condition.  Experiments  1,  2,  and  3  indicate  that  the  response  of  the  accessory  sex 
organs  may  lag  behind  spermatogenesis.  Further,  in  many  species  the  male  normally 
stores  sperm  during  a  non-breeding  stage  of  the  reproductive  cycle  (Fox,  1952; 
Harrington,  1956).  To  determine  the  true  breeding  status  some  reliable  test  for 
the  presence  of  androgenic  secretions  should  accompany  tests  for  viable  sperm. 
In  the  English  sparrow  the  color  of  the  bill  serves  this  purpose  (Keck,  1933).  In 
many  species  the  emergence  of  characteristic  behavioral  patterns  or,  more  directly, 
the  histology  of  the  interstitial  cells  has  been  correlated  with  androgenic  activity. 
We  have  made  only  cursory  observations  on  the  above  criteria  in  this  study.  The 
nuchal  crest,  a  secondary  sex  character,  appeared  to  enlarge  in  animals  brought 
into  full  breeding  condition.  Although  no  detailed  records  of  behavior  were  made, 
we  observed  frequent  attempted  copulations  between  males  exposed  to  long  days  in 
the  winter  and  spring  experiments. 

The  histology  of  the  sexual  segment  of  the  kidney  in  lizards  can  be  used  as  an 
accurate  measure  of  androgenic  activity  and  the  stage  of  the  breeding  cycle.  First, 
cell  height  and  tubule  diameter  can  be  measured  with  precision.  Second,  the 
progressive  spread  of  secretion  granules  can  be  traced  both  intracellularly  (from 
the  apices  to  the  basal  nuclei  of  the  tall  columnar  cells)  and  from  the  ureter  and 
distal  ends  of  the  collecting  ducts  to  the  proximal  collecting  ducts. 

If  the  presence  of  viable  sperm  alone  were  used  as  an  indicator  it  would  appear 
that  male  anoles  were  brought  into  breeding  condition  by  exposure  to  18L  for  60 
days  at  any  season  of  the  year.  However,  examination  of  the  sexual  segment 
reveals  that  none  were  brought  into  breeding  condition  in  experiments  started  in 
September  and  October  and  only  half  of  those  in  experiments  started  in  November. 

Refractoriness  to  photoperiodic  stimulation.     Passerine  birds  characteristically 


DAY-LENGTH  AND  MALE  ANOLIS  CYCLES  435 

-exhibit  a  refractoriness  to  stimulus  by  long  photoperiods  at  the  close  of  the  breeding 
season.  This  refractoriness  persists  until  there  has  been  a  period  of  exposure  to 
long  nights  (Wolfson,  1952).  The  refractory  period  allows  for  the  physiological 
reorganization  of  the  gonadal  and  fat  cycles  (Wolfson,  1954  )  and  is  probably  caused 
by  seasonal  reduction  in  pituitary  activity  (Miller,  1948.  1949  ;  Farner  and  Mewaldt, 
1955). 

We  have  been  unable  to  find  a  period  during  which  Anolis  is  completely  refrac- 
tory to  photoperiodic  stimulation.  The  testes  seem  to  respond,  to  some  extent,  at  all 
seasons  of  the  year  (Fig.  1,  Table  II).  In  all  experiments  except  Nos.  5  and  6, 
which  were  started  near  the  peak  of  testicular  development  (Stage  III),  a  longer 
photoperiod  resulted  in  either  greater  mean  or  maximum  testis  weights.  In  Ex- 
periments 5  and  6,  although  there  was  no  quantitative  increase,  there  was  a  prema- 
ture progression  to  a  more  advanced  stage  of  spermatogenesis.  Experiments 
8,  9,  and  10,  initiated  after  the  peak  of  testicular  size  had  been  achieved, 
yielded  variable  results.  Normally,  animals  at  this  time  should  have  progressed 
from  spermatogenic  Stages  III  or  IV  towards  Stages  V  or  VI  with  the  number  of 
dividing  spermatogonia  and  spermatocytes  rapidly  decreasing.  In  some  individuals, 
however,  all  classes  of  germinal  cells  were  well  represented  so  that  the  gonad 
appeared  to  be  in  Stage  III.  In  other  individuals  spermatogenesis  was  maintained 
at  about  a  Stage  IV  level  without  showing  the  expected  decline.  Tn  still  others, 
germinal  elements  characteristic  of  Stages  I  and  V  were  present  in  the  same  semi- 
niferous tubule,  although  intervening  stages  were  missing.  This  suggests  a  pre- 
mature beginning  of  a  new  cycle  before  the  completion  of  the  old. 

The  sexual  segment  of  the  kidney  appeared  to  be  refractive  in  experiments 
started  in  September  and  only  mildly  responsive  in  October.  Although  this  could 
be  due  to  a  difference  in  the  cyclic  nature  of  the  two  pituitary  gonadotropins  as 
suggested  by  Farner  and  Mewaldt  (1955),  we  believe  that  the  poor  response  of  the 
sexual  segment  can  be  accounted  for  by  the  delay  in  arousing  the  atrophic 
interstitial  cells. 

Infierent  rhythm.  In  studying  the  natural  reproductive  cycle  of  male  anoles, 
Fox  (1958)  noted  that  spermatogenesis  is  initiated  in  the  fall  and  makes  con- 
siderable progress  during  the  winter  despite  the  short  day-lengths.  We  sought 
to  determine  if  there  was  any  period  during  the  year  in  which  constant  exposure 
to  short  days  would  disrupt  the  normal  cycle.  Nine  hours  of  artificial  light  were 
chosen  instead  of  10  (day-length  at  time  of  the  winter  solstice  at  New  Orleans) 
since  10  hours  of  bright  light  obviously  are  not  available  during  the  winter.  Nine 
hours  did  not  disrupt  the  cycle  of  immature  lizards  exposed  from  October  through 
December  and  from  January  through  March,  nor  that  of  adults  exposed  from 
December  through  February.  The  accessory  sex  organs  likewise  were  not  re- 
tarded by  the  short  days.  In  fact,  in  adults  of  the  9L  sample  killed  in  February, 
both  the  testes  and  accessory  sex  organs  were  more  advanced  than  those  of  wild 
lizards  at  that  date.  At  the  time  of  writing,  additional  data  were  furnished  us 
by  Anthony  Dimaggio  of  our  Biochemistry  Department.  Six  immature  males 
(49-57  mm.),  maintained  at  6L  for  60  days  ending  March  1,  appeared  to  have 
normally  active  testes  (left  testis  ranged  12-40  mg.,  mean  =  24.3  ±  11.4  mg.). 
One  must  conclude  that  reasonably  short  day-lengths  are  not  very  effective  in 
disrupting  the  inherent  reproductive  rhythm  of  male  anoles  in  the  fall,  winter  and 
early  spring  when  gametogenesis  is  on  the  upswing. 


436  WADE  FOX  AND  HERBERT  C.  DESSAUER 

Fox  (1958)  also  stated  that  the  peak  of  spermatogenesis  for  most  individuals 
of  this  species  was  achieved  in  April.  Normally,  all  maintained  quite  active 
spermatogenesis  through  July.  Experiment  7  showed  that  six-hour  day-lengths, 
initiated  at  the  end  of  April,  produced  a  significant  reduction  in  testis  weight  within 
34  days,  and  highly  significant  atrophy  in  both  the  testes  and  accessory  sex  organs 
within  52  days.  Nine  hours  of  light  (Experiment  8)  from  May  to  July  also  pre- 
cipitated complete  involution.  Thus,  it  appears  that  short  days  will  end  a  reproduc- 
tive cycle  prematurely  after  the  cycle  has  neared  or  passed  maximum  development. 

The  stimulating  effects  of  different  day-lengths.  Since  our  original  choice  of 
18  hours  for  a  long  day  stimulus  was  arbitrary,  we  designed  three  experiments 
to  test  whether  shorter  periods  might  be  equally  stimulating  but  have  fewer  detri- 
mental effects  upon  the  animals.  In  Experiment  3  all  average  measurements  of 
the  16L  sample  tended  to  be  greater  than  the  18L  sample.  However,  the  larger 
gonads  and  more  secretory  sexual  segments  occurred  in  the  18L  sample.  In 
Experiment  5  a  few  adults  on  16L  gave  a  better  response  than  any  on  18L.  The 
smaller  gonads  of  the  18L  sample  were  judged  on  a  histological  basis  to  be 
more  advanced  than  those  of  the  controls,  but  there  does  not  appear  to  be 
justification  to  similarly  appraise  the  smaller  accessory  sex  organs.  We  believe 
the  data  for  the  18L  sample  reflect  the  exhausting  features  of  long  hours  of  wake- 
fulness  imposed  upon  animals  with  initially  low  fat  reserves,  rather  than  a  lack  of 
stimulation.  Experiment  10  demonstrated  that  exposure  to  14L  from  August  to 
October  was  stimulating  (Fig.  1)  to  the  testes  but  had  little  effect  on  the  accessory 
sex  organs.  On  the  other  hand,  18L  was  very  stimulating  to  both  gonads  and 
accessory  sex  organs. 

These  data  indicate  that,  in  general,  the  longer  the  day-length  the  greater  the 
response  of  the  reproductive  organs  of  Anolis.  A  similar  relationship  between 
day-length  and  gonadal  size  of  the  white-crowned  sparrow  has  been  thoroughly 
analyzed  recently  by  Farner  and  Wilson  (1957).  Eighteen-hour  day-lengths  ap- 
peared detrimental  to  many  anoles  (see  mortalities  and  starvation,  Table  II). 
For  this  reason  a  16-hour  day-length  is  probably  more  satisfactory  to  use  as  a  long 
day  stimulus  for  Anolis.  Fourteen-hour  day-lengths  (the  maximum  day-length 
for  New  Orleans)  are  definitely  stimulatory  to  Anolis,  but  the  response  may  not 
be  sufficiently  rapid  to  be  detected  in  short-term  experiments.  Dessauer  (1953) 
found  no  differences  in  the  metabolism  of  anoles  exposed  for  three  weeks  to  10L 
and  14L.  It  now  seems  likely,  in  view  of  the  considerable  individual  variation 
he  obtained,  that  three  weeks  may  not  have  been  sufficient  or  the  light  regimes  not 
sufficiently  different  to  produce  marked  contrasts  in  metabolism. 

The  response  of  immature  lizards  compared  to  adults.  The  usefulness  of 
immature  anoles  as  experimental  animals  was  impaired  by  their  high  mortality 
rate  which  resulted  from  starvation.  However,  since  they  were  more  abundant 
than  adults  in  the  wild  it  was  easier  to  collect  a  large  series.  They  responded  to 
the  long  day-length  exposure  very  much  as  did  the  adults  without  any  indication 
of  a  refractory  period.  Comparison  of  the  September  experiments  (I  and  II) 
indicates  that  the  non-starving  immature  lizards  responded  as  well  as  or  better  than 
the  adults.  Likewise,  the  maximal  measurements  recorded  in  Experiment  6 
(Table  II,  Fig.  1)  compare  favorably  with  the  best  results  on  adults  in  any  experi- 
ment. The  most  striking  responses  occurred  in  animals  above  55  mm.  (snout-vent 


DAY-LENGTH  AND  MALE  ANOLIS  CYCLES 


437 


mg. 
40- 

35- 

30- 

25- 

20- 

15- 

10- 

5- 


Captive  lizards 
Wild  lizards 


Jan.      Feb.    Mar.    April     May    June    July      Aug.   Sept.    Oct.     Nov.     Dec. 

FIGURE  2.  Curves  comparing  the  annual  variation  in  testis  weights  of  freshly  captured 
wild  anoles  (Fox,  1958)  with  those  of  laboratory  controls  exposed  to  natural  day-lengths  for 
60  days. 


length).  All  individuals  with  markedly  active  reproductive  organs  grew  at  least 
several  millimeters  and  usually  reached  60  mm.  in  snout-vent  length. 

Data  on  animals  maintained  for  periods  longer  than  two  months  suggest  that 
unseasonal  long  photoperiods  are  most  stimulating  during  the  first  two  months. 
Over  a  period  of  four  months  the  controls  matched  the  earlier  achievements  of 
the  experimental  and  the  experimentals  regressed. 

Captive  lizards  exposed  to  natural  day-lengths  compared  to  wild  lizards.  The 
marked  weight  difference  between  testes  of  two-month  captive  lizards  exposed  to 
natural  day-lengths  and  wild  lizards  can  be  seen  in  Figure  2.  From  February 
through  most  of  July  the  testes  of  captive  controls  usually  averaged  significantly 
heavier  than  those  of  wild  animals  sacrificed  at  approximately  the  same  date. 
Bartholomew  (1950)  found  similar  differences  in  captive  and  wild  yucca  night 
lizards  (Xantusia  vigilis}.  He  suggested  that  the  differences  were  due  to  the 
higher  temperature  of  the  laboratory.  In  the  case  of  Anolis,  this  could  easily 
account  for  the  differences  during  the  winter  and  spring  but  it  is  doubtful  whether 
it  would  account  for  the  differences  in  June  and  July. 

SUMMARY 

1.  The  reproductive  system  of  adult  male  anoles  was  stimulated  by  artificially 
lengthened  days  at  all  seasons  of  the  year.  At  least  a  few  males  were  brought 
into  full  breeding  condition  in  experiments  initiated  from  November  through 
August.  Sperm  were  produced  at  all  seasons. 


438  WADE  FOX  AND  HERBERT  C.  DESSAUER 

2.  A  refractory  period,  such  as  occurs  in  passerine  birds,  did  not  appear  to 
be  present  in  Anolis. 

3.  A  previous  exposure  to  short  days  was  not  essential  for  obtaining  a  response 
to  a  long  photoperiod. 

4.  Both  the  testes  and  accessory  sex  organs  exhibited  progressively  greater 
responses  in  successive  experiments  through  the  fall,  winter,  and  spring. 

5.  The  seasonal  gradient  of  response  which  produced  increasingly  larger  testes 
could  be  accounted  for  by  the  increasingly  larger  number  of  spermatogonia  at  the 
beginning  of  each  experiment.     The  gradient   in   response  of   the  accessory   sex 
organs  was  dependent  on  the  normal  cyclic  fluctuations  of  their  histology  and  the 
abundance  of  hypertrophied  interstitial  cells. 

6.  The  inherent  rhythms  of  both  the  gonad  and  accessory  sex  organs  were  not 
disturbed  by  60  days'  exposure  to  9-  or  6-hour  photoperiods  during  the  fall  and 
winter.     However,  such  exposures  near  or  after  the  peak  of  spennatogenesis  re- 
sulted in  premature  atrophy  of  all  reproductive  organs. 

7.  Comparison  of  the  effects  of  14-,  16-,  and  18-hour  day-lengths  suggested  that 
the  longer  the  day-length  the  more  rapid  the  response.     Extremely   long   days, 
however,  appeared  to  have  detrimental  effects  upon  some  individuals. 

8.  Most  immature  lizards  of  less  than   55   mm.   snout-vent   length  responded 
poorly  to  the  experimental   conditions.     All   that   responded   well   were   growing 
rapidly.     Those  that  grew  to  60  mm.  or  more  had  reproductive  organs  as  large  or 
larger  than  older  adults. 

9.  Captive  lizards  exposed  to  natural  day-length  for  60  days  at  any  time  be- 
tween February  and  July  tended  to  have  larger  testes  than  wild  lizards  sacrificed 
on  the  same  date. 

LITERATURE  CITED 

BARTHOLOMEW,  G.  A.,  JR.,   1950.     The  effects  of  artificially  controlled  temperature  and  day 

length  on  gonadal  development  in  a  lizard,  Xantusia  vigilis.    Anat.  Rec.,  106:  49-59. 
BARTHOLOMEW,  G.  A.,  JR.,  1953.     The  modification  by  temperature  of  the  photoperiodic  control 

of  gonadal  development  in  the  lizard  Xantusia  vigilis.     Copeia,  1953:  45-50. 
BURGER,   J.   W.,    1937.     Experimental   sexual   photoperiodicity   in   the   male   turtle,   Pseudcmys 

elcgans   (Wied.).     Amcr.  Nat.,  71:  481-487. 
BURGER,  J.  W.,   1949.     A  review  of  experimental   investigations  on   seasonal   reproduction   in 

birds.     Wilson  Bull.,  61:  211-230. 
CLAUSEN,  H.  J.,  AND  E.  G.  PORIS,  1937.     The  effect  of  light  upon  sexual  activity  in  the  lizard, 

Anolis  carollnensis,  with  especial  reference  to  the  pineal  body.     Anat.  Rec.,  69:  39-50. 
DESSAUER,  H.  C.,  1953.     Hibernation  of  the  lizard,  Anolis  carollnensis.    Proc.  Soc.  Exp.  Biol. 

Mcd.,  82:   351-353. 
EARNER,    D.    S.,    1955.     The    annual    stimulus    for    migration :    experimental    and    physiologic 

aspects.     In:   Recent  Studies  in  Avian  Biology,  Ed.  by  A.  Wolf  son,  Publ.  by  Amer. 

Ornithol.  Union,  Univ.   111.   Press,  Urbana. 
EARNER,  D.  S.,  AND  L.  R.  MEWALDT,  1955.     The  natural  termination  of  the  refractory  period 

in  the  white-crowned  sparrow.     Condor,  57:  112-116. 
EARNER,  D.  S.,  AND  A.  C.  WILSON,  1957.     A  quantitative  examination  of  testicular  growth  in 

the  white-crowned  sparrow.     Biol.  Bull.,  113:  254-267. 
FORBES,  T.  R.,  1941.     Observations  on  the  urogenital  anatomy  of  the  adult  male  lizard,  Scelo- 

porus,  and  on  the  action  of  implanted  pellets  of  testosterone  and  esterone.     /.  Morph., 

68:  31-69. 
Fox,  W.,  1952.     Seasonal  variation  in  the  male  reproductive  system  of  Pacific  Coast  garter 

snakes.     /.  Morph.,  90:  481-553. 
Fox,  W.,  1958.     Sexual  Cycle  of  the  male  lizard,  Anolis  carolinensis.     Copeia,   1958:  22-29. 


DAY-LENGTH  AND  MALE  ANOLIS  CYCLES  439 

Fox,  W.,  AND  H.  C.  DESSAUER,  1957.     Photoperiodic  stimulation  of  appetite  and  growth  in  the 

male  lizard,  Anolis  carolinensis.    J.  Exp.  Zool.,  134 :  557-575. 
HAMMOND,  J.,  JR.,   1954.     Light  regulation   of  hormone   secretion.     Vitamins  and  Hormones, 

12:   157-206.     Academic  Press   Inc.,  N.  Y. 
HARRINGTON,  R.  W.,  JR.,  1956.     An  experiment  on  the  effects  of  contrasting  daily  photoperiods 

on    gametogenesis    and    reproduction    in    the    centrarchid    fish,    Enneacanthus    obcsus 

(Girard).     /.  Exp.  Zool.,  131:  203-223. 
KECK,  W.  N.,  1933.     Control  of  bill  color  of  the  male  English  sparrow  by  injection  of  male 

hormone.     Proc.  Soc.  Exp.  Biol.  Med.,  30:  1140-1141. 
MILLER,   A.    H.,    1948.     The    refractory   period   in   light-induced    reproductive    development    of 

golden-crowned  sparrows.     /.  Exp.  Zool.,  109:  1-11. 
MILLER,  A.  H.,   1949.     Potentiality  for  testicular  recrudescence  during  the  annual   refractory 

period  of  the  golden-crowned  sparrow.     Science,  109 :   546. 
MILLER,  A.  H.,  1954.     The  occurrence  and  maintenance  of  the  refractory  period  in  crowned 

sparrows.     Condor,  56:  13-20. 
REYNOLDS,  A.  E.,  1943.     The  normal  seasonal  reproductive  cycle  in  the  male  Eumeces  fasciatus 

together  with  some  observations  on  the  effects  of  castration  and  hormone  administra- 
tion.    /.  Morph.,  72 :  331-377. 
VAN  OORDT,  P.  G.  W.  J.,  1956.     The  role  of  temperature  in  regulating  the  spermatogenetic 

cycles  in  the  common  frog  (Rana  temporaria) .    Ada  Endocrinol.,  23:  251-264. 
VAUGIEN,  L.,   1955.     Sur  les  reactions  testiculaires  du  jeune  moineau  domestique   illumine  a 

diverses  epoques  de  la  mauvaise  saison.     Bull.  Biol.  France  et  Bclgiquc,  89:  218-243. 
WOLFSON,  A.,   1952.     The  occurrence  and  regulation   of  the   refractory   period   in   the   gonadal 

and  fat  cycles  of  the  junco.     /.  Exp.  Zool,  121 :  311-326. 
WOLFSON,  A.,  1954.     Production  of  repeated  gonadal,  fat,  and  molt  cycles  within  one  year  in 

the  junco  and  white-crowned  sparrow  by  manipulation  of  day  length.     /.   Exp.  Zool., 

125  :  353-376. 


A  PERSISTENT  DIURNAL  RHYTHM  OF  LUMINESCENCE 
IN  GONYAULAX  POLYEDRA x 

J.  WOODLAND   HASTINGS  AND  BEATRICE  M.  SWEENEY 

Division  of  Biochemistry,  Noyes  Laboratory  of  Chemistry,   University  of  Illinois, 

Urbana,  Illinois,  and  Division  of  Marine  Biology,  The  Scripts  Institution 

of  Oceanography,  La  Jolla,  California 

The  photosynthetic  marine  dinoflagellate,  Gonyaulax  polyedra,  emits  a  brief 
flash  of  light  (duration,  about  90  milliseconds)  when  stimulated  by  agitation.  It 
is  one  of  the  many  organisms  responsible  for  the  luminescent  display  sometimes 
observed  in  the  ocean  at  night  when  the  water  is  disturbed  (see  Harvey,  1952). 
Previous  studies  with  this  organism  (Haxo  and  Sweeney.  1955;  Sweeney  and 
Hastings,  1957a)  have  shown  that  the  luminescent  response  to  stimulation  varies 
rhythmically  in  a  diurnal  fashion.  Cultures  grown  in  natural  illumination,  or  in 
artificial  lights  with  alternating  light  and  dark  periods  of  12  hours  each  (==  LD), 
display  a  much  greater  luminescence  during  the  dark  period  (Fig.  2). 

When  LD  cultures  are  transferred  to  a  dark  chamber,  the  rhythm  continues 
but  its  amplitude  decreases  progressively.  By  action  spectra  studies,  it  has  been 
found  (Sweeney,  Haxo  and  Hastings,  unpublished  data)  that  this  decrease  in 
amplitude  arises  from  the  need  for  light  in  the  organic  nutrition  of  Gonyaula.r, 
via  photosynthesis.  This  rinding  prompted  the  search  for  constant  environmental 
conditions  under  which  the  endogenous  rhythm  would  persist,  without  the  loss 
of  amplitude  which  occurs  in  continuous  darkness. 

The  possibility  of  maintaining  the  cells  heterotrophically  was  explored,  but  the 
consistently  negative  results  obtained  indicated  that  Gonyanla.r  is  an  obligate 
photo-auxotroph.  Continuous  bright  light  inhibits  the  rhythmic  fluctuations  in 
luminescence,  and  it  has  not  been  possible  to  separate,  by  using  light  of  different 
colors,  the  photosynthetic  requirements  for  light  from  the  inhibitory  action  of 
light  on  rhythmicity.  It  has  been  found,  however,  that  if  LD  cultures  are  placed 
in  a  continuous  dim  light,  the  rhythm  of  luminescence  persists  without  loss  of 
amplitude.  It  has  thus  been  possible  to  investigate  in  some  detail  the  nature  of 


this  endogenous  rhythm. 


MATERIALS  AND  METHODS 


G.  polyedra  has  been  maintained  in  a  modified  sea  water  medium  described 
previously  (Sweeney  and  Hastings,  1957a).  The  growth  rate  is  dependent  upon 
light,  temperature,  and  the  concentrations  of  mineral  nutrients.  The  maximum 
growth  rate  which  we  have  measured  is  one  division  per  day,  but  under  the  condi- 

1  This  research  has  been  supported  in  part  by  grants  from  The  National  Science  Founda- 
tion, the  Graduate  Schools  of  Northwestern  University  and  the  University  of  Illinois,  and  the 
Marine  Life  Research  Program  of  the  California  Cooperative  Oceanic  Fisheries  Investigation. 
Part  of  the  research  was  carried  out  at  Northwestern  University,  Evanston,  Illinois.  Contri- 
bution from  the  Scripps  Institution  of  Oceanography,  new  series. 

440 


RHYTHM  OF  LUMINESCENCE  441 

tions  used  in  the  experiments  to  be  described  the  rates  were  always  less  than  this. 
The  illumination  was  provided  by  "cool  white"  fluorescent  lamps,  the  intensity 
being  measured  in  foot-candles  with  a  Weston  illumination  meter. 

The  experimental  procedure  was  as  follows :  stock  cultures  were  maintained 
in  Fernbach  flasks  containing  1500  ml.  of  medium.  In  preparation  for  an  experi- 
ment, 2-ml.  aliquots  from  these  cultures  were  pipetted  into  each  of  several  hundred 
test  tubes  at  cell  densities  between  2000  and  7500  cells  per  ml.  All  tubes  were 
then  subjected  to  the  appropriate  conditions  of  light  and  temperature.  To  measure 
the  luminescence  at  any  given  time,  two  tubes  were  removed,  assayed,  and  then 
discarded.  The  cells  were  stimulated  to  luminesce  by  bubbling  air  through  the 
cell  suspension,  and  the  resulting  phototube  current  was  accumulated  on  a  ca- 
pacitor. Luminescence  is  expressed  in  terms  of  the  total  amount  of  light  emitted 
during  one  minute  of  stimulation,  at  the  end  of  which  time  essentially  all  lumines- 
cence has  ceased.  Additional  details  of  the  light  measurement  procedure  may  be 
found  elsewhere  (Sweeney  and  Hastings,  1957a). 

RESULTS 

Demonstration  of  the  persistent  rhythm.  A  persistent  rhythm  of  luminescence 
may  be  observed  if  cells  which  have  been  kept  for  a  time  under  LD  conditions  are 
transferred  to  continuous  dim  light  (about  100  foot-candles).  A  typical  example 
of  the  persistent  rhythm  under  conditions  of  constant  light  and  constant  tempera- 
ture is  shown  in  Figure  5.  In  similar  experiments,  we  have  continued  measure- 
ments for  as  long  as  14  days ;  the  rhythmic  pattern  continues  undamped  during 
this  time.  At  the  light  intensity  used  in  such  experiments  there  was  little  growth. 

The  natural  period  of  the  rhythm.  The  period  of  the  rhythm  is  measured  by 
the  time  between  successive  maxima  in  luminescence.  When  the  cells  are  sub- 
jected to  alternating  light  and  dark  periods  on  a  daily  (24-hour)  schedule,  the 
period  of  the  rhythm  is  24  hours  ( Fig.  2 ) .  Under  conditions  of  constant  illumina- 
tion, however,  the  rhythmic  changes  have  a  period  which  is  close  to,  but  not  neces- 
sarily exactly  24  hours.  Pittendrigh  and  Bruce  (1957)  have  referred  to  this  as 
the  natural  period,  or  the  innate  period  of  an  endogenous  rhythm  when  light  and 
temperature  are  held  constant. 

The  natural  period  in  Gonyaula.v  is  a  function  of  at  least  two  environmental 
factors,  light  intensity  and  temperature.  The  effect  of  light  intensity  upon  the 
period  is  illustrated  in  Figure  1.  Cells  were  placed  in  continuous  light  at  three 
different  intensities,  and  it  is  evident  that  the  natural  period  was  shorter  at  higher 
intensities.  These  experiments  also  illustrate  the  light  intensity  dependence  of 
the  inhibitory  effect  of  continuous  illumination  upon  the  rhythm.  At  the  two 
higher  light  intensities  the  amplitude  of  the  rhythm  was  progressively  damped, 
while  at  the  lowest  light  intensity  no  marked  damping  of  the  amplitude  of  the 
rhythm  was  evident. 

The  effect  of  temperature  upon  the  natural  period  is  not  large  but,  contrary 
to  expectation,  the  period  becomes  longer  rather  than  shorter  as  the  temperature 
is  raised  (Hastings  and  Sweeney,  1957b).  At  16°  C.  the  period  was  found  to 
be  22.8  hours  while  at  26.7°  C.  it  was  26.5  hours.  A  O10  of  less  than  1.0  is 
unusual,  and  the  results  were  interpreted  as  evidence  for  a  compensation  mech- 
anism which  functions  to  keep  the  period  approximately  temperature-independent. 


442 

12 


J.  WOODLAND  HASTINGS  AND  BEATRICE  M.  SWEENEY 


20 


~AO 


60  80 

TIME-  HOURS 


10O 


120 


145 


FIGURE  1.  The  effect  of  light  intensity  upon  the  natural  period  at  constant  temperature 
(21°  C.)-  The  cells  were  grown  in  LD  conditions  (800  foot-candles  during  the  light  period). 
The  beginning  of  the  experiment,  shown  on  the  graph  as  0  time,  fell  at  the  end  of  a  normal 
light  period.  At  this  time,  some  cells  were  placed  in  the  dark,  and  others  in  light  of  120  foot- 
candles  (upper  curve),  380  foot-candles  (middle)  and  680  foot-candles  (bottom).  The  average 
periods  were  as  follows :  680  foot-candles,  22.0  hours ;  380  foot-candles,  22.8  hours ;  120  foot- 
candles,  24.5  hours;  dark,  24.5  hours  (not  shown  on  graph;  one  period  measured). 

In  view  of  the  relatively  small  temperature  effect,  the  period  of  this  rhythm  may 
be  characterized  as  essentially  temperature-independent. 

The  endogenous  nature  of  the  diurnal  rhythm.  The  persistence  of  the  rhythm 
of  luminescence  under  conditions  of  constant  temperature  and  light  intensity  indi- 
cates that  the  mechanism  of  the  rhythmicity  is  endogenous.  Several  other  experi- 
ments serve  to  support  this  conclusion. 

Figure  2  illustrates  one  of  many  experiments  in  which  the  phase  of  the  rhythm 
was  shifted  by  changing  the  time  at  which  the  light  and  dark  periods  occurred. 
In  such  experiments  the  phase  (i.e.,  the  solar  time  at  which  the  maximum  in 
luminescence  occurs)  may  be  shifted  so  that  it  will  bear  any  desired  relationship 
to  the  solar  day.  In  cultures  which  are  subsequently  transferred  to  constant  con- 
ditions of  dim  light  or  darkness,  the  phase  of  the  persistent  rhythm  is  related  to 
the  previous  light  and  dark  program  rather  than  to  solar  time,  or  any  other  factor. 
Changes  in  the  phase  of  the  endogenous  rhythm  have  not  been  observed  when 
light  and  temperatures  were  held  constant. 


RHYTHM  OF  LUMINESCENCE 


443 


o 

o    3 
en 

UJ 

I ' 

->   5 
3 


12          24          36          48          60 

HOURS 


72 


84 


96 


FIGURE  2.  This  experiment  illustrates  the  effect  of  changing  the  solar  time  at  which 
tli  light  and  dark  periods  occur.  The  upper  curve  shows  the  pattern  of  luminescence  changes 
in  an  LD  culture  which  had  been  on  the  schedule  indicated  for  some  time.  The  black  bars 
on  the  time  axis  indicate  dark  periods.  The  lower  two  graphs  illustrate  the  effect  of  imposing 
upon  cultures  (which  were  previously  on  the  schedule  shown  in  the  top  graph)  an  LD  schedule 
in  which  the  light  and  dark  periods  were  at  a  different  time  of  day.  The  new  schedules  were 
started  at  zero  hours  on  the  graph.  Temperature,  about  26°  C.  Light  intensities  used,  about 
250  foot-candles. 


A  series  of  experiments  has  been  carried  out  from  which  it  is  evident  that 
pre-treatment  with  diurnal  light  and  dark  periods  (i.e.,  one  dark  plus  one  light 
period  equals  24  hours)  is  not  necessary  in  order  to  demonstrate  an  endogenous 
rhythm.  That  is  to  say,  there  is  no  evidence  that  a  "learning"  or  "memory" 
process  is  involved.  For  example,  cells  have  been  exposed  to  "non-diurnal"  light 
and  dark  periods  which  together  add  up  to  greater  or  less  than  24  hours,  followed 
by  conditions  of  either  constant  light  or  constant  dark.  An  experiment  of  this 
sort  is  shown  in  Figure  3.  In  this  experiment,  cells  were  exposed  to  alternating 
light  and  dark  periods  of  7  hours  each  for  about  100  hours.  During  this  period 
the  luminescence  changes  were  quite  evidently  governed  by  these  light  and  dark 
periods  so  that  there  was  a  maximum  in  luminescence  every  14  hours.  At  the 
end  of  this  treatment,  some  cells  were  placed  in  constant  dim  light  and  others  in 
darkness.  In  both  cases  a  diurnal  rhythm  with  a  period  of  approximately  24  hours 
was  evident.  The  14-hour  cycle  had  not  been  "learned,"  even  though  it  had  been 


444 


J.  WOODLAND  HASTINGS  AND  BEATRICE  M.  SWEENEY 


possible  to  entrain  the  luminescence  rhythm  to  the  14-hour  cycle.  A  difference 
between  those  placed  in  darkness  and  those  in  dim  light  was  that  the  amplitude 
of  the  rhythm  in  darkness  progressively  decreased  as  a  result  of  the  lack  of  light 
(see  introduction). 

Similar  experiments  have  been  carried  out  in  which  the  alternating  light  and 
dark  periods  were  6  hours  each,  8  hours  each,  and  16  hours  each,  giving  cycles 
of  12,  16  and  32  hours,  respectively.  The  results  were  similar  to  those  shown 
in  Figure  3.  After  about  100  hours  of  such  a  non-diurnal  light-dark  cycle  the 
cells  were  placed  in  constant  dim  light  and  a  rhythm  of  luminescence  having  a 
period  close  to  24  hours  was  evident. 

Another  series  of  experiments  has  shown  that  it  is  not  necessary  to  pre-treat 
the  cells  with  any  sort  of  alternating  light  and  dark  periods  in  order  to  demonstrate 
endogenous  diurnal  rhythmicity.  As  mentioned  previously,  if  cells  are  grown  in 
continuous  bright  light  (ca.  800-1500  foot-candles)  there  is  no  detectible  rhyth- 
micity. Cells  maintained  in  this  way  for  several  months,  or  for  as  long  as  several 
years,  have  been  found  to  exhibit  a  diurnal  rhythmicity  when  they  are  placed  in 
darkness  (Haxo  and  Sweeney,  1955;  Sweeney  and  Hastings,  1957a).  The  phase 
of  the  rhythm  which  is  initiated  when  the  cells  are  moved  from  bright  light  to 
darkness  is  independent  of  the  solar  time,  and  related  only  to  the  time  at  which 
the  light-to-dark  transition  is  made. 

A  similar  result  was  obtained  when  cells  which  had  been  grown  in  bright  light 
for  almost  one  year  were  merely  transferred  to  dim  light.  This  experiment  is 


32 

28- 

24 


220 

UJ 

O 
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y  16 


3,2 


10        20        30       40        50       60       70 


80       90       100     110 
TIME    -  HOURS 


120      130      140     150      160      170      180      190 


FIGURE  3.  This  illustrates  the  entrainment  of  the  luminescence  rhythm  to  a  14-hour  cycle 
and  the  manifestation  of  an  endogenous  diurnal  rhythm  when  the  cells  are  placed  in  constant 
conditions  subsequent  to  the  treatment.  Dark  periods  are  indicated  by  black  bars  on  the  time 
axis.  The  cells  were  on  an  LD  schedule  previous  to  the  time  when  the  14-hour  cycle  was 
started  (at  26  hours).  Light  intensity  throughout  the  14-hour  cycling  was  800  foot-candles. 
At  117  hours  some  aliquots  were  removed  from  the  dark  and  placed  in  constant  light  at  230 
foot-candles.  The  luminescence  changes  in  these  cultures  are  shown  by  the  circles.  From 
124  hours  on,  the  other  aliquots  were  left  in  the  dark  and  the  luminescence  changes  are  plotted 
with  solid  dots.  Temperature,  21°  C. 


RHYTHM  OF  LUMINESCENCE 


445 


O 


O 

in 
LJ 


40 


60  80 

TIME  -  HOURS 


100 


120 


140 


FIGURE  4.  The  initiation  of  an  endogenous  diurnal  rhythm  of  luminescence  by  means  of 
a  one-step  change  in  illumination.  Cultures  which  had  been  grown  in  bright  light  for  one 
year  were  moved  from  bright  light  (800  foot-candles)  to  dim  light  (90  foot-candles)  at  the 
time  indicated  on  the  graph  as  0  hours.  Luminescence  measurements  were  made  approximately 
every  two  hours  thereafter.  Temperature,  21.0°  C.  Average  period,  24.5  hours. 

illustrated  in  Figure  4.  It  differs  from  the  previously  mentioned  experiment 
(in  which  cultures  were  moved  from  bright  light  to  darkness)  in  that  the  ampli- 
tude does  not  decrease  with  time,  since  light  is  available  for  the  nutrition  of  the 
cells.  The  precise  phase  relationship  to  the  time  of  transfer  from  bright  light 
is  somewhat  different,  but  here  also  it  is  not  related  to  solar  time. 

Phase  shift  by  light  perturbation.  It  is  clear  from  Figure  2  that  the  phase 
of  the  rhythm  may  readily  be  shifted  by  an  appropriate  manipulation  of  the  light 
and  dark  periods  to  which  the  cells  are  exposed.  It  is  not  necessary,  however, 
to  expose  the  cells  to  a  new  light-dark  cycle  in  order  to  reset  the  phase  of  the 
rhythm.  A  single  exposure  to  a  different  light  intensity  can  result  in  a  stable 
phase  shift.  Pittendrigh  and  Bruce  (1957)  have  discussed  the  significance  of 
phase  resetting  of  biological  rhythms  by  single  light  perturbations.  If  rhythmicity 
results  from  an  innate  oscillatory  mechanism  characterized  by  its  own  natural 
period,  and  the  phase  (but  not  the  period)  is  determined  by  the  sequence  of  light 
and  darkness,  then  it  is  to  be  expected  that  non-repeated  light  changes  should 
suffice  to  change  the  phase.  The  perturbation  therefore  need  not  contain  any 
information  concerning  period. 

The  experiment  shown  in  Figure  4  illustrates  phase  setting  by  a  single  step- 
type  light  perturbation.  The  phase  of  the  previously  aperiodic  cells  was  deter- 
mined by  the  time  at  which  the  light  intensity  was  changed.  The  shifting  of  phase 
in  already  rhythmic  cultures  is  evident  in  the  experiments  shown  in  Figure  3. 
The  entrainment  of  the  rhythm  to  a  14-hour  cycle  may  be  explained  by  assuming 
that  each  transition,  either  from  darkness  to  light  or  from  light  to  darkness,  serves 
to  shift  the  phase,  so  that  repetitive  phase  resetting  occurs. 

A  phase  shift  in  the  Gonyaula.v  rhythm  by  single  light  perturbations  has  also 
been  demonstrated  in  other  ways.  Figure  5  illustrates  a  shift  in  the  phase  of 
rhythmic  cells  which  were  given  a  single  exposure  to  either  bright  light  or  darkness. 
The  phase  shift  which  results  in  such  experiments  has  been  found  to  be  stable 
since,  in  experiments  where  measurements  were  continued  for  an  additional  48 
hours,  the  phase  difference  between  the  controls  and  the  treated  cells  remained 
unchanged. 


446 


I.  WOODLAND  HASTINGS  AND  BEATRICE  M.  SWEENEY 


BRIGHT  LIGHT 


40        50        60 
TIME -HOURS 


100 


FIGURE  5.  This  experiment  illustrates  a  phase  shift  in  the  rhythm  following  changes  in 
light  intensity.  Cells  previously  kept  under  LD  conditions  were  placed  at  constant  tempera- 
ture (23.5°  C.)  and  constant  light  intensity  (100  foot-candles)  at  the  end  of  a  12-hour  dark 
period.  Two  days  later  (zero  time  on  the  graph)  measurements  of  luminescence  were  begun 
and  the  endogenous  rhythm  was  apparent.  Some  cultures  (upper  curve)  were  transferred  to 
bright  light  (1400  foot-candles)  for  a  period  of  6  hours  and  then  returned  to  the  previous 
condition  (100  foot-candles).  Other  cultures  (middle  curve)  were  transferred  to  darkness 
for  6  hours  and  returned  to  dim  light  at  200  foot-candles.  The  time  at  which  treatment  was 
given  is  indicated  by  bars  on  the  time  axis.  In  both  cases  a  marked  phase  shift  in  the  rhythm  is 
evident.  The  control  (lower  curve)  was  left  in  dim  light  all  the  while.  Average  period  in 
control :  25.7  hours. 

Figure  6  shows  another  technique  which  has  been  used  in  the  study  of  phase 
shifting  by  single  perturbations.  Rhythmic  cells  were  placed  in  the  dark  and, 
at  a  later  time,  received  an  exposure  to  light.  Although  the  amplitude  of  the 
rhythm  decreases  over  the  next  few  days,  the  times  at  which  maxima  in  lumines- 
cence occur  are  evident,  so  that  the  phase  may  be  determined.  The  number  of 
hours  by  which  the  phase  is  shifted  would  be  expected  to  be  some  function  of  both 
the  magnitude  of  the  perturbation,  and  the  time  in  the  old  cycle  at  which  it  is 
administered.  The  technique  of  interrupting  darkness  by  light  has  been  used  to 
investigate  these  parameters. 


RHYTHM  OF  LUMINESCENCE 


447 


LJ 
O 
Z 
LiJ 
O 
(f) 
LL) 


380  FOOT  CANDLES 


FOOT 
CANDLES 


120  FOOT  CANDLES 


200   FOOT  CANDLES 


45   FOOT 

CANDLES 


DARK    CONTROL 


0 


50       60 


70 


TIME  -HOURS 


FIGURE  6.  This  illustrates  phase  shifting  in  a  rhythmic  culture  by  a  single  2% -hour 
exposure  to  light,  and  the  effect  of  intensity  upon  the  magnitude  of  the  phase  shift.  Prior  to 
the  time  shown  on  the  graph,  all  cultures  were  in  LD  conditions,  and  "two  hours"  on  the  time 
axis  was  the  end  of  the  last  12-hour  light  period.  All  cultures  were  put  in  the  dark  at  that 
time  and  the  control  was  left  in  the  dark  thereafter.  The  remaining  cells  were  exposed  to 
a  2%-hour  illumination  beginning  6  hours  after  the  light-to-dark  transition  (indicated  by  the 
rectangle  on  the  time  axis).  Following  this  2%-hour  illumination  they  were  returned  to 
darkness  for  the  remaining  time.  The  intensities  used  are  shown  in  the  figure.  A  21/L>-hour 
exposure  to  1400  foot-candles  (not  plotted)  was  found  to  be  no  more  effective  than  the  ex- 
posure to  660  foot-candles  (Fig.  7).  Temperature  during  experiment,  21°  C. 

The  effect  of  varying  the  light  intensity  was  determined  in  experiments  such 
as  the  one  shown  in  Figure  6.  The  amount  of  phase  shift  was  found  to  increase 
with  increasing  light  intensities,  up  to  a  "saturation"  value  of  about  800  foot- 
candles.  This  relationship  is  illustrated  in  Figure  7,  and  the  stability  of  the  re- 
setting is  shown  by  plotting  on  the  same  graph  the  phase  shift  measured  at  each 
of  the  subsequent  cycles.  Several  experiments  of  this  sort  have  been  carried  out 
and  the  same  type  of  relationship  has  been  observed.  The  quantitative  values  ob- 
tained in  separate  experiments  were  somewhat  different,  however,  and  the  reason 
for  this  variation  has  not  been  determined. 


448 


J.  WOODLAND  HASTINGS  AND  BEATRICE  M.  SWEENEY 


The  magnitude  of  the  perturbation  may  also  be  changed  by  varying  the  dura- 
tion of  light  exposure.  In  an  experiment  similar  to  that  shown  in  Figure  6, 
the  duration  instead  of  the  intensity  was  varied.  All  exposures  (at  800  foot- 
candles)  were  started  simultaneously,  six  hours  after  the  cells  were  placed  in 
darkness.  A  longer  exposure  to  such  a  light  perturbation  was  found  to  be  more 
effective  than  a  shorter  exposure.  The  amount  of  phase  shift  was  found  to  be 
proportional  to  the  duration  of  the  exposure,  up  to  a  maximum  phase  shift  of  about 
111/2  hours,  which  was  achieved  with  2I/1.  hours  exposure.  The  relationship  be- 
tween phase  shift  and  duration  might  be  expected  to  be  different,  depending  upon 
the  time  in  the  old  cycle  at  which  the  perturbations  were  given,  as  discussed  below. 
This  aspect  has  not  been  studied,  however. 

The  effect  of  varying  the  time  in  the  cycle  at  which  the  perturbation  is  given 
has  been  studied  by  again  using  a  procedure  similar  to  that  used  in  the  experiments 
shown  in  Figure  6.  Cells  grown  in  LD  conditions  were  transferred  to  a  dark 


12 


10 


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en 

UJ 

en 
<  4 

Q_ 


o    FIRST    MAXIMUM 
•    SECOND  MAXIMUM 
A  THIRD   MAXIMUM 


200      400      600       800       1000     1200 
LIGHT  INTENSITY -FOOT  CANDLES 


1400 


FIGURE  7.  The  relationship  between  the  intensity  of  a  single  21X>-hour  light  perturbation 
and  the  number  of  hours  by  which  the  phase  is  shifted.  Data  taken  from  the  experiments 
shown  in  Figure  6.  Different  symbols,  as  marked  on  the  graph,  give  the  phase  difference 
between  the  control  and  the  experimentals,  measured  at  each  of  the  three  maxima  in  lumi- 
nescence subsequent  to  the  perturbation. 


RHYTHM  OF  LUMINESCENCE 


449 


10 


TIME  -HOURS 


FIGURE  8.  The  effect  of  light  perturbations  (1400  foot-candles  for  3  hours)  given  at  dif- 
ferent times  during  the  cycle,  upon  the  phase  of  the  endogenous  rhythm.  Cells  which  had 
been  kept  under  LD  conditions  were  placed  in  the  dark  at  zero  time  on  the  graph,  which  was 
the  end  of  a  12-hour  light  period.  The  times  at  which  the  maxima  in  luminescence  occurred 
in  the  control,  which  remained  in  the  dark  all  the  while,  are  indicated  by  vertical  lines.  In 
the  experimentals,  a  triangular  symbol  shows  a  time  at  which  a  maximum  in  luminescence 
occurred,  and  thus  represents  phase.  The  experiments  were  carried  out  in  a  way  similar  to 
those  illustrated  in  Figure  6.  Each  horizontal  line  represents  a  different  experiment.  For  ex- 
ample, the  line  at  11  hours  on  the  ordinate  was  an  experiment  in  which  a  maximum  in 
luminescence  occurred  at  7  hours.  A  light  perturbation  was  begun  at  11  hours  and  terminated 
at  14  hours.  Maxima  in  luminescence  occurred  subsequently  at  29M>  hours  and  52%  hours. 
The  other  experiments  are  represented  in  a  similar  way.  The  relationship  between  the  time 
in  the  cycle  at  which  the  light  perturbation  was  administered  and  the  number  of  hours  by 
which  the  phase  was  changed  may  be  better  visualized  by  rotating  the  figure  by  90°. 

chamber  at  the  end  of  a  light  period.  At  regular  time  intervals  thereafter,  some 
of  the  cells  were  removed  and  exposed  for  three  hours  to  light  at  an  intensity  of 
1400  foot-candles,  and  then  returned  to  darkness.  Times  at  which  exposures  to 
light  were  made  were  selected  so  that  the  experiment  served  to  scan  somewhat 
more  than  a  full  24-hour  cycle.  A  control  received  no  exposure  to  light,  and  the 
times  at  which  maxima  in  luminescence  occurred  in  this  control  are  indicated  by 
the  vertical  lines  in  Figure  8. 

The  results  of  the  experiments  are  summarized  in  Figure  8.  First  of  all,  it 
may  be  noted  that  the  new  phase,  following  a  light  perturbation,  was  not  directly 
related  to  the  time  at  which  the  light  perturbation  was  administered.  That  is,  the 
maxima  in  luminescence  did  not  occur  at  a  fixed  time  interval  following  the  light 
treatment.  If  that  had  been  the  case,  the  symbols  indicating  phase  would  fall  along 
a  line  at  45°,  parallel  to  the  lines  representing  the  times  at  which  light  exposures 
occurred.  This  latter  type  of  result  was  obtained  in  experiments  mentioned  pre- 
viously (Figure  4,  for  example)  where  a  rhythm  was  initiated  in  an  arrhythmic 
culture,  and  the  phase  was  determined  only  by  the  time  at  which  the  light  intensity 
was  changed. 


450  J.  WOODLAND  HASTINGS  AND  BEATRICE  M.  SWEENEY 

Secondly,  it  is  apparent  that  the  sensitivity  to  light  perturbations  was  greater 
during  the  first  12  hours  (Fig.  8)  than  during  the  second  12  hours  of  the  cycle. 
During  the  first  12  hours  a  rather  pronounced  phase  shift  resulted,  whereas  during 
the  second  12  hours  there  was  little  or  no  phase  shift.  In  other  longer  term  ex- 
periments it  has  been  found  that  this  variation  in  sensitivity  continues  in  a  rhythmic 
way.  It  may  therefore  be  stated  that,  in  general,  the  cells  are  maximally  sensitive 
to  a  light  perturbation  at  a  time  when  luminescence  is  near  maximum,  and  that 
this  sensitivity  declines  to  a  minimum  at  a  time  when  luminescence  is  minimum. 

Finally,  however,  it  may  be  noted  from  Figure  8  that  a  light  exposure  given 
before  the  maximum  in  luminescence  results  in  a  phase  delay,  so  that  the  time 
between  the  light  perturbation  and  the  subsequent  maximum  in  luminescence  is 
greater  than  24  hours.  On  the  other  hand,  a  light  exposure  given  after  the  maxi- 
mum in  luminescence  results  in  a  phase  advance,  such  that  the  next  maximum  in 
luminescence  occurs  in  less  than  24  hours.  This  difference  is  illustrated  by  the 
light  perturbations  which  start  at  three  hours  and  at  seven  hours  in  Figure  8. 

Perturbation  by  mechanical  stimulation.  It  is  of  interest  to  consider  the  nature 
of  the  cellular  component  or  components  which,  being  modified  as  a  result  of  the 
light  perturbation,  result  in  the  observed  phase  shift.  If  perturbation  by  means 
other  than  light  also  resulted  in  a  change  in  the  components  of  the  rhythmic  mech- 
anism, then  a  phase  shift  would  be  similarly  expected.  It  seemed  possible  that 
mechanical  stimulation  might  be  effective  in  this  regard.  Consequently,  a  perturba- 
tion experiment  was  carried  out,  in  which  air  was  bubbled  through  the  cell  sus- 
pensions instead  of  exposing  the  cells  to  light  (Fig.  9).  No  phase  shift  occurred; 
the  cells  which  had  been  stimulated  retained  the  same  phase  as  the  unstimulated 
controls. 

The  experiment  also  shows  that  it  is  possible  to  modify  the  concentrations  of 
compounds  which  are  involved  in  the  luminescence  rhythm  without  having  any 
effect  upon  the  phase  of  the  rhythmic  mechanism  itself.  It  was  found  previously 
(Hastings  and  Sweeney,  1957a)  that  the  rhythm  of  luminescence  involves  a  daily 
variation  in  the  amount  of  extractable  components  of  the  luminescent  system 
(luciferin  and  lucif erase).  Mechanical  stimulation  causes  the  luminescent  reaction 
to  occur,  so  that  one  would  suppose  that  the  concentrations  of  components  in  the 
luminescent  system  (and  other  biochemical  systems  coupled  to  it)  might  be 
changed.  In  fact,  the  apparent  effect  of  stimulation  is  similar  to  the  effect  of 
light ;  the  luminescence  decreases  to  a  low  level  in  both  cases.  But  since  no  phase 
shift  occurred  following  stimulation,  it  does  not  seem  likely  that  the  luminescent 
system  could  be  directly  involved  in  the  basic  rhythmic  mechanism,  although  it  is 
clearly  coupled  to  such  a  mechanism.  Moreover,  it  is  evident  that  there  is  no 
feedback  from  the  luminescent  system  to  the  system  controlling  the  phase  of  the 
rhythm.  From  previous  evidence  we  had  suggested  that  the  luminescent  system 
might  itself  constitute  an  autonomous  chemical  oscillation  (Hastings  and  Sweeney, 
1957b).  The  results  described  above,  however,  favor  a  hypothesis  which  proposes 
a  basic  mechanism  of  cellular  rh'ythmicity  to  which  various  physiological  and  bio- 
chemical processes,  such  as  luminescence  or  cell  division  (Sweeney  and  Hastings, 
1957b;  1958),  could  be  coupled. 

Cellular  interaction.  Since  all  the  experiments  which  have  been  described 
are  carried  out  with  large  cell  populations  (4000-15,000  cells  per  tube),  the  ques- 


RHYTHM  OF  LUMINESCENCE  451 


60 


50 


UJ 

o 

§40 


30 


20 


10 


I  T  I 

o    FIRST    STIMULATION 
•    SECOND    STIMULATION 


5  10  15  20  25  30  35  40 

HOURS   IN    DARKNESS 

FIGURE  9.  The  effect  of  perturbation  by  mechanical  stimulation  upon  the  phase  of  the 
rhythm.  Previous  to  the  time  shown,  all  cultures  were  in  LD  conditions,  and  zero  time  on 
the  graph  was  the  end  of  a  light  period.  At  this  time  all  aliquots  were  placed  in  the  dark. 
Six  hours  later  a  large  number  of  aliquots  were  stimulated  by  bubbling  air,  but  were  not 
exposed  to  light.  The  luminescence  changes  of  both  these  and  the  controls  were  determined 
through  the  subsequent  maximum  in  luminescence.  No  significant  change  in  the  phase  of  the 
stimulated  cultures  was  observed. 

tion  arises  as  to  whether  or  not  some  cellular  interaction  might  occur.  Since  the 
rhythmic  mechanism  involves  fluctuations  in  the  concentrations  of  chemical  com- 
ponents within  the  cells,  it  is  conceivable  that  certain  diffusible  compounds  might 
escape  into  the  medium,  and  that  their  concentrations  might  also  fluctuate  in  a 
diurnal  fashion.  The  importance  of  such  a  phenomenon  would  be  evident  if  the 
supposed  compound  or  compounds  could  function,  as  in  a  feedback  mechanism, 
for  stabilizing  the  frequency  and/or  phase  of  the  rhythm.  It  is  also  possible  that 
some  other  phenomenon,  such  as  cellular  motility,  could  be  involved  in  such  a 
feedback  mechanism.  This  latter  possibility  seems  unlikely,  however,  in  view 
of  the  fact  that  mechanical  stimulation,  with  its  attendant  violent  motion  and  dis- 
turbance of  cellular  motility,  did  not  result  in  a  phase  change. 

An  experiment  in  which  this  question  was  investigated  is  illustrated  in  Figure 
10.  Two  cultures  were  maintained  under  LD  conditions  for  several  weeks  with 


452 


J.  WOODLAND  HASTINGS  AND  BEATRICE  M.  SWEENEY 


their  phases  different  by  5  hours.  Samples  were  pipetted  from  each  culture 
and  moved  to  constant  dim  light  at  the  end  of  a  dark  period.  After  each  had  been 
under  constant  conditions  for  several  days  (their  phases  still  being  different  by 


0 


•   STANDARD  CURVE 

°   5  HR.  PHASE   DIFFERENCE 
SUMMATION 


LL) 


LU 
O 
CO 


0 


0         10         20        30      40        50       60        70       80 

TIME -HOURS 


90 


FIGURE  10.  The  effect  of  mixing  two  rhythmic  cultures  which  were  out  of  phase  with 
one  another.  Cultures  which  had  been  in  constant  dim  light  for  several  days,  having  a  5-hour 
phase  difference  as  shown  (bottom  curves),  were  mixed  at  the  time  indicated  by  the  arrow. 
The  rhythm  continued,  with  a  phase  having  its  maximum  at  a  time  precisely  halfway  between 
the  maxima  of  the  two  original  cultures.  The  middle  curve  shows  the  result  of  mixing  cul- 
tures having  the  same  phase,  done  at  the  time  indicated  by  the  arrow.  No  change  of  phase 
was  observed.  The  upper  graph  shows  the  result  which  would  be  theoretically  expected 
upon  mixing  two  cultures  5  hours  out  of  phase,  on  the  assumption  that  no  interaction  was 
involved.  Two  "standard"  luminescence  curves,  which  were  measured  from  a  culture  which 
had  not  been  mixed,  were  summated  with  a  5-hour  phase  difference.  For  purposes  of  com- 
parison, the  resultant  curve  is  plotted  on  the  graph  along  with  the  original  standard  curve, 
the  latter  having  been  displaced  by  2V-2  hours  on  the  time  axis  and  normalized  to  the  calculated 
curve.  It  may  be  seen  that  the  shape  of  the  calculated  curve  does  not  differ  greatly  from  that 
of  the  original  "standard"  luminescence  curve. 


RHYTHM  OF  LUMINESCENCE  45J 

5  hours),  the  cultures  were  mixed  in  equal  proportions,  and  the  luminescence 
changes  in  the  mixed  cultures  were  measured. 

If  two  typical  curves  showing  the  luminescence  rhythm  are  summated,  the 
phase  of  the  two  curves  being  different  by  five  hours  (75°),  the  resultant  curve 
differs  only  slightly  in  shape  from  the  original  curves  (Fig.  10,  top).  The  maxi- 
mum of  the  resultant  curve  lies  precisely  midway  between  the  maxima  of  the: 
two  original  curves. 

In  the  actual  mixing  experiment,  the  maximum  in  luminescence  of  the  mixed 
cultures  occurred  halfway  between  the  maxima  of  the  two  separate  unmixed  cul- 
tures. Moreover,  the  shape  of  the  curve  from  the  mixed  cultures  was  very  similar 
to  that  which  was  obtained  when  the  measured  luminescence  of  the  separate  cul- 
tures was  summated.  The  mixing  experiment  therefore  indicates  that  no  cellular 
interaction  was  involved. 

DISCUSSION 

The  subject  of  persistent  endogenous  rhythms  has  been  recently  reviewed  by 
Harker  (1958),  Pittendrigh  and  Bruce  (1957),  and  Biinning  (1956).  These 
reviewers,  as  well  as  other  authors,  have  taken  the  view  that  the  property  of 
rhythmicity  may  be  a  nearly  universal  feature  of  organisms.  This  view  is  derived, 
largely,  from  the  observation  that  endogenous  rhythms  are  extremely  widespread, 
having  been  reported  from  a  large  variety  of  both  plants  and  animals.  Further- 
more, Pittendrigh  and  Bruce  develop  the  generalization  that  most,  if  not  all  organ- 
isms can  measure  time ;  that  they  possess  clocks.  They  consider  that  the  basic 
mechanism  evolved  early,  and  that  it  has  been  retained  in  the  course  of  evolution 
as  a  part  of  the  adaptive  organization  of  all  organisms.  Their  use  of  the  word 
"clock"  refers  to  the  basic  mechanism  involved  in  cellular  rhythmicity,  and  the 
essential  properties  of  this  mechanism  are  considered  to  be  similar  in  different 
organisms. 

Pittendrigh  and  Bruce  (1957)  thus  distinguish  between  the  clock  as  the  basic 
mechanism,  and  the  persistent  rhythms  which  are  presumed  to  be  controlled  by 
the  clock.  Other  authors  (Brown,  Hines,  Webb  and  Fingerman,  1950;  Stephens, 
1957a;  Harker,  1958)  have  similarly  concluded  that  an  overt  persistent  rhythm 
may  be  distinguished  from  an  underlying  mechanism,  and  our  studies  with  Gonyau- 
lax  give  support  to  this  thesis.  For  example,  since  it  was  found  that  concentrations 
of  compounds  taking  part  in  the  luminescent  reaction  could  be  changed  without 
shifting  the  phase  of  the  rhythm,  it  is  probable  that  the  luminescence  rhythm  does 
not  in  itself  constitute  the  basic  mechanism.  Furthermore,  we  have  recently 
reported  a  persistent  rhythm  of  cell  division  in  Gonyaula.v  (Sweeney  and  Hastings, 
1957b).  The  luminescence  rhythm  and  the  cell  division  rhythm  have  essentially 
identical  properties.  Moreover,  we  have  not  been  able  to  demonstrate  a  phase 
shift  in  one  rhythm  which  is  not  accompanied  by  a  similar  phase  shift  in  the 
other  rhythm.  These  findings  give  additional  support  to  the  hypothesis  that  one 
basic  mechanism  controls  both  rhythms. 

The  identity  and  physico-chemical  nature  of  the  presumed  basic  clock  mech- 
anism in  persistent  rhythms  remains  undefined.  But  if  the  properties  of  this 
basic  mechanism  in  Gonyaulax  may  be  deduced  from  the  rhythm  of  luminescence, 
then  it  is  evident  that  the  mechanism  possesses  essential  clock-like  properties;  the 


454  J.  WOODLAND  HASTINGS  AND  BEATRICE  M.  SWEENEY 

period  is  not  greatly  affected  by  environmental  factors,  but  the  phase  is  labile  to 
resetting  by  the  appropriate  external  changes.  We  may  note,  in  addition,  that 
light  emission  in  Gonyaulax  is  clocked  so  that  it  is  maximal  during  the  night  phase, 
when  it  is  visible ;  and  without  environmental  inhibition,  luminescence  is  minimal 
during  the  day  phase.  However,  since  the  possible  utility  of  the  light  emission 
is  not  known,  the  functional  significance  of  clocked  luminescence  is  not  apparent. 

Many  of  the  characteristics  of  the  rhythm  of  luminescence  which  we  have  de- 
scribed are  similar  to  the  characteristics  of  persistent  rhythms  in  a  variety  of  other 
organisms,  ranging  from  other  unicellular  forms  to  mammals.  The  comparisons 
outlined  below  do  not  pretend  to  be  complete,  but  they  serve  to  illustrate  the  point. 
The  remarkable  similarities  found  support  the  view  of  Pittendrigh  and  Bruce 
(1957),  that  the  basic  mechanism  involved  in  rhythmicity  is  the  same  in  all 
organisms. 

Practically  all  the  persistent  diurnal  rhythms  described  have  natural  periods 
which  are  close  to  but  different  from  24  hours.  This  includes  rhythms  in  Dro- 
sophila  (Pittendrigh,  1954),  Uca  (Webb,  Brown  and  Sandeen,  1954),  Oedogbnium 
(Biihnemann,  1955a),  Euglena  (Bruce  and  Pittendrigh,  1956),  and  many  others. 
The  natural  period  may  range,  in  different  organisms,  from  about  21  to  27  hours. 
In  fact,  significant  differences  in  the  natural  periods  in  different  individual  mice 
are  well  documented  (Pittendrigh  and  Bruce,  1957). 

Studies  of  rhythms  in  a  variety  of  organisms,  including  the  bee  (Wahl,  1932), 
Uca  (Brown  and  Webb,  1948),  Avena  (Ball  and  Dyke,  1954),  Drosophila  (Pit- 
tendrigh, 1954),  and  Euglena  (Bruce  and  Pittendrigh,  1956),  have  shown  that  in 
each  case  the  period  is  nearly  the  same  at  temperatures  which  differ  by  15°  C, 
or  more.  It  is  interesting  to  note  that  the  effect  of  temperature  upon  the  period 
of  the  Gonyaulax  rhythm  is  similar  to  that  reported  by  Biihnemann  (1955b)  for 
the  rhythm  of  sporulation  in  Oedogoniuni,  in  that  the  apparent  Oin  for  both  is  less 
than  1.0.  Two  cases  may  therefore  be  interpreted  as  the  result  of  an  over-compen- 
sation in  the  mechanism  responsible  for  temperature  independence  (Hastings  and 
Sweeney,  1957b). 

Only  a  few  experiments  have  been  specifically  designed  to  detect  the  effect 
of  different  light  intensities  upon  the  natural  period  of  persistent  rhythms.  In 
those  cases  which  have  been  reported  (see  Harker,  1958),  the  natural  period  has 
been  found  to  change  no  more  than  an  hour  or  two  under  different  light  intensities. 

The  entrainment  of  rhythms  to  periods  different  from  24  hours  has  been 
reported  in  several  organisms,  including  Euglena  (Bruce  and  Pittendrigh,  1956) 
and  Oedogonium  (Biihnemann,  1955a).  In  these  and  other  cases,  as  in  Gonyaulax, 
the  rhythms  return  to  the  characteristic  natural  period  when  the  organisms  are 
returned  to  constant  conditions. 

On  the  other  hand,  several  experiments  have  been  reported  in  which  rhythmic 
organisms  still  continue  to  show  a  24-hour  rhythm  while  being  subjected  to  light- 
dark  cycles  which  differ  from  24  hours.  For  example,  Webb  (1950)  found  that 
the  period  of  the  Uca  rhythm  was  not  changed  while  the  organisms  were  subjected 
to  light  (95  foot-candles)  and  dark  periods  of  16  hours  each,  and  Tribukait  (1954) 
found  that  entrainment  to  an  imposed  light-dark  cycle  occurred  in  the  mouse  only 
so  long  as  the  imposed  cycles  did  not  differ  greatly  from  the  natural  period. 


RHYTHM  OF  LUMINESCENCE  455 

Studies  with  Gonyaulax  suggest  a  possible  reason  for  the  lack  of  apparent  en- 
trainment  in  experiments  such  as  those  cited  above :  the  light  intensities  used  may 
not  have  been  sufficiently  bright.  In  Gonyaulax,  the  luminescence  rhythm  may 
be  entrained  to  periods  which  differ  greatly  from  the  natural  period.  Our  inter- 
pretation of  this  entrainment  is  that  repetitive  phase  resetting  results  in  a  period 
corresponding  to  the  imposed  schedule.  The  importance  of  light  intensity  as  a 
parameter  in  phase  shifting  by  single  light  perturbations  has  been  documented  in 
experiments  with  Gonyaulax.  That  it  is  equally  important  in  entrainment  has  been 
shown  in  an  experiment  with  Gonyaulax  described  elsewhere  (Hastings  and 
Sweeney,  1958),  in  which  it  was  found  that  entrainment  occurred  at  a  light  inten- 
sity of  800  foot-candles,  but  not  at  200  foot-candles. 

Entrainment  of  rhythms  to  imposed  cycles  which  are  only  slightly  longer  or 
shorter  than  the  natural  period  has  been  discussed  by  Pittendrigh  and  Bruce 
(1957).  Their  interpretation  suggests  that  the  mechanism  may  be  different  from 
that  involved  in  entrainment  to  cycles  differing  greatly  from  the  natural  period. 

The  role  of  24-hour  light-dark  cycles  in  establishing  the  phase  of  diurnal 
rhythms  has  long  been  recognized,  and  experiments  with  many  organisms  have 
demonstrated  that,  as  in  Gonyaulax,  the  phase  shifts  in  response  to  a  new  light- 
dark  cycle  which  is  out  of  phase  with  solar  night  and  day.  The  fact  that  the 
light  intensity  used  in  such  experiments  is  of  importance  has  been  shown  by 
Brown,  Fingerman  and  Hines  (1954). 

That  non-repeated  light  perturbations  are  capable  of  establishing  or  changing 
the  phase  of  a  persistent  rhythm  has  been  stated  as  an  important  generalization 
only  in  recent  years  (Pittendrigh  and  Bruce,  1957),  although  some  previous 
studies  (Kalmus,  1940;  Webb,  1950)  do  provide  examples  of  the  phenomenon.  The 
phenomenon  provides  another  analogy  between  the  characteristics  of  persistent 
rhythms  and  the  known  properties  of  physical  oscillators.  It  is  well  known  that  a 
single  disturbance  or  perturbation  applied  to  an  oscillating  system  will  quite  gen- 
erally shift  its  phase  without  any  modification  to  the  period,  and  the  behavior  of 
a  simple  pendulum  is  a  good  example.  Pittendrigh  and  Bruce  (1957)  have  found 
phase  shifts  following  single  light  perturbations  in  persistent  rhythms  of  Englena 
and  Drosophila,  and  the  rhythm  in  Gonyaulax  provides  another  example  of  the 
phenomenon. 

Detailed  studies  on  the  effect  of  the  duration  and  intensity  of  single  perturba- 
tions have  not  yet  been  reported  in  other  organisms,  but  it  appears  that  the  nature 
of  the  phase  shift  in  Gonyaulax  may  differ  in  one  respect  from  that  reported  for 
Drosophila  (Pittendrigh  and  Bruce,  1957).  Following  a  single  light  perturbation 
in  Drosophila  there  may  occur  "transients,"  so  that  the  phase  is  not  reset  immedi- 
ately but  comes  to  its  stable  position  only  after  several  cycles.  In  Gonyaulax,  on 
the  other  hand,  phase  has  been  found  to  be  reset  immediately.  The  reason  for 
this  difference  is  not  known,  but  it  may  be  related  to  the  relative  complexity  of 
the  organisms  involved. 

With  respect  to  the  phenomenon  of  phase  shifting,  Bruce  and  Pittendrigh 
(1957)  have  discussed  whether  the  resetting  signal  is  the  step-up  in  light  intensity 
(dawn)  or  the  step-down  in  light  intensity  (dusk).  Several  experiments  with 
Gonyaulax  have  adequately  illustrated  that  the  phase  is  labile  to  both,  so  that 


456  |.  WOODLAND  HASTINGS  AND  BEATRICE  M.  SWEENEY 

neither  event  may  be  said  to  be  the  timing  cue  to  the  exclusion  of  the  other.  For 
example,  the  experiments  shown  in  Figure  3  illustrate  both  a  light-to-dark  transi- 
tion followed  by  constant  darkness,  and  a  dark-to-light  transition  followed  by  con- 
stant light.  In  both  cases,  the  last  transition  resulted  in  a  phase  shift. 

The  action  spectrum  for  shifting  the  phase  of  the  luminescence  rhythm  by  a 
single  light  perturbation  shows  relatively  sharp  maxima  in  effectiveness  at  475  m/i 
and  650  mp.  (Hastings  and  Sweeney,  unpublished).  The  red  maximum,  in  par- 
ticular, suggests  that  chlorophyll  acts  as  a  photosensitizer  for  phase  shifting.  Since 
the  effects  of  single  light  perturbations  are  essentially  the  same  in  plants  and  ani- 
mals, we  may  conclude  that  in  Gonyaulax  the  photosensitizers  involved  in  phase 
determination  are  not  a  part  of  the  basic  mechanism  of  rhythmicity.  In  animals, 
also,  the  photoreceptor  pigments  of  the  eye  are  not  a  part  of  the  basic  mechanism, 
although  they  function  in  phase  determination  by  light.  Whitaker  (1940)  re- 
ported that  blinded  mice  possess  a  natural  period  of  about  24  hours  in  their  activity 
rhythm,  but  that  the  rhythm  could  not  be  entrained  by  24-hour  light-dark  cycles 
to  correspond  with  solar  night  and  day,  as  in  normal  mice. 

It  is  known  that  temperature  changes  (Pittendrigh,  1954;  Stephens,  1957a), 
and  perhaps  certain  other  factors  (Harker,  1958)  may  also  serve  to  establish  or 
reset  phase.  There  is  no  report,  however,  that  mechanical  disturbances  can  be 
effective  in  other  organisms  in  this  regard. 

The  possibility  that  individuals  in  a  population  may  entrain  each  other  was 
suggested  by  Pittendrigh  and  Bruce  (1957).  However,  Stephens  (1957b)  was 
unable  to  demonstrate  any  significant  phase  modification  in  individual  fiddler  crabs 
when  they  were  placed  together  with  crabs  possessing  a  different  phase.  A  similar 
result  was  found  in  the  present  studies  with  Gonyaulax. 

It  is  of  interest  to  note  that  the  shape  of  the  luminescence  curve  obtained  in 
experiments  where  Gonyaulax  cultures  possessing  different  phases  were  mixed  is 
not  greatly  different  from  that  for  the  unmixed  cultures.  Indeed,  as  already 
pointed  out,  this  is  the  expected  result  of  adding  two  luminescence  curves  which 
are  five  hours  out  of  phase  with  one  another.  Thus,  a  population  composed  of 
cells  having  at  least  two  different  phases  is  difficult  to  distinguish  from  the  usual 
experimental  populations,  in  which  we  have  assumed  that  all  cells  possess  the  same 
phase.  This  experiment  serves  to  caution  us.  In  a  biological  rhythm  having  a 
sinusoidal  shape,  measurements  from  populations  may  not  accurately  represent  the 
behavior  of  individual  cells. 

We  do  not  know  how  the  luminescence  of  the  individual  Gonyaulax  cell  at 
different  times  in  the  cycle  compares  with  that  measured  in  a  population.  The 
question  is  an  important  one,  and  there  are  several  possibilities  which,  in  the 
absence  of  any  relevant  data,  need  not  be  discussed  here.  This  problem  is  being 
investigated  utilizing  measurements  of  the  rhythm  of  cell  division,  where  the  per- 
formance of  an  individual  cell  may  be  repeatedly  and  relatively  easily  scored. 

Although  several  suggestions  have  been  made  concerning  the  physico-chemical 
nature  of  the  basic  mechanism  involved  in  persistent  diurnal  rhythmicity  (Pitten- 
drigh and  Bruce,  1957;  Hastings  and  Sweeney,  1958),  none  has  received  any 
substantial  support.  It  is  hoped  that  information  concerning  the  extent  and  kind 
of  biochemical  changes  associated  with  the  rhythms  will  be  of  value  in  under- 
standing this  basic  problem.  Studies  of  this  nature  are  in  progress  with  Gonyaulax.. 


RHYTHM  OF  LUMINESCENCE  457 

SUMMARY 

1.  The  characteristics  of  a  persistent  diurnal  rhythm  of  luminescence  in  the 
dinoflagellate  Gonyaulax  polyedra  are  described. 

2.  The  light  emission  upon  stimulation,  from  cultures  which  are  kept  in  alter- 
nating light  and  dark  periods  of  12  hours  each  (=  LD),  is  40  to  60  times  greater 
during  the  dark  period  than  during  the  light  period.     If  LD  cultures  are  placed  in 
continuous  dim  light  (100  foot-candles)  a  diurnal  rhythm  of  luminescence  persists. 
If  LD  cultures  are  placed  in  continuous  bright  light   (>  1500  foot-candles)   the 
rhythm  is  damped,  and  no  fluctuations  occur  in  the  amount  of  light  emitted. 

3.  The  occurrence  of  rhythmicity   is  not  dependent   upon  prior  exposure  to 
LD  conditions.     Cultures  which  have  been  grown  in  bright  light  for  as  long  as  one 
year  show  a  diurnal  rhythm  when  placed  in  constant  dim  light  or  darkness.     Cul- 
tures kept  in  alternating  light  and  dark  cycles  which  are  greater  or  less  than  24 
hours  similarly  show  a  diurnal  rhythm  when  returned  to  constant  dim  light  or 
darkness.     "Training"  or  "memory"  is  therefore  not  involved. 

4.  The  rhythm  can  be  entrained  by  light-dark  cycles  which  are  different  from 
24  hours.     The  period  of  the  luminescence  rhythm  corresponds  to  light-dark  cycles 
which  have  periods  ranging  between  12  and  32  hours. 

5.  The  period  of  the  rhythm  is  always  close  to  24  hours  when  the  cells  are  kept 
under  constant  conditions,  but  it  varies  slightly  depending  upon  the  temperature 
and  light  intensity. 

6.  The  phase  of  the  rhythm  under  constant  conditions  is  related  to  the  time  at 
which  the  previous  light  and  dark  periods  occurred.     Moreover,  the  phase  may  be 
shifted  by  interposing  a  non-repeated  exposure  to  a  different  light  intensity.     The 
number  of  hours  by  which  the  phase  is  shifted  in  such  an  experiment  is  dependent 
upon  the  intensity  and  duration  of  the  light  treatment,  and  the  time  in  the  cycle 
when  it  is  administered. 

7 .  Exhaustive  mechanical  stimulation  does  not  alter  the  phase  of  the  rhythm. 

8.  When  cultures  having  different  phases  were  mixed,  no  evidence  was  found 
which  would  indicate  that  there  was  any  interaction  between  them. 

9.  The  evidence  presented  indicates  that  the  diurnal  rhythmicity  is  the  conse- 
quence of  a  basic  oscillatory  mechanism  which  is  inherent  to  the  cell. 

LITERATURE  CITED 

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illumination  upon  the  magnitude  of  the  diurnal  rhythm  of  Uca.  Anat.  Rec.,  108:  604. 

BROWN,  F.  A.,  JR.,  M.  FINGERMAN  AND  M.  N.  HINES,  1954.  A  study  of  the  mechanism  in- 
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BRUCE,  V.  G.,  AND  C.  S.  PITTENDRIGH,  1956.  Temperature  independence  in  a  unicellular 
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BRUCE,  V.  G.,  AND  C.  S.  PITTENDRIGH,  1957.  Endogenous  rhythms  in  insects  and  micro- 
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BUHNEMANN,  F.,  1955a.  Die  rhythmische  Sporenbildung  von  Ocdogonium  cardiacum  Wittr. 
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BUHNEMANN,  F.,  1955b.  Das  endodiurnale  System  der  Oedogonium-Zelle.  III.  t)ber  den 
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HARVEY,  E.  N.,  1952.     Bioluminescence.     Academic  Press,  New  York,  New  York. 
HASTINGS,  J.  W.,  AND  B.  M.  SWEENEY,  1957a.     The  luminescent  reaction  in  extracts  of  the 

marine  dinoflagellate  Gonyaulax  polyedra.     J.   Cell.   Comp.  Physiol.,  49 :   209-226. 
HASTINGS,  J.  W.,  AND  B.  M.  SWEENEY,  1957b.     On  the  mechanism  of  temperature  independence 

in  a  biological  clock.     Proc.  Nat.  Acad.  Sci.,  43:  804-811. 
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related  phenomena  in  plants  and  animals    (Ed.,  Alice  P.   Withrow),  A.A.A.S.   Press, 

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luminescence  of  biological  systems,  pp.  415-420  (Ed.,  F.  H.  Johnson),  A.A.A.S.  Press, 

Washington,  D.  C. 
KALMUS,  H.,   1940.     Diurnal   rhythms   in   the   axolotl   larva  and   in   Drosophila.     Nature,   145 : 

72-73. 
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emergence  time  in  Drosophila.     Proc.  Nat.  Acad  Sci.,  40:   1018-1029. 
PITTENDRIGH,  C.  S.,  AND  V.  G.  BRUCE,  1957.     An  oscillator  model  for  biological  clocks.     In: 

Rhythmic   and   synthetic   processes   in   growth,   pp.   75-109    (Ed.,   Dorothea    Rudnick ) , 

Princeton  University  Press. 
STEPHENS,   G.   C.,    1957a.     Influence   of   temperature    fluctuations   on    the    diurnal    melanophore 

rhythm  of  the  fiddler  crab,  Uca.    Physiol.  ZooL,  30:  55-69. 
STEPHENS,   G.    C.,   1957b.     Twenty-four   hour   cycles    in   marine   organisms.     Amer.    Nat.     91  : 

135-151. 

SWEENEY,  B.  M.,  AND  J.  W.  HASTINGS,  1957a.     Characteristics  of  the  diurnal  rhythm  of  lumi- 
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SWEENEY,  B.  M.,  AND  J.  W.  HASTINGS,  1957b.     A  persistent  rhythm  of  cell  division  in  popu- 
lations of  Gonyaulax  polyedra.     Plant  Physiol.,  32:  XXV    (Suppl.) 
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Gonyaulax  polyedra.       J.  Protozoolof/v,  5:  217—224. 
TRIBUKAIT,  B.,  1954.     Aktivitatsperiodik  der  Maus  im  kunstlich  verkurtzen  Tag.     Naturu'iss., 

41 :  92-93. 
WAHL,  O.,  1932.     Neue  Untersuchungen  uher  das  Zeitgedachtnis  der  Bienen.     Zeitschr.  vergl. 

Physiol.,  16 :  529-589. 
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Zool.,  23:  316-337. 
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of  the  persistent  daily  rhythm  of  the  fiddler  crab.     Anat.  Rec.,  120:  796. 

WHITAKER,  W.  L.,  1940.     Some  effects  of  artificial  illumination  on  reproduction  in  the  white- 
footed  mouse,  Pcromyscus  I  cue  opus  noveboracensis.     J.  Exp.  ZooL,  83 :  33-60. 


THE  ACTION  OF  INSULIN  ON  CELLS  AND  PROTOPLASM  x 

L.  V.  HEILBRUNN,  FRANCIS  T.  ASHTON,  CARL  FELDHERR  AND 

WALTER  L.  WILSON 

Department  of  Zoology,  University  of  Pennsylvania,  Philadelphia,  Pa.;  Department  of 

Physiology  and  Biophysics,  University  of  Vermont,  Burlington,  Ft.;  and  the 

Marine  Biological  Laboratory,  Woods  Hole,  Mass. 

In  a  lecture  he  gave  in  1947,  Best  (1948)  stated,  "We  often  hear  the  statement 
made  that  we  have  had  insulin  for  25  years  and  still  do  not  know  exactly  how  it 
acts.  This  is  quite  true,  but  we  know  more  about  the  action  of  insulin  than  about 
any  other  hormone." 

Strangely  enough,  the  great  science  of  endocrinology,  with  its  vast  body  of 
information  concerning  the  chemistry  and  the  ultimate  effects  of  various  hormones, 
has  not  been  able  to  solve  the  basic  problem  of  why  the  different  hormones  act  as 
they  do.  In  recent  years  in  the  attempt  to  understand  the  action  of  insulin,  there 
has  been  more  and  more  emphasis  on  studies  of  the  cell  as  a  whole  rather  than  on 
studies  of  fragments  or  extracts  of  cells.  In  a  thoughtful  review  of  the  literature, 
Ross  (1956)  is  led  to  comment  (p.  364),  "It  is  apparent  that  no  consistent  effect 
of  insulin  has  been  demonstrated  in  cell-free  systems."  And  the  biochemist  Levine, 
a  leading  authority  in  the  field  of  carbohydrate  metabolism  and  long  a  student  of 
insulin,  wrote  recently  (Levine  and  Goldstein,  1955)  (p.  344),  "It  would  be 
expected  therefore  that  a  certain  degree  of  morphological  intactness  is  necessary 
to  demonstrate  hormonal  effects  and  actions.  Otherwise  we  would  be  pulling  the 
trigger  of  an  unassembled  gun."  Levine  himself  has  turned  to  the  ways  of  thought 
and  the  methods  of  cell  physiology  in  order  to  obtain  a  solution  of  the  insulin 
problem. 

Thus  it  may  not  be  too  presumptuous  for  cell  physiologists  to  express  ideas 
about  the  mechanism  of  insulin  action.  Indeed  back  in  1914,  Hober  suggested  that 
diabetes  might  be  due  to  a  change  in  the  permeability  of  cells  for  sugar.  This  idea 
was  then  taken  up  by  Wiechmann  (1924,  1926),  and  by  Hausler  and  Loewi  (1925 ; 
see  also  Loewi,  1927).  However,  the  older  evidence  in  favor  of  the  permeability 
theory  was  not  very  convincing,  and  interpretations  in  terms  of  the  cell  as  a  whole 
were  pushed  into  the  background  by  chemical  work  which  seemed  to  show  that 
insulin  had  a  specific  effect  on  certain  isolated  enzyme  systems.  More  recently, 
and  on  the  basis  of  newer  evidence,  the  permeability  theory  has  been  revived  and 
modernized,  and  it  is  finding  wide  support. 

No  attempt  will  be  made  here  to  review  the  enormous  mass  of  literature  on 
insulin.  Books  on  endocrinology  contain  a  great  deal  of  information  and  there 
have  been  a  number  of  recent  reviews  by  authorities  in  the  field  (Haugaard  and 
Marsh,  1953;  Stadie,  1954;  Levine  and' Goldstein,  1955;  Weil-Malherbe,  1955; 
Ross,  1956;  Stich  and  Maske,  1956). 

1  Supported  by  a  grant  from  the  National  Science  Foundation. 

459 


460     L.  V.  HEILBRUNN,  F.  T.  ASHTON,  C.  FELDHERR  AND  W.  L.  WILSON 

A  few  facts  stand  out.  Some  of  these  have  been  long  known  and  were 
mentioned  in  a  review  published  by  Macleod  in  1924.  For  our  purposes,  it  may 
be  well  to  remind  the  reader  that : 

1.  The  action  of  insulin  in  reducing  the  concentration  of  sugar  in  the  blood 
is  not  due  to  an  action  on  the  blood  itself,  for  insulin  does  not  have  this  effect  on 
blood  withdrawn  from  the  body.     Hence  the  cells  and  the  protoplasm  they  contain 
must  play  a  part  in  the  lowering  of  sugar  concentration  in  the  blood. 

2.  The  convulsive  action  caused  by  excess  insulin  is  not  due  to  an  effect  on 
the  cerebrum,  for  it  occurs  in  decerebrate  animals. 

3.  Lowering  of  the  sugar  concentration  in  the  medium   surrounding  isolated 
nerve  or  muscle  has  no  effect  in  stimulating  either  nerve  or  muscle. 

4.  When  a  rabbit  is  given  a  lethal  dose  of  insulin,  violent  convulsions  occur ; 
these  are  then  followed  by  a  comatose  stage,  and  convulsions  and  coma  continue  in 
alternate  phases  until,  after  an  hour  or  more,  the  animal  dies. 

5.  The  harmful  results  of  excess  insulin  can  be  relieved  by  the  injection  into  the 
blood  stream  of  a  small  amount  of  glucose. 

6.  Lack  of  insulin  causes  a  failure  of  fat,  carbohydrate  and  protein  formation. 
Best  (1953)   sums  up  the  situation  (p.  434)   by  stating  that  "insulin  is  a  central 
anabolic    hormone    without    which    many  of    the    building    processes  .  .  .  cannot 
proceed  at  the  physiologic  rate." 

7.  Insulin  also  causes  an  increase  in  the  rate  of  oxidation  of  sugar.     Thus  it  is 
a  catabolic  hormone  as  well  as  an  anabolic  hormone. 

It  is  clear  that  insulin  markedly  increases  the  rate  of  activity  of  various  enzymic 
actions,  and  yet  when  purified  preparations  of  these  enzymes  have  been  tested, 
there  seems  to  be  little  or  no  effect  of  insulin  upon  them.  Moreover  it  would  be 
hard  to  explain  how  it  would  be  possible  for  a  single  substance  to  have  a  direct 
effect  on  all  the  various  enzymes  responsible  for  the  synthesis  of  carbohydrates, 
proteins  and  fats,  as  well  as  those  responsible  for  the  oxidation  of  sugar.  Hence 
we  apparently  must  conclude  that  in  one  way  or  another  some  change  in  the  cell 
or  its  protoplasm  has  an  accelerating  effect  on  many  types  of  enzyme  activity. 

At  the  present  time,  what  is  doubtless  the  leading  theory  of  insulin  action  holds 
that  the  primary  effect  of  insulin  is  to  change  the  cell  in  such  a  way  as  to  facilitate 
the  passage  into  it  of  various  sugars.  This  theory,  due  in  its  present  form  to  Levine 
and  his  collaborators,  has  been  supported  not  only  by  the  work  of  Levine  and  his 
group,  but  also  by  the  careful  and  ingenious  experiments  of  various  other  investi- 
gators. Perhaps  the  most  impressive  work  is  that  of  Park,  Bornstein  and  Post 
(1955).  The  entire  subject  is  ably  reviewed  by  Ross  (1956),  and  this  review 
should  be  consulted  by  anyone  interested  in  details  or  references  to  the  rather 
extensive  literature.  The  permeability  theory  has  been  enthusiastically  endorsed 
by  Stadie  (1957),  who  is  certainly  one  of  the  outstanding  investigators  in  the  field. 

Scarcely  anyone  has  attempted  to  criticize  the  permeability  theory,  although 
such  criticism  is  possible,  both  from  the  standpoint  of  our  knowledge  of  permeability 
and  transport  mechanisms,  and  also  because  the  theory  can  scarcely  offer  an 
explanation  of  some  of  the  basic  known  facts  of  insulin  action.  This  latter  point 
will  be  discussed  in  a  later  section. 


ACTION  OF  INSULIN  ON  PROTOPLASM  461 

EFFECT  OF  INSULIN  ON  THE  PERMEABILITY  TO  GLUCOSE 

Our  experiments  with  insulin  were  done  entirely  on  relatively  simple,  isolated 
cells  of  lower  organisms.  These  cells  offer  exceptionally  favorable  material  for 
the  cell  physiologist,  but  work  with  such  cells  is  open  to  the  criticism  that  the 
action  of  insulin  may  be  confined  to  the  cells  of  vertebrate  animals.  Such  an  opinion 
is  held  by  Ross  in  the  review  cited  above,  and  it  was  expressed  forcibly  by  Best, 
Jephcott  and  Scott  (1932).  However,  there  are  reports  of  insulin  action  on  the 
cells  or  tissues  of  protozoa,  sponges,  flatworms,  crabs  and  insects ;  on  yeast  cells, 
and  on  various  kinds  of  bacteria.  Whether  this  literature  is  sound  or  not  is  a 
question  we  do  not  care  to  discuss.  Certainly  there  can  be  no  objection  to  our 
using  simple  living  cells  to  explain  insulin  action,  for  if  we  can  obtain  effects  on 
these  cells  with  insulin  and  if  such  effects  can  be  used  to  interpret  the  action  of 
insulin  in  higher  animals,  we  may  be  able  to  offer  suggestions  of  some  value. 

For  the  study  of  cells  and  protoplasm  the  eggs  of  marine  invertebrates  offer 
many  advantages  and  this  type  of  material  has  often  been  used  by  cell  physiologists. 
We  used  the  eggs  of  the  sea  urchin  Arbacia  punctulata,  the  surf  clam  Spisula 
solidissima,  and  the  annelid  worm  Chaetopterus  pergamentaceus.  All  of  these  eggs 
are  readily  available  at  Woods  Hole.  If  insulin  directly  favors  the  entrance  of 
glucose  into  cells  (quite  apart  from  any  indirect  effect  it  might  have  as  a  result 
of  the  utilization  or  combination  of  glucose  within  the  cell),  we  thought  that  perhaps 
we  might  be  able  to  find  evidence  for  such  a  direct  effect  on  marine  egg  material. 
This  we  were  unable  to  do,  and  our  results  were  wholly  negative.  For  this  reason 
we  shall  not  attempt  to  report  them  in  any  detail,  but  will  merely  cite  a  few  of  our 
experiments  as  briefly  as  possible. 

One  of  the  standard  ways  of  determining  the  ease  with  which  dissolved  sub- 
stances pass  through  cell  membranes  is  to  study  the  osmotic  behavior  of  cells  in 
relation  to  solutions  of  the  substances  in  question.  There  are  a  variety  of  such 
osmotic  methods.  One  of  the  simplest  of  these  methods  depends  on  the  fact  that 
the  more  readily  a  substance  penetrates,  the  less  osmotic  pressure  it  can  exert 
against  the  plasma  membrane  of  the  cell.  By  observing  the  changes  in  volume  of 
the  cells  when  they  are  immersed  in  various  concentrations  of  a  given  substance, 
one  can  obtain  a  rapid  measure  of  the  ease  with  which  a  substance  enters.  In  our 
particular  case,  if  insulin  favored  the  entrance  of  glucose,  a  solution  of  glucose 
containing  insulin  would  be  less  potent  osmotically  than  a  similar  control  solution 
which  differed  only  in  lacking  insulin.  In  order  to  obtain  as  great  an  effect  as 
possible,  we  used  saturated  solutions  of  insulin.  These  were  obtained  by  dissolving 
0.2  mg.  of  insulin  in  one  ml.  of  the  glucose  solution.  Actually  not  all  of  the  insulin 
went  into  solution.  All  the  solutions  were  brought  to  the  pH  of  sea  water.  The 
insulin  we  used  was  a  preparation  which  was  relatively  zinc-free;  it  was  kindly 
supplied  by  the  Eli  Lilly  Company  through  the  kindness  and  courtesy  of  Dr.  G.  H. 
A.  Clowes.  In  view  of  the  fact  that  many  types  of  protoplasm  are  very  sensitive 
to  zinc,  we  were  indeed  fortunate  to  obtain  this  preparation  from  which  98.8%  of 
the  zinc  ordinarily  present  in  crystalline  insulin  had  been  removed.  Actually  an 
assay  made  by  the  Eli  Lilly  Company  showed  only  0.0061%  zinc  in  the  dry  material. 

Table  I  shows  the  results  of  our  experiments  on  sea  urchin  eggs.  In  this  table, 
as  also  in  Tables  II  and  III,  the  ±  sign  indicates  standard  deviation.  A  molar 
solution  of  glucose  caused  a  slight  decrease  in  the  volume  of  the  eggs.  In  the 


462      L.  V.  HEILBRUNN,  F.  T.  ASHTON,  C.  FELDHERR  AND  W.  L.  WILSON 


TABLE  I 

Effect  of  glucose  and  glucose  +  insulin  on  the  osmotic  behavior  of  Arbacia  eggs.     Measurements 

of  diameters  were  made  after  the  eggs  had  been  immersed  in  the  solutions  for  5  minutes. 

The  values  show  the  average  of  10  measurements 


Diameter  of  eggs  in  sea  water 

Diameter  of  eggs  in  molar  glucose  solution 

Diameter  of  eggs  in  molar  glucose  solution  containing  insulin 


77.8  ±  2.02  microns 
71.5  ±  2. 18  microns 
70.7  ±  2.29  microns 


presence  of  insulin  approximately  the  same  decrease  occurred.  If  insulin  had 
favored  the  entrance  of  the  glucose,  then  the  solution  of  glucose  containing  the 
insulin  should  not  have  caused  as  great  a  shrinkage. 

Similar  results  were  obtained  with  eggs  of  the  clam  Spisula,  as  is  shown  in 
Table  II.  Again  there  is  no  indication  that  insulin  favors  the  entrance  of  glucose. 

In  our  experiments  with  eggs  of  the  worm  Chaetopterus,  we  ran  into  difficulty. 
When  we  immersed  these  eggs  in  solutions  of  glucose,  the  sugar  entered  rapidly, 
so  rapidly  in  fact  that  even  solutions  are  strong  as  2  M  caused  no  shrinkage  of  the 
eggs.  We  experimented  with  2  M  and  1.75  M  and  1.5  M  glucose  solutions  with  and 
without  insulin.  In  all  cases,  in  the  absence  of  insulin  the  glucose  entered  more 
rapidly  than  when  it  was  present.  In  the  1.75  M  and  1.5  M  glucose,  frequently  the 
eggs  swelled  so  rapidly  that  they  broke.  This  sometimes  made  measurements  un- 
certain. Apparently  in  the  glucose  solutions,  absence  of  ions  like  calcium  changed  the 
semipermeable  membrane  of  the  cell  in  such  a  way  that  it  became  permeable  to  glu- 
cose. Perhaps  the  small  amount  of  zinc  in  the  insulin  helped  partially  to  stabilize  the 
membrane.  Because  of  the  increased  permeability  of  the  cell  membrane  in  the 
absence  of  the  salts  of  sea  water,  we  decided  to  compare  the  behavior  of  glucose 
and  glucose  +  insulin  in  the  presence  of  an  appreciable  amount  of  sea  water.  The 
results  are  shown  in  Table  III.  In  this  case  also,  presence  of  insulin  does  not  favor 
the  entrance  of  glucose. 

Our  results  with  marine  eggs  lead  to  the  conclusion  that  the  effect  of  insulin 
in  increasing  the  rate  of  entrance  of  glucose  into  cells  is  not  a  general  phenomenon 
true  for  all  types  of  living  material.  Other  authors  in  the  past  have  reached  the 
same  conclusion.  Thus  it  is  now  commonly  held  that  insulin  does  not  increase 
the  rate  of  entrance  of  glucose  into  the  erythrocytes  of  man  and  mammals.  However, 
the  literature  on  erythrocytes  is,  or  at  least  has  been,  highly  controversial  (see 
Foshay,  1925 ;  Hausler  and  Loewi,  1925 ;  Loewi,  1927 ;  Hogler,  Thomann  and 
Uberrack.  1929;  Himmerich  and  Tschernjak,  1936;  also  many  papers  cited  by 
them;  Wilbrandt,  1947;  Guensberg.  1947;  Pletscher,  von  Planta  and  Hunzinger. 

TABLE  II 

Effect   of  solutions   of  glucose  and  glucose   +  insulin   on  the  osmotic  behavior  of  Spisula   egg*. 

Measurements  of  diameters  were  made  after  the  eggs  had  been  immersed  in  the  solutions  for 

5  minutes.     The  values  are  in  microns ;  they  show  the  average  of  10  meastirements 


0.9  M 

0.8  M 

0.7  M 

Diameter  of  eggs  in  sea  water 
Diameter  of  eggs  in  glucose 
Diameter  of  eggs  in  glucose  +  insulin 

61.0  ±  2.28 
56.8  ±  1.78 
53.7  ±  1.77 

56.8  ±  1.67 
56.4  ±  1.52 
53.5  ±  3.80 

56.4  ±  1.07 
58.9  ±  0.83 
54.1  ±  2.87 

ACTION  OF  INSULIN  ON  PROTOPLASM 


463 


TABLE  III 

Effect  of  glucose  and  glucose  +  insulin  on  the  osmotic  behavior  of  Chaetopterus  eggs. 

Solution  A  =  4  parts  molar  glucose  solution  +  1  part  sea  water. 

Solution  B  =  4  parts  molar  glucose  solution  containing 

insulin  +  1  part  sea  water 

Control  eggs  in  sea  water  measure  99.4  ±  2.06  microns 


Eggs  in  A  (glucose  alone)  measure 

Eggs  in  B  (glucose  +  insulin)  measure 

After  3  minutes 
After  136  minutes 

94.0  ±  1.63  microns 
95.4  ±  2.50  microns 

After  3  minutes 
After  135  minutes 

94.2  ±  1.83  microns 
94.0  ±  2.58  microns 

1955).  Any  interpretation  of  this  literature  is  complicated  by  the  fact  that  glycol- 
ysis  and  other  changes  in  carbohydrates  may  well  occur  in  blood  cells.  Park  and 
Johnson  (1955)  failed  to  find  any  increase  in  the  rate  of  entrance  of  glucose  and 
galactose  into  rat  brain  cells  when  insulin  was  present,  but  here,  too,  the  results 
are  based  on  the  assumption  that  under  the  conditions  of  the  experiment  both 
glucose  and  galactose  remained  unaltered  when  they  entered  the  cells,  and  this 
conclusion  may  not  be  entirely  warranted  (compare  Sols  and  Crane,  1954). 

If,  as  is  now  commonly  believed,  the  favorable  effect  of  insulin  on  the  transport 
of  sugar  into  cells  is  due  to  some  sort  of  an  enzyme-controlled  reaction,  then 
acceleration  of  this  transport  promoting  enzyme  might  well  be  the  cause  of  the 
increase  in  the  rate  of  entrance  of  sugar.  Thus  the  more  rapid  transport  of  sugar 
into  cells  in  the  presence  of  insulin  might  merely  represent  one  aspect  of  the  general 
effect  of  insulin  in  accelerating  diverse  types  of  enzyme  activity.  In  other  words, 
the  more  rapid  entrance  of  sugar,  instead  of  being  the  basic  reason  for  insulin 
action,  might  be  a  result  rather  than  a  cause  of  some  underlying  change  that  is 
responsible  for  a  general  increase  in  enzymic  activity.  What  could  such  a  cause  be  ? 

EFFECT  OF  INSULIN  ON  COLLOIDAL  CHANGES  IN  PROTOPLASM 

When  various  types  of  cells  are  excited  by  stimuli  of  one  sort  or  another,  calcium 
is  released  from  the  cell  cortex,  and  this  calcium  then  activates  a  proteolytic  enzyme 
system.  The  proteolytic  enzyme  also  serves  as  a  clotting  enzyme  and  produces  a 
gelation  of  protoplasm  in  the  interior  of  the  cell.  This  gelation  involves  an  oxida- 
tion of  -SH  to  S-S  groups.  Thus  the  release  of  calcium  can  result  in  an  increase 
in  protease  activity  and  also  an  increase  in  cellular  oxidations.  In  other  words, 
calcium  release  is  the  trigger  that  starts  off  a  number  of  enzymic  reactions.  The 
evidence  on  which  these  statements  is  based  has  been  presented  in  considerable 
detail  in  recent  books  (Heilbrunn,  1956,  1958)  ;  see  also  Wilson  and  Heilbrunn, 
1957. 

Could  it  be  possible  that  in  one  way  or  another  insulin  might  act  in  somewhat 
the  same  way  that  stimulating  agents  do,  and  what  could  conceivably  be  the  reason 
for  such  an  action  ?  This  possibility  is  what  intrigued  us  and  induced  us  to  under- 
take the  work  that  is  described  below. 

If  gelation  and  the  reactions  underlying  gelation  in  protoplasm  constitute  the 
trigger  for  protoplasmic  activity,  then  presumably  protoplasm  must  have  some 


464     L.  V.  HEILBRUNN,  F.  T.  ASHTON,  C.  FELDHERR  AND  W.  L.  WILSON 

method  of  braking  or  inhibiting  the  gelation.  In  the  books  just  referred  to,  strong 
evidence  is  presented  to  show  that  heparin  and  heparin-like  substances  can  constitute 
such  a  brake.  Heparin  not  only  can  inhibit  protoplasmic  gelation  in  much  the  same 
way  that  it  inhibits  blood  clotting,  it  can  also  inhibit  the  action  of  various  types  of 
enzymes.  In  living  cells  generally,  there  seems  to  be  a  balance  between  the  factors 
which  tend  to  induce  gelation  or  clotting  and  those  which  tend  to  prevent  it. 
Heparin  (and/or  heparin-like  substances)  is  one  of  the  inhibiting  factors.  If  we 
could  imagine  a  substance  which  would  antagonize  or  neutralize  the  effect  of 
heparin  and  similar  substances,  then  it  might  well  act  to  accelerate  various  enzyme 
systems  in  the  protoplasm.  Insulin  is  such  a  substance,  as  we  shall  now  attempt 
to  show. 

Sol-gel  reactions  undoubtedly  occur  in  many,  if  not  all  types  of  protoplasm,  but 
they  are  especially  evident  in  the  ameba.  Moreover,  in  the  ameba,  a  small  amount 
of  heparin  can  be  shown  to  prevent  the  clotting  reaction  which  normally  occurs 
whenever  the  cell  is  torn  or  broken,  that  is  to  say  the  surface  precipitation  reaction. 
We  use  the  giant  ameba,  Chaos  cJwos,  and  the  heparin  we  used  in  our  experiments 
was  kindly  supplied  by  the  Upjohn  Company.  If  an  ameba  is  immersed  in  a 
dilute  solution  of  heparin,  say  a  0.01  %  solution,  and  the  ameba  is  crushed  by 
exerting  pressure  on  the  coverslip  over  the  animal,  no  surface  precipitation  reaction 
occurs  and  the  contents  of  the  ameba  flow  out  through  the  solution.  However,  if 
the  heparin  solution  is  also  made  to  contain  a  0.01%  solution  of  insulin,  there  is 
an  excellent  surface  precipitation  reaction  and  the  exuding  protoplasm  forms  a 
distinct  membrane  about  itself. 

We  performed  a  series  of  experiments  in  which  various  concentrations  of  insulin 
were  balanced  against  various  concentrations  of  heparin.  In  deciding  whether  or 
not  a  surface  precipitation  reaction  occurs,  it  is  important  not  to  vary  too  greatly 
the  amount  of  pressure  with  which  the  ameba  is  broken.  For  with  too  great 
pressure  and  with  too  rapid  emergence  of  the  interior  protoplasm,  there  is  scarcely 
time  for  a  proper  reaction  to  occur.  It  is  difficult  to  measure  the  amount  of  pressure 
applied  to  a  coverslip.  In  order  to  measure  this  pressure,  one  of  us  (Ashton) 
devised  an  apparatus  in  which  a  small  rectangular  piece  of  glass  was  attached  to  a 
lever  which  in  turn  was  attached  to  a  DeNouy  tensiometer.  With  this  apparatus  it 
is  possible  to  measure  the  amount  of  pressure  applied  before  a  cell  breaks.  The 
measurements  are  not  very  exact,  but  they  have  the  advantage  of  being  objective. 
For  cells  which  do  not  vary  greatly  in  volume,  as  for  example  sea  urchin  eggs, 
the  pressure  required  to  break  them,  as  indicated  by  our  apparatus,  is  reasonably 
constant.  However,  for  amebae  which  differ  markedly  in  size,  as  do  our  specimens 
of  Chaos  chaos,  the  breaking  pressure  varies  more  widely,  for  with  the  larger  amebae 
there  is  more  resistance  to  the  pressure  imposed  upon  them.  Table  IV  shows 
what  happens  when  amebae  are  broken  in  mixtures  of  insulin  and  heparin.  The 
last  column  indicates  whether  or  not  a  surface  precipitation  reaction  occurred  and 
whether  it  was  a  strong  or  a  weak  reaction.  The  amount  of  pressure  required  to 
break  the  amebae  is  also  recorded.  As  was  to  be  expected,  this  pressure  varied 
widely,  but  whether  the  pressure  was  relatively  great  or  relatively  small,  the  results 
were  always  the  same.  It  is  clear,  therefore,  that  the  effect  of  heparin  in  preventing 
the  protoplasmic  clotting  necessary  for  the  surface  precipitation  reaction  can  be 
completely  blocked  by  the  addition  of  insulin.  Moreover,  a  control  test  showed 


ACTION  OF  INSULIN  ON  PROTOPLASM 


465 


that  the  amount  of  zinc  present  in  our  solutions  had  no  such  effect.  It  should  be 
noted  that  a  given  amount  of  insulin  can  neutralize  four  times  as  much  heparin. 
Further  evidence  of  a  combination  between  insulin  and  heparin  is  provided  by 
experiments  in  which  it  was  shown  that  the  metachromatic  reaction  of  heparin  with 
toluidine  blue  was  prevented  by  solutions  of  insulin.  In  these  experiments,  shown 
in  Table  V,  relatively  large  amounts  of  insulin  were  necessary  to  block  completely 
the  metachromatic  reaction.  Here  we  are  dealing  with  a  system  in  which  only  insulin 
and  heparin  are  present,  whereas  in  the  earlier  experiments  the  system  included  not 
only  insulin  and  heparin,  but  also  the  protoplasm  of  the  ameba.  Probably  the 
protoplasm,  or  rather  some  proteins  contained  in  it,  have  an  affinity  for  heparin 
and  can  unite  with  it  in  spite  of  the  presence  of  insulin.  At  any  rate,  this  might 
constitute  an  explanation  of  the  different  types  of  ratios  obtained  in  the  two 
experiments.  Another  explanation  might  be  that  it  may  take  more  insulin  to  block 


TABLE  IV 
The  effect  of  mixtures  of  heparin  and  insulin  on  the  surface  precipitation  reaction  of  Chaos  chaos 


'  ',    insulin 

%  heparin 

Ratio  insulin/heparin 

Pressure  in  milligrams 

Spr 

0.01 

0.01 

1-1 

120 

strong 

1-1 

88 

strong 

1-1 

92 

strong 

1-1 

120 

strong 

1-1 

160 

strong 

0.01 

0.02 

1-2 

208? 

strong 

1-2 

140 

strong 

1-2 

116 

strong 

1-2 

52 

strong 

1-2 

96 

strong 

0.01 

0.03 

1-3 

116 

weak 

1-3 

124 

strong- 

1-3 

188 

weak 

1-3 

48 

weak 

1-3 

116 

weak 

0.01 

0.04 

1-4 

48 

wea  k 

1-4 

168 

weak 

1-4 

128 

weak 

1-4 

252? 

very  weak 

1-4 

76 

very  weak 

0.01 

0.05 

15 

96 

none 

1-5 

60 

none 

1-5 

134 

none 

1-5 

68 

none 

2  X  10~7  M  zinc 

0.01 

124 

none 

125 

none 

144 

none 

172 

none 

145 

none 

466     L.  V.  HEILBRUNN,  F.  T.  ASHTON,  C.  FELDHERR  AND  W.  L.  WILSON 


TABLE  V 

Metachromatic  reaction  of  mixtures  of  heparin  and  insulin.     One  milliliter  of  a  0.02%  insulin 

solution  was  mixed  with  an  equal  volume  of  various  concentrations  of  heparin,  and  the 

various  mixtures  -were  then  tested  for  metachromasia  with  6  drops  of  a 

0.01%  solution  of  toluidine  blue 


%  insulin 

%  heparin 

Ratio  insulin/heparin 

Reaction 

0.02 

0.02 

1 

+ 

0.02 

0.01 

2 

+ 

0.02 

0.0067 

3 

+ 

0.02 

0.005 

4 

+ 

0.02 

0.004 

5 

+ 

0.02 

0.0033 

6 

+ 

0.02 

0.00286 

7 

+ 

0.02 

0.0025 

8 

+ 

0.02 

0.0022 

9 

+ 

002 

0.002 

10 

+ 

0.02 

000182 

11 

+  ? 

0.02 

0.00167 

12 

+  ? 

0.02 

0.00154 

13 

— 

0.02 

0.00142 

14 

— 

0.02 

0.00134 

15 

— 

the  metachromatic  reaction  of  heparin  than  it  does  to  block  its  effect  on  clotting 
or  on  enzymic  action. 

When  amebae  are  stained  with  toluidine  blue,  the  outer  region  of  the  cell  gives 
a  beautiful  metachromatic  color,  a  color  such  as  that  which  would  be  given  by 
heparin  or  a  heparin-like  substance.  But  if  amebae  are  immersed  in  solutions  of 
insulin  for  some  hours,  staining  with  toluidine  blue  no  longer  gives  a  metachromatic 
reaction.  Such  a  loss  of  the  metachromatic  reaction  occurs  even  in  very  dilute 
solutions  of  insulin.  This  is  shown  in  Table  VI.  In  interpreting  this  table,  it 
should  be  remembered  that  the  amebae  were  immersed  in  solutions  whose  volume 
was  very  large  in  comparison  with  the  volume  of  the  amebae.  Actually  in  the 
experiments  reported  in  the  table,  10  ml.  of  solution  were  used  and  only  a  few  drops 
of  a  concentrated  suspension  of  amebae. 

Our  experiments  indicate  that  insulin  can  and  does  combine  with  heparin. 
There  is  some  indication  in  the  chemical  literature  in  support  of  this  view.  Ac- 
cording to  Gorter  (1954),  heparin  can  combine  with  various  proteins,  including 
insulin.  In  Gorter's  experiment,  the  insulin  was  combined  with  the  lipid  cephalin 


TABLE  VI 

Metachromatic  reaction  of  Chaos  chaos  after  the  amebae  were  immersed  for  16  hours 
in  various  concentrations  of  insulin  solution 


Concentration 
of  insulin,  % 

0 

0.000625 

0.00125 

0.0025 

0.005 


Reaction 

+ 


ACTION  OF  INSULIN  ON  PROTOPLASM  467 

(phosphatidyl-serine),  and  Gorter  believes  that  as  a  result  of  the  complex  formed 
between  insulin  and  heparin,  the  lipid  is  set  free.  The  reaction  between  heparin 
and  insulin  is  strongly  influenced  by  hydrogen  ion  concentration,  a  fact  which  may 
be  of  considerable  importance  in  the  interpretation  of  biological  phenomena. 

DISCUSSION 

Although  the  permeability  theory  of  insulin  action  has  been  so  widely  accepted, 
as  already  noted,  the  fact  that  insulin  increases  the  rate  of  passage  of  sugar  into  a 
cell  could  well  be  the  result  of  some  acceleration  of  an  enzyme  responsible  for  such 
transport,  so  that  the  more  rapid  entrance  of  the  sugar  would  really  be  a  result 
rather  than  a  cause  of  enzyme  action.  A  somewhat  similar  idea  was  expressed 
many  years  ago  by  Staub  (1927).  But  this  type  of  objection  is  perhaps  not  too 
serious.  The  value  of  a  theory  lies  in  the  extent  to  which  it  can  explain  and 
interpret  known  facts.  Perhaps  the  most  important  fact  about  the  action  of  insulin 
is  that  it  behaves  as  an  anabolic  hormone  and  produces  an  increase  in  the  synthesis 
of  carbohydrates,  proteins  and  fats.  It  could  of  course  be  claimed  that  inasmuch 
as  insulin  hastens  the  entrance  of  sugar  into  a  cell,  this  fact  in  itself  might  favor 
the  synthesis  of  proteins,  for  the  energy  for  such  syntheses  is  now  believed  to  come 
from  the  oxidation  of  carbohydrate.  This  may  well  be  the  correct  explanation. 
However,  according  to  Sinex,  MacMullen  and  Hastings  (1952),  the  addition  of 
glucose  tends  to  prevent  the  insulin-induced  synthesis  of  protein  in  the  rat  dia- 
phragm. And  if  we  consider  the  long-known  facts  concerning  the  effects  of  insulin, 
the  permeability  theory  would  have  some  difficulty  in  offering  a  complete  explana- 
tion. For  if  the  primary  effect  of  the  hormone  is  to  increase  the  sugar  content  of 
a  cell,  then  logically  one  should  be  able  to  imitate  the  effects  of  insulin  merely  by 
feeding  or  injecting  excess  glucose,  for  such  glucose  would  also  increase  the  cellular 
sugar.  But  administration  of  excess  sugar  is  hardly  a  cure  for  diabetes.  Moreover, 
under  conditions  in  which  there  is  an  excess  of  insulin  in  the  blood,  if  the  sugar 
permeability  theory  were  correct,  from  a  logical  standpoint  the  very  worst  possible 
treatment  to  offset  the  harmful  effects  of  the  excess  would  be  to  feed  or  inject  glucose  ; 
for  the  permeability  theory  assumes  that  insulin  acts  by  introducing  more  glucose 
into  the  cells.  And  yet,  as  is  standard  knowledge,  the  convulsions  and  other  adverse 
symptoms  induced  by  excess  insulin  can  be  cured  readily  enough  by  the  administra- 
tion of  glucose. 

Of  course  in  an  animal  as  complicated  as  a  mammal,  there  are  too  many  interac- 
tions of  various  organ  systems  to  enable  one  to  reach  unassailable  conclusions  by 
logic  based  on  the  behavior  of  any  one  organ  system.  It  is  quite  possible  that 
insulin  and  sugar  have  one  effect  on  muscle  and  another  on  the  cells  of  the  medulla 
and  spinal  cord.  Thus  it  might  be  postulated  that  in  insulin  shock,  the  nerve  cells 
in  the  basal  part  of  the  brain  come  to  lack  glucose  and  that  this  lack  is  responsible 
for  the  convulsions  and  the  coma.  Then  the  administration  of  sugar  might  quickly 
restore  the  nerve  cells  to  their  normal  state. 

On  the  basis  of  our  theory,  if  insulin  acts  primarily  by  combining  with  heparin 
and  counteracting  its  effects,  then  such  an  action  would  immediately  accelerate 
various  types  of  enzymic  activity.  For,  as  is  well  known,  heparin  is  an  inhibitor  of 
some  proteases.  Thus,  it  inhibits  the  action  of  trypsin  (Horwitt,  1940),  and 
pepsin  (Marini  and  Levey,  1955).  It  also  inhibits  the  action  of  ribonuclease 


468     L.  V.  HEILBRUNN,  F.  T.  ASHTON,  C.  FELDHERR  AND  W.  L.  WILSON 

(Zollner  and  Fellig,  1953),  and  of  amylase  (Myrback  and  Persson,  1952).  Because 
it  is  a  polyanion,  it  can  have  a  retarding  effect  on  the  activity  of  various  enzymes 
(Spensley  and  Rogers,  1954).  When  injected  into  the  blood  stream,  it  tends  to 
extract  Upases  from  tissues  (Iselin  and  Schuler,  1957),  and  presumably  this  would 
retard  cellular  lipase  activity.  And  inasmuch  as  it  retards  the  activity  of  ribonuc- 
lease,  it  might  also  tend  to  prevent  the  synthesis  of  ribonucleoproteins ;  this  might 
be  a  factor  in  retarding  the  formation  of  proteins  with  enzymic  activity.  Finally, 
because  heparin  prevents  the  clotting  of  protoplasm,  and  such  clotting,  as  stated 
previously,  acts  as  a  trigger  for  oxidative  reactions,  it  might  also  retard  the  oxidative 
activity  of  a  cell.  Hence  by  the  simple  combination  with  heparin,  insulin  could 
exert  many  of  the  effects  we  know  it  to  have.  In  support  of  our  point  of  view,  it 
might  be  noted  that  according  to  Bond  and  Spitzer  (1955),  much  of  the  hypo- 
glycemic  effect  of  insulin  is  lost  if  it  is  injected  into  rabbits  previously  injected  with 
heparin.  Bond  and  Spitzer  do  not  believe  that  this  phenomenon  is  due  to  any 
combination  of  heparin  with  insulin,  for  when  they  vinjected  a  mixture  of  the  two 
substances,  insulin  action  remained  unimpaired.  But  within  blood,  and  even  more 
within  cells,  various  factors  such  as  pH,  ionic  strength  or  even  the  presence  of 
protein  co-factors,  might  have  an  influence  on  any  possible  combination. 

As  recent  authors  are  coming  to  realize  (Weissbecker  and  Hitzelberger,  1953; 
Riley,  Shepherd,  West  and  Stroud,  1955),  heparin  has  many  physiological  actions 
in  addition  to  its  effect  on  blood  clotting.  Thus  Riley  ct  al.  suggest  "that  the 
function  of  heparin  may  be  concerned  rather  with  events  in  the  tissues  than  with  the 
coagulability  of  the  circulating  blood,"  and  a  similar  statement  is  also  made  by 
Weissbecker  and  Hitzelberger.  One  interesting  phenomenon  is  the  fact  that 
heparin  antagonizes  the  effects  of  ACTH  and  cortisone.  As  we  learn  more  about 
the  heparin  and  heparin-like  substances  that  are  found  in  cells,  we  may  gain  addi- 
tional insight  into  life  processes  and  the  action  of  various  drugs  on  these  processes. 

Obviously  the  work  we  have  done  represents  only  a  beginning.  If  the  theory 
we  propose  is  correct,  then  much  more  wrork  needs  to  be  done  in  order  to  place  it 
on  a  firm  footing.  Any  theory  which  attempts  to  give  a  complete  explanation  of 
insulin  activity  is  faced  with  many  difficulties. 

SUMMARY 

1.  Insulin  does  not  speed  the  entrance  of  glucose  into  the  eggs  of  a  sea  urchin, 
a  clam  and  a  worm. 

2.  Dilute  solutions  of  heparin  prevent  protoplasmic  clotting  in  ameba.     This 
action  of  heparin  is  blocked  by  insulin. 

3.  Evidence  is  presented  to  show  that  insulin  combines  with  heparin.     It  blocks 
the  metachromatic  reaction  that  heparin  gives  with  toluidine  blue.     This  can  clearly 
be  shown  in  vitro,  and  it  is  also  indicated  by  studies  on  living  amebae. 

4.  Earlier  work  has  shown  that  heparin  acts  as  an  inhibitor  of  various  enzymes, 
and  in  general  it  may  be  thought  of  as  constituting  a  brake  on  many  of  the  chemical 
activities  of  a  cell.     By  preventing  this  inhibiting  action,  insulin  is  able  to  promote 
the  synthesis  of  various  essential  constituents  of  the  protoplasm. 

5.  Also,  in  view  of  the  fact  that  protoplasmic  clotting  involves  oxidation  and 
can  act  as  a  trigger  for  oxidative  activity,  insulin  by  preventing  the  anticlotting 
action  of  heparin  can  promote  oxidations. 


ACTION  OF  INSULIN  ON  PROTOPLASM  469 

LITERATURE  CITED 

BEST,  C.  H.,  1948.     Diabetes  and  Insulin  and  the  Lipotropic  Factors.     The  Beaumont  Lecture. 

Thomas,  Springfield. 

BEST,  C.  H.,  1953.     Aspects  of  the  action  of  insulin.     Ann.  Int.  Mcd.,  39:  433-443. 
BEST,  C.  H.,  C.  M.  JEPHCOTT  AND  D.  A.  SCOTT,  1932.     Insulin  in  tissues  other  than  the  pancreas. 

Amcr.  J.   PhysioL,  100:   285-294. 
BOND,  B.  D.,  AND  J.  j.  SPITZER,  1955.     Effects  of  heparin  on  carbohydrate  metabolism  in  the 

rabbit.    Amcr.  J.  Physio!.,  180:  575-579. 
FOSHAY,  L.,  1925.     Observations  upon  the  action  of  insulin  on  the  blood,  with  special  reference 

to  the  cause  of  the  condition  known  as  hypoglycemia.     Amcr.  J.  PhysioL,  73  :  470-479. 
GORTER,  E.,  1954.     Heparin  und  Eiweiss.     Kolloid-Zeitschr.,  136:   102-106. 
GUENSBERG,  E.,  1947.     Die  Glukoseaufnahme  in  menschliche  rote  Blutkorperchen.     Inaug.  Diss., 

Bern. 

HAUGAARD,  N.,  AND  J.  B.  MARSH,  1953.     The  Action  of  Insulin.     Thomas,  Springfield. 
HAUSLER,  H.,  AND  O.  LOEWI,  1925.     Zur  Frage  der  Wirkungsweise  des  Insulins.     I.     Insulin 

und  die   Glucoseverteilung  zwischen   fliissigen  und  nicht-flussigen   Systemen.     Arch.  f. 

d.  gcs.  PhysioL,  210  :  238-279. 

HEILBRUNN,  L.  V.,  1956.     The  Dynamics  of  Living  Protoplasm.     Academic  Press,  New  York. 
HEILBRUNN,  L.  V.,  1958.     The  Viscosity  of  Protoplasm.     Springer- Verlag,  Vienna. 
HIMMERICH,  F.,  AND  F.  S.  Tsci-iERNjAK,  1936.     Die  Regulierung  der  Sauerstoffaufgabe  von 

Erythrocyten.     III.     Blutglykolyse,  Insulin,  und  Adrenalin.     Biochem.  Zeitschr.,  286 : 

344-359. 

HOBER,  R.,  1944.     (Appendix  to  a  paper  by  S.  Kozawa.)     Biochem.  Zeitschr.,  60:  253-256. 
HOGLER,  F.,  A.   THOMANN   AND  K.  UBERRACK,   1929.     liber   die   Glucosefixation   durch   Blut- 
korperchen.    Biochem.  Zeitschr.,  209 :   1-31. 

HORWITT,  M.  K.,  1940.     The  anti-tryptic  properties  of  heparin.     Science,  92  :  89-90. 
ISELIN,  B.,  AND  W.  SCHULER,  1957.     Uber  die  Einwirkung  von  Heparin  auf  Lipoprotein-Lipase 

(Clearing  Factor)  aus  Gewebe.     Helvet.  PhysioL  ct  PliarmacoL  Ada,  15:  14-24. 
LEVINE,   R.,   AND   M.   S.   GOLDSTEIN,   1955.     On   the   mechanism   of  action   of   insulin.     Recent 

Pi'iii/rcss  in  Hormone  Research,  11 :  343-380. 

LOEWI,  O.,   1927.     Glykamin  und  Insulin.     Klin.   Wochcnschr.,  6:  2169-2176. 
MACLEOD,  J.  J.  R.,  1924.     Insulin.     PhysioL  Rev.,  4:  21-68. 

MARINI,  M.,  AND  S.  LEVEY,  1955.     Effect  of  pepsin  inhibitors  on  milk  clotting  activity  of  crys- 
talline pepsin.     Proc.  Soc.  Exp.  Biol.  Mcd.,  88:  611-613. 
MYRBACK,  K.,  AND  B.  PERSSON,  1952a.     Uber  die  Inaktivierung  der  Malzamylase.     II.     Inakti- 

vierung  durch  Heparin.     Arkiv  Kemi,  5 :  177-185. 
MYRBACK,  K.,  AND  B.  PERSSON,  1952b.     Action  of  heparin  on  barley  (3-amylase.  Arkiv.  Kemi, 

5:  477-488. 
PARK,  C.  R.,  J.  BORNSTEIN  AND  R.  L.  POST,  1955.     Effect  of  insulin  on  free  glucose  content  of 

rat  diaphragm  in  vitro.     Amer.  J.  PhysioL,  182  :    12-16. 
PARK,  C.  R.,  AND  L.  H.  JOHNSON,  1955.     Effect  of  insulin  on  transport  of  glucose  and  galactose 

into  cells  of  rat  muscle  and  brain.     Amcr.  J.  PhysioL,  182:  17-23. 
PLETSCHEK,  A.,  P.  VON  PLANTA  AND  W.  A.  HUNZINGER,  1955.     Beeinflussung  der  Fructose-  und 

Glukosepermeabilitat    von    Erythrocyten    durch    Temperatur,    Cortison    und    Insulin. 

Helvet.  PhysioL  et  Pharmacol.  Ada,  13 :  18-24. 
RILEY,  J.  F.,  D.  M.  SHEPHERD,  G.  B.  WEST  AND  S.  W.  STROUD,  1955.     Function  of  Heparin. 

Nature,  176:  1123. 
Ross,   E.   J.,    1956.     The    "permeability"   hypothesis   of   the   action    of   insulin.     Medicine,   35 : 

355-388. 

SINEX,  F.  M.,  J.  MACMULLEN  AND  A.  B.  HASTINGS,  1952.     The  effect  of  insulin  on  the  incor- 
poration of  C14  into  the  protein  of  rat  diaphragm.     /.  Biol.  Chem.,  198 :  615-619. 
SOLS,  A.,  AND  R.  K.  CRANE,  1954.     Substrate  specificity  of  brain  hexokinase.     /.  Biol.  Chem., 

210:  581-595. 

SPENSLEY,  P.  C.,  AND  H.  J.  ROGERS,  1954.     Enzyme  inhibition.     Nature,  173:  1190. 
STADIE,  W.  C.,   1954.     Current  concepts  of  the  action  of  insulin.     PhysioL  Rev.,  34:   52-100. 
STADIE,  W.   C.,   1957.     The   "permeability"   hypothesis   of  the   action   of   insulin.     Diabetes,   6: 

446-447. 


470     L.  V.  HEILBRUNN,  F.  T.  ASHTON,  C.  FELDHERR  AND  W.  L.  WILSON 

STAUB,    H.,    1927.     t)ber    Insulin    uncl    seinen    Wirkungsmechanismus.     Ergeb.    inn.    Med,,    31 : 

121-164. 
STICK,  W.,  AND  H.   MASKE,   1956.     Insulin   und   Insulintherapie.     Urban   und   Schwarzenberg, 

Munchen-Berlin. 
WEIL-MALHERBE,   H.,    1955.     The   mechanism   of   action   of   insulin.     Ergeb.   d.    Physio].,   48: 

54-111. 
WEISSBECKER,  L.,  AND  A.  HITZELBERGER,  1953.     Gibt  es  ein  Regulationssystem  ACTH-Heparin? 

Klin.  Wochenschr.,  31 :  288-289. 
WIECHMANN,  E.,  1924.     Zur  Frage  der  Permeabilitat  der  roten  Blutkorperchen  fiir  Trauben- 

zucker  unter  besonderer  Beriicksichtigung  des  Diabetes.     Zcitschr.  /.  d.  ges.  exp.  Med., 

41 :  462-492. 
WIECHMANN,  E.,  1926.     Zur  Permeabilitatstheorie  des  Diabetes  mellitus.     Dcutschcs  Arch.  f. 

klin.  Med.,  150:  186-207. 
WILBRANDT,  W.,   1947.     Die  Wirkung  des   Phlorizins   auf  die   Permeabilitat   der   menschlichen 

Erythrocyten.     Helvet.  Physio] .   ct  Pharmacol.   Ada,  5:   C64-C65. 
WILSON,  W.  L.,  AND  L.  V.  HEILBRUNN,  1957.     The  relation  of  protoplasmic  gelation  to  oxidative 

processes.     Exp.  Cell  Res.,  13:  "234-243. 
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PhysioL,  173:  223-228. 


THE  EFFECTS  OF  CERTAIN  NEUROHUMORS  AND  OF  OTHER 

DRUGS  ON  THE  VENTRICLE  AND  RADULA  PROTRACTOR 

OF  BUSYCON  CANALICULATUM  AND  ON  THE 

VENTRICLE  OF  STROMBUS  GIGAS  *• 2 

ROBERT  B.  HILL 

Biological  Laboratories,  Harvard   University,  Cambridge  38,  Massachusetts,5  and   the 
Bermuda    Biological  Station  for  Research,  Inc.* 

Krijgsman  and  Divaris  (1955)  called  attention  to  the  need  for  pharmacological 
information  about  the  heart  of  Biisycon  canaliculatwn.  Such  information  was 
gained  in  the  course  of  investigations  of  the  physiology  of  Busycon,  carried  out 
between  1953  and  1956,  and  is  presented  here.  For  the  sake  of  comparison, 
experiments  on  the  ventricle  of  Husvcon  were  repeated  on  the  ventricle  of  Strombus 
i/if/us.  Further  experiments  with  the  Strombus  heart  are  also  reported  here.  The 
/-•nsycon  raclula  protractor  (recommended  for  physiological  investigation  by  Her- 
rick,  1906)  was  used  for  a  comparison  of  the  effects  of  the  same  drugs  on  non- 
cardiac  muscle. 

I  wish  to  thank  Professor  John  H.  Welsh  for  the  suggestion  which  led  to  this 
study  and  for  his  guidance. 

Pharmacology  of  the  ventricle 

METHODS 

The  amplitude  of  heart  beat  was  measured  on  kymograph  records  from  isolated 
ventricles,  perfused  with  sea  water  through  the  auricle  in  a  manner  similar  to  that 
described  by  Welsh  and  Smith  ( 1949)  for  larger  crustacean  hearts.  The  bath  was 
so  arranged  that  it  could  be  flushed  with  sea  water  while  the  ventricle  was  washed 
through  the  cannula  between  tests.  Drugs  in  sea  water  solution  were  applied  by 
substitution  for  the  perfusion  fluid.  Experiments  on  the  Busycon  ventricle  were 
carried  out  at  room  temperature  of  23°  C,  and  experiments  on  the  Strombus 
ventricle  were  carried  out  at  room  temperature  which  varied  between  20°  and  25°  C. 

RESULTS 

Acetylcholine  produced  a  decrease  in  amplitude  of  beat  in  the  Busycon  ventricle 
at  a  10~0  molar  concentration,  with  diastolic  arrest  at  1OT  molar  (Fig.  1,  A).  The 

1  This  investigation  was  supported  in  part  by  a  predoctoral  fellowship,  HF-5211,  from 
the  National  Heart  Institute,  Public  Health  Service. 

-  This  paper,  in  slightly  different  form,  comprised  part  of  a  thesis  presented  in  partial 
fulfillment  of  the  requirements  for  the  degree  of  Doctor  of  Philosophy  at  Harvard  University. 

3  Present   address :    Department   of   Zoology,    Coburn    Hall,    University    of    Maine,    Orono, 
Maine. 

4  Contribution  Number  249  from  the  Bermuda  Biological  Station.     Assisted  by  a  National 
Science  Foundation  grant-in-aid  through  the  Bermuda  Biological  Station. 

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FIGURE  1.  A.  The  effect  of  acetylcholine  on  three  Busy  con  canaliculatum  ventricles. 
Each  point  represents  the  average  of  the  responses  of  a  ventricle  to  two  exposures  to  the  same 
concentration,  once  in  an  ascending  series,  and  once  in  a  descending  series.  B.  The  effect  of 
acetylcholine  (solid  lines),  carbamylcholine  (dotted  line),  and  acetyl-beta-methylcholine  (broken 
line),  on  the  Strombus  gigas  ventricle.  C.  The  effect  of  eserine  on  the  concentration-action 
curve  of  the  Strombus  gigas  ventricle  for  acetylcholine  (ACH),  carbamylcholine  (CCH),  and 
acetyl-beta-methylcholine  (MCH).  In  each  case  the  solid  line  represents  the  effect  of  the 
ACH  or  ACH  analogue  in  the  perfusion  sea  water  of  an  uneserinized  heart.  The  broken 
line  represents  the  effect  of  the  ACH  or  ACH  analogue  applied  in  perfusion  fluid,  consisting 
of  a  10~5  molar  sea  water  solution  of  eserine,  to  a  ventricle  previously  soaked  for  an  hour  in 
10~r'  molar  eserine. 


PHARMACOLOGY  OF  BUSYCON  473 

threshold  to  acetylcholine  was  found  to  be  approximately  the  same  for  Strombus, 
but  the  response  to  a  concentration  just  above  threshold  was  an  increase  in  ampli- 
tude, with  a  decrease  in  amplitude  elicited  by  concentration  ten  times  threshold 
(Fig.  1,  B).  In  both  Strombus  and  Busycon,  acetylcholine  improved  irregular 
beating  at  concentrations  ten  times  less  than  the  level  of  the  threshold  for  an  effect 
on  amplitude. 

Although  carbamylcholine  in  low  concentrations  failed  to  produce  the  increase 
in  amplitude  of  the  Strombus  ventricle  beat  that  was  seen  with  acetylcholine,  it 
produced  decrease  in  amplitude  in  the  neighborhood  of  5  X  10"8  molar  concentration 
(Fig.  1,  B). 

Acetyl-beta-methylcholine  has  an  acetylcholine- like  effect  on  the  Strombus  ven- 
tricle but  with  a  threshold  concentration  one  thousand  times  greater  (Fig.  1,  B). 

Eserine  failed  to  potentiate  the  action  of  acetylcholine,  carbamylcholine,  or 
acetyl-beta-methylcholine  on  the  Strombus  ventricle.  It  did  regularly  abolish  the 
excitatory  effect  of  low  concentrations  of  acetylcholine  (Fig.  1,  C). 

Both  adrenalin  and  noradrenalin  proved  to  have  a  positive  tonotropic  effect  on 
the  Busycon  ventricle  but  the  effective  concentrations  were  not  in  the  extremely 
dilute  range  at  which  acetylcholine  became  effective.  At  a  10"5  molar  concentration 
either  neurohumor  increased  the  amplitude  of  beat  about  fifty  per  cent,  but  the  am- 
plitude was  increased  nearly  one  hundred  per  cent  when  a  10~5  molar  concentration 
was  obtained  as  the  sum  of  the  molarities  of  adrenalin  and  noradrenalin  added 
simultaneously  (Fig.  2,  A). 

5-Hydroxytryptamine  was  found  to  have  an  action  on  the  Busycon  heart  similar 
to  that  of  adrenalin  but  with  a  threshold  in  the  vicinity  of  10  9  molar,  and  is  thus 
active  in  dilutions  comparable  to  acetylcholine  dilutions.  The  Busycon  ventricle 
is  a  thousand  times  less  sensitive  to  tryptamine  than  to  5-hydroxytryptamine  (Fig. 
2,  C).  Adrenalin  or  noradrenalin  concentrations  fifty  times  greater  than  thresh- 
old concentration  for  a  particular  heart  irreversibly  stop  the  heart,  but  5-hydroxy- 
tryptamine at  10~2  molar,  ten  million  times  the  threshold  concentration,  does  not 
even  produce  systolic  arrest. 

In  contrast  to  the  synergistic  effect  of  simultaneous  addition  of  adrenalin  and 
noradrenalin  to  the  perfusion  fluid,  when  adrenalin  and  tryptamine  are  added  to  the 
perfusion  fluid  simultaneously  the  effect  is  not  significantly  greater  than  if  the  same 
molar  concentration  were  made  up  of  one  drug  (Fig.  2,  B). 

5-Hydroxytryptamine  acts  on  the  Strombus  ventricle  over  a  wide  range  of 
concentrations  with  a  threshold  at  1O10  molar  (Fig.  3,  A). 

The  antagonism  between  the  negative  tonotropic  effect  of  acetylcholine  and  its 
analogs,  and  the  positive  tonotropic  effect  of  5-hydroxytryptamine,  on  the  Strombus 
ventricle,  is  plotted  in  Figure  3.  B,  in  terms  of  the  reduction  by  acetylcholine,  car- 
bamylcholine, or  acetyl-beta-methylcholine  of  the  amplitude  maintained  by  10" 
molar  5-hydroxytryptamine.  Acetylcholine  acts  on  the  5-hydroxytryptamine 
excited  ventricle  much  as  on  the  normal  ventricle,  but  carbamylcholine,  which  is  less 
effective  than  acetylcholine  in  depressing  the  spontaneous  heart  beat,  is  almost  as 
effective  an  antagonist  of  5-hydroxytryptamine  as  is  acetylcholine.  Gramme  is 
another  antagonist  of  the  action  of  5-hydroxptryptamine  on  the  Strombus  ventricle, 
and  will  completely  block  the  action  of  10"  molar  5-hydroxytryptamine  at  a 
5  X  10~5  molar  gramine  concentration. 


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FIGURE  2.  A.  The  effects  of  adrenalin,  of  noradrenalin,  and  of  the  same  molar  concentra- 
tions made  up  with  equal  amounts  of  the  two  amines,  on  the  amplitude  of  beat  of  the  isolated 
Kiisycon  canaliculatitm  ventricle.  Each  curve  ends  at  the  concentration  at  which  the  ventricle 
stopped  in  systole.  B.  A  similar  comparison  of  the  effects  of  tryptamine,  of  noradrenalin,  and 
of  both  simultaneously  on  the  Busycon  canaliculatum  ventricle.  C.  The  effects  of  tryptamine 
and  5-hydroxytryptamine  on  the  amplitude  of  beat  of  the  isolated  Busycon  canaliculatitm 
ventricle. 

DISCUSSION 

The  pharmacological  relations  of  the  Busycon  canaliculatum  ventricle  resemble 
those  of  other  gastropod  hearts.  Acetylcholine  has  been  shown  to  depress  the  beat 
of  the  hearts  of  the  gastropods  Bncchuun  iindatiiin  and  Cyprina  islandica  (Welsh, 
1956),  Dolabclla  auricula  (Ebara,  1955),  Cochlitoma  zebra  (Divaris  and  Krijgs- 
man,  1954),  Helix  pomatia  (Jullien  and  Ripplinger,  1950),  and  Mure.v  irunculus 
(Jullienand  Morin,  1931). 

Among  the  hearts  listed  above,  Mure.v  tntncnlns  has  been  reported  by  Morin 
and  Jullien  (1930)  to  have  a  small  group  of  nerve  cell  bodies  near  the  location 
where  Carlson  (1905)  reports  a  ganglion  in  Bnsycon.  However,  Divaris  and 
Krijgsman  (1954)  not  only  found  no  nervous  elements  in  the  white  spot  at  the 
Cochlitoma  scbra  ventriculo-aortic  junction,  but  also  demonstrated  the  existence  of 


PHARMACOLOGY  OF  BUSYCON 


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oi 


100 


75 


50 


-- 


10 


-11 


10 


-10 


10'9  10'8  ID'7 

MOLAR     CONCENTRATION 


10 


-6 


10 


-5 


LlJ 
(S) 

<    -25 

LlJ 

a: 
o 

Ld 

a  -50 

h- 

LJ 

o   -75 

LU 

a. 

-100 


MCH 


10 


'9 


10' 


10 


'7 


10 


'6 


10 


'5 


10 


'4 


10 


-3 


MOLAR     CONCENTRATION 


FIGURE  3.  A.  The  effect  of  5-hydroxytryptamine  on  the  amplitude  of  beat  of  the  isolated 
ventricles  of  Strombus  f/ifias  (solid  line)  and  Aplysia  protect,  (broken  line).  B.  The  effects  of 
acetylcholine,  carbamylcholine,  and  acetylbetamethyl  choline,  on  the  amplitude  of  beat,  of  the 
Strombus  gigas  ventricle,  which  is  maintained  by  perfusion  with  10~7  molar  5-hydroxytryptamine. 

myogenic  pacemakers.  A  myogenic  origin  for  the  beat  of  the  heart  of  Mnrc.v 
trunciilns  was  indicated  when  Cardot,  Jullien  and  Morin  (1929)  showed  that 
isolated  fragments  would  beat  in  sea  water.  Thus,  the  Busycon  ventricle  reacts  to 
acetylcholine  like  the  hearts  of  other  gastropods  which  have  been  demonstrated  to 
have  myogenic  pacemakers. 

The  extremely  low  concentrations  at  which  acetylcholine  and  5-hydroxy- 
tryptamine are  effective  on  the  B-nsycon  ventricle  are  in  accordance  with  Welsh's 
(1957)  rinding  that  they  act  as  neurohumors  in  Venus  uierccnaria.  That  5- 
hydroxytryptamine  is  effective  in  lower  concentration  than  adrenalin  or  noradren- 
alin.  with  an  equally  rapid  onset  of  action,  suggests  that  5-hydroxytryptamine  might 


476  ROBERT  B.  HILL 

be  closer  in  structure  to  the  natural  cardio-regulatory  neurohumor  of  Busycon  than 
are  the  mammalian  neurohumors.  5-Hydroxytryptamine  has,  in  fact,  been  found 
in  the  pooled  ganglia  of  Busycon  by  Welsh  (1954). 

The  response  of  the  Busycon  canaliculatum  ventricle  to  adrenalin  and  noradrena- 
lin  is  evidence  that  it  is  more  similar  pharmacologically  to  the  hearts  of  the  molluscs 
Eledone  cirrosa  and  Anodonta  (Fange  and  Ostlund,  1954),  which  respond  by  an 
increase  in  amplitude  of  beat,  than  to  that  of  Aplysia  dactylomela,  which  is  insensitive 
to  the  two  neurohumors,  as  reported  by  von  Euler,  Chavez  and  Teodosio  (1952). 
That  the  isolated  Busycon  heart  beats  so  well  is  also  a  contrast  to  the  Aplysia 
dactylomela  heart  wrhich  will  beat  spontaneously  only  if  adjacent  ganglia  are  isolated 
with  it  (von  Euler,  Chavez,  and  Teodosio,  1952).  However,  I  have  found  the 
isolated  ventricle  of  Aplysia  to  beat  spontaneously,  although  not  well.  Its  beat  may 
lie  sustained  by  ergonovine  or  by  5-hydroxytryptamine,  but  its  threshold  to  5- 
hydroxtryptamine  is  between  10~s  and  10  7,  which  is  considerably  higher  than  the 
thresholds  of  the  Busycon  and  Stronibns  ventricles  (Fig.  3,  A). 

Pharmacology  of  the  leached  ventricle 

Following  Burn's  theory  (1950)  of  the  relation  of  local  hormones  to  cardiac 
automatism,  it  might  be  expected  that  a  dener vnted  ventricle,  which  had  been 
deprived,  by  leaching,  of  previously  synthesized  neurohumors,  would  respond  by 
contraction  to  either  acetylcholine  or  5-hydroxytryptamine. 

METHODS 

In  order  to  ascertain  the  upper  limit  to  the  time  an  isolated  Busycon  canalicu- 
latum ventricle  may  remain  viable  and  useful  for  bioassay,  six  isolated  entire  hearts 
were  set  aside  in  sea  water  at  9°  C.  for  periods  ranging  from  one  to  six  weeks.  At 
intervals,  a  ventricle  was  removed  to  room  temperature,  allowed  five  hours  for 
adjustment,  and  then  perfused.  It  would  seem  probable  that  after  a  week  the  cut 
distal  portions  of  the  cardioregulatory  nerves  (from  the  visceral  ganglion;  Carlson. 
1905)  would  have  degenerated,  so  that  the  leached  ventricle  might  react  to  pharma- 
cological agents  primarily  as  a  muscle  preparation. 

RESULTS 

Of  two  hearts  kept  at  9°  C.  for  one  week,  both  survived  and  both  beat  normally 
when  perfused.  That  is,  after  one-half  hour  one  was  beating  at  18  systoles  per 
minute,  the  other  at  21  and  both  were  emptying  completely  at  each  systole.  Each 
continued  at  its  original  rate  for  5  hours,  at  the  end  of  which  time  one  showed  a 
threshold  response  to  5  X  10~9  M  5-hydroxytryptamine  and  10~9  M  acetylcholine, 
and  the  other  a  threshold  response  to  10~9  M  acetylcholine  and  to  10~'J  M  5- 
hydroxytryptamine. 

Of  the  two  hearts  set  aside  for  three  weeks,  only  one  survived.  The  other,  after 
one-half  hour  of  perfusion,  was  beating  regularly  at  a  beat  of  22  systoles  per  minute. 
After  three  hours  it  had  slowed  down  to  12  systoles  per  minute  but  was  still  beating 
regularly.  Now,  however,  the  ventricle  was  no  longer  emptying  completely  and 
relaxed  to  three  times  its  previous  volume  at  diastole.  By  increasing  the  pressure 
of  perfusion  the  rate  was  increased  to  18  systoles  per  minute  and,  while  the  ventricle 


PHARMACOLOGY  OF  BUSYCON  477 

retained  the  full  relaxation  at  diastole,  it  returned  to  complete  emptying  at  systole. 
After  eight  hours  of  perfusion,  the  rate  was  down  to  nine  systoles  per  minute  but 
was  restored  to  eighteen  by  an  increase  in  pressure.  After  ten  hours  of  perfusion 
the  rate  had  dropped  again  to  ten  systoles  per  minute  and  a  further  increase  in 
pressure  was  required  to  raise  it  to  sixteen.  After  this  ventricle,  which  had  been 
set  aside  for  three  weeks  in  sea  water  at  9°  C.,  had  maintained  an  uninterrupted 
rhythm  for  fourteen  hours  and  sixteen  minutes  its  threshold  for  5HT  was  found  to 
be  in  the  neighborhood  of  a  5  X  10~9  molar  concentration  and  its  threshold  for 
acetylcholine  was  10~10  molar. 

Of  the  two  hearts  set  aside  for  six  weeks  at  9°  C,  one  survived.  When  set  up 
in  a  heart  bath  and  perfused  with  sea  water  the  ventricle  showed  no  sign  of 
spontaneous  activity,  but  it  did  react  to  10"7  molar  5HT  by  beating  at  the  rate  of  18 
systoles  per  minute  as  long  as  it  was  subjected  to  5HT. 

Subsequently,  four  hearts  were  taken  which  had  survived  four  weeks  at  11°  C. 
but  which  did  not  beat  spontaneously  when  perfused.  Each  was  subjected  to 
concentrations  of  acetylcholine  and  of  5-hydroxytryptamine  from  1O1-  to  10~2  molar 
at  half-molar  intervals.  No  concentration  of  acetylcholine  provoked  beating.  All 
four  hearts  beat,  when  perfused  with  10~7  molar  5-hydroxytryptamine,  at  a  normal 
rate  but  at  an  amplitude  much  less  than  that  which  had  been  elicited  by  5-hydroxy- 
tryptamine after  soaking  at  9°  C.  for  six  weeks. 

DISCUSSION 

The  failure  of  acetylcholine  to  restore  automatism  is  in  accord  with  the  similar 
findings  of  Jensen  (1957)  with  several  lamellibranch  hearts. 

Pharmacology  of  the  radula  protractor 

METHODS 

The  radula  protractor  was  isolated  intact,  attached  to  a  bit  of  radula  sac  at  <mc 
end,  and  to  a  fragment  of  odontophore  at  the  other,  and  set  up  in  a  sea  water  bath 
at  19°  to  21°  C.  Drugs  in  sea  water  solution  were  added  to  the  bath  to  produce 
the  desired  molar  concentration.  Ejection  from  a  syringe  assured  thorough  mixing. 
Air  was  bubbled  through  the  bath  from  a  capillary  tube  entering  at  the  bottom. 

RESULTS 

Spontaneous  contractions  do  not  occur  in  a  Busycon  canalicnlatmn  radula 
protractor  isolated  and  maintained  under  slight  tension  in  a  constant  temperature 
sea  water  bath.  This  makes  it  a  more  favorable  preparation  for  the  study  of 
induced  contractions  than  the  more  commonly  used  snail  retractor  pharyngis,  where 
rhythmicity  obscures  the  effect  of  stimulation  (Masai,  1951). 

Acetylcholine  at  10~5  molar  concentration  will  induce  a  contraction  which 
reaches  full  amplitude  immediately.  When  the  acetylcholine  is  washed  off,  the 
muscle  relaxes  immediately.  If  the  acetylcholine  solution  is  left  in  contact  with 
the  muscle,  the  contraction  declines  slightly  for  five  or  ten  minutes,  and  then  the 
rate  of  relaxation  accelerates,  and  by  forty-five  minutes  after  adding  acetylcholine 


478 


ROBERT  B.  HILL 


the  muscle  is  relaxed  again.  The  bathing  solution  has  then  lost  its  ability  to  con- 
tract a  fresh  muscle.  Normal  responsiveness  of  the  muscle  is  restored  by  ten 
minutes  of  washing  with  aerated  sea  water. 

When  1O5  M  acetylcholine  is  followed  in  three  minutes  by  sufficient  tryptamine 
to  produce  a  concentration  in  the  bath  of  10~3  M,  relaxation  is  not  an  immediate 
fall  in  tension  such  as  is  seen  when  acetylcholine  is  washed  off,  but  is  gradual  at 
first  and  then  develops  into  rhythmic  pulsation  with  a  slowly  declining  base  line 
(Fig.  4,  A).  The  rhythmicity  may  be  as  regular  in  rate  and  amplitude  as  a  heart- 
beat and  ceases  abruptly  with  the  relaxation  that  follows  when  the  mixture  of  the 
two  drugs  is  washed  off.  In  each  of  the  rhythmic  contractions  the  radula-  pro- 
tractor shortened  to  about  one-third  of  its  resting  length.  Figure  4,  B,  shows  the 
similar  response  when  the  muscle  is  subjected  first  to  10  r>  M  acetylcholine  and  then 
in  two  minutes  to  the  combination  of  1O5M  acetvlcholine  and  10~3  M  5-hvdroxv- 


ACH        5HT 


FIGURE  4.  Responses  of  the  Busycon  canaliculatnni  radula  protractor  to  10~r'  molar  acetyl- 
choline (ACH)  followed  by  10~3  molar  tryptamine  (TRYP),  5 -hydroxy tryptamine  (5-HT), 
or  adrenalin  (EPN). 


PHARMACOLOGY  OF  BUSYCON 


479 


tryptamine.  Figure  4,  C,  shows  the  response  obtained  when  the  radula  protractor 
is  subjected  first  to  10~5  M  acetylcholine  and  then  in  two  minutes  to  10~3  M  adrena- 
lin, also.  These  combinations  were  found  to  be  optimum  for  regularity  and  ampli- 
tude of  "beat,"  and  it  may  be  seen  that  tryptamine  was  the  most  effective  of  the 
three  amines. 

Figure  5,  A,  shows  that  in  the  radula  retractor,  tryptamine  following  acetylcho- 
line produces  a  similar  rhythmic  decline  of  tension.  The  same  is  true  for  the 
odontophore  retractor  (Fig.  5,  B).  It  may  be  seen  that  in  Figure  5,  A,  the  first 
effect  of  tryptamine  was  a  slight  decline  in  tension  while  in  Figure  5,  B,  it  was  a 
slight  increase.  The  first  effect  seems  to  vary  randomly  for  all  three  radula 
apparatus  muscles,  but  is  always  followed  by  the  rhythmic  relaxation. 

In  Figure  5,  A,  at  Tx,  10~5  M  tryptamine  was  added  in  the  absence  of  prior 
stimulation  by  acetylcholine,  and  at  T2,  the  concentration  was  brought  to  10~4  M 
tryptamine.  Neither  contraction  nor  relaxation  was  elicited,  yet  at  the  second  T2 
the  same  concentration  (following  acetylcholine)  produced  relaxation.  At  the 
temperature  of  these  experiments,  19-21°  C.,  no  concentration  of  tryptamine,  5- 
hydroxytryptamine,  or  adrenalin  relaxed  a  radula  apparatus  muscle  that  had  not 
been  previously  excited  to  contract.  Later  it  was  found  that  at  27°  C.  5-hydroxy- 
tryptamine  would  cause  a  previously  unstimulated  muscle  to  contract  slowly  and 
irregularly,  but  never  to  relax. 

A  radula  protractor  in  10"3  M  5-hydroxy tryptamine  develops  a  sensitivity  to 
stretching  not  shown  in  the  muscle  simply  isolated  in  sea  water.  It  responds  to  a 
sharp  tug  by  a  quick  contraction  followed  by  slower  relaxation. 


FIGURE  5.  Busycon  canaliculatiim.  A.  Radula  retractor :  Ti  =  10  5  molar  tryptamine, 
T2  =  10"*  molar  tryptamine,  Ai  =  10~5  molar  acetylcholine.  B.  Odontophore  retractor :  ACH  = 
10~4  molar  acetylcholine,  TRYP  =  10~3  molar  tryptamine,  M  =  10~5  molar  Mytolon  for  a  half 
hour.  A  plain  arrow  indicates  that  the  muscle  was  washed  with  sea  water  with  the  drum 
moving,  and  a  double-ended  arrow  indicates  that  the  muscle  was  washed  with  sea  water  for 
an  hour  with  the  drum  stopped. 


480  ROBERT  B.  HILL 

Acetylcholine  contraction  of  the  radula  protractor  may  be  blocked  both  by  an 
agent  active  at  motor  end  plates,  d-tubocurarine,  and  by  the  most  effective  antagonist 
of  acetylcholine  on  the  Venus  nierccnaria  heart  (Luduena  and  Brown,  1952), 
Mytolon.  When  10~3  molar  d-tubocurarine  is  applied  for  half  an  hour  the  con- 
traction elicited  by  1O5  molar  acetylcholine  is  blocked  but  not  that  due  to  0.5% 
KH2PO4.  Similarly,  1 : 10,000  Mytolon  applied  for  a  half  hour  greatly  reduces  the 
contraction  elicited  by  10~5  molar  acetylcholine  but  has  no  effect  on  the  contraction 
following  0.5%  KH2PO4. 

Acetylcholine  contraction  of  the  radula  protractor  is  potentiated  by  eserine. 
When  the  graded  responses  of  the  same  muscle  to  an  increasing  series  of  acetylcho- 
line concentrations,  before  and  after  soaking  for  an  hour  in  1  : 10,000  eserine,  are 
compared,  it  is  found  that  the  response  at  each  concentration  is  augmented  although 
the  threshold  to  acetylcholine  is  not  altered.  Prior  soaking  in  eserine  has  the  same 
effect  on  the  rhythmicity  obtained  with  acetylcholine  and  tryptamine  as  has 
increasing  the  concentration  of  acetylcholine  used. 

Lysergic  acid  diethylamide  is  antagonistic  toward  the  production  of  a  "beat" 
by  the  combined  action  of  acetylcholine  and  tryptamine,  but  does  not  itself  cause 
contraction  or  relaxation  at  concentrations  from  10~5  molar  to  10^10  molar. 

DISCUSSION 

The  rhythmic  "beat"  of  the  radula  protractor  suggests  a  model  of  the  heart 
beat.  Acetylcholine  and  5-hydroxytryptamine  both  occur  as  natural  neurohumors, 
both  will  regulate  the  Busycon  heart,  and  together  they  induce  a  rhythmicity  in 
the  Busycon  radula  protractor  comparable  to  the  automatic  rhythmicity  of  the  heart. 
Tryptamine,  however,  is  more  effective  than  5-hydroxytryptamine  in  inducing 
rhythmicity,  whereas  5-hydroxytryptamine  is  more  effective  on  the  heart.  Heart 
strips,  when  cut  to  dimensions  similar  to  the  radula  protractor  and  set  up  on  the 
same  apparatus,  respond  to  5-hydroxytryptamine  with  rhythmic  contractions,  which 
are  opposed  by  acetylcholine. 

It  is  tempting  to  speculate  that  the  radula  protractor  "beat"  might  originate  in 
the  presence  at  the  cell  surface  of  the  opposing  neurohumors  in  the  right  proportions 
for  alternate  action.  Welsh  and  Slocombe  (1952)  suggest  that  released  acetylcho- 
line depresses  the  Venus  mercenaria  heart  by  changing  the  membrane  polarization 
of  muscle  fibers  and  thus  interfering  with  normal  contraction  and  the  normal  spread 
of  excitation.  The  effects  of  acetylcholine  and  5-hydroxytryptamine  on  the  surface 
membrane  polarity  of  a  non-cardiac  molluscan  smooth  muscle  have  been  investigated 
by  Twarog  (1954).  She  found  that  acetylcholine  depolarized  the  Mytiliis  cdnlis 
anterior  byssus  retractor  and  initiated  contraction.  5-Hydroxytryptamine  caused 
immediate  relaxation  but  produced  no  change  in  membrane  polarization.  Further- 
more, when  the  acetylcholine  was  washed  off,  the  muscle  immediately  repolarized, 
but  the  contraction  persisted.  (It  may  be  recalled  that  when  acetylcholine  was 
washed  off  the  radula  protractor,  the  muscle  relaxed  immediately).  Twarog 
suggests  that  it  is  probable  that  the  depolarization  induced  by  acetylcholine  is 
directly  related  to  the  ensuing  contraction.  The  failure  of  5-hydroxytryptamine 
to  produce  membrane  changes  while  relaxing  the  muscle  could  be  attributed  to  a 
direct  action  on  the  contractile  element. 

A  possible  explanation  for  the  rhythmicity  induced  in  the  radula  protractor  by 


PHARMACOLOGY  OF  BUSYCON  481 

acetylcholine  and  tryptamine  could  be  based  on  Twarog's  byssus  retractor  results. 
It  could  be  supposed  that  the  acetylcholine  in  the  bath  kept  the  muscle  cells 
depolarized,  which  would  lead  to  contraction.  The  tryptamine  also  in  the  bath 
would  relax  the  contractile  elements  and  a  second  contraction  would  then  occur 
in  response  to  the  surface  depolarization. 

One  alternative  hypothesis  would  be  that  acetylcholine  depolarized  the  muscle 
fiber  surface  membrane  and  that  tryptamine  then  repolarized  it.  If  it  is  supposed 
that  contraction  follows  depolarization  and  relaxation  follows  repolarization,  the 
"beat"  might  be  explained.  That  the  applied  acetylcholine  and  tryptamine  act  on 
muscle  rather  than  on  nerve  may  be  indicated  by  the  persistence  of  susceptibility  to 
induced  "beating"  in  isolated  radula  protractors  stored  for  a  week. 

SUMMARY 

1.  The  hearts  of  Busycon  canaliculatwn  and  S  trombus  gigas  were  found  to 
respond  to  applied  neurohumors  as  do  the  myogenic  hearts  of  other  gastropods. 
Acetylcholine  was  cardio-inhibitory,  and  5-hydroxytryptamine  was  cardio-accelera- 
tory,   in   concentrations   low   enough   to   suggest   that   they   might   be  the   normal 
regulatory  neurohumors. 

2.  The  Busycon  canaliculatum  radula  protractor  was  contracted  by  acetylcho- 
line, and  could  then  be  relaxed  rhythmically  by  5-hydroxytryptamine,  tryptamine, 
and  adrenalin,  all  of  which  raise  the  tonus  of  the  ventricle. 

LITERATURE  CITED 

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'  30:  177-193. 
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de  Murex  trunculus  places  dans  1'eau  de  mer.     C.  R.  Soc.  Biol.,  Paris,  102:  441. 
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482 


ROBERT  B.  HILL 


MASAI,  S.,  1951.     Tonic  and  Phasic  contractions  of  the  retractor  pharyngis  of  snail    (Euhadra 

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RIJLANT,  P.,  1931.     L'automatisme  du  coeur  des  Gasteropodes  Fulgur  carica  et  Fulgur  canalicu- 

latum,  Polyniccs.     C.  R.  Soc.  Biol,  Paris,  108:   1150-1152. 
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tryptamine.     /.   Cell.   Coinp.  Physiol.,  44:    141-164. 
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13:  162-163. 
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Buccimtm.     J.  Mar.  Biol.  Assoc.,  35:  193-201. 
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animals.     Ann.  N.  V.  Acad.  Sci.,  66:  618-630. 
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Venus  heart.     Biol.  Bull,  102  :  48-57. 
WELSH,   J.    H.,   AND   R.    I.    SMITH,    1949.     Laboratory   Exercises    in    Invertebrate    Physiology. 

Burgess   Publishing   Company,   Minneapolis,    Minnesota. 


OXYGEN  UTILIZATION  IN  THE  SYMBIOSIS  OF  EMBRYOS  OF 

THE  SALAMANDER,  AMBYSTOMA  MACULATUM  AND 

THE  ALGA,  OOPHILA     AMBLYSTOMATIS 

VICTOR  H.  HUTCHISON  AND  CARL  S.  HAMMEN 
Department  of  Zoology,  Duke    University,  Durham,  North   Carolina 

Unicellular  algae  occur  as  symbionts  in  a  great  number  of  aquatic  invertebrates, 
in  protozoans,  sponges,  coelenterates,  turbellarians,  bryozoans,  rotifers,  gastropods, 
lamellibranchs,  annelids,  and  ascidians  (Yonge,  1944).  An  association  of  unicellu- 
lar algae  and  vertebrates  has  been  known  only  since  1888  when  Orr  reported  a 
spherical  green  alga  in  the  egg  envelopes  of  Ambystoma  maculatum  (Shaw).  The 
symbiotic  relationship  between  these  salamander  eggs  and  algae  was  established  by 
Gilbert  (1942,  1944)  who  performed  several  experiments  and  made  several 
interesting  observations  on  egg  masses  of  A.  maculatum. 

Masses  of  these  salamander  eggs  inhabited  by  the  unicellular  alga  Oophila 
amblystomatis  (Lambert)  1  were  demonstrated  to  have  lower  mortality,  an  earlier 
hatching  time,  and  a  faster  growth  rate  than  algae-free  masses :  the  newly-hatched 
larvae  averaged  1.3  mm.  more  in  length  and  were  more  than  two  Harrison  stages 
in  advance  of  larvae  from  algae-free  egg  masses  of  the  same  age.  Gilbert 
demonstrated  that  the  results  were  clue  to  the  presence  of  the  algae  and  not  to  the 
presence  of  light.  It  was  also  shown  that  the  algae  grow  more  vigorously  in  the 
presence  of  the  embryo  than  in  its  absence.  Other  organisms  in  addition  to  the 
alga  inhabit  the  egg  envelopes.  These  include  a  protozoan  (Haptophyra  sp.) 
which  feeds  on  the  algae,  other  protozoa,  nematodes,  rotifers,  acanthocephalans, 
diatoms  (usually  on  the  surface  of  the  egg  masses),  and  even  an  occasional  annelid. 

A  more  rapid  growth  rate  and  better  viability  of  embryos  from  algae-inhabited 
complexes,  as  reported  by  Gilbert,  were  confirmed  during  the  course  of  this  inves- 
tigation. These  observations  suggest  that  the  symbiotic  relationship  may  consist 
of  the  utilization  by  the  embryo  of  the  O2  produced  in  photosynthesis  by  the 
algae  and  the  utilization  by  the  algae  of  CO2  produced  by  the  embryo. 

Eggs  of  Ambystoma  maculatum  are  often  laid  in  small,  temporary,  woodland 
ponds  usually  containing  decaying  leaves  and  other  detritus.  Such  a  pond  often 
has  a  low  O2  content,  and  any  method  which  would  increase  the  O2  supply  to  the 
embryo  might  aid  in  its  development.  The  following  experiments  were  designed 
to  determine  if  the  algae  inhabiting  the  egg  membranes  produced  O2  in  a  sufficient 
quantity  to  be  of  value  to  the  developing  embryo. 

MATERIALS  AND  METHODS 

Eggs  of  A.  maculatum  in  the  early  stages  of  development  (Harrison  Stages 
9-13)  were  collected  from  a  small,  detritus-filled,  temporary  pond  in  Duke  Forest 

1  See  Gilbert  (1942)  and  Smith  (1950)  for  description  of  the  alga  and  discussions  of 
correct  nomenclature. 

483 


484  VICTOR  H.  HUTCHISON  AND  CARL  S.  HAMMEN 

near  Durham,  North  Carolina,  on  February  26  and  27,  1958.  Several  days  of  cold 
rains  and  snow  preceded  the  time  of  collection.  The  temperature  of  the  water  was 
between  5°  and  6°  C.  at  the  time  the  eggs  were  taken.  Algal  growth  was  not 
evident  in  these  early  stages. 

The  egg  masses  were  separated  into  approximately  equal  halves.  One  half  of 
each  mass  was  then  allowed  to  develop  in  the  light  in  pond  water  collected  with 
the  eggs ;  thick  algal  growths  developed  in  these  eggs.  The  other  half  was  removed 
from  the  pond  water,  placed  in  spring  water,  and  kept  in  the  dark ;  no  algal  growths 
were  visible  in  these  eggs.  Egg  masses  utilized  in  preliminary  experiments  were 
kept  at  6°  C.,  but  all  other  eggs  used  were  kept  in  a  constant  temperature  cabinet 
at  20  ±  1°  C. 

All  oxygen  consumption  measurements  were  made  in  a  Warburg  respirometer 
at  25°  C.  Slightly  different  amounts  of  water  were  added  to  the  flasks  to  equalize 
the  volume  of  their  contents.  A  period  of  two  hours  was  allowed  for  temperature 
equilibration  before  O2  consumption  was  measured.  Flasks  were  not  shaken  in 
the  experiments  reported  here. 

Light  was  excluded  from  the  flasks  in  some  of  the  experiments  by  wrapping 
aluminum  foil  around  the  flask  and  manometer  stern  or  by  cutting  off  all  lights  in 
the  laboratory  during  hours  of  darkness.  All  other  determinations  were  made  with 
approximately  150  foot  candles  of  light  at  the  level  of  the  flasks,  measured  with  a 
Weston  Model  603  Illumination  Meter. 

Embryos  were  separated  from  their  surrounding  envelopes  by  placing  them  in 
a  measuring  spoon  slightly  smaller  than  the  eggs  and  pressing  them  gently  against 
the  side  of  a  finger  bowl,  rupturing  the  membranes  and  allowing  the  embryo  to 
drop  out  when  the  spoon  contents  were  submerged  in  the  water  of  the  finger  bowl. 
Six  to  nine  eggs,  embryos,  or  envelopes  were  then  placed  in  each  experimental  flask. 

All  measurements  in  each  category  of  experiments  were  made  in  duplicate, 
reduced  to  standard  temperature  and  pressure,  and  expressed  as  microliters  (/xl) 
per  hour  per  embryo,  envelope,  or  a  complex  of  both  embryo  and  envelope.  All 
determinations  were  made  over  a  period  of  at  least  ten  hours. 

Although  the  experiments  reported  here  involve,  primarily,  the  algae  and 
embryo,  many  other  inhabitants  are  present  in  the  egg  envelopes.  For  this  reason 
we  refer  to  the  "complex,"  meaning  the  entire  egg  envelopes  together  with  their 
contents — embryo,  algae,  protozoa,  bacteria,  etc. 

Availability  of  materials  in  the  proper  stages  of  development  sometimes  limited 
categories  of  experiments  which  could  be  performed.  Oxygen  consumption  deter- 
minations were  made  on  the  following  categories  of  experimental  materials : 

I.  Algae-free  complexes 

1.  Stages  12-13 

2.  Stages  32-34 

II.  Algae-inhabited  complexes 

1.  Stages  12-13 

2.  Stages  32-34 

a.  Light 

b.  Dark 


AMPHIBIAN  EMBRYO-ALGA  SYMBIOSIS  485 

III.  Isolated  embryos 

1.  From  algae-free,  stages  39-40 

2.  From  algae-inhabited 

a.  Stages  12-13 

b.  Stages  39-40 

IV.  Isolated  envelopes 

1.  Algae-free,  stages  39-40 

2.  Algae-inhabited,  stages  39-40 

RESULTS 

Oxygen  consumption  was  linear  over  the  entire  periods  of  observation  as  seen 
in  Figure  1,  where  each  point  represents  the  average  of  duplicate  flasks  in  five  of 
the  experiments.  Little  variation  was  obtained  among  flasks  containing  similar 
experimental  material. 

Mean  rates  of  O2  consumption  for  each  category  of  experiments  are  shown  in 
Figure  2.  Differences  in  rate  of  oxygen  consumption  at  different  stages  must  be 
considered  in  interpreting  these  results.  According  to  Hopkins  and  Handford 
(1943),  the  rate  of  oxygen  consumption  begins  with  a  low  value  and  rises  gradually 
to  stages  immediately  preceding  hatching  (Stages  37-38).  At  this  time  the  rate 
increases  rapidly  to  a  maximum  and  then  falls  off  during  the  last  few  stages  of 
development. 

The  O2  consumption  of  the  algae-inhabited  complex  (Stages  12-13)  averaged 
1 .41  {A  and  for  the  complex  without  algae,  0.97  ju,l.  This  represents  an  increase  of 
45.4%  when  algae  are  present.  In  later  stages  (32-34)  of  development  the  values 
were  1.91  /A  for  the  complex  with  algae  and  1.11  /A  for  the  algae-free  complex,  an 
increase  of  74.8%  when  algae  are  present.  Thus,  the  difference  between  algae-free 
and  algae-inhabited  complexes  is  in  the  direction  of  greater  consumption  by  the 
latter  and  increases  with  stages  in  development  of  the  embryo. 

Oxygen  consumption  of  algae-inhabited  complexes  (Stages  32-34)  determined 
under  conditions  of  darkness  averaged  2.50  p.1,  an  increase  of  0.56  /*!  (28.9%)  over 
that  of  similar  complexes  in  the  light.  This  latter  value  can  be  taken  to  approximate 
the  respiration  of  the  algae  in  the  dark  and  may  be  assumed  to  represent  the  net 
amount  of  O2  evolved  during  photosynthesis  of  the  algae,  provided  the  assumption 
is  correct  that  the  respiration  of  the  algae  is  balanced  by  O2  production  during 
photosynthesis.  As  pointed  out  below,  if  the  algae  are  facultative  heterotrophs, 
this  may  be  an  incorrect  assumption. 

Algae-free  envelopes  consumed  0.84  /u.1,  while  envelopes  with  algae  present 
consumed  only  0.41  /J,  a  difference  of  104%.  The  consumption  by  envelopes  can 
be  accounted  for  by  the  respiration  of  the  bacteria,  protozoa,  etc.,  living  within  the 
envelopes.  The  difference  in  O2  consumption  evidently  is  due  to  O2  production 
during  photosynthesis  of  the  algae.  This  net  reduction  of  O2  consumption  in  the 
envelopes  may  be  of  value  to  the  embryo  by  decreasing  the  removal  of  O2  from  the 
immediate  environment.  However,  there  is  a  net  consumption  of  O2  by  all  envelopes, 
indicating  that  there  is  no  surplus  of  photosynthetically  produced  O2  available  to 
the  embryo.  Therefore,  a  higher  O2  tension  could  not  account  for  the  faster 
development  and  greater  respiratory  rates  of  embryos  associated  with  algae. 


486 


VICTOR  H.  HUTCHISON  AND  CARL  S.  HAMMEN 


FIGURE  1.  Oxygen  consumption  in  five  categories  of  experiments.  A,  complex  with  algae 
in  the  dark.  B,  complex  with  algae  in  the  light.  C,  complex  without  algae.  D,  envelopes 
without  algae.  E,  envelopes  with  algae.  Embryos  in  A,  B,  and  C  were  in  stages  32-34. 


The  difference  between  O.2  consumption  of  the  isolated  envelopes  with  and 
without  algae  in  the  light  was  0.43  /A.  This  value  should  approximate  the  amount 
of  O.2  produced  by  the  algae.  If  the  symbiotic  relationship  of  algae  and  embryo 
were  only  one  of  respiratory  gas  exchange,  then  the  consumption  of  a  complex 
without  algae  should  be  greater  than  a  complex  with  algae,  and  the  subtraction 
of  the  amount  of  O2  supplied  by  the  algae  from  the  O2  consumption  of  an  algae-free 
complex  should  give  a  value  approximately  equal  to  that  of  an  algae-inhabited 
complex.  The  data  presented  here  show  that  this  is  not  the  case.  Indeed,  such 


AMPHIBIAN  EMBRYO-ALGA  SYMBIOSIS 


487 


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FIGURE  2.     Mean  rates  of  oxygen  consumption  for  each  category  of  experiments. 

a  subtraction  only  serves  to  increase  the  difference  in  respiration  rates  of  algae-free 
and  algae-inhabited  complexes. 

Isolated  embryos  of  Stages  12-13  consumed  an  average  of  0.94  p.\,  practically 
identical  with  that  of  intact  complexes  without  algae,  containing  embryos  in  the 
same  stages  of  development.  This  suggests  that  the  greater  consumption  of  a 
complex  with  algae  is  due  primarily  to  respiration  of  the  embryos  themselves,  rather 
than  to  inhabitants  of  the  envelopes. 

Embryos  alone  in  Stages  39-40  consumed  the  same  amount  of  O2,  whether 
from  algae-free  or  from  algae-inhabited  envelopes.  At  first  glance  this  would 
seem  to  contradict  the  results  of  other  experiments.  The  fact  that  these  embryos 
were  in  the  hatching  stage  probably  lends  no  support  to  conclusions  about  their 
metabolic  rates.  Developmental  changes  during  this  period  are  rapid,  and  activity 
of  individual  embryos  varies  widely.  Hopkins  and  Handford  (1943)  found  that 
the  O2  consumption  of  Ambystoma  tigrinum  was  consistently  higher  than  that  of 
A.  punctatum  during  stages  of  development  up  to  41-42.  At  this  stage  the  varia- 
tions in  results  were  wide,  and  species  differences  were  obscured. 


DISCUSSION 

If  photosynthesis  by  the  algae  resulted  in  a  supply  of  O2  for  the  embryo,  it  is 
reasonable  to  assume,  given  equal  requirements  by  the  embryos,  that  the  net  O2 
consumption  of  algae-inhabited  egg  masses  in  the  light  would  be  less  than  the  con- 
sumption of  algae-free  complexes.  However,  the  opposite  results  were  obtained. 


488  VICTOR  H.  HUTCHISON  AND  CARL  S.  HAMMEN 

The  results  given  above  indicate  that  embryos  with  envelopes  containing  algae 
respire  at  a  greater  rate  than  embryos  in  the  same  stage  of  development  without 
the  benefit  of  algae.  Furthermore,  this  increase  in  O2  demand  is  not  equaled  by 
the  smaller  amounts  of  O2  evolved  by  the  algae  during  photosynthesis.  These  re- 
sults lead  to  the  conclusion  that  the  symbiotic  relationship  of  embryos  and  algae 
is  not  one  of  respiratory  gas  exchange  alone,  and  imply  some  other  factor — possibly 
a  growth-stimulating  substance,  which  would  account  for  higher  respiratory  rates, 
faster  growth,  better  survival,  and  larger  size  at  hatching.  The  fact  that  a  complex 
with  algae  consumes  more  O2  (and  linearly)  in  the  dark  than  a  similar  complex  in 
the  light  as  compared  with  an  algae-free  complex,  indicates  that  this  growth  factor 
is  probably  supplied  in  equal  amounts  during  photosynthesis  and  in  darkness,  since 
the  rate  of  consumption  continues  at  the  same  high  level  throughout  the  ten  hours 
of  measurements. 

The  isolation  and  identification  of  such  a  growth  factor  might  require  the  use 
of  extracts  from  the  algae  grown  in  pure  culture.  The  fact  that  the  alga  involved 
inhabits  only  Ambystoma  egg  envelopes  (with  one  exception,  where  it  was  found 
in  Rana  sylvatica  envelopes)  suggests  that  the  determination  of  a  proper  culture 
medium  would  not  be  easily  accomplished.  If  such  culture  media  were  developed, 
the  different  components  of  algal  extracts  added  to  eggs  without  algae  might  lead 
to  the  proper  identification  of  the  substances  involved.  The  presence  of  numerous 
bacteria  within  the  envelopes  indicates  that  the  factor  involved  is  probably  not  an 
antibiotic. 

Several  problems  are  suggested  by  the  presence  and  better  growth  of  the  algae 
associated  with  embryos.  It  is  likely  that  the  algae  obtain  organic  nitrogenous 
substances  from  the  embryo.  The  observation  by  Gilbert  (1942)  that  the  alga 
is  thickest  in  the  inner  envelope  and  is  especially  numerous  around  the  proctodaeum 
of  the  embryo  suggests  that  nitrogenous  wastes  or  bicarbonates  are  being  utilized 
by  the  algae.  Experiments  by  Gregg  and  Ballentine  (1946)  indicate  the  presence 
of  CO2  as  bicarbonate  in  the  blastocoel  and  archenteron  fluid  of  amphibian  embryos. 
If  the  alga  is  a  facultative  heterotroph,  then  it  is  inaccurate  to  assume  that  its  photo- 
synthesis balances  or  exceeds  its  respiration.  In  addition,  the  alga  is  unknown 
from  outside  egg  masses,  and  the  process  used  to  migrate  into  and  through  the 
gelatinous  egg  coverings  is  also  unknown. 

Buchsbaum  (1937)  has  shown  that  a  metabolic  mutualism  occurs  when  a  cul- 
ture of  the  green  alga  Chlorella  is  combined  with  embryonic  chick  tissue  cells, 
where  both  were  favored  over  algae  alone  or  chick  tissues  alone.  He  found  that 
tissues  without  algae  grew  better  in  the  dark  than  those  with  algae  and  concluded 
that  the  results  were  "best  explained  on  the  basis  of  a  photosynthetic  mutualism." 
These  conclusions  were  based  on  measurements  of  growth  alone  and  not  on  dif- 
ferences in  respiratory  rates. 

Buchsbaum's  data  suggest  that  the  mutual  benefits  derived  from  an  association 
of  Chlorella  and  chick  tissue  cells  are  due  to  gas  exchanges  involved  in  photo- 
synthesis, or  if  some  other  factors  are  responsible,  they  are  produced  only  during 
photosynthesis.  Our  results  indicate  the  opposite — that  photosynthesis  alone  can- 
not accovmt  for  all  of  the  increased  respiration  and  growth  of  the  embryos,  but 
that  other  factors  are  involved  in  the  facultative  mutualism  of  Ambystoma  embryos 
and  the  alga  Oophila. 


AMPHIBIAN  EMBRYO-ALGA  SYMBIOSIS  489 

We  wish  to  express  our  appreciation  to  Dr.  H.  J.  Humm,  Dr.  I.  E.  Gray,  and 
Dr.  J.  R.  Gregg  for  helpful  suggestions  and  critical  reading  of  the  manuscript. 

SUMMARY 

1.  Oxygen  consumption   measurements   were   made   on   embryos   of   the   sala- 
mander, Ambystoma  tnaculatum,  which  develop  within  a  gelatinous  envelope  usu- 
ally inhabited  by  a  unicellular  green  alga,  Oophila  amblystomatis,  and  numerous- 
protozoa  and  bacteria. 

2.  A  comparison  of  embryos  associated  with  the  alga,  and  others  of  the  same 
developmental  stage  but  lacking  visible  signs  of  the  alga,  reveal  that  the  former 
respire  at  a  greater  rate  in  both  early  and  later  stages. 

3.  In  darkness  the  oxygen  consumption  of  the  algae-containing  complexes  is 
greater  than  in  the  light,  suggesting  that  photosynthesis  by  the  algae  answers  a 
portion  of  the  oxygen  demand.     This  is  also  indicated  by  the  fact  that  isolated 
green  envelopes  consume  only  half  as  much  oxygen  as  isolated  colorless  envelopes. 
Net  consumption   by  the  isolated  green   envelopes   is   taken   to   mean   that   algal 
oxygen  production  is  not  sufficient  to  meet  the  requirements  of  the  heterotrophic 
inhabitants  of  the  envelope,   and  therefore   could   not   provide   a   surplus   to   the 
embryo.     It  is  suggested  that  the  higher  metabolic  rates  and  more  rapid  develop- 
ment of  embryos  associated  with  algae  must  depend  in  part  on  some  factor  other 
than  oxygen  supplied  by  photosynthesis. 

4.  The  problem  of  proving  the  existence  of  a  growth  factor  is  pointed  out, 
and  some  additional  unsolved  problems  regarding  the  life  of  the  alga  and  its  means 
of  penetration  into  the  egg  envelope  are  indicated. 

LITERATURE  CITED 

BUCHSBAUM,  R.,  1937.  Chick  tissue  cells  and  Clilorella  in  mixed  cultures.  Physiol.  Zoo/., 
10:  373-379. 

GILBERT,  P.  W.,  1942.  Observations  on  the  eggs  of  Ambystoma  tnaculatum  with  especial 
reference  to  the  green  algae  found  within  the  egg  envelopes.  Ecol.,  23 :  215-227. 

GILBERT,  P.  W.,  1944.  The  alga-egg  relationship  in  Ambystoma  maculatum,  a  case  of  symbiosis. 
Ecol.,  25 :  366-369. 

GREGG,  J.  R.,  AND  ROBERT  BALLENTINE,  1946.  Nitrogen  metabolism  of  Rama  pipicns  during 
embryonic  development.  /.  Exp.  Zool.,  103 :  143-168. 

HOPKINS,  H.  S.,  AND  S.  W.  HANDFORD,  1943.  Respiratory  metabolism  during  development  in 
two  species  of  Amblystoma.  J.  Exp.  Zoo!.,  93 :  403-414. 

ORR,  H.,  1888.  Note  on  the  development  of  amphibians,  chiefly  concerning  the  central  nervous 
system ;  with  additional  observations  on  the  hypophysis,  mouth,  and  the  appendages 
and  skeleton  of  the  head.  Quart.  J.  Micr.  Set.,  N.S.,  29 :  295-324. 

SMITH,  G.  M.,  1950.  The  Fresh  Water  Algae  of  the  United  States.  Second  Ed.  New  York : 
McGraw-Hill  Book  Co. 

YONGE,  C.  M.,  1944.  Experimental  analysis  of  the  association  between  invertebrates  and  uni- 
cellular algae.  Biol.  Rev.,  19 :  68-80. 


A  COMPARISON  BETWEEN  TASTE  RECEPTORS  AND  OTHER 

NERVE  TISSUES  OF  THE  COCKROACH  IN  THEIR 

RESPONSES  TO  GUSTATORY  STIMULI J 

CHESTER  C.  ROYS 
Department  of  Biolot/y,  Tufts  Unhrrsity.  Mcdford  55,  Mass. 

It  has  long  been  known  from  behavioral  studies  that  some  butterflies,  flies,  and 
bees  have  taste  receptors  on  the  tarsi  (Minnich,  1921,  1929,  1932).  The  electro- 
physiological  studies  reported  here  were  begun  to  determine  whether  or  not  such 
tarsal  taste  receptors  are  present  in  the  American  cockroach,  Pcriplaneta  ainen'cana. 
In  the  course  of  these  studies  a  clue  was  found  to  a  fundamental  characteristic  of 
all  taste  perception  in  the  cockroach.  This  is  the  similarity  between  the  responses 
from  recognized  taste  receptors  and  from  nerve  tissue  unspecialized  for  taste  percep- 
tion. It  is  this  relationship,  briefly  described  earlier  (Roys,  1956),  which  is  the 
principal  subject  of  this  paper. 

MATERIALS 

Adult  male  and  female  American  cockroaches,  Periplaneta  americana,  of  various 
ages  were  used  for  all  the  electrophysiological  experiments,  while  the  behavioral 
experiments  included  nymphs  of  both  sexes  as  well.  Sex  differences  did  not  seem 
to  have  any  effect  on  the  responses  in  the  experiments.  They  were  all  kept  at 
room  temperature  and  fed  on  powdered  dog  biscuit  (Purina  chow). 

The  chemicals  used  in  the  experiment  were  all  of  reagent  grade  except  for  the 
quinine  which  was  U.  S.  P. 

Nerve  action  potentials  were  picked  up  from  the  tarsal  preparations  through 
tungsten  electrodes  drawn  to  fine  points  in  a  gas-oxygen  flame.  The  electrodes 
were  connected  by  copper  leads  to  a  Grass  P-3A  amplifier  and  a  Dumont  208B 
oscilloscope.  A  Grass  cathode  follower  was  also  used  in  some  of  the  experiments. 
In  nerve  cord  preparations,  bare  silver  electrodes  were  substituted  for  tungsten. 
Changes  in  nerve  activity  were  measured  with  an  Electrodyne  decade  impulse 
counter  which  recorded  the  number  of  nerve  impulses  per  second.  This  was 
connected  to  the  output  of  the  oscilloscope  amplifier. 

EXPERIMENTS  AND  RESULTS 

Seven  types  of  experiments  were  carried  out — six  on  the  response  of  various 
types  of  nerve  preparations  to  a  test  substance  and  one  on  the  behavioral  response 
to  the  same  substance  presented  in  the  drinking  water.  Four  test  substances  were 
used — sodium  chloride,  hydrochloric  acid,  sucrose  and  quinine,  corresponding  to 
the  four  accepted  taste  sensations  of  salt,  sour,  sweet  and  bitter.  First,  sodium 

1  This  work  was  made  possible  by  a  grant  from  the  U.  S.  Public  Health  Service  to  Tufts 
University.  Some  of  the  apparatus  used  was  obtained  under  a  previous  contract  between  the 
U.  S.  Chemical  Corps  and  Tufts  University. 

490 


TASTE  PERCEPTION  IN  THE  COCKROACH  491 

chloride  was  used  in  all  seven  types  of  experiments,  then  hydrochloric  acid,  sucrose 
and  quinine,  making  a  total  of  28  different  experiments.  Each  of  these  28  was 
repeated  at  least  five  times  to  insure  the  validity  of  the  results.  In  the  following 
sections  A  through  G,  the  rationale,  techniques  and  results  of  the  experiments  with 
sodium  chloride  are  discussed  in  some  detail.  Section  H  deals  with  substitution 
of  hydrochloric  acid,  sucrose  and  quinine  for  sodium  chloride  in  these  same  experi- 
ments. 

A.     Intact  tarsus 

To  test  whether  application  of  sodium  chloride  solutions  to  the  tarsus  would 
produce  any  afferent  activity  in  the  nerve  of  the  leg,  a  prothoracic  leg  was  cut  off 
at  the  femoro-tibial  joint  and  mounted  with  two  electrodes  in  contact  with  the  nerve. 
One  was  inserted  into  the  opening  at  the  cut  end  of  the  leg,  the  other  pushed  in 
through  the  membrane  at  the  tibio-tarsal  joint  until  the  tips  were  about  one  milli- 
meter apart  within  the  tibia.  The  electrodes  supported  the  leg,  and  the  tarsus 
extended  down  into  a  wax  cup  filled  with  water  or  test  solution  which  could  be 
changed  with  a  pipette.  Because  of  the  large  diameter  of  the  electrodes  relative 
to  the  size  of  the  tibia,  they  usually  detected  afferent  impulses  without  special  care 
as  to  their  exact  position.  The  tungsten  electrodes  were  connected  to  the  amplifier 
and  oscilloscope. 

When  the  tarsus  was  submerged  in  water  in  the  wax  cup,  the  nerve  showed  a 
steady  discharge  of  typical  nerve  spikes  which  probably  originated  in  mechano- 
receptors  of  the  leg.  This  basal  activity  was  measured  by  counting  the  number  of 
spikes  per  second  with  an  electronic  counter  set  to  count  all  spikes  above  the 
noise  level.  Ten  consecutive  counts  were  taken  in  a  group  and  averaged.  When 
two  or  more  successive  groups  of  counts  showed  the  same  level  of  activity,  it  was 
considered  that  a  satisfactory  base  had  been  established.  Then  the  water  was  re- 
placed with  sodium  chloride  in  successively  higher  concentrations  of  1,  2,  3,  4  and  5 
M.  Each  concentration  was  left  in  contact  with  the  tarsus  for  approximately  one 
minute.  The  nerve  activity  continued  at  about  the  same  level  until  a  sudden 
increase  showed  that  the  threshold  had  been  reached,  i.e.,  that  tarsal  stimulation 
occurred  at  that  concentration. 

Selection  of  the  "threshold"  response  must,  from  the  nature  of  the  experiments, 
be  somewhat  arbitrary,  since  the  basal  activity  showed  continual  small  fluctuations. 
To  be  certain  that  the  threshold  was  a  valid  one,  a  point  was  selected  where  the 
activity  clearly  exceeded  any  of  the  preceding  base  line  fluctuations  or  any  probable 
instrumental  changes  caused  by  shifts  in  line  voltage,  etc.  Usually  when  a  clear 
increase  of  100%  or  more  in  the  number  of  spikes  per  second  occurred  immediately 
after  a  change  in  concentration  of  the  chemical  under  test  and  lasted  longer  than  the 
quick-adapting  tactile  response,  it  was  considered  to  be  the  threshold  point.  In 
some  instances  lower  percentage  increases  were  accepted  as  thresholds  due  to 
special  circumstances  such  as  an  unusually  even  base  line.  Experiments  were 
continued  until  five  or  more  such  thresholds  at  the  lower  end  of  the  range  coincided 
within  half  a  log  unit  of  one  another.  The  range  of  the  five  is  given  in  Table  I 
and  the  lowest  one  is  plotted  as  the  threshold  indicated  by  the  bar  graph  in  Fig- 
ure 1-A. 

The  threshold  was  usually  reached  at  5  -M  sodium  chloride.     However,  it  was 


492 


CHESTER  C  ROYS 


variable  and  in  one  instance  occurred  at  a  concentration  as  low  as  1  M.  The  roaches 
used  in  this  series  of  experiments  were  all  adult  females,  but  their  age  as  adults 
was  unknown.  If  the  age  was  the  variable  factor,  then  it  seemed  possible  that 
the  work  of  Slifer  (1950)  on  locusts  offered  a  further  explanation  of  this  variation 
in  thresholds.  She  found  that  water  permeability  of  the  cuticle  on  locust  tarsi 
increased  with  age  because  the  impermeable  outer  layer  was  abraded  away  in  older 
individuals.  This  suggested  that  permeability  of  the  tarsal  cuticle  might  be  the 


4OO. 
300. 


uj  400. 

CO 

z 

2  300. 

CO 


A.  TARSUS,  INTACT 

B.  TARSUS,  PADS   SLIT 

C.  NERVE  CORD,   INTACT 


NERVE  CORD,    DESHEATHED        D. 


NERVE  CORD,   DESHEATHED 
INITIAL    RESPONSE    TO 
VARIOUS      CONCENTRATIONS 


NERVE  CORD,   EXCISED 


F. 


G.   ORAL,   BEHAVIORAL 


i\\\\\\\\\\\\\\\\\\\\\ 


ACCEPTANCE       PARTIAL         COMPLETE 
REJECTION       REJECTION 


.000001         .00001  .0001  .001  .01 

MOLAR   CONCENTRATION   OF    SODIUM    CHLORIDE 


1.0 


10.0 


FIGURE  1.     Responses  of  all  types  of  preparations  to  sodium  chloride 


TASTE  PERCEPTION  IN  THE  COCKROACH 


493 


TABLE  I 

Thresholds  for  the  four  major  taste  qualities  in  different  types  of  preparation 


Thresholds 

Calculated 

osmotic 

| 

pressure 

Tarsus 
intact 

Tarsus 
pads  slit 

Xerve  cord 
intact 

Nerve  cord 
desheatlied 

Nerve  cord 
excised 

Behavioral 
oral 

at  slit 
tarsus 
thresholds 

Salt 

1-5  M 

0.2-0.4  .17 

0.08- 

0.0002 

0.0002- 

0.004- 

9.76  Atm. 

Sodium 

0.1  M 

0.0006  .17 

0.0006  ,17 

0.008  .17 

chloride 

Sour 

0.1-0.6 

0.006- 

0.001- 

0.0002- 

0.00001- 

0.0006- 

.29 

1  fydrochloric 

0.008 

0.004 

0.0004 

0.00004 

0.0008 

acid 

Sweet 

No 

0.1    0.4 

0.1-0.6 

0.002 

0.001- 

0.006 

2.44 

Sucrose 

response 

0.004 

0.004 

at  2.5  M 

Bitter 

No 

0.001- 

0.001 

0.00008 

0.000006- 

0.0002- 

.02 

Quinine 

response 

0.008 

0.006 

0.0001 

0.000008 

0.0004 

at  0.01   ,17 

factor  controlling  the  tarsal  thresholds  in  cockroaches.     If  so,  slitting  the  tarsal 
pads  to  allow  free  entry  of  the  test  solution  could  be  expected  to  lower  the  threshold. 

B.  Tarsus  with  pads  slit 

The  tests  were  repeated  using  tarsi  the  five  tarsal  pads  of  which  had  been  slit 
longitudinally  with  a  razor  blade.  Concentrations  for  these  experiments  were 
increased  by  steps  in  accord  with  the  following  series:  .01,  .02,  .04,  .06,  .08,  .1, 
.2,  .4,  .6,  .8,  1.0,  etc.  This  progression  of  concentrations  was  also  used  for  most 
subsequent  experiments,  but  in  some  it  was  shortened  to  .02,  .04,  .08,  .2,  .4,  .8, 
2.0,  etc.,  which  approximates  a  doubling  of  concentration  at  each  step.  Because 
the  test  solutions  would  enter  the  tissue,  they  were  made  up  in  saline  solution 
(9.0  g.  NaCl,  0.2  g.  KG,  0.2  g.  CaCL  per  liter  of  solution ;  proportions  from 
Pringle,  1938)  instead  of  water.  Thus  in  the  sodium  chloride  of  the  test  solution 
was  included  the  amount  of  sodium  chloride  in  saline  solution  plus  the  sodium 
chloride  added.  For  example,  at  a  threshold  of  0.2  M  sodium  chloride  in  saline 
solution,  the  sodium  chloride  present  was  0.15  M  (from  9.0  g.  per  liter  in  the 
saline  solution)  plus  the  additional  0.2  M,  giving  a  total  concentration  of  0.35  M. 
The  same  criteria  for  thresholds  were  used  as  for  intact  tarsi  in  the  preceding 
series.  The  results  are  shown  in  Table  I  and  Figure  1-B  and  it  can  be  seen  that 
the  thresholds  are  much  lower  than  for  the  normal  intact  tarsi. 

•C.  Nerve  cord  in  situ,  intact 

However,  when  the  test  solution  entered  the  tissue  of  the  tarsus  it  reached 
all  types  of  nerve  endings.  Did  the  afferent  response  originate  in  special  subcuticu- 
lar  taste  receptors  or  in  nerve  endings  unspecialized  for  taste  ?  One  way  to  answer 


494  CHESTER  C.  ROYS 

this  question  was  to  compare  the  responses  of  the  tarsi  to  those  from  a  nerve  located 
where  it  could  not  normally  be  concerned  with  taste  perception.  Such  nerves  are 
found  in  the  connectives  and  ganglia  of  the  ventral  nerve  cord.  If  they  respond 
to  the  same  concentrations  of  sodium  chloride  it  would  indicate  that  the  response 
of  the  tarsi  was  not  dependent  on  special  taste  receptors. 

It  was  found  to  be  possible  to  pick  up  impulses  from  the  ventral  nerve  cord  in 
the  intact  roach  simply  by  slipping  the  active  electrode  between  the  sternal  plates 
and  into  the  abdominal  cavity  just  ventral  to  the  abdominal  cord.  For  routine 
experiments  the  roach  was  anesthetized  with  carbon  dioxide,  laid  on  its  back  on 
a  small  lucite  block  and  secured  in  place  with  strips  of  Cenco  Tackiwax.  A  bare 
tungsten  electrode  was  inserted  between  the  second  and  third  abdominal  sternites. 
The  indifferent  electrode  was  inserted  in  the  opening  formed  by  cutting  off  the  tip 
of  an  antenna.  Since  the  roach  antenna  contains  no  muscles  or  efferent  nerves, 
an  electrode  placed  there  does  not  pick  up  any  extraneous  nerve  or  muscle  poten- 
tials. The  test  solution  was  injected  close  to  the  cord  and  posterior  to  the  pickup 
electrode  with  a  fine  glass  pipette.  Base  line  and  threshold  were  established  as 
before  and  the  thresholds  obtained  are  shown  in  Table  I  and  Figure  1-C.  They 
are  slightly  lower  than  those  from  the  tarsi,  indicating  that  the  tarsal  response  is 
not  entirely  dependent  on  special  taste  receptors. 

D.  Nerve  cord  in  situ,  dcshcathed 

Twarog  and  Roeder  (1956)  showed  that  the  sensitivity  of  the  roach  nerve 
cord  to  chemicals  was  greatly  increased  when  the  connective  tissue  sheath  was 
removed.  To  test  the  effect  of  this  sheath  on  the  sodium  chloride  threshold,  the 
experiments  were  repeated  using  a  modification  of  the  Twarog  window  preparation. 
For  this  preparation  the  head  and  hind  legs  of  the  roach  were  removed,  and  the 
roach  laid  on  its  back  on  a  plastic  block  and  held  in  place  by  a  band  of  Tackiwax 
across  the  thorax,  leaving  the  ventral  side  of  the  abdomen  exposed.  With  a  fine 
pair  of  scissors  two  windows  were  cut  in  the  ventral  abdominal  wall.  For  one, 
the  central  section  of  the  second  sternite  was  cut  away  to  expose  the  ventral  nerve 
cord.  A  loop  of  the  exposed  cord  was  lifted  out  and  hung  over  a  bare  silver 
wire  which  formed  the  pickup  electrode.  As  soon  as  the  nerve  cord  had  dried 
enough  to  stick  to  the  wire  a  little,  it  was  cut  on  the  anterior  side  of  the  electrode 
to  limit  activity  to  that  originating  in  the  abdomen.  When  a  cathode  follower  was 
used  with  this  type  of  preparation  it  was  possible  to  let  the  exposed  section  of  nerve 
cord  dry  completely,  the  pickup  then  being  through  the  dry  dead  section  to  the 
living  cord  inside  the  body  (Roeder  and  Treat,  1957).  The  indifferent  electrode 
was  inserted  into  the  body  cavity  at  the  neck.  The  second  window  was  formed 
by  cutting  away  the  central  portion  of  the  fourth,  fifth  and  sixth  sternites  to  expose 
a  nerve  cord  ganglion  and  its  adjacent  connectives.  Under  this  section  of  cord 
was  placed  a  narrow  strip  of  Parafilm  or  wax  paper  to  separate  it  from  the 
underlying  tissue  and  body  fluid.  The  connective  tissue  sheath  was  torn  with 
fine  forceps  (see  Twarog  and  Roeder,  1956)  and  the  exposed  section  perfused 
with  saline  followed  by  a  series  of  test  solutions.  The  perfusing  fluid  was  supplied 
from  a  small  reservoir  through  a  fine  glass  tube  and  carried  away  by  a  wick  of 
absorbent  paper. 

The  results  are  shown  in  Table  I  and  Figure  1-D.     The  base  line  of  this  and  all 


TASTE  PERCEPTION  IN  THE  COCKROACH  495 

subsequent  curves  is  adjusted  to  100  for  convenient  comparison  with  other  experi- 
ments. Curve  D  is  taken  from  a  single  experiment  considered  typical  of  those 
done.  The  salient  peaks  and  hollows  were  found  in  curves  from  all  the  experi- 
ments of  the  series,  but  they  varied  in  their  exact  position  on  the  x-axis  so  that 
an  addition  of  several  curves  to  form  a  composite  would  conceal  the  true  form  of 
the  curve  through  cancellation.  Hence,  the  single  representative  curve  is  given 
instead  of  a  composite  based  on  several  experiments. 

It  will  be  noted  that  the  thresholds  are  much  lower  than  those  of  either  intact 
sheathed  cord  or  tarsus,  clearly  showing  that  the  sheath  checks  penetration  of 
sodium  chloride  and  conversely  that  activity  stimulated  by  these  low  concentrations 
must  come  from  within  that  part  of  the  cord  normally  enclosed  by  the  sheath. 

E.  Nerve  cord  in  situ,  deslicatlicd.     Initial  response  to  various  concentrations 

The  preceding  curve  D,  showing  the  response  to  steadily  increasing  concentra- 
tions of  sodium  chloride,  has  a  characteristic  succession  of  peaks  and  depressions. 
On  first  thought,  one  might  interpret  this  to  mean  that  the  nerve  does  not  respond 
to  certain  concentrations  of  sodium  chloride,  e.g.,  between  .0008  M  and  .008  M. 
However,  it  seemed  more  likely  that  these  depressions  represented  some  sort  of 
adaptation  -to  continued  exposure  to  sodium  chloride,  and  that  a  fresh  nerve  cord 
would  respond  initially  to  any  concentration  of  sodium  chloride  above  the  threshold 
value  of  .0002  M.  To  check  this  a  series  of  experiments  was  run  to  determine  the 
response  of  a  freshly  dissected  nerve  cord  in  saline  to  each  concentration  shown  on 
the  curve.  The  results  are  shown  in  Figure  1-E.  At  the  highest  concentrations, 
blocking  of  all  nerve  activity  began  before  the  ten  counts  were  completed  so  that 
the  end  of  the  curve,  based  on  an  average  of  ten  readings,  shows  a  drop  even  though 
the  first  one  or  two  readings  are  higher  than  any  preceding  ones. 

It  can  be  seen  from  the  figure  that  there  are  no  concentrations  above  threshold 
to  which  the  nerve  does  not  initially  respond,  and  that  the  extent  of  response 
generally  increases  with  the  concentration  of  sodium  choride,  particularly  at  high 
concentrations.  This  seems  to  support  the  inference  that  the  depressions  in  curve 
D  are  due  to  the  cumulative  affect  of  previous  treatment  and  do  not  result  solely 
from  the  concentrations  at  which  they  appear. 

F.  Excised  nerve  cord 

While  the  experiments  with  the  normal  and  desheathed  cord  showed  a  clear 
response  to  rather  low  concentrations  of  sodium  chloride,  it  may  be  questioned 
whether  this  response  was  due  to  direct  action  on  the  nerve  cord.  Possibly  the 
perfusing  fluid  leaked  down  onto  some  unsuspected  area  of  taste  receptors.  To 
check  this  possibility  the  abdominal  section  of  the  nerve  cord  was  removed  from  the 
roach  and  tested  alone.  This  consists  of  a  chain  of  six  ganglia  joined  by  paired  con- 
nectives and  is  about  one  centimeter  long.  It  was  dissected  out  and  laid  across 
silver  wire  electrodes  in  a  small  depression  in  a  lucite  block,  in  a  modification  of  the 
preparation  described  by  Roeder  and  Roeder  (1939).  When  this  depression  was 
filled  with  saline  or  test  solution,  the  cord  was  submerged,  and  no  impulses  were 
picked  up  because  of  electrical  shunting  through  the  saline  solution.  However, 
when  the  solution  was  drawn  out  of  the  depression  with  a  small  piece  of  absorbent 


496  CHESTER  C.  ROYS 

paper,  the  moist  cord  was  left  hanging  in  the  air  across  the  two  electrodes  and 
impulses  were  picked  up  for  viewing  on  the  oscilloscope  and  for  counting. 

To  establish  the  base  line  activity  of  the  cord  in  saline  solution,  it  was  first 
submerged  in  saline  for  several  minutes  to  equilibrate  with  the  new  medium,  then 
the  solution  was  drawn  off,  ten  counts  of  the  number  of  spikes  per  second  taken, 
and  the  cord  submerged  in  saline  again  for  one  minute  before  another  count  was 
taken.  As  soon  as  the  base  level  of  activity  had  been  established  as  for  the  intact 
tarsus,  test  solutions  of  increasing  concentrations  of  sodium  chloride  were  substi- 
tuted for  pure  saline.  Thus  the  cord  was  alternately  submerged  and  exposed  for 
periods  of  one  minute  each  during  the  experiments,  in  contrast  to  the  preceding- 
experiments  where  the  nerve  was  continually  perfused.  However,  length  of 
exposure  to  each  test  solution  was  approximately  the  same  in  both  types  of 
experiment. 

Although  the  excised  nerve  cord  was  not  desheathed.  numerous  openings  were 
left  wherever  connectives  were  cut  away  and  where  the  whole  cord  was  cut  at 
the  anterior  end  in  the  process  of  excision.  Therefore,  it  seems  probable  that  the 
protective  function  of  the  sheath  was  reduced  almost  as  much  as  by  stripping  it 
away. 

In  this  preparation  all  six  ganglia  and  their  connectives  were  simultaneously 
exposed  to  each  change  of  solution,  in  contrast  to  the  preparations  with  the  nerve 
cord  in  situ  where  only  a  single  ganglion  and  its  connectives  were  exposed.  This 
produced  responses  in  the  excised  cord  which  were  more  sharply  defined  and  the 
thresholds  were  more  easily  determined  than  with  the  cord  in  situ.  It  also  resulted 
in  blocking  at  a  lower  concentration  than  that  which  blocked  the  cord  in  situ,  where 
some  of  the  ganglia  and  connectives  were  protected  from  direct  exposure  to  the 
salt. 

From  Table  I  and  Figure  1-F  it  is  clear  that  the  threshold  is  at  least  as  low 
as  that  of  the  cord  in  situ,  further  confirming  that  no  special  taste  receptors  are 
needed  to  account  for  the  response.  Considering  the  preceding  six  types  of 
experiment  in  retrospect,  it  is  now  clear  that  as  the  protective  coverings  are 
stripped  away — first  the  cuticle  to  expose  the  leg  nerve  and  intact  cord  to  chemical 
action,  then  the  sheath  from  the  exposed  cord — the  sensitivity  increases.  Receptors 
specialized  for  taste  are  usually  assumed  to  be  more  sensitive  to  chemicals  than 
other  nerves.  However,  in  view  of  the  low  threshold  of  the  desheathed  nerve  cord 
it  seemed  worth  while  to  check  the  sensitivity  of  recognized  taste  receptors  for 
comparison. 

G.     Oral  taste,  behavioral 

Evidence  from  various  sources  and  confirmed  by  Frings  and  Frings  (1949) 
indicates  that  the  maxillary  and  labial  palpi  of  the  cockroach  carry  the  oral  taste 
receptors.  Therefore  the  palpi  were  set  up  in  the  same  way  as  the  tarsal  prepara- 
tions, in  the  expectation  of  getting  responses  in  line  with  the  behavioral  thresholds 
reported  by  Frings  (1946).  However,  the  thresholds  obtained  were  only  slightly 
lower  than  those  from  the  intact  tarsi.  It  seems  probable  that  this  discrepancy  was 
due  to  the  very  small  size  of  the  fibers  which  carry  the  normal  gustatory  responses 
—a  condition  which  makes  recording  very  difficult.  To  avoid  this  difficulty  the 
method  developed  by  Hodgson,  Lettvin  and  Roeder  (1955)  was  tried.  Working 


TASTE  PERCEPTION  IN  THE  COCKROACH  497 

with  flies  they  were  able  to  make  electrical  contact  with  single  sensory  hairs  in 
the  oral  region  which  responded  clearly  to  chemical  stimuli.  However,  this  method 
proved  unsuccessful  when  applied  to  the  cockroach.  The  longer  hairs  at  the  tips 
of  the  palpi  did  not  give  any  clear  responses  to  gustatory  stimuli,  and  it  may  be 
that  gustatory  perception  is  through  short  bristles  or  pegs  which  lie  between  the 
longer  hairs  and  are  therefore  rather  difficult  to  reach  with  the  electrode.  There- 
fore, for  the  present  we  must  rely  on  the  older  method  of  behavioral  response. 

Behavioral  thresholds  for  sodium  chloride  were  determined  by  a  modification  of 
the  method  used  by  Dethier  and  Rhoades  in  1954  for  flies.  This  method  offers  the 
test  population  a  choice  between  flavored  and  unflavored  drinking  water  and  meas- 
ures the  amount  of  each  kind  consumed  in  each  of  a  succession  of  trial  periods. 
In  each  trial  the  concentration  of  flavored  material  is  greater  than  in  the  preceding 
one.  Any  change  from  a  one-to-one  ratio  of  consumption  indicates  ability  to  dis- 
tinguish between  the  two,  i.e.,  the  threshold  concentration  of  the  flavoring  material. 

In  the  first  of  these  experiments  a  colony  of  about  100  adult  roaches  of  both 
sexes  was  used  for  one  series  of  experiments  and  a  colony  of  nymphs  for  another 
series.  However,  no  differences  between  the  responses  of  nymphs  and  adults 
were  noted  and  in  subsequent  experiments  a  breeding  colony  of  mixed  nymphs  and 
adults  of  both  sexes  was  used.  The  roaches  were  confined  in  15-gallon  aquaria 
containing  cardboard  shelters,  but  were  not  restricted  in  any  other  way  and  were 
free  to  eat  and  drink  whenever  they  chose.  Temperature  ranged  from  22°  to 
26°  C.  and  the  cages  were  lighted  during  the  day  by  room  illumination  and  were 
dark  at  night.  This  near-normal  environment  and  lack  of  any  restriction  to  move- 
ment of  the  experimental  animal  are  notable  advantages  of  this  method. 

The  drinking  water  for  each  colony  was  supplied  from  two  identical  glass  tubes 
of  6  mm.  inside  diameter  and  about  50  cm.  long.  These  tubes  lay  parallel  on  the 
bottom  of  the  aquarium  except  for  the  ends  which  were  bent  up  to  prevent  the 
water  from  running  out.  At  one  end  the  bent  sections  came  up  at  right  angles 
for  12  cm.  and  were  taped  to  the  wall  of  the  aquarium  to  hold  them  in  position. 
The  other  two  ends  sloped  up  at  30°  to  a  height  of  1.5  cm.  above  the  aquarium 
floor  and  were  plugged  with  rolled  cylinders  of  lens  paper  which  acted  as  wicks  to 
draw  the  water  from  the  long  horizontal  reservoirs  and  make  it  available  to  the 
roaches.  It  was  found  best  to  put  the  ends  of  the  tubes  with  the  wicks  about  one 
centimeter  apart,  fastening  them  to  a  spacer  block  with  Tackiwax  to  hold  them 
firm.  They  were  in  an  open  area  of  the  aquarium  floor  and  in  the  light.  The  lens 
paper  plugs  were  changed  daily.  Each  day  each  tube  was  filled  with  water  from 
a  graduated  syringe  to  a  mark  1.5  cm.  up  on  the  vertical  section.  Thus  the  amount 
of  water  consumed  in  the  preceding  24  hours  was  determined  by  measuring  the 
amount  needed  to  refill  the  tube  to  the  mark.  A  colony  of  100  roaches  took  about 
10  ml.  of  water  a  day  or  5  ml.  from  each  tube  if  the  tubes  were  equally  preferred. 
Evaporation,  checked  in  a  separate  tube,  was  nearly  constant  at  0.5  ml.  per  day. 
Day-to-day  fluctuations  in  consumption  from  the  two  tubes  were  erratic  and  ran 
as  high  as  20%  difference  between  the  two  with  water  in  both  tubes.  To  deter- 
mine the  salt  threshold,  increasing  concentrations  of  sodium  chloride  (in  the  same 
steps  used  in  the  nerve  preparations)  were  substituted  for  water  in  one  of  the 
tubes.  The  threshold  concentration  was  clearly  marked  by  a  sharp  increase  in 
preference  for  the  tube  containing  sodium  chloride,  i.e.,  an  acceptance  threshold. 


498  CHESTER  C.  ROYS 

This  was  followed  by  a  continued  preference  for  the  sodium  chloride  tube  at  higher 
concentrations  until  the  rejection  threshold  was  reached  when  there  was  a  sharp 
change  in  preference  from  sodium  chloride  to  water.  Higher  concentrations  of 
salt  were  progressively  less  and  less  acceptable  until  a  concentration  was  reached 
at  which  no  salt  solution  at  all  was  taken,  i.e.,  salt  was  completely  rejected  at  or 
above  that  concentration.  Supplementary  experiments  showed  that  previous  con- 
ditioning had  little  effect  on  any  given  trial.  For  example,  in  a  given  pair  of  tubes 
when  an  unacceptable  sodium  chloride  solution  was  replaced  by  water,  there  was 
an  immediate  return  to  an  approximately  one-to-one  ratio  of  preference  and  the 
same  appeared  to  be  true  in  shifting  from  an  acceptable  solution  to  w^ater. 

Two  complete  series,  ranging  from  well  below  the  acceptance  threshold  to  well 
above  the  point  of  complete  rejection,  were  run.  In  addition  three  short  series 
were  run  in  the  ranges  of  the  acceptance  and  rejection  thresholds.  The  results 
are  shown  in  Table  I  and  the  bar  graph  in  Figure  1-G,  and  it  is  clear  from  these 
that  the  behavioral  threshold  is  well  above  that  of  any  of  the  nerve  cord  preparations. 

H.     Responses  from  all  types  of  preparation  to  sour,  sweet  and  bitter  stimuli 

To  test  whether  comparable  thresholds  and  curves  could  also  be  obtained  from 
sour,  sweet  and  bitter  substances,  similar  series  of  tests  were  carried  out  with 
hydrochloric  acid,  sucrose  and  quinine.  Quinine  monohydrochloride  was  substi- 
tuted for  quinine  at  concentrations  above  0.001  M  because  of  its  greater  solubility. 
At  lower  concentration  it  had  the  same  threshold  as  the  pure  alkaloid.  The  experi- 
ments with  sucrose  and  quinine  were  not  carried  out  to  5.0  M  because  sucrose 
solution  becomes  a  thick  syrup  above  2.0  M  concentration,  while  quinine  mono- 
hydrochloride tends  to  precipitate  out  of  solution  at  concentrations  much  above 
0.01  M  in  water.  In  every  other  way  the  same  procedure  was  followed  as  for 
sodium  chloride. 

The  results  of  these  experiments  are  shown  in  Table  I  and  Figures  2,  3  and  4. 
Figure  5  is  a  summary  of  all  the  thresholds  for  all  four  substances,  plotted  on  a 
single  graph  for  comparison. 

In  comparing  the  responses  to  hydrochloric  acid  with  those  to  sodium  chloride 
it  is  at  once  apparent  that  most  of  the  hydrochloric  acid  thresholds  are  lower.  An 
apparent  exception  is  found  in  the  thresholds  of  the  desheathed  nerve  cord  in  situ. 
This  discrepancy  is  probably  due  to  a  limitation  of  technique  rather  than  a  real 
physiological  difference.  This  technical  limitation  lies  in  the  fact  that  much  less 
area  is  exposed  to  test  solutions  with  the  cord  /;;  situ  than  with  it  excised  (see 
section  F).  For  this  reason  the  responses  from  the  cord  in  situ  were  less  clearly 
defined  and  the  threshold  less  easily  determined.  In  the  cases  of  sodium  chloride 
and  sucrose  the  experimental  evidence  seemed  to  support  thresholds  of  the  cord 
in  situ  as  low  as  those  of  the  excised  cord.  With  hydrochloric  acid  and  quinine 
the  evidence  at  hand  did  not  seem  to  warrant  a  firm  statement  to  this  effect,  although 
there  is  a  strong  probability  that  it  is  true  for  these  substances  also. 

As  with  sodium  chloride,  the  behavioral  tests  with  hydrochloric  acid  showed 
both  acceptance  and  rejection  thresholds,  and  concentrations  causing  rejection  also 
stimulated  the  tarsi  and  produced  a  strong  response  in  the  nerve  cord. 

Sucrose  did  not  stimulate  the  intact  tarsus  in  concentrations  up  to  2.0  M,  and 


TASTE  PERCEPTION  IN  THE  COCKROACH 


499 


thresholds  for  all  types  of  preparation  were  rather  high.     In  the  behavioral  tests 
sucrose  was  acceptable  at  all  concentrations  from  threshold  to  2.0  M. 

Quinine  also  failed  to  stimulate  the  intact  tarsi,  suggesting  that  the  cuticle  is  less 
permeable  to  the  large  molecules  of  sucrose  and  quinine  than  to  the  ionized  salts 
and  acids.  In  behavioral  tests  quinine  was  rejected  in  all  concentrations  above 
the  threshold. 


A.  TARSUS,   INTACT 

B.  TARSUS,    PADS  SLIT 

C.  NERVE  CORD,   INTACT 


o 

o 

LJ 
CO 


400. 
300. 
200. 
100. 


NERVE  CORD,    DESHEATHED          D. 


Q- 

co 

LU 
CO 


o. 


o 

UJ 

cr 
3 

CO 


400_ 

300- 
200. 
100. 
0 


NERVE  CORD,  DESHEATHED 
INITIAL  RESPONSE  TO 
VARIOUS  CONCENTRATIONS 


LJ  400., 
to 


NERVE  CORD,  EXCISED 


F. 


G.  ORAL,  BEHAVIORAL 


.000001    .00001     .0001      .001       .01 
MOLAR  CONCENTRATION  OF  HYDROCHLORIC  ACID 


1.0 


i.O 


FIGURE  2.  Responses  of  all  types  of  preparations  to  hydrochloric  acid.  Legend  for 
bar:  unshaded  area,  acceptance;  cross-hatched  area,  partial  rejection;  black  area,  complete 
rejection. 


500 


CHESTER  C.  ROYS 


400. 
300_ 
200. 

Q 

I    IOOJ 

UJ 
CO 

cr        0 

UJ 
Q. 

CO 
UJ 

^400J 

CL 

-  300. 
a:  200. 

LJ 

Z 

?    100  J 

Q 

UJ 

o:         0 

CO 

UJ 

2 

uj   400. 
co 


A.  TARSUS.  INTACT 

B.  TARSUS.  PADS  SLIT 

C.  NERVE  CORD.  INTACT 


NO  RESPONSE 


NERVE   CORD.    DESHEATHED 


BASE  LINE 


NERVE  CORD.    DESHEATHED 
INITIAL  RESPONSE    TO 
VARIOUS  CONCENTRATIONS 


BASE  LINE 


300. 


CO 


NERVE    CORD.    EXCISED 


G.    ORAL.     BEHAVIORAL 


ACCEPTANCE 


.000001         .00001  .0001  .001 

MOLAR   CONCENTRATION    OF   SUCROSE 


.01 


1.0 


10.0 


FIGURE  3.     Responses  of  all  types  of  preparations  to  sucrose. 


DISCUSSION 

The  foregoing  sets  of  experiments  seem  to  indicate  that  the  chemical  sensitivity 
of  recognized  oral  taste  receptors,  as  well  as  receptors  on  the  tarsi,  is  always  less 
than  that  of  many  other  nerves  in  the  body  not  normally  concerned  with  taste. 
However,  there  are  a  number  of  points  in  the  work  which  may  need  clarification 
or  can  profitably  be  amplified. 

The  first  consideration  is  whether  the  nerve  responses  obtained  are  true  chem- 


TASTE  PERCEPTION  IN  THE  COCKROACH 


501 


A.  TARSUS,    INTACT 

B.  TARSUS,  PADS  SLIT 

C.  NERVE  CORD.  INTACT 


400. 
300. 
200- 


z 
o 


co 

o: 

UJ 
Q. 

CO 

Ul 
CO 


NO  RESPONSE 


NERVE  CORD,  DESHEATHED 


D. 


BASE    LINE 


400. 


200. 
100. 
0 


NERVE  CORD.  DESHEATHED 
INITIAL  RESPONSE   TO 
VARIOUS   CONCENTRATIONS 


E. 


BASE    LINE 


CO 

< 

UJ 


CO 


CO 
UJ 


400. 
300. 
200. 
100. 
0 


NERVE   CORD,   EXCISED 


F. 


BASE    LINE 


G.    ORAL,  BEHAVIORAL 


COMPLETE 
REJECTION 

.000001 

.00001            .0001 

.001                  .01 

.1                     1.0                10.0 

MOLAR    CONCENTRATION    OF   QUININE 

FIGURE  4.     Responses  of  all  types  of  preparations  to  quinine. 


jcal  thresholds  or  whether  they  are  due  to  osmotic  or  other  physical  changes.  If 
they  are  due  simply  to  increased  osmotic  pressure,  then,  conversely,  threshold  con- 
centrations of  all  four  substances  should  produce  the  same  osmotic  pressure. 
However,  the  computed  osmotic  pressures  at  threshold  concentrations  for  sodium 
chloride,  hydrochloric  acid,  sucrose  and  quinine  are  given  in  Table  I  and  show  a 
wide  range  of  values  for  the  four  substances.  This  would  seem  to  show  conclusively 
.that  osmotic  pressure  was  not  the  principal  cause  of  the  responses.  The  wide  range 


502 


CHESTER  C.  ROYS 


SODIUM  CHLORIDE 


TARSUS,  INTACT 
TARSUS,  PADS  SLIT 
NERVE  CORD,  INTACT 
NERVE  CORD,  DESHEATHED 

NERVE  CORD,  DESHEATHED, 
INITIAL  RESPONSE 
NERVE  CORD,  EXCISED 

ORAL,  BEHAVIORAL 


HYDROCHLORIC  ACID 


K\\\\\\\\\\\\\\\\\\\ 


TARSUS,  INTACT 
TARSUS,  PADS  SLIT 
NERVE  CORD,  INTACT 
NERVE  CORD,  DESHEATHED 

NERVE  CORD,  DESHEATHED, 
INITIAL  RESPONSE 
NERVE  CORD,  EXCISED 

ORAL,  BEHAVIORAL 


SUCROSE 
NO  RESPONSE 


J 


QUININE 
NO   RESPONSE 


.000001  .0001     .00! 

MOLAR    CONCENTRATION 


.01 


TARSUS,  INTACT 
TARSUS,  PADS  SLIT 
NERVE  CORD,  INTACT 
NERVE  CORD,  DESHEATHED 

NERVE  CORD,  DESHEATHED, 
INITIAL  RESPONSE 
NERVE  CORD,  EXCISED 

ORAL,  BEHAVIORAL 


TARSUS,  INTACT 
TARSUS,  PADS  SLIT 
NERVE  CORD,  INTACT 
NERVE  CORD,  DESHEATHED 

NERVE  CORD,  DESHEATHED, 
INITIAL  RESPONSE 
NERVE  CORD,  EXCISED 

ORAL,  BEHAVIORAL 


1.0   10.0 


FIGURE  5.     Responses  of  all  types  of  preparations  to  sodium  chloride,  hydrochloric  acid, 

sucrose  and  quinine 


TASTE  PERCEPTION  IN  THE  COCKROACH  503 

over  which  the  thresholds  extend  seems  to  be  further  evidence  against  a  single 
physical  factor  being  responsible. 

Secondly,  some  may  question  the  physiological  validity  of  certain  of  these 
thresholds.  In  particular  the  response  of  the  nerves  to  sucrose  may  seem  to  be 
in  direct  conflict  with  the  widespread  practice  among  neurophysiologists  of  using 
"inert"  sucrose  to  maintain  the  osmotic  pressure  of  physiological  saline  solution  in 
the  absence  of  certain  salts.  However,  it  should  be  emphasized  that  at  threshold 
concentrations  the  effects  of  these  solutions  are  transitory.  It  is  customary  to 
allow  a  short  period  of  time  for  any  nerve  preparation  to  recover  from  injuries  of 
surgery,  small  osmotic  changes  produced  when  saline  solution  is  substituted  for 
blood,  etc.,  before  the  activity  is  studied.  Thus  these  transitory  threshold  responses 
might  easily  be  overlooked  or  masked  by  other  adaptive  changes  in  activity.  How- 
ever, they  show  up  clearly  when  separated  experimentally  from  other  possible 
causes  of  nerve  activity.  Moreover,  when  used  in  adjusting  osmotic  pressure  the 
sucrose  is  substituted  for  some  constituent  of  the  solution,  not  added  to  the  solu- 
tion as  was  done  in  these  experiments. 

It  is  also  possible  that  injury  from  operative  techniques  may  have  made  the 
nerves  more  sensitive  to  chemical  action.  This  seems  unlikely  in  the  tarsal  prepara- 
tions where  injury  was  minimal  and  in  the  normal  nerve  cord  where  test  substances 
were  injected  into  an  otherwise  intact  animal.  Also,  the  Twarog  window  prepara- 
tion was  particularly  designed  to  do  a  minimal  amount  of  damage,  and  this  is 
supported  by  the  fact  that  thresholds  for  nerve  cord  exposed  in  the  window  with 
the  sheath  intact  were  no  lower  than  those  obtained  by  injection  of  the  same  sub- 
stance. Desheathing  may  have  torn  some  of  the  nerve  fibers,  but  it  seems  very 
unlikely  that  these  could  have  amounted  to  more  than  a  small  fraction  of  the  total 

J 

which  responded  to  threshold  concentrations.  However,  in  the  excised  nerve  cord 
a  large  number  of  connecting  fibers  were  cut  to  remove  it  and  these  cut  ends  may 
have  been  very  sensitive.  This  may  have  been  a  factor  in  the  very  low  thresholds 
obtained  from  this  type  of  preparation  in  response  to  stimulation  by  hydrochloric 
acid  and  quinine.  If  so,  this  would  be  interesting  in  itself,  but  these  two  thresholds 
are  not  essential  to  the  general  thesis  of  this  paper. 

Another  important  consideration  is  whether  or  not  the  thresholds  from  the  intact 
tarsi  and  from  exposed  nerves  are  truly  comparable  to  oral  taste.  The  strongest 
argument  in  favor  of  this  is  the  close  correlation  between  the  behavioral  threshold 
and  the  thresholds  of  the  various  nerve  preparations  in  response  to  a  given  chem- 
ical. This  is  most  clearly  shown  in  the  responses  to  sucrose  and  quinine  (see 
Figure  5)  where  the  thresholds  of  the  various  nerve  preparations  for  quinine  are 
much  closer  to  the  behavioral  threshold  for  quinine  than  to  the  behavioral  threshold 
for  sucrose  and  vice  versa.  The  same  may  be  said  for  responses  to  any  other 
pair  of  test  chemicals  though  it  is  more  conspicuous  in  some  pairs  than  in  others. 

Further  correlation  is  found  between  the  behavioral  rejection  thresholds  for 
sodium  chloride  and  hydrochloric  acid  and  the  activity  produced  in  the  nerve 
preparations  by  the  same  concentrations  of  the  salt  or  acid.  These  concentrations 
suffice  to  raise  the  activity  in  desheathed  nerves  to  a  very  high  level  and  initiate 
activity  in  those  nerves  protected  by  a  sheath  or  even  by  a  sheath  plus  cuticle. 
This  correlation  seems  to  lend  additional  support  to  the  relation  between  behavioral 
responses  and  the  responses  from  nerve  preparations.  Further,  it  suggests  that 


504  CHESTER  C.  ROYS 

when  the  stimulating  chemical  reaches  a  concentration  which  produces  violent 
activity  in  the  special  taste  receptor  and  begins  to  produce  activity  in  all  other 
nerves  in  the  area,  behavioral  rejection  sets  in. 

Results  from  the  experiments  with  quinine  do  not  show  this  correlation  as 
clearly,  while  with  sucrose  we  find  violent  activity  in  the  nerve  cord  correlated 
with  behavioral  acceptance.  However,  this  need  not  detract  from  the  significance 
of  the  correlation  between  behavioral  rejection  and  nerve  response  with  salts  and 
acids,  since  it  is  probable  that  the  bitter  and  sweet  molecules  act  on  the  receptors, 
through  quite  different  mechanisms  than  the  salt  and  acid  ions. 

The  protection  afforded  by  the  sheath  in  these  cases  brings  us  to  a  general 
summary  of  the  factors  which  determine  thresholds  in  different  types  of  prepara- 
tion. In  the  leg  nerve  preparation  the  tarsal  cuticle  was  ruptured  with  the  result 
that  a  lower  concentration  of  chemical  was  needed  to  initiate  nerve  action.  How- 
ever, the  sheath  remained  as  a  second  line  of  protection.  It  was  not  practical  to 
remove  the  sheath  from  the  leg  nerve,  but  a  switch  to  the  nerve  cord  showed  similar 
thresholds.  When  the  nerve  cord  sheath  was  removed,  the  concentration  required 
for  stimulation  again  dropped  markedly.  Viewing  these  experiments  in  general 
terms,  it  now  seems  probable  that  the  true  nerve  threshold  is  the  concentration  of 
test  substance  which,  when  acting  directly  on  the  unprotected  nerve,  will  produce 
a  response.  Any  higher  thresholds  for  protected  nerves  are  measures  of  the  con- 
centration which  must  be  applied  outside  the  sheath  or  cuticle  to  produce  the  true 
threshold  concentration  at  the  nerve.  Extrapolating  to  the  normal  oral  taste  re- 
ceptors which  control  the  behavioral  thresholds,  we  may  infer  from  the  position  of 
these  behavioral  thresholds,  intermediate  between  those  of  sheath-protected  and 
of  desheathed  nerves,  that  the  nerves  which  govern  them  are  not  enclosed  by  normal 
sheath  or  cuticle  but  have  more  protection  than  bare  nerve.  This  barrier  may  be 
to  protect  them  from  damage  by  high  concentrations  of  chemicals,  or  it  may  be  con- 
cerned with  selection  or  differentiation  between  different  types  of  taste  stimulation. 

In  examining  the  curves  lettered  D  and  F,  which  represent  the  responses  of 
desheathed  nerve  to  the  four  chemicals  tested,  the  most  striking  characteristic  is 
the  sharp  drop  in  activity  first  seen  after  the  threshold  response.  This  rise  to  a 
peak  of  activity  followed  by  a  period  of  depression  is  repeated  one  or  more  times 
in  varying  degrees  in  all  the  curves  before  blocking  occurs.  A  possible  explanation 
is  that  the  three  or  more  peaks,  particularly  clear  in  the  excised  nerve  cord  prepara- 
tions, may  come  from  different  groups  of  fibers  each  less  sensitive  or  less  exposed 
than  the  preceding.  Each  of  these  groups  in  turn  could  become  active,  reach  a 
maximum,  and  then  adapt  or  partially  block  to  account  for  the  peaks  and  depressions 
as  the  concentration  of  stimulating  chemical  is  increased.  In  other  words,  parts 
of  the  nerve  cord  may  be  protected  from  chemical  action  by  barriers  comparable 
to  the  connective  tissue  sheath  and  the  cuticle. 

It  is  interesting  to  compare  the  behavioral  thresholds  with  those  obtained  by 
Frings  (1946)  who  also  worked  with  cockroaches,  using  a  different  test  method. 
In  testing  whether  individual  roaches  would  accept  or  reject  test  solutions  offered 
them,  Frings  first  determined  the  sucrose  threshold  by  offering  increasing  concen- 
trations of  sucrose  in  water  until  an  acceptable  concentration  was  reached.  This 
acceptance  threshold  was  clear,  but  since  individual  roaches  often  refuse  pure  water, 
he  had  to  use  a  different  method  to  determine  rejection  thresholds.  For  this  he- 


TASTE  PERCEPTION  IN  THE  COCKROACH 


505 


TABLE  II 

Behavioral  thresholds  determined  by  tu'o  methods 


Thresholds 

Sucrose 

Sodium  chloride 

Hydrochloric  acid 

Acceptance 

Acceptance 

Rejection 

Acceptance 

Rejection 

Partial 

Complete 

Partial 

Complete 

Reported  here 
Frings 

.006 
.007 

.004 

.04 

.6 

.2 

.0006 

.006 

.4 
.02 

selected  a  sucrose  concentration  far  enough  above  threshold  to  be  always  acceptable 
(0.1  M)  and  offered  this  with  the  addition  of  increasing  concentrations  of  sodium 
chloride  until  it  was  rejected.  This  he  took  as  the  rejection  threshold  for  sodium 
chloride.  He  repeated  the  experiments  with  hydrochloric  acid  and  a  large  number 
of  other  salts  and  acids,  and  Table  II  shows  a  comparison  of  his  results  with  the 
behavioral  thresholds  determined  in  these  experiments. 

It  is  at  once  apparent  that  there  is  very  good  agreement  on  the  determination  of 
sucrose  against  water,  but  the  present  method  gives  much  more  information  about 
the  sodium  chloride  and  hydrochloric  acid.  As  might  be  expected,  Frings'  lowest 
records  for  rejection  lie  within  the  range  reported  here  as  partial  rejection.  Frings 
was  aware  of  the  existence  of  the  lower  acceptance  thresholds  reported  here  but 
was  unable  to  determine  them  because  he  could  not  get  consistent  feeding  responses 
without  the  use  of  sucrose.  It  was  the  use  of  a  large  population  of  roaches  instead 
of  individuals  which  made  it  possible  to  get  consistent  feeding  responses  in  these 
experiments  without  the  use  of  sucrose. 

It  is  quite  possible  that  these  thresholds  as  well  as  those  of  the  nerve  prepara- 
tions may  vary  with  different  diets.  In  these  experiments  the  same  diet  was  sup- 
plied throughout,  but  it  seems  probable  that  any  dietary  change  would  affect  both 
behavioral  and  nerve  thresholds  equally  so  that  the  same  relationships  would 
remain. 

The  methods  used  do  not  show  whether  the  impulses  from  the  nerve  cord 
arise  in  the  axons,  dendrites  or  somata  of  the  activated  nerve  cells.  Since  all 
impulses  above  the  amplifier  noise  level  were  counted,  it  is  not  possible  from  the 
present  data  to  estimate  either  the  size  of  the  responding  elements  or  their  relative 
number  in  the  total  population  of  active  neurons.  From  other  studies  (Roeder, 
1948)  it  seems  probable  that  many  compounds  exert  their  effects  on  the  dendritic 
or  somatic  regions  of  the  central  neurons. 

These  studies  seem  to  indicate  that  the  chemical  thresholds  for  nerves  unspe- 
cialized  for  gustatory  reception  are  as  low  as.  or  lower  than,  those  of  the  specialized 
receptors.  On  this  basis  we  may  conclude  that  taste  or  contact  chemoreception 
depends  on  two  factors.  One  of  these,  sensitivity,  is  also  held  by  the  neurons 
unspecialized  for  gustatory  perception.  Therefore,  we  can  study  it  in  nerve  tissue 
other  than  the  complex  receptors  which  offer  considerable  technical  difficulty. 
Furthermore,  we  can  bring  to  bear  on  this  study  the  vast  amount  of  work  which 
has  previously  been  done  on  nerve  tissue.  The  other  quality,  discrimination  be- 


506  CHESTER  C.  ROYS 

tween  different  substances,  remains  a  property  of  the  receptor  mechanism.  How- 
ever, it  is  hoped  that  its  study  has  been  simplified  slightly  by  separating  from  it 
the  factor  of  sensitivity  which  has  often  been  considered  an  integral  part  of  this 
mechanism. 

SUMMARY 

1.  Application  of  increasing  concentrations  of  sodium  chloride  to  the  normal 
intact  tarsi  of  the  American  cockroach  resulted  in  increased  activity  in  the  afferent 
fibers  of  the  leg  nerve  when  the  threshold  concentration  was  reached. 

2.  The  threshold  for  this  response  was  lowered  by  slitting  the  tarsal  pads. 

3.  Although  it  may  be  presumed  that  there  are  no  taste  receptors  on  the  nerve 
cord,  when  increasing  concentrations  of  sodium  chloride  in  saline  solution  were 
applied  to  an  exposed  section  of  intact  nerve  cord,  it  responded  to  a  lower  concen- 
tration than  did  the  tarsal  preparations. 

4.  The  threshold  of  the  nerve  cord  was  further  lowered  by  removing  the  con- 
nective tissue  sheath  which  normally  encloses  it. 

5.  A  section  of  the  nerve  cord,  completely  removed  from  the  roach  and  exposed 
to  the  same  concentrations  of  sodium  chloride,  responded  at  the  same  threshold 
concentration  as  the  exposed  nerve  cord  in  situ,  showing  conclusively  that  the  re- 
sponse did  come  from  the  nerve  cord  itself,  not  from  adjacent  chemoreceptors. 

6.  Behavioral  experiments  showed  a  response  to  the  taste  of  sodium  chloride 
at  a  threshold  higher  than  that  of  the  nerve  cord  preparations. 

7.  There  \vas  also  an  increase  in  the  nerve  activity  from  leg  and  nerve  cord 
preparations  in  the  same  range  of  concentrations  of  sodium  chloride  which  pro- 
duced behavioral  rejection. 

8.  Similar  experiments  with  hydrochloric  acid,  sucrose  and  quinine,  represent- 
ing the  sour,  sweet  and  bitter  sensations,  also  showed  behavioral  thresholds  higher 
than  those  from  the   nerve  cord   preparations,   and  hydrochloric   acid   showed   a 
correlation  between  nerve  activity  and  behavioral  rejection  similar  to  that  of  sodium 
chloride. 

9.  It  was  concluded  that  high  sensitivity  to  the  four  types  of  substances  which 
produce  the  four  taste  sensations  is  inherent  in  nerves  not  normally  connected 
with  taste  rather  than  being  a  special  feature  of  the  taste  receptor,  and  that  the 
basis  for  behavioral  rejection  may  also  be  found  in  nerves  not  normally  concerned 
with  taste. 

LITERATURE  CITED 

DETHIER,   V.   G.,   AND   M.    V.    RHOADES,    1954.     Sugar    preference-aversion    functions    for   the 

blowfly.     /.  Exp.  ZooL,  126:  177-204. 
FRINGS,  H.,  1946.     Gustatory  thresholds  for  sucrose  and  electrolytes  for  the  cockroach,  Peri- 

plancta  americana  (Linn.).     /.  Exp.  ZooL,  102:  23-50. 
FRINGS,  H.,  AND  M.  FRINGS,  1949.     The  loci  of  contact  chemoreceptors  in  insects.     Amcr.  Midi. 

Nat.,  41 :  602-658. 
HODGSON,  E.  S.,  J.  Y.  LETTVIN  AND  K.  D.  ROEDER,  1955.     Physiology  of  a  primary  chemore- 

ceptor  unit.     Science,  122:  417-418. 
MINNICH,  D.  E.,  1921.     An  experimental  study  of  the  tarsal  chemoreceptors  of  two  nymphalid 

butterflies.    /.  Exp.  ZooL,  33:  173-203. 
MINNICH,  D.  E.,  1929.     The  chemical  sensitivity  of  the  legs  of  the  blowfly,  Calliphora  vomitoria 

Linn.,  to  various  sugars.     Zcitschr.  vcrgl.  PhysioL,  11:   1-55. 


TASTE  PERCEPTION  IN  THE  COCKROACH  507 

MINNICH,  D.  E.,  1932.     The  contact  chemoreceptors  of  the  honey  bee,  Apis  mcllifcra  Linn. 

/.  Exp.  Zoo}.,  61 :  375-393. 
PRINGLE,  J.  W.  S.,  1938.     Proprioception  in  insects.     I.     A  new  type  of  mechano  receptor  from 

the  palps  of  the  cockroach.     /.  Exp.  Biol.,  15:  101-113. 
ROEDER,    K.    D.,    1948.     The    effect   of   anticholinesterases    and    related    substances    on    nervous 

activity  in  the  cockroach.     Bull.  Johns  Hopkins  Hosp..  83:  587-600. 
ROEDER,  K.  D.,  AND  S.  ROEDER,  1939.     Electrical  activity  in  the  isolated  ventral  nerve  cord  of 

the    cockroach.     I.     The    action    of    pilocarpine,    nicotine,    eserine    and    acetylcholine. 

/.  Cell.  Comp.  Physiol.,  14:   1-12. 
ROEDER,  K.  D.,  AND  A.  E.  TREAT,  1957.     Ultrasonic  reception  by  the  tympanic  organ  of  noctuid 

moths.     /.  Exp.  Zool,  134:   127-157. 
ROYS,  C.  C,  1956.     A  comparison  between  the  thresholds  of  taste  receptors  and  of  non-gustatory 

nerve  tissue  to  taste  stimuli  in  the  cockroach.     Anat.  Rcc.,  125:  555. 

SLIFER,  E.  H.,  1950.     Vulnerable  areas  on  the  surface  of  the  tarsus  and  pretarsus  of  the  grass- 
hopper  (Acrididae,  Orthoptera).     Ann.  Ent.  Soc.  Amcr.,  43:   173-188. 
TWAROG,   B.   M.,  AND  K.  D.   ROEDER,   1956.     Properties  of  the  connective  tissue   sheath  of  the 

cockroach  abdominal  nerve  cord.     Biol.   Bull.,   Ill  :   278-286. 


HISTOPHYSIOLOGICAL  STUDIES  ON  THE  CORPUS  ALLATUM 

OF  LEUCOPHAEA  MADERAE.     I.  NORMAL  LIFE  CYCLE 

IN  MALE  AND  FEMALE  ADULTS  l 

BERTA  SCHARRER  AND  MARIANNE  VON  HARNACK  - 
Department  of  Anatomy,  Albert  Einstein  College  of  Medicine,  New  York  61,  New  York 

In  the  course  of  previous  work  in  our  laboratory  dealing  with  the  functional 
and  cytological  properties  of  neuroendocrine  systems,  in  particular  the  inter- 
cerebralis-cardiacum-allatum  system  of  the  insect,  Leucophaea  maderae  (Scharrer, 
1946b;  Scharrer  and  Scharrer,  1944),  it  was  observed  that  the  corpora  allata 
display  an  impressive  variability  in  their  morphology.  The  question  arose  whether 
differences  in  the  size  and  histological  appearance  of  these  glands  reflect  differences 
in  age,  sex,  functional  state,  etc.,  as  is  the  case  in  an  analogous  organ,  the  anterior 
pituitary,  or  merely  constitute  the  range  of  individual  variation,  or  perhaps  both. 

The  first  indication  that  physiological  state  determines  structure  in  the  corpus 
allatum  of  Leucophaea  was  obtained  by  an  analysis  of  the  results  of  experiments 
in  which  nerves  connecting  this  organ  with  the  brain  were  severed.  Following  this 
operation,  when  performed  at  the  appropriate  time,  the  functional  capacity  of  the 
gland  was  stepped  up  as  demonstrated  in  last  instar  nymphs  (Scharrer,  1946a), 
and  there  was  a  marked  increase  in  glandular  volume  and  relative  cytoplasmic 
content  (Scharrer,  1952).  The  conclusion  seemed  justified  that,  at  least  under 
the  conditions  of  these  experiments,  a  large  gland  in  which  the  nuclei  are  loosely 
distributed  represents  a  physiologically  active  gland. 

This  conclusion  was  borne  out  further  by  more  recent  studies  (Engelmann, 
1957)  describing  cyclic  changes  in  the  size  of  the  corpora  allata  of  normal  adult 
females  of  the  same  species,  Leucophaea  maderae,  in  conjunction  with  reproductive 
processes. 

Another  investigation  in  which  corpus  allatum  volume  could  be  correlated  with 
functional  change  concerns  the  effect  of  gonadectomy  (von  Harnack  and  Scharrer, 
1956).  These  observations  gave  rise  to  two  questions:  (1)  Are  the  increase  in  the 
size  of  the  corpus  allatum  following  gonadectomy  and  that  occurring  after  nerve 
severance  unrelated  though  comparable  phenomena,  or  is  there  a  mechanism 
involved  which  operates  in  both  instances,  resulting  in  a  similar  histophysiological 
response?  (2)  Is  the  volumetric  increase  observed  in  the  corpora  allata  after  both 
types  of  operation  equivalent  to  that  occurring  under  normal  physiological  condi- 
tions, i.e.,  merely  a  sign  of  "activation,"  or  does  the  response  of  the  operated  speci- 
mens go  beyond  the  normal  physiological  range  characteristic  of  the  corpora  allata? 

In  order  to  answer  these  questions  data  are  needed,  in  addition  to  those  reported 
by  Engelmann  (1957),  concerning  variations  in  the  morphology  of  the  corpora 

1  Supported  by  research  grants  from  the  American  Cancer  Society  and  the  U.  S.  Public 
Health  Service. 

-  Grantee  of  the  American  Association  of  University  Women. 

508 


CORPUS  ALLATUM  OF  NORMAL  LEUCOPHAEA          509 

allata  in  conjunction  with  physiological  states  throughout  the  adult  life  span  of 
both  males  and  females.  Therefore,  prior  to  reports  on  experimental  results,  the 
present  paper  deals  with  cytological  evidence  of  secretory  activity  and  variations 
in  cell  size,  cell  number,  and  nuclear-cytoplasmic  ratio  in  the  normal  animal  to  the 
extent  to  which  they  can  be  examined  with  conventional  techniques  of  light 
microscopy.  These  observations  will  serve  as  points  of  reference  for  subsequent 
papers  which  will  deal  with  experimental  observations. 

MATERIAL  AND  METHODS 

The  material  on  which  this  investigation  is  based  consists  of  88  female  and  46 
male  normal  animals  of  known  adult  age.  They  were  removed  from  stock  colonies 
on  the  day  of  their  emergence  and  isolated  in  pairs  in  pint-size  jars.  They  were 
kept  at  room  temperature  on  a  routine  diet  of  dog  chow  and  fresh  apple  until  the 
day  of  fixation.  The  animals  were  killed  at  selected  intervals  ranging  from  0  to 
599  days  in  the  female,  and  from  0  to  471  days  in  the  male  series.  In  the  majority 
of  cases  an  autopsy  was  performed  to  determine  the  condition  of  the  internal 
organs,  particularly  that  of  the  reproductive  organs  of  the  females. 

For  the  histological  study  of  the  corpora  allata  and  associated  endocrine  organs, 
the  heads  were  fixed  in  Zenker-formol.  Among  a  variety  of  staining  techniques 
tested  the  aldehyde  fuchsin  method  (Gomori,  1950)  proved  most  useful,  when 
modified  according  to  Halmi  (1952)  and  Dawson  (1953).  The  addition  of 
Weigert's  hematoxylin  as  a  nuclear  stain  permitted  the  use  of  the  sections  for 
nuclear  counts  as  well  as  a  study  of  secretory  products.  Most  of  the  tissues  were 
cut  serially  at  7  /x,  a  small  number  of  cases  at  5  ju. 

As  a  basis  for  comparison  of  the  morphological  characteristics  of  the  corpora 
allata,  three  values  were  determined:  the  volume  of  both  glands,  the  total  number 
of  nuclei,  and  their  relative  number  per  unit  of  tissue  (nuclear-cytoplasmic  ratio) . 
In  addition,  nuclear  size  was  measured  in  representative  cases  of  large  and  small 
glands.  Since  it  was  considered  desirable  to  include  a  large  number  of  animals 
in  the  normal,  as  well  as  the  experimental  series  which  will  be  treated  in  subsequent 
papers,  it  became  necessary  to  select  methods  of  determining  these  quantitative 
values  which  afford  a  sufficient  degree  of  accuracy  without  excessive  expenditure 
of  time. 

(a)  Determination  of  volume 

The  conventional  method  of  estimating  organ  volume  by  measurements  of  each 
consecutive  section  in  a  series,  when  carried  out  in  hundreds  of  specimens  would 
be  a  staggering  task.  Therefore,  methods  based  on  the  measurement  of  representa- 
tive sections  were  explored  for  their  validity  in  expressing  organ  volume.  This 
seemed  feasible  because  in  our  studies  the  aim  was  not  so  much  to  determine 
glandular  volume  as  accurately  as  possible,  but  to  select  a  convenient  and  reasonably 
valid  numerical  expression  of  quantitative  differences.  An  additional  advantage  of 
such  a  simplified  procedure  is  the  possibility  of  using  incomplete  series  of  histological 
slides,  a  not  infrequent  result  of  the  technical  difficulties  encountered  with  chitinous 
material. 

The  validity  of  the  method  to  be  adopted  was  tested  as  follows.  To  serve  as 
a  basis  for  comparison,  the  volumes  of  the  corpora  allata  of  20  representative  cases 


510  BERTA  SCHARRER  AND  MARIANNE  VON  HARNACK 

were  calculated  in  ju,3  as  accurately  as  possible  by  drawing  each  consecutive  section 
with  the  aid  of  a  camera  lucida  and  measuring  the  drawings  with  a  planimeter. 
By  the  inclusion  of  the  extremes  in  this  group  of  cases  the  range  of  variability  was 
accurately  established.  Next,  the  same  20  cases  were  evaluated  by  measuring  only 
intermittent  sections.  A  comparison  of  results  showed  inaccuracies  to  be  still 
rather  small  when  only  every  ninth  section  was  measured.  The  average  number 
of  sections  per  corpus  allatum  being  40  to  50,  approximately  6  measurements  were 
available  for  the  calculation  of  the  volume  of  each  gland.  Estimated  values  obtained 
by  calculating  the  means  between  each  of  two  consecutive  measured  sections  were 
substituted  for  the  values  of  the  "skipped"  sections.  The  inaccuracy  of  this  pro- 
cedure when  compared  with  the  results  of  measuring  every  section  turned  out  to  be 
small  enough  to  permit  the  adoption  of  the  abbreviated  method  for  this  and  the 
following  papers  of  this  series. 

(b)  Nuclear  counts 

An  accurate  determination  of  the  density  of  nuclear   distribution  meets  with 

j 

certain  difficulties  especially  in  small  corpora  allata  poor  in  cytoplasm.  In  addition 
to  the  crowding  of  nuclei,  a  certain  degree  of  irregularity  in  nuclear  distribution  has 
to  be  taken  into  consideration.  Different  methods  of  estimation  were  tested ;  the  most 
reliable  figures  were  obtained  by  direct  counts  under  the  microscope  with  the  aid 
of  a  micrometer  disc  added  to  the  eyepiece  and  of  a  tally  counter.  In  each  corpus 
allatum  three  representative  sections  were  selected  in  approximately  the  same 
location.  I.e.,  the  largest  section  in  the  middle  of  the  series  plus  the  fifth  section 
from  the  middle  section  on  either  side.  The  total  area  of  the  three  sections 
representing  each  corpus  allatum  and  the  sum  of  nuclei  counted  in  this  area 
permitted  the  calculation  of  the  number  of  nuclei  per  mm.2,  a  figure  which  represents 
a  fairly  good  index  of  nuclear  density.  These  values  are  recorded  in  Figures  1 
and  2. 

In  order  to  determine  whether  a  volumetric  increase  in  the  corpora  allata  is  due 
solely  to  a  rise  in  cytoplasmic  content  or  whether  this  is  accompanied  by  an  increase 
in  nuclear  number,  an  at  least  rough  calculation  of  the  absolute  number  of  nuclei 
present  in  each  pair  of  corpora  allata  studied  became  desirable.  For  this  the 
following  formula  suggested  by  Engelmann  (1957)  was  used: 

N'XV 


A  X  (T  +  2r) 

where   N  =  total   number  of  nuclei   per   pair   of  corpora   allata,   N'  —  number  of 
nuclei  counted  in  A  (total  area  of  three  sections  selected  for  nuclear  counts),  V 
volume  of  both  corpora  allata,   T    -  thickness  of  sections,  2  r  —  average  nuclear 
diameter. 

RESULTS 
a.     Females 

The  aim  of  this  study  was  to  analyze  in  detail  the  periodic  changes  in  the 
corpora  allata  in  the  course  of  one  reproductive  cycle,  and  to  determine  whether 
essentially  the  same  pattern  obtains  in  successive  cycles  throughout  the  adult  life 
span.  It  soon  became  apparent  (and  was  subsequently  substantiated  in  our 


CORPUS  ALLATUM  OF  NORMAL  LEUCOPHAEA 


511 


TABLE  I 

Quantitative  changes  in  the  corpora  allata  of  26  females  of  Leucophaea  maderae  of  varying 

adult  age  (maximum  599  days)  grouped  according  to  different  stages 

in  the  reproductive  cycle 


Number 
of  animals 

Range  in  volume 
of  corpora  allata 
(in  mill,  /i3) 

Range  in  number 
of  nuclei 

Range  in  nuclear- 
cytoplasmic  ratio 
(nuclei/mm.2) 

Group  A  (small  eggs  in  ovary  ; 

8 

5.0-10.8 

3657-6411 

733-2034 

beginning  of  cycle) 

Group  B  (growing  eggs  in  ovary) 

3 

9.0-12.4 

4137-9488 

864-1558 

Group  C  (pre-ovulatory  stage) 

3 

15.3-37.0 

7017-15,591 

825-987 

Group  D  (pregnancy) 

12 

3.9-17.3 

3124-11,361 

1155-1978 

experimental  work)  that  the  corpus  allatum  of  Leucophaea  shows  a  fair  degree 
of  structural  variability  within  groups  of  specimens  selected  under  comparable 
physiological  conditions.  This  fact  had  to  be  taken  into  account  in  the  search  for 
significant  correlations  with  functional  states  and  necessitated  the  use  of  larger 
samples  than  wroulcl  otherwise  be  called  for. 

There  is  also  a  certain  degree  of  individual  variation  in  the  timing  of  the 
alternating  periods  of  ovarian  activity  and  quiescence  characteristic  of  the  repro- 
ductive activity  of  Leucophaea.  Therefore,  an  analysis  of  the  first  cycle  which 
begins  after  the  emergence  of  the  adult  offers  advantages  because  of  the  greater 
ease  of  dating.  For  the  study  of  subsequent  cycles  the  "adult  age,"  i.e.,  the  time 
elapsed  since  emergence,  is  less  significant  than  the  conditions  of  the  ovary  at  the 
time  of  fixation.  Consequently  the  selection  of  appropriate  stages  in  these  later 
cycles  is  facilitated  by  the  recording  of  preceding  parturitions.  This  is  taken  into 
account  in  the  arrangement  of  the  data  summarized  in  Figure  1.  These  include 
62  females  killed  at  intervals  of  a  few  days  during  the  first  and  second  reproductive 
cycles.  The  cases  illustrating  the  first  cycle  are  arranged  according  to  adult  age 
(days  elapsed  between  emergence  and  fixation  of  the  animal),  those  of  the  second 
cycle  are  grouped  in  reference  to  the  interval  between  preceding  (i.e.,  first)  parturi- 
tion and  fixation. 

An  additional  26  animals  studied  in  this  series  encompass  the  remainder  of  the 
entire  adult  life  span.  The  oldest  specimens  represent  extremes  in  longevity 
obtained  from  a  large  collection  of  dated  females  and  are  of  particular  value  in  the 
search  for  possible  changes  in  corpus  allatum  function  with  increasing  age.  This 
group  of  older  females,  dated  according  to  adult  age,  does  not  lend  itself  for  the 
same  graphic  representation  as  the  younger  specimens,  since  the  number  of 
reproductive  cycles  they  had  completed  was  not  recorded  in  all  cases.  This  group 
is,  therefore,  not  included  in  Figure  1.  The  values  obtained  for  these  animals 
grouped  according  to  phases  of  the  reproductive  cycle  are  summarized  in  Table  I. 
As  can  be  seen  from  Figure  1,  within  30  days  after  emergence  the  volume 
of  the  corpora  allata  rose  from  an  average  value  of  4.2  million  ^3  (minimum  3.5) 
in  the  very  young  female  to  an  average  value  of  15.3  million  //,3,  (maximum  16.6) 
corresponding  to  the  time  when  the  largest  oocytes  had  almost  reached  their 
maximal  size. 

After  ovulation,  the  corpora  allata  returned  to  approximately  the   same   size 


512 


BERTA  SCHARRER  AND  MARIANNE  VON  HARNACK 


26 
24 
22 

20 
—     18 

_•     16 

1 


c     14 
1     '2 

:>   10 

8 

6 
4 

2 

_     16.000 

°j=  14.000 

4[  12.000 

•J>  10.000 

:   I  8.000 

'S  6.000 

c  4. 000 


Ovarian 
growth 


First  pregnancy 


12.000 
II 

10    J> 
o 

9    Z 
8    ^ 

7      ° 

6    I 

^     § 
4   Z 

3 

2.000 


10         20        30        40        50        60         70        80        90        100 
Adult    Age  (days) 


Interval  after  First  Parturition  (days) 
II 1 


FIGURE  1.  Graphic  representation  of  morphological  changes  in  the  corpora  aliata  of  adult 
females  of  Lcucophaca  maderac  in  conjunction  with  reproductive  cycles.  -  — •—  — •—  -=  vol- 
ume of  both  corpora  aliata  in  million  /j.3;  -  — O O—  -  —  number  of  nuclei,  calculated  for 

both  glands;  -  -  =  nuclear-cytoplasmic  ratio  (number  of  nuclei/mm.2).     Diagram 

includes  (I)  entire  period  of  first  reproductive  cycle  (from  emergence  of  adult  to  first  parturi- 
tion at  about  100  days  of  adult  age),  (II)  second  reproductive  cycle  (from  first  parturition  to 
early  part  of  second  pregnancy).  In  this  and  subsequent  figures  of  this  series  the  curves  do 
not  represent  mathematically  correct  summaries  of  the  quantitative  data  which  do  not  lend 
themselves  to  such  treatment ;  they  are  merely  intended  to  facilitate  the  visualization  of  the 
changes  over  the  periods  indicated.  Note  that  volumetric  changes  are  paralleled  by  changes  in 
nuclear  numbers  and  in  nuclear-cytoplasmic  ratios.  For  details  see  text. 

as  those  of  newly  emerged  females.  This  level  was  maintained  throughout 
"pregnancy"  to  be  followed  by  a  more  pronounced  and  more  rapid  rise  (maximum 
volume  25.5  million  ^3)  reached  after  15  days  in  the  pre-ovulation  period  of  the 
second  cycle.  A  study  of  this  and  older  groups  (Table  I)  indicated  that  essentially 
the  same  periodic  changes  in  corpus  allatum  volume  in  conjunction  with  alternating 
phases  of  activity  and  inactivity  occur  in  consecutive  reproductive  cycles.  The 
first  cycle  differs  from  the  subsequent  ones  only  in  degree  in  that  the  volumetric 
maximum  is  lower  and  is  reached  more  slowly.  In  this  respect  our  data  agree 
with  those  of  Engelmann  (1957). 

We  came  to  different  results,  however,  with  respect  to  the  remarkable  cellular 
changes  accompanying  the  periodic  increase  and  decrease  in  corpus  allatum  volume. 
While  it  is  true  that  the  nuclei  are  more  widely  spaced  in  large,  active  corpora 
aliata  than  in  small,  the  rise  in  glandular  volume  is  not  exclusively  due  to  an 
increase  in  the  amount  of  cytoplasm.  The  contribution  made  by  the  nuclei  in  this 
growth  process  is  primarily  by  a  rise  in  their  number,  and  less  by  an  increase  in 


CORPUS  ALLATUM  OF  NORMAL  LEUCOPHAEA          513 

their  size.  In  our  series,  the  total  number  of  nuclei  estimated  per  pair  of  corpora 
allata  rose  from  a  minimum  of  3128  in  newly  emerged  females  to  a  maximum  of 
9220  in  the  first,  and  of  8236  in  the  second  pre-ovulation  period. 

The  highest  nuclear  count  of  the  whole  series  (15,591)  belongs  to  a  female 
which  also  has  the  highest  volumetric  value  for  normal  corpora  allata  (37  million 
/A3).  This  case  is  noteworthy  in  that  it  is  a  female  with  the  exceptional  adult 
age  of  599  days  carrying  large  ova  at  the  time  of  fixation.  Its  corpora  allata,  in 
addition  to  the  large  number  of  normal  sized  nuclei,  contain  a  giant  nucleus 
(diameter  46  //.)  comparable  to  those  described  by  DeLerma  (1932)  and  Palm 
(1947)  in  Gryllotalpa.  The  smallest  number  of  nuclei  (2782)  occurred  in  small 
corpora  allata  of  a  90-day-old  female  fixed  before  the  onset  of  the  second  cycle. 
This  shows  the  interesting  fact  that,  with  decreasing  organ  volume  in  pregnant 
females,  the  number  of  nuclei  falls  to  reach  again  the  level  characteristic  of  very 
young  adult  specimens. 

To  sum  up,  during  subsequent  periods  of  activation  and  quiescence  of  the 
corpora  allata  the  increase  and  decrease  in  organ  volume  is  accompanied  by  periodic 
changes  in  nuclear  numbers.  The  fact  that  the  volumetric  range  (3.0-37.0  mil- 
lion /j?  for  both  corpora  allata)  surpasses  that  of  the  nuclear  numbers  (2782- 
15,591)  expresses  itself  in  marked  differences  in  nuclear  distribution:  Generally 
speaking,  the  larger  the  glands,  the  fewer  the  nuclei  counted  per  mm.2  (range: 
3698-16,765),  and  the  higher  the  absolute  and  relative  cytoplasmic  content  of  the 
corpus  allatum  tissue.  The  cyclic  fluctuations  in  nuclear  numbers  are  so  pro- 
nounced that  cytological  manifestations  of  these  changes  should  be  expected.  Signs  of 
mitotic  activity,  to  account  for  the  cell  multiplication  calculated,  have  been  observed 
both  in  our  normal  and  colchicine-treated  adult  specimens.  The  number  of  mitotic 
figures  counted  in  the  corpora  allata  is  altogether  small ;  while  they  appear  to  be 
more  frequent  during  organ  growth,  mitoses  are  not  entirely  restricted  to  this 
period. 

Conversely,  signs  of  nuclear  destruction  (pycnosis)  seem  to  be  more  pro- 
nounced in  corpora  allata  returning  to  the  inactive  state,  but  are  also  occasionally 
found  in  growing  or  maximally  active  glands.  This  means  that  growth  and 
regression  of  the  corpora  allata  are  not  solely  responsible  for  the  fluctuations  in 
nuclear  numbers.  The  shifts  in  the  frequency  of  mitotic  and  pycnotic  nuclei  can 
perhaps  be  better  understood  in  conjunction  with  the  cytological  manifestations  of 
the  secretory  activity  of  these  glands. 

While  no  comprehensive  analysis  of  the  elaboration  of  the  secretory  product 
by  the  corpora  allata  of  Lcucopliaea  is  intended  in  this  paper,  certain  statements 
may  be  made.  Methods  such  as  Gomori's  chrome  hematoxylin  phloxine,  Foot's 
modification  of  Masson's  trichrome,  or  hematoxylin  and  eosin  stain  are  not  suitable 
for  the  demonstration  of  secretory  materials  in  these  glands.  With  the  aldehyde 
fuchsin  technique,  distinct  small  granules  can  be  demonstrated  within  the  cytoplasm 
which  stain  from  a  rather  deep  purple  to  lavender.  On  occasion  larger  green 
staining  droplets  are  observed.  Newly  emerged  animals  in  our  material  do  not 
show  these  granules ;  in  older  specimens  their  number  and  distribution  appear 
to  depend  on  the  functional  state. 

During  what  seems  to  be  a  rather  short  and  early  phase  in  the  secretory  cycle 
certain  corpus  allatum  cells  stand  out  because  their  cytoplasm  is  densely  packed 


514  BERTA  SCHARRER  AND  MARIANNE  VON  HARNACK 

with  stainable  granules.  Their  presence  permits  the  tracing  of  cellular  processes 
which  are  the  longer  the  more  central  the  location  of  the  cell  within  the  gland. 
In  other  words,  the  corpus  allatum  cells  in  their  mature  form  are  stellate,  and 
obviously  release  their  secretory  products  by  means  of  processes  which  end 
perpendicularly  to  the  surface  of  the  organ.  This  stellate  cell  shape  cannot  be 
readily  ascertained  in  the  absence  of  secretory  granules,  since  cell  boundaries  are 
not  always  easily  observed  in  the  corpora  allata.  Many  specimens  show  a  more 
widespread  distribution  of  granules  which  in  sections  can  no  longer  be  brought 
in  spatial  relationship  with  specific  gland  cells. 

In  cells  presumably  representing  later  stages  in  the  secretory  cycle  the  cyto- 
plasm, instead  of  being  homogeneous,  assumes  a  more  or  less  "stringy"  appearance 
interspersed  with  vacuoles.  These  strands  of  cytoplasm  form  a  characteristic 
pattern  as  though  applied  by  strokes  of  a  brush  in  a  direction  from  the  center  to 
the  periphery  of  the  gland.  It  is  along  these  "lines  of  flow"  (Mendes,  1948; 
Ozbas,  1957)  that  the  secretory  granules  are  now  oriented,  and  their  direction 
corresponds  to  that  of  the  cell  processes  mentioned  above.  The  fact  that  the 
granules  tend  to  become  lined  up  in  greater  numbers  in  the  periphery  of  the  corpora 
allata  also  speaks  for  their  eventual  release  into  the  surrounding  body  fluid. 

A  further  point  of  interest  is  that  the  nuclei  of  cells  containing  many  secretory 
granules  often  appear  pycnotic.  These  and  additional  pycnotic  nuclei,  not  sur- 
rounded by  secretory  granules,  range  from  slightly  shrunken  structures  to  homo- 
geneous intensely  staining  bodies.  Thus,  there  appears  to  be  in  adult  corpora 
allata  a  continuous  cellular  turnover,  whereby  cells  becoming  exhausted  in  the 
process  of  their  secretory  function  are  replaced  by  the  mitotic  activity  of  younger 
probably  non-secreting  cell  elements.  The  rate  of  cellular  turnover  seems  to 
fluctuate  in  the  course  of  a  reproductive  cycle,  with  the  result  that  activation  of 
the  corpora  allata  is  accompanied  by  increase,  and  return  to  inactivity  by  a  decrease 
in  cell  numbers. 

A  further  question  concerns  possible  fluctuations  in  nuclear  diameters  in 
relation  to  periodic  changes  in  organ  volume.  Measurements  of  representative 
cases  have  shown  the  nuclear  diameter  to  vary  only  moderately  in  normal  specimens 
(from  6.4  p.  to  8.2  //,;  mean  7.0  p.}.  The  nuclei  of  any  given  specimen  may  fall 
within  this  range,  and  no  definite  relationship  between  organ  volume  and  average 
nuclear  size  could  be  established.  The  measurements  given  do  not  include  those 
of  rarely  found  giant  nuclei  which  seem  to  occur  characteristically  in  old  specimens 
(see  above). 

Of  all  the  periodic  changes  in  the  appearance  of  the  corpus  allatum  of 
Leucophaea  the  one  most  readily  observed  is  that  of  the  nuclear-cytoplasmic  ratio. 
Thus,  even  without  quantitative  determinations,  the  large,  i.e.,  active  corpus  allatum 
can  be  easily  distinguished  from  the  inactive  on  the  basis  of  its  histological 
appearance. 

b.     Males 

The  46  male  specimens  studied  range  from  an  adult  age  of  0  to  471  days  (Fig. 
2).  As  in  the  female  series,  the  corpora  allata  of  animals  of  the  same  age  may 
show  a  certain  variability  in  size  and  nuclear  distribution.  The  smaller  average 
body  size  of  males  is  reflected  in  lower  corpus  allatum  values.  Shortly  after 


CORPUS  ALLATUM  OF  NORMAL  LEUCOPHAEA 


515 


emergence  the  glands  begin  to  grow,  but  this  period  is  shorter  than  in  females. 
After  about  10  days  a  peak  is  reached  which,  on  the  average,  amounts  to  a 
volumetric  increase  of  2^  times  over  the  initial  volume.  After  this,  the  values 
level  off  to  fluctuate  only  mildly  throughout  the  adult  stage.  The  volume  of  the 
largest  pair  of  glands  measured  in  this  series  (9.0  million  p?)  is  about  four  times 
that  of  the  smallest  (2.1  million  /A3).  As  one  might  expect,  in  analogy  to  the 
situation  in  females,  the  variability  in  total  nuclear  counts  is  somewhat  lower  in 
degree  than  that  in  volume,  but  it  is  nevertheless  considerable  (range:  2032  to 
7000). 

Thus,  if  arranged  in  the  order  of  increasing  volume,  male  corpora  allata  also 
show  a  gradual  increase  in  the  relative  amount  of  cytoplasm.  The  number  of 
nuclei  per  mm.-  ranges  from  7696  to  17,131.  Inasmuch  as  male  corpora  allata 
are  on  the  average  smaller  than  female,  their  nuclei  are  generally  more  crowded. 
The  highest  density  occurred  in  a  case  (adult  age:  51  days)  which  also  had  the 
largest  absolute  number  of  nuclei ;  the  corpora  allata  were  of  medium  size.  The 
loosest  arrangement  of  nuclei  was  observed  in  an  old  specimen  (adult  age :  396 
days)  with  large  corpora  allata  (7.4  million  /x3).  The  relative  cytoplasmic  content 
was  almost  as  high  in  a  male  (adult  age:  10  days)  which  had  the  largest  corpora 
allata  in  the  entire  series. 

Figure  2  shows  quite  clearly  that  the  rise  in  corpus  allatum  volume  during  the 
first  ten  days  of  adult  life  is  accompanied  by  an  increase  in  nuclear  numbers  and 
a  decrease  in  the  number  of  nuclei  per  unit  of  tissue.  The  reverse  trend  occurs 
after  the  peak,  i.e.,  in  males  older  than  10  days,  but  as  a  group  these  males  do 
not  entirely  return  to  the  situation  characteristic  of  newly  emerged  animals.  In 
principle,  the  male  corpora  allata  show  the  same  relationships  between  organ  size, 
number  of  nuclei,  and  density  of  nuclear  arrangement  as  the  females,  but  this 
relationship  is  not  so  pronounced.  It  is  interesting,  for  example,  that  within  a 
medium  size  range  (4-5  million  ^3)  which  encompasses  the  majority  of  males 


9 

_  e 
V 


—  3 


-      8 


17.000 

I   -|  "o5^  15.000 
O  jj        ^  13.  000 


~u  o"  o  J> 


9.000 
7.000 


7.000 
6-S 

y 

2.000 


8       16      24      32      40      48      56      64      72      80      88      96     104    112 


200    250   300   350  400  450   5OO 


Adult  Age   (days) 


FIGURE  2.  Diagram  showing  morphological  characteristics  of  corpora  allata  of  males  of 
Lcucophaea  maderae  ranging  in  adult  age  from  0  to  471  days.  After  a  small  initial  rise  (maxi- 
mum at  10  days)  the  volumes  (—  — •—  — •—  — )  level  off  to  fluctuate  around  a  mean  value 
maintained  throughout  the  adult  life  span.  A  lack  of  distinct  cyclic  activity  is  also  apparent 

from  the  values  expressing  nuclear  numbers   (--  —  O O )   and  nuclear-cytoplasmic  ratios 

( X X ). 


516  BERTA  SCHARRER  AND  MARIANNE  VON  HARNACK 

studied,  the  nuclear-cytoplasmic  ratio  shows  the  highest  degree   of  variability  as 
compared  to  that  in  groups  with  smaller  or  larger  glands. 

The  data  mentioned  so  far  do  not  provide  evidence  for  the  existence  of  cyclic 
activity  changes  in  the  corpora  allata  of  adult  males.  This  also  applies  to  the 
cytology  of  secretory  processes.  Purple-staining  granules  in  the  cytoplasm  have 
been  found  in  varying  amounts  in  animals  of  different  ages  with  the  exception  of 
newly  emerged  specimens.  While  indicating  the  existence  of  a  secretory  function 
in  male  adult  corpora  allata,  these  cytological  phenomena  are  less  impressive  than 
those  in  females  which  they  resemble  in  principle.  The  fact  that  signs  of  secretory 
cycles  as  well  as  volumetric  and  nuclear  changes  are  less  apparent  in  males  is 
related  to  the  greater  range  in  the  response  of  female  corpora  allata  to  varying 
physiological  conditions. 

DISCUSSION 

In  most  species  of  insects  studied,  extirpation  and  implantation  experiments 
have  established  the  control  of  reproductive  processes  by  a  hormone  of  the  corpora 
allata  (for  review  see  Scharrer,  1955).  There  are  also  numerous  indications  that, 
at  the  height  of  their  "gonadotropic"  activity,  the  corpora  allata  of  females  are 
larger  than  when  they  are  inactive.  This  was  reported  among  others  by  Ito  (1918), 
Wigglesworth  (1936),  Thomsen  (1942),  Palm  (1947),  Mendes  (1948)  and  Kaiser 
(1949).  However,  most  of  these  and  more  recent  studies  (Miissbichler,  1952; 
Nayar,  1956;  DeWilde,  1954;  Brandenburg,  1956;  Lukoschus,  1956;  Lhoste, 
1957)  do  not  extend  beyond  a  relatively  short  interval  between  emergence  and 
oviposition.  A  more  detailed  analysis  of  this  relationship  was  carried  out  by 
Engelmann  (1957)  who  correlated  corpus  allatum  volume  and  structure  with 
ovarian  activity  in  Lcucophaea  maderae,  the  species  which  was  also  used  in  the 
present  study.  During  the  long  life  span  of  this  "ovo-viviparous"  species,  periods 
of  ovarian  quiescence  during  "pregnancy"  alternate  with  those  of  activity  in  wrhich 
the  terminal  oocytes  grow  and  deposit  yolk,  and  the  accessory  sex  glands  produce 
secretory  material.  Only  this  active  phase  in  the  reproductive  cycle  requires  the 
presence  of  the  corpus  allatum  hormone  while  the  maturation  of  the  embryos  pro- 
ceeds without  it  (Scharrer,  1946b).  When  Engelmann  found  large  corpora  allata 
with  relatively  high  cytoplasmic  content  in  females  approaching  ovulation,  and 
small  glands  with  densely  packed  nuclei  during  pregnancy,  he  concluded  that  "acti- 
vation" of  the  corpora  allata  is  characterized  by  an  increase  in  the  amount  of  cyto- 
plasm while  the  number  of  nuclei  remains  constant.  The  present  study,  based  on 
a  larger  material,  confirms  these  earlier  results  as  far  as  the  periodic  changes  in 
corpus  allatum  volume  are  concerned ;  it  also  offers  evidence  that  these  changes 
continue  beyond  the  period  (135  days  after  emergence)  analyzed  in  Engelmann's 
study,  i.e.,  throughout  the  adult  (reproductive)  phase  of  the  insect.  In  addition, 
our  results  demonstrate  a  participation  of  the  nuclei  in  the  activity  cycles  of  the 
corpora  allata.  The  role  of  the  nuclei  could  be  ascertained  only  by  quantitative 
methods  in  a  sufficiently  large  material,  since  cell  divisions  are  not  observed  fre- 
quently enough  in  normal  adult  specimens.  Nuclear  counts  have  shown  that,  in 
the  first  as  well  as  subsequent  reproductive  cycles,  an  up  to  four-fold  increase  in 
the  number  of  nuclei  may  take  place  during  the  phase  of  growth  and  activation  of 
the  corpora  allata.  The  decrease  in  organ  volume  which  follows  is  accompanied 
by  a  corresponding  decrease  in  nuclear  number.  These  differences  in  total  nuclear 


CORPUS  ALLATUM  OF  NORMAL  LEUCOPHAEA          517 

counts  are  so  large  that  they  allow  a  considerable  margin  of  error  which  cannot 
be  avoided  in  the  calculation  of  these  figures. 

While  our  cytological  observations  do  not  lend  themselves  to  a  quantitative 
evaluation  of  nuclear  changes  during  the  activity  cycles  of  the  corpora  allata,  it  is 
significant  that  signs  of  mitotic  activity  are  more  prominent  in  growing  glands  and 
pycnotic  nuclei  are  more  conspicuous  in  corpora  allata  returning  to  the  inactive 
state.  These  observations  in  correlation  with  the  study  of  the  cytology  of  the 
secretory  cycle  lead  to  the  conclusion  that  corpus  allatum  cells  are  used  up  and 
replaced  during  the  adult  life  of  the  animal.  Cyclic  changes  in  the  rate  of  this 
cellular  turnover  which  accompany  secretory  cycles  in  these  glands  account  for 
the  periodic  increase  and  decrease  in  nuclear  numbers  and  accompanying  fluctua- 
tions in  organ  volume. 

By  comparison,  nuclear  size  showed  less  variability  in  our  material,  so  that  the 
main  contribution  of  the  nuclei  toward  increase  in  organ  volume  is  due  to  a  rise 
in  their  number.  Taken  as  a  whole,  the  participation  of  the  nuclear  component 
stays  behind  that  of  the  cytoplasm  w^hich  shows  not  only  pronounced  quantitative 
changes  but  undergoes  qualitative  transformations  during  the  process  of  elabora- 
tion of  secretory  granules. 

The  use  of  the  aldehyde  fuchsin  technique  in  the  present  study  for  the  first 
time  permitted  the  demonstration  of  secretion  granules  in  the  corpora  allata  of 
Leucoplmca.  Although  details  of  the  whole  secretory  cycle  still  need  to  be  worked 
out,  present  evidence  supports  the  view  that  the  active  principle  is  elaborated  in 
the  cytoplasm  of  stellate  cells  and  released  into  the  body  fluid  surrounding  the 
surface  of  the  gland.  Depending  on  the  more  peripheral  or  central  location  of  a 
cell,  its  processes  may  be  short  or  long.  They  account  for  the  characteristic  struc- 
tural pattern  of  the  gland  during  certain  stages  of  the  cycle  in  which  strands  of 
cytoplasm  directed  toward  the  periphery  and  interspersed  with  vacuoles  are  deline- 
ated by  rows  of  secretory  granules  which  become  more  numerous  in  the  periphery 
of  the  gland.  It  is  uncertain  whether  a  gland  cell  remains  active  only  during  one 
secretory  cycle  or  not,  but  the  frequent  observations  of  more  or  less  pycnotic 
nuclei  in  cells  filled  with  the  secretory  product  indicate  that  the  cells  may  become 
exhausted  and  are  replaced  by  cells  resulting  from  mitotic  divisions  in  the  adult 
gland.  The  observation  of  cytoplasm  becoming  vacuolized  during  phases  of  activity 
as  well  as  the  arrangement  of  secretory  granules  along  "lines  of  flow"  (determined 
by  the  peripheral  direction  of  the  cell  processes)  is  in  agreement  with  the  findings 
of  other  authors  in  different  species  of  insects  (Mendes,  1948;  Ozbas,  1957). 

Thus,  in  the  cyclic  activity  of  the  corpora  allata  of  adult  females  of  Leucophaea 
a  number  of  factors  are  involved.  The  question  arises  which  of  the  changes 
observed  express  the  physiological  activation  of  the  corpus  allatum.  This  question 
cannot  be  fully  answered  as  yet,  but  certain  known  data  are  of  interest  in  this  con- 
nection. Increase  in  nuclear  number  without  relative  cytoplasmic  increase  can 
be  achieved  in  adult  female  glands  by  the  implantation  of  prothoracic  glands  (Engel- 
mann,  personal  communication).  The  absence  of  ovarian  stimulation  in  animals 
thus  treated  indicates  that  in  the  corpus  allatum  of  adult  Leucophaea  the  relative 
cytoplasmic  increase  is  an  important  prerequisite  for  its  activation.  The  possibility 
that  it  is  the  only  one  is  illustrated  by  the  situation  in  those  species  where  corpus  al- 
latum growth  in  adults  is  said  to  take  place  solely  by  an  increase  in  cell  volume  and 


518  BERTA  SCHARRER  AND  MARIANNE  VON  HARNACK 

nuclear  volume,  but  not  in  cell  number  (Brandenburg,  1956;  Lukoschus,  1956). 
It  is  evident  from  Engelmann's  data  as  well  as  our  own  (Fig.  1)  that  at  the  first 
peak  of  activity  the  corpus  allatum  volume  does  not  reach  the  same  level  as  at  the 
second  and  subsequent  ones.  Conversely,  more  time  is  required  for  the  stimula- 
tion of  the  ovary  during  the  first  reproductive  cycle  than  later  on.  It  seems  that 
the  corpus  allatum  may  require  a  short  period  after  emergence  to  complete  its 
adult  development,  as  has  been  postulated  also  for  other  organs  (Rockstein,  1956). 
The  gland  may,  therefore,  not  be  capable  of  complete  "activation"  until  after  the 
first  cycle. 

The  possibility  that  such  post-emergence  maturation  takes  place  is  also  indi- 
cated by  the  moderate  rise  in  volume  and  nuclear  numbers  occurring  in  male 
corpora  allata  within  the  first  ten  days  of  adult  life.  Our  own  observations  in  this 
respect  are  in  line  with  those  of  Engelmann  (personal  communication)  in  Leuco- 
phaea  and  by  Mendes  (1948)  in  Mclanophts.  At  any  rate,  no  interpretation  other 
than  one  of  tissue  maturation  can  be  given  for  the  initial  volumetric  increase  of 
the  corpora  allata  of  adult  males,  as  long  as  their  functional  role  is  so  little  under- 
stood. All  we  know  is  that  the  male  reproductive  activity  in  Leucophaca  is  undis- 
turbed after  allatectomy  (Scharrer,  1946b),  and  that  certain  data  suggest  a  rela- 
tionship between  corpora  allata  and  metabolic  processes  (Samuels,  1956).  A 
sustained  control  of  metabolic  functions  by  the  corpora  allata  might  well  account 
for  the  picture  of  "mild  activity"  frequently  observed  in  histological  preparations 
of  adult  male  glands  of  every  age.  This  steady  appearance,  which  is  in  contrast  to 
the  cyclic  pattern  in  the  corpora  allata  of  adult  females,  concerns  range  in  organ 
volume  and  nuclear  number  as  well  as  cytological  manifestations  of  secretory 
activity. 

In  connection  with  the  observation  in  both  sexes  of  corpus  allatum  stimulation 
following  emergence  it  is  of  interest  that  this  early  adult  period  in  Leucophaea  also 
differs  from  later  ones  with  respect  to  certain  metabolic  data,  such  as  the  animal's 
lipid  content  (Scharrer  and  Wilson,  unpublished  data). 

The  present  study  illustrates  that  the  histophysiological  approach  whose  value 
is  well  recognized  in  vertebrate  endocrinology  is  equally  fruitful  in  the  exploration 
of  endocrine  mechanisms  in  insects.  In  the  special  case  of  the  corpora  allata  of 
Leucophaea,  known  variations  of  their  activity  are  paralleled  by  marked  changes 
in  the  volume  of  the  entire  organ,  the  number  of  cells,  the  nuclear-cytoplasmic  ratio, 
and  the  cytology  of  the  secretory  process.  The  subsequent  papers  will  be  con- 
cerned with  corpus  allatum  structure  following  experimentally  induced  changes  in 
the  normal  pattern  of  activity  and  quiescence  of  this  gland. 

SUMMARY 

1.  The  corpora  allata  of  Leucophaca  madcrac  display  a  remarkable  degree  of 
structural  variability  in  conjunction  with  changing  functional  states.     This  is  par- 
ticularly apparent  in  adult  females,  where  a  regular  sequence  of  activity  and  inac- 
tivity of  these   glands   parallels   alternating   phases   of   ovarian   development   and 
quiescence. 

2.  The  volume  of  active  corpora  allata  surpasses  that  of  inactive  glands  beyond 
the   range   of  individual   variation,    which   is   considerable.     The   volumetric    rise 
signalling  activation  is  accomplished  to  a  large  extent  by  an  absolute  and  relative 


CORPUS  ALLATUM  OF  NORMAL  LEUCOPHAEA 


519 


increase  in  cytoplasmic  content  which  results  in  a  characteristic  "loose"  distribution 
of  the  nuclei. 

3.  The  present  study  shows  further  that  the  nuclei  participate  significantly  in 
the  cyclic  changes  of  these  organs.     During  each  growth  period,  the  nuclear  num- 
bers increase  up  to  several  times  the  original  values.     When,  after  ovulation,  the 
corpus  allatum  returns  to  a  state  of  inactivity  which  is  maintained  during  pregnancy, 
the  nuclear-cytoplasmic  ratio  returns  to  a  level  characteristic  of  the  newly  emerged 
female.     The  accompanying  reduction  in  cell  number  to  the  initial  level  is  evident 
not  only  from  a  drop  in  nuclear  counts  but  from  the  observation  of  pycnotic  nuclei. 

4.  The  differences  in  the  frequencies  with  which  nuclear  pycnosis  on  one  hand, 
and  mitotic  figures  on  the  other  are  observed  in  various  stages  suggest  the  existence 
of  a  cellular  turnover  which  seems  continuous  but  whose  rate  changes  periodically. 
During  activation  of  the  corpus  allatum,  when  secretory  products  are  elaborated, 
the  increase  in  cell  number  surpasses  the  rate  of  cell  destruction.     In  the  regressing 
gland  the  latter  process  predominates  over  that  of  cell  replacement. 

5.  With  the  use  of  a  modified  aldehyde  fuchsin  technique  distinct  secretory 
granules  have  been  demonstrated  in  the  corpus  allatum  cells  of  Leucophaea.     The 
granules  line  up  along  processes  of  the  cells  which  are  directed  to  the  surface  of 
the  corpus  allatum.     This  fact,  as  well  as  the  accumulation  of  stainable  granules 
in  the  periphery  of  the  gland,  speaks  for  the  release  of  the  active  substance  into 
the  surrounding  hemolymph.     The  occurrence  of  pycnotic  nuclei  in  cells  filled  with 
secretion  granules  suggests  that  these  gland  cells  may  become  exhausted  fairly 
quickly,  perhaps  in  the  course  of  one  secretory  cycle. 

6.  By  comparison  with  the  situation  in  the  females,  the  corpora  allata  of  adult 
males  show  considerably  less  variability.     Soon  after  emergence,  a  short  period  of 
"activation"  seems  to  occur,  as  judged  by  the  same  structural  characteristics  as 
in  the  females.     After  that  a  fairly  constant  level  of  presumably  mild  activity  ap- 
pears to  be  maintained  throughout  adult  life.     Since  the  available  information  on 
the  functional  role  of  the  corpora  allata  in  male  adult  animals  suggests  no  pattern 
of  periodicity,  the  lack  of  distinct  cyclic  changes  in  the  morphology  of  these  glands 
is  not  surprising. 

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der  Lepidopteren  in  Bezug  auf  ihre  Funktion.     Arch.  f.  Entzv.,  144 :  99-131. 
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J.  Micr.  Sci.,  97:  83-88. 
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Orthoptera.     Commun.  Fac.  Sci.    Univ.  Ankara,  8 :    19-44. 
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Fysiogr.  Sdllsk.  Lund  ForhandL,  17:  Nr.   13,   1-11. 
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SAMUELS,   A.,   1956.     The   effect   of   sex   and   allatectomy   on   the   oxygen   consumption   of   the 

thoracic   musculature  of  the   insect,   Leucophaca  maderae.     Biol.   Bull.,   110:    179-183. 
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SCHARRER,  B.,  1946b.     The  relationship  between  corpora  allata  and  reproductive  organs  in  adult 

Leucophaca  maderae  (Orthoptera).     Endocrinol.,  38:  46-55. 
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cardiacum-allatum  system  of  the  insect  Leucophaca  maderae.     Biol.  Bull.,  102 :  261-272. 
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New  York,  Academic  Press. 
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320-405. 

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HISTOPHYSIOLOGICAL  STUDIES  ON  THE  CORPUS  ALLATUM  OF 
LEUCOPHAEA  MADERAE.     II.  THE  EFFECT  OF  STARVATION  1 

MARIANNE  VON  HARNACK  2 
Department  of  Anatomy,  Albert  Einstein  College  of  Medicine,  Nciv  York  61,  New  York 

The  corpora  allata  of  Leucophaca  maderae  undergo  marked  histological  changes 
in  conjunction  with  various  phases  of  the  reproductive  cycle  (Engelmann,  1957; 
Scharrer  and  von  Harnack,  1958).  Activation  of  the  corpus  allatum  is  character- 
ized by  an  increase  in  organ  volume,  in  nuclear  number,  and  in  absolute  and 
relative  cytoplasmic  content.  These  changes,  as  well  as  the  cytological  manifes- 
tations of  secretory  activity,  reach  a  peak  shortly  before  ovulation.  During  the 
subsequent  period  of  pregnancy,  when  the  ovaries  presumably  receive  no  hormonal 
stimulation,  the  corpora  allata  return  to  the  inactive  condition  (small  size,  dense 
nuclear  arrangement)  in  which  they  remain  until  the  onset  of  the  next  reproductive 
cycle. 

The  decision  as  to  whether  or  not  the  corpora  allata  become  activated  at  any 
given  time  resides  in  the  central  nervous  system  which,  under  certain  conditions, 
exerts  a  restraining  influence  on  these  glands.  In  addition,  nervous  or  neuro- 
humoral  stimuli  appear  to  be  necessary  to  sustain  the  activity  of  the  corpora  allata 
(Scharrer,  1952;  Engelmann,  1957).  The  type  of  message  sent  to  the  corpora 
allata  is  determined  by  a  variety  of  afferent  impulses  from  the  external  and  internal 
milieu  (see  Scharrer,  1958,  1959).  One  of  these  is  the  nutritional  state  of  the 
animal. 

During  a  period  of  total  starvation  egg  development  is  suppressed  in  Leucophaea 
(Scharrer,  1946)  ;  however,  the  ovary  remains  capable  of  responding  to  implanted 
active  corpora  allata  (Johansson,  1955).  From  this  result  one  can  conclude  that 
the  absence  of  nutrients  acts  as  a  stimulus  to  the  brain  eliciting  an  inhibitory 
message  to  the  corpora  allata. 

It  was  of  interest,  therefore,  to  examine  the  effects  of  inanition  on  the 
morphology  of  the  corpora  allata  of  adult  females  of  Leucophaca  maderae  (von 
Harnack,  1958).  The  present  study  is  concerned  with  two  aspects  of  this  problem : 
(1)  Does  prolonged  total  starvation  affect  the  structure  of  the  corpora  allata, 
either  to  or  perhaps  beyond  the  point  of  preventing  their  activation?  (2)  How 
do  corpora  allata,  kept  inactive  by  starvation  for  a  considerable  period,  respond 
to  the  resumption  of  a  normal  diet? 

MATERIAL  AND  METHODS 

The  material  on  which  this  investigation  is  based  consists  of  two  series  (A  and 
B),  one  in  which  the  animals  were  subjected  to  total  starvation  for  various  periods 

1  Supported  by  a  Research  Grant  from  the  U.  S.  Public  Health  Service  administered  by 
Dr.  Berta  Scharrer. 

-  Grantee  of  the  American  Association  of  University  Women. 

521 


522  MARIANNE  VON   HARNACK 

of  time,  and  another  in  which  a  period  of  starvation  was  followed  by  return  to  a 
normal  diet.  All  animals  used  were  adult  females  removed  from  the  stock  colonies 
on  the  day  of  emergence  and  isolated  in  pint-size  jars.  At  the  same  time  the 
period  of  starvation  was  initiated  during  which  the  majority  of  animals  received 
only  water;  a  small  number  was  deprived  of  water  as  well  as  food. 

Series  A  was  used  for  the  study  of  the  influence  of  prolonged  total  starvation 
on  the  morphology  and  function  of  the  corpora  allata.  It  consisted  of  52  females 
on  a  water  diet,  and  of  31  females  starved  and  dehydrated  at  the  same  time.  The 
animals  were  fixed  at  intervals  ranging  from  5  to  95  days  following  the  beginning 
of  the  starvation  period,  i.e.,  the  specimens  surviving  longest  had  reached  an  adult 
age  at  which  normally  the  first  reproductive  cycle  would  have  been  completed. 
Animals  showing  increasing  signs  of  weakness  before  being  fixed  could  be  presumed 
to  have  largely  exhausted  their  nutritional  resources.  As  might  be  expected, 
the  point  of  exhaustion  was  reached  sooner  in  the  dehydrated  group. 

Series  B  gives  information  on  the  response  of  the  corpora  allata  to  the 
resumption  of  a  normal  food  intake  following  an  extended  starvation  period.  In 
this  group  51  newly  emerged  females  received  nothing  but  water  for  30  days. 
They  were  then  returned  to  a  regular  diet  of  dog  chow  and  apple ;  they  were 
kept  together  with  normal  males,  and  allowed  to  survive  for  up  to  an  additional 
90  days.  In  both  experimental  series,  fixation  was  scheduled  at  five-day  intervals, 
except  during  periods  when  more  pronounced  structural  changes  of  the  corpora 
allata  were  observed  and,  therefore,  one-  or  two-day  intervals  became  desirable. 

Autopsies  were  performed  in  all  cases  in  order  to  ascertain  the  condition  of 
the  reproductive  organs.  The  histological  procedure,  and  the  method  of 
quantitative  evaluation  of  the  corpora  allata  were  the  same  as  reported  in  the 
preceding  paper  of  this  series  (Scharrer  and  von  Harnack,  1958). 

RESULTS 
Scries  A:  Starved  animals 

In  contrast  to  the  corpora  allata  of  normally  fed  females  which  reach  a  four- 
fold increase  in  volume  within  30  days  after  emergence,  the  corpora  allata  of 
starved  females  are  noticeably  suppressed  (Fig.  1  and  Fig.  3).  The  situation 
in  the  group  of  52  animals  kept  on  a  water  diet  was  as  follows.  During  an  initial 
period  of  15  days,  when  inanition  had  not  yet  become  effective,  the  corpora  allata 
grew  at  a  rate  comparable  to  that  in  normally  fed  animals,  i.e.,  their  volume 
doubled.  After  that  the  corpus  allatum  volume  of  starved  females  showed  a 
gradual  but  continuous  decrease.  At  30  days  of  adult  age  the  corpora  allata  had 
returned  to  the  level  of  the  newly  emerged  female,  and  the  longest  survivor,  fixed 
after  95  days,  had  reached  a  minimal  corpus  allatum  volume  of  1.7  million  /A3. 
This  means  that  prolonged  starvation  had  caused  a  decrease  of  corpus  allatum 
volume  to  about  one-half  of  the  minimal  size  found  in  normally  fed  adult  females. 

The  initial  rise  in  corpus  allatum  volume  of  the  starvation  series  is  paralleled 
by  an  increase  in  nuclear  numbers  which  compares  with  that  in  the  normal  control 
series.  The  turning  point  at  which  the  nuclei  begin  to  decrease  in  number  occurs 
sooner  in  the  experimental  group  (Fig.  1)  than  in  the  controls.  Subsequently  the 
nuclear  counts  return  to  a  value  characteristic  of  the  normal  inactive  gland,  while 
the  cytoplasmic  content  drops  considerably  below  the  normal  baseline.  The 


CORPUS  ALLATUM  OF  STARVED  LEUCOPHAEA 


523 


—  10r 


9.000 


J2.000 


10 


20 


30        40        50         60        70 
Adult  Age    (days) 


90       100 


FIGURE  1.  Diagram  indicating  morphological  changes  in  the  corpora  allata  of  adult  females 
of  Lcucophaca  maderae  which  received  only  water  from  the  day  of  emergence.  Note  that  the 

volume  of  the  corpora  allata  (—  — •—  — •—  — )  and  their  number  of  nuclei  ( O O ) 

fall   after    a   moderate   initial    rise.         —X—  =  nuclear-cytoplasmic    ratio.     For    com- 

parison  with  normal   controls  see  Figure  3. 

result  is  a  very  crowded  nuclear  arrangement  in  the  corpora  allata  of  specimens 
fixed  after  excessively  long  periods  of  starvation. 

These  stepwise  histological  changes  are  enhanced  in  the  starved-dehydated 
group.  When  combined  with  dehydration,  the  first  manifestations  of  inanition 
become  evident  earlier ;  the  turning  point  in  corpus  allatum  volume  occurs  already 
at  about  12  days  of  adult  age  and  appears  accordingly  somewhat  lower  on  the 
curve  (Fig.  3)  than  that  in  the  starved-hydrated  group.  Thus  the  decline  in 
corpus  allatum  volume  which  follows  the  moderate  peak  in  the  dehydrated  group 
runs  roughly  parallel  to  that  in  the  hydrated  group  and  reaches  the  endpoint  sooner 
(Fig.  3).  Another  indication  of  the  aggravating  effect  of  dehydration  on  starving 
animals  was  the  extreme  crowding  of  nuclei  which  made  quantitative  estimates 
of  reasonable  accuracy  impossible.  Nuclear  counts  were,  therefore,  omitted  in 
the  starved-dehydrated  group  of  animals.  In  the  present  as  well  as  earlier 
experimental  series  survival  rates  were  consistently  lower  in  starved-dehydrated 
than  in  starved-hydrated  groups.  The  longest  survival  recorded  in  the  dehydrated 
group  was  only  65  days. 

As  was  to  be  expected  from  preceding  studies  (Scharrer,  1946;  Johansson, 
1955),  none  of  the  females  deprived  of  food  from  the  day  of  their  emergence 
showed  signs  of  reproductive  activity.  On  autopsy,  only  small  undeveloped  eggs 
were  observed  in  the  ovaries,  and  the  accessory  sex  glands  contained  no  appreciable 
amount  of  secretory  material. 

Series  B:  Starved  and  re-fed  animals 

In  this  group  of  females  the  return  to  a  normal  diet  occurred  after  a  starvation 
period  of  30  days  in  which  only  water  was  available ;  i.e.,  at  a  time  when  the 


524 


MARIANNE  VON   HARNACK 


o 

o: 


a  ^ 

Q-  _*> 
_O      O 

%     C 

u  ^ 


36 
34 
32 
30 
28 
26 
24 


120 

c 
— '    18 

Q) 

I'6 

14 


12 
10 
8 
6 
4 
2 


24.000 

22.000 

20.000 

18.000 

16.000 

14.000 

12.000 

10.000 

8,000 

6.000 

4.000 

2.000 


9.000 

8  ^ 

o 

7   _2 


4-i 

3  J 

2.000 


_  L 


30        40         50 

starv.  t   start  of 
period  I  refeeding 


60         70        80         90 
Adult  Age    (days) 


100       110        120 


FIGURE  2.     Diagram    showing    the    response    of    the    corpora    allata    in    adult    females    of 
Leucophaea  maderae  which  were  starved,  with  access  to  water,  for  30  days  following  emergence 

and  were  then  returned  to  a  normal  diet.         —  • • =  volume  of  both  corpora  allata; 

O O--  -  =  number    of    nuclei    of    both    corpora    allata  ;         —X—  -  —  nuclear- 

cytoplasmic  ratio  (number  of  nuclei/mm2).     Compare  volumetric  changes  with  those  of  normal 
controls  (Fig.  4). 


CORPUS  ALLATUM  OF  STARVED  LEUCOPHAEA 


525 


18r 


10 


20 


30       40        50        60        70 
Adult  Age  (days) 


80 


90        100 


FIGURE  3.  Diagram  illustrating  changes  in  the  volume  of  both  corpora  allata  of  females 
of  Leucophaea  madcrac  which  were  starved  and  dehydrated  following  emergence  (curve  A). 
Compare  with  group  of  starved-hydrated  animals  (curve  B)  and  with  normally  fed  control 
group  (curve  C). 

corpus  allatum  volume  as  well  as  the  nuclear  counts  had  almost  returned  to  baseline 
levels  characteristic  of  the  normal  inactive  gland.  Re-feeding  resulted  in  a  rather 
dramatic  response  of  the  corpora  allata.  Within  15-20  days,  a  peak  was  reached 
which  represented  a  seven-fold  increase  in  volume  (Fig.  2).  This  considerably  sur- 
passes the  peak  characteristic  of  the  growth  curve  of  the  first  reproductive  cycle  under 
normal  feeding  conditions  (four-fold  increase  in  volume).  It  compares  favorably 
with  that  of  the  second  normal  cycle  which  it  also  resembles  with  respect  to  the 
steepness  of  the  slopes  (Fig.  4).  It  appears  as  though  the  starved  animal,  on 
resumption  of  feeding,  is  capable  of  "making  up  for  lost  time"  and  does  not  have 
to  start  its  reproductive  period  as  "gradually"  as  the  normal  animal. 

The  rapid  rise  of  corpus  allatum  volume  in  the  starved  and  re-fed  series  is  not 
accompanied  by  a  correspondingly  high  increase  in  nuclear  number  (Fig.  2). 
Consequently,  the  largest  corpora  allata  have  an  exceptionally  high  content  in 
cytoplasm,  and  accordingly  the  nuclei  are  more  widely  spaced  than  in  the  most 
active  glands  of  the  first  reproductive  cycle  of  the  normal  series. 

The  full-scale  activation  of  the  corpora  allata  in  this  first  reproductive  cycle  of 
the  starved-re-fed  animals  promptly  led  to  stimulation  of  the  ovaries ;  ovulation 
occurred  within  about  25  days  after  the  resumption  of  feeding. 

DISCUSSION 

In  the  present  as  well  as  earlier  experiments  (Scharrer,  1946;  Johansson,  1955  ; 
Willis  and  Lewis,  1957),  the  roach  Leucophaea  maderae,  when  subjected  to  total 
starvation,  showed  a  considerable  capacity  to  survive.  If  water  was  provided, 
adult  females  which  were  starved  from  the  day  they  emerged  lived  for  up  to  three 
months  at  room  temperature.  This  period  was  shortened  by  about  three  weeks, 
when  the  insects  were  deprived  of  water  as  well  as  solid  food.  In  Leucophaea 
maderae,  as  in  certain  other  insect  species,  eggs  do  not  develop  in  the  total 


526 


MARIANNE  VON   HARNACK 


10        20       30 


40        50        60        70        80 
Adult  Age   (days) 


90       100       110.120 


0         10        20       30       40 

Interval  after  First  Parturition 

(days) 


FIGURE  4.  Diagram  showing  the  pronounced  rise  in  corpus  allatum  volume  of  starved-re-fed 
females  (curve  B),  in  comparison  with  corpus  allatum  growth  in  first  normal  (curve  A)  and 
second  normal  reproductive  cycle  (curve  C). 


absence  of  nutrients  (Scharrer,  1946),  unless  active  corpora  allata  are  implanted 
(Johansson,  1954;  1955),  or  the  starving  insect's  own  corpora  allata  are  released 
by  surgery  from  the  action  of  the  restraining  nerves  (Johansson,  personal  com- 
munication). These  results  demonstrate  that  (a)  the  gonads  of  starved  animals 
have  the  capacity  to  respond  to  gonadotropic  stimulation,  and  (b)  the  initial 
moderate  rise  in  corpus  allatum  volume  observed  in  starving  Leucophaca  females 
seems  to  represent  a  degree  of  activation  insufficient  to  elicit  ovarian  response. 

The  longer  the  period  of  inanition,  the  smaller  and  the  poorer  in  cytoplasmic 
content  are  the  corpora  allata.  It  is  difficult  to  estimate  the  number  of  nuclei 
present  in  these  small  organs,  and  to  demonstrate  possible  signs  of  secretory 
activity.  At  the  endpoint,  beyond  which  survival  was  no  longer  possible,  the 
corpora  allata  had  only  about  one-half  the  volume  of  normal  female  "inactive" 
glands.  It  cannot  be  determined  with  certainty  whether  or  not  this  decrease  below 
the  normal  range  of  corpus  allatum  size  is  nothing  more  than  a  general  effect 


CORPUS  ALLATUM  OF  STARVED  LEUCOPHAEA          527 

-of  inanition  shared  by  other  organs  of  the  body.  An  attempt  was  made  to 
measure  organs  in  the  vicinity  of  the  corpora  allata,  such  as  the  musculature  of  the 
head.  A  comparison  of  the  diameters  of  muscle  fibers  in  normal  and  starved 
specimens  suggests  that  some  "wasting"  occurs  in  the  latter.  This  is  also  evident 
from  the  larger  spaces  between  muscle  fibers  in  drastically  starved  animals.  How- 
ever, since  the  individual  muscle  elements  vary  in  width,  a  reasonably  accurate 
estimation  of  the  degree  of  shrinkage  is  not  possible. 

What  was  said  so  far  applies  to  starved  animals  receiving  water,  and  to  an 
even  larger  degree  to  starved-dehydrated  specimens.  Their  corpora  allata  do  not 
even  reach  the  size  of  the  starved-hydrated  group,  and  the  decline  in  volume 
(and  activity)  occurs  proportionately  sooner.  A  comparable  dependency  of  the 
morphology  and  function  of  the  corpus  allatum  on  the  nutritional  state  has  been 
observed  also  in  several  other  insect  species  (Wiggles worth,  1936;  Schwinck,  1951 ; 
Miissbichler,  1952;  Engelmann,  1957). 

In  a  general  way,  the  effects  of  nutritional  deficiency  on  the  corpus  allatum  of 
the  insects  compare  with  those  on  its  analogue,  the  anterior  pituitary  of  the 
vertebrates. 

A  variety  of  studies  in  mammals  have  shown  that,  under  conditions  of 
starvation,  (a)  distinct  structural  and  functional  changes  occur  in  the  pars 
anterior,  (b)  gonadal  malfunction  is  attributable  to  suppression  of  gonadotropic 
activity"  and  (c)  the  reproductive  system  deficient  because  of  starvation  responds 
to  the  administration  of  pituitary  material. 

The  morphological  changes  observed  in  pituitaries  of  starving  laboratory 
mammals  as  well  as  human  patients,  such  as  decrease  in  the  volume  or  weight 
of  the  anterior  lobe  (Jackson,  1917),  in  the  size  and  number  of  parenchymal 
(acidophilic)  cells  (Sedlezky,  1924;  Schubothe,  1940;  D'Angelo  ct  al,  1948), 
and  in  relative  cytoplasmic  content  (Jackson,  1917),  are  in  line  with  the  changes 
described  in  the  present  study  for  the  corpora  allata  of  the  insect,  Leucophaea. 

As  in  the  insect,  the  concomitant  disturbance  of  gonadotropic  activity  in  the 
mammals  studied  led  to  depression  of  gonadal  function  (Mulinos  and  Pomerantz, 
1940;  Rinaldini,  1949/50)  which  could  be  remedied  by  the  administration  of 
gonad-stimulating  substances  (Boutwell  ct  al.,  1948;  Rinaldini,  1949/50). 

It  was  of  particular  interest  to  observe  the  effects  of  the  return  to  a  normal  diet 
in  females  of  Leucophaea  whose  ovarian  activity  had  been  restrained  by  a  30-day 
starvation  period  following  emergence.  Instead  of  beginning  their  reproductive 
period  in  the  same  manner  as  young  normal  adults,  even  though  belatedly,  starved- 
re-fed  females  "skipped  the  first  cycle"  for  which  a  slower  and  more  moderate  acti- 
vation of  the  corpora  allata  is  typical.  At  once  they  acted  at  full  capacity.  Thus, 
the  return  to  ample  food  supplies  may  represent  a  powerful  stimulus  for  corpus 
allatnm  growth.  Within  a  short  period  of  20-25  days  an  up  to  seven-fold  volumetric 
increase  occurred.  The  subsequent  decline  from  these  high  values  was  equally 
rapid.  From  every  point  of  view,  the  curves  illustrating  this  reproductive  cycle 
do  not  differ  essentially  from  those  characteristic  of  the  second  or  subsequent  cycles 
in  normal  specimens  (Figs.  2,  4).  In  fact,  the  peak  illustrating  the  range  of 
corpus  allatum  activation  is  higher  in  the  starved-fed  group  than  in  any  cycle  of 
our  normal  series.  However,  this  difference  might  perhaps  be  due  to  individual 
variation,  since  Engelmann's  (1957)  values  for  the  second  reproductive  cycle  of 


MARIANNE  VON   HARNACK 

normal  females  reach  approximately  the  same  maximum  as  that  obtained  in  the 
present  series  of  experimental  animals.  On  the  other  hand,  the  values  obtained  by 
Engelmann  are  not  entirely  comparable  to  those  reported  here,  because  his  experi- 
ments were  conducted  under  different  conditions  of  temperature  and  humidity. 
At  any  rate,  there  can  be  no  doubt  that  corpora  allata  of  animals  having  been  starved 
for  some  time  and  thai  returned  to  normal  food  supply,  respond  with  great  readi- 
ness and  display  pronounced  signs  of  activation.  The  same  observation  was  made 
in  nymphs  of  Panorpa  (Schwinck,  1951).  In  contrast  to  a  newly  emerged  animal, 
a  starved-fed  female  undergoing  its  first  reproductive  cycle  has  had  time  to  adjust 
to  the  changes  connected  with  "metamorphosis."  This  may  be  the  reason  why 
its  corpora  allata,  like  those  of  older  normal  specimens,  respond  maximally  as  soon 
as  the  restraining  effect  of  the  brain  is  lifted.  This  would  mean  that  the  "post- 
emergence  maturation"  postulated  by  Rockstein  (1956)  can  take  place  under 
conditions  of  total  starvation. 

Be  this  as  it  may,  within  certain  limits  the  degree  of  corpus  allatum  activation 
obtained  under  various  normal  and  experimental  conditions  is  perhaps  not  too 
significant.  One  must  keep  in  mind  that  the  more  moderate  activation  of  the 
corpora  allata  in  the  first  normal  cycle  suffices  for  the  development  of  a  full  set  of 
eggs.  The  more  pronounced  response  of  the  corpora  allata  in  more  mature  adults, 
normal  as  well  as  starved-fed  may  be  the  result  of  a  higher  metabolic  rate  of  their 
tissues.  This  possibility  is  suggested  by  comparable  data  in  mammals.  •  Here, 
re-feeding  after  starvation  resulted  in  an  increased  metabolic  rate  (Quimby  et  al., 
1948)  and  in  a  prompt  response  of  the  anterior  lobe  (Jackson,  1917). 

SUMMARY 

1.  Adult  females  of  LcitcopJiaca  inadcrae  were  subjected  to  total   starvation, 
with  or  without  dehydration,  following  their  emergence.     Throughout  the  period 
of  survival,  i.e.,  up  to  95  days,  no  egg  development  occurred  as  a  consequence  of 
the  failure  of  the  corpora  allata  to  become  properly  activated.     The  corpora  allata 
of  a  series  of  starved  animals  fixed  after  varying  intervals  showed  only  a  small 
initial  rise  in  volume  which  was  followed  by  a  gradual  decrease  reaching  a  minimum 
below  that  of  normal  controls. 

2.  In  another  series  of  animals,  starved  for  30  days,  the  return  to  a  normal  diet 
promptly  initiated  a  growth  phase  in  the  corpora  allata  which  considerably  surpassed 
that  characteristic  of  the  first  reproductive  cycle  in  normal  animals    (seven-fold, 
instead  of  four-fold  volumetric  increase).     As  to  speed  and  degree,  this  period  of 
activation  compared  favorably  with  that  of  the   second  cycle   in  normal  females. 
Thus  the  delay  in  reproductive  activity,  caused  by  starvation,  was  at  least  in  part 
compensated  for  by  a  more  rapid  and  complete  activation  of  the  corpora  allata 
which  in  turn  promptly  led  to  ovarian  development. 

3.  The  effects  of  starvation  and  re-feeding  on  the  structure  and  function  of  the 
corpora  allata  of  LeucopJiaca  are  in  line  with  those  described  in  the  literature  for  the 
analogous  organ  in  mammals,  i.e.,  the  anterior  lobe  of  the  pituitary. 


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62-108. 

WIGGLESWORTH,  V.  B.,  1936.     The  function  of  the  corpus  allatum  in  the  growth  and  reproduc- 
tion of  Rhoduius  proli.rus  (Hemiptera).     Quart.  J.  Micr.  Sci.,  79:  91-121. 
WILLIS,  E.  R.,  AND  N.  LEWIS,  1957.     The  longevity  of  starved  cockroaches.     /.  Econ.  Entom., 

50 :  438-440. 


SUBSTANCES  WITH  JUVENILE  HORMONE  ACTIVITY  IN 
CRUSTACEA  AND  OTHER  INVERTEBRATES  l 

HOWARD  A.  SCHNEIDERMAN  AND  LAWRENCE  I.  GILBERT  - 

Department  of  Zoology,  Cornell  University,  Ithaca,  N.  Y .,  and  Marine  Biological 

Laboratory,  Woods  Hole,  Mass. 

The  cyclical  growth  and  molting  of  immature  insects  is  brought  about  by  two- 
hormones,  one  secreted  by  the  insect's  brain  and  the  other  by  the  prothoracic  glands. 
A  third  hormone,  the  juvenile  hormone,  is  secreted  by  the  corpora  allata,  endocrine 
glands  in  the  head  or  prothorax  of  the  insect.  This  hormone  promotes  larval  de- 
velopment but  prevents  metamorphosis  (Wigglesworth,  1957).  Its  presence  in 
the  immature  insect  guarantees  that  when  the  larva  molts  it  will  retain  its  juvenile 
characters  and  not  differentiate  into  an  adult.  The  juvenile  hormone  is  thus  a 
remarkable  molecule  that  permits  growth  but  prevents  maturation.  So  far  as  we 
are  aware  it  has  no  functional  counterpart  in  the  vertebrates.  Recently  Williams 
(1956)  has  reported  that  ether  extracts  of  the  abdomens  of  male  Cecropia  moths 
(Hyalophora  cccropia  L.)  contain  large  amounts  of  juvenile  hormone.  When  this 
extract  was  injected  into  lepidopterous  pupae,  they  molted  into  second  pupae  in- 
stead of  molting  into  adults.  This,  of  course,  is  precisely  what  occurs  when  active 
corpora  allata  are  implanted  into  pupae  (Piepho,  1951  ;  Williams,  1952). 

Although  initial  experiments  demonstrated  juvenile  hormone  only  in  extracts 
of  male  Cecropia  moths,  we  have  since  extracted  it  from  both  males  and  females 
of  22  species  of  Lepidoptera  representing  6  families  (Schneiderman  and  Gilbert, 
1957;  Gilbert  and  Schneiderman,  1958a).  This  result  suggested  that  the  hormone 
could  have  a  wider  distribution  in  the  animal  kingdom.  The  experiments  to  be 
reported  wrere  conducted  to  determine  whether  substances  with  juvenile  hormone 
activity  could  be  extracted  from  other  insect  orders  besides  Lepidoptera,  from  other 
classes  of  arthropods  and  from  other  phyla. 

MATERIALS  AND  METHODS 

1.  Experimental  animals 

Pupae  of  the  polyphemus  silkworm  (Anthcraca  polyphemus  Cram.)  were  used 
as  test-objects  for  assay  of  juvenile  hormone  activity.  They  were  stored  for  about 
thirty  weeks  at  6°  C.  prior  to  use. 

2.  Preparation  and  assay  of  extracts 

Animals  representing  most  of  the  major  groups  of  invertebrates  were  collected 
at  Woods  Hole,  preserved  in  methanol  and  shipped  to  Cornell  University  for  ex- 

1  This  investigation  was  supported  by  grant  H-1887  from  the  National  Heart  Institute  of 
the  U.  S.  Public  Health  Service. 

-  Present   address :    Department   of   Biology,   Northwestern   University,    Evanston,    Illinois. 

530 


JUVENILE  HORMONE  IN  INVERTEBRATES 


531 


traction.  Some  animals  (e.g.,  earthworms  and  slugs)  were  collected  locally  and 
extracted  immediately.  The  tissues  were  homogenized  in  ethyl  ether  and  the 
homogenates  and  methanolic  extracts  vigorously  re-extracted  with  ether  in  a  con- 
tinuous extractor.  The  ether  extracts  were  washed  several  times  with  water,  the 
ether  evaporated  off  and  the  oily  or  waxy  residue  dried  in  vacua  at  60°  C. 

To  test  for  juvenile  hormone  activity  in  the  resulting  extracts,  many  of  which 
were  toxic  and  waxy,  a  new  and  exceedingly  sensitive  assay  procedure  was  devel- 
oped which  permitted  detection  of  traces  of  juvenile  hormone  activity  in  crude 
extracts.  The  assay  takes  advantage  of  the  extraordinary  sensitivity  of  regenerat- 
ing epidermal  tissue  to  juvenile  hormone  (Piepho,  1950;  Piepho  and  Heims,  1952). 
The  extract  to  be  assayed  is  mixed  with  peanut  oil  and  paraffin  wax.  A  small 
rectangle  of  integument  is  excised  from  the  thorax  of  a  Polyphemus  pupa,  a  few 
crystals  of  streptomycin  and  phenylthiourea  (an  anti-tyrosinase)  placed  in  the 
wound,  and  the  wound  sealed  with  a  few  milligrams  of  melted  wax-peanut  oil- 
extract  mixture.  When  the  adult  moth  emerges  three  to  four  weeks  later,  the 
wound  area  is  examined.  In  the  case  of  inactive  extracts,  the  only  evidence  of 
the  former  wound  is  a  small  indentation  covered  with  adult  cuticle.  However,  if 
the  extract  is  active,  then  an  island  of  pupal  cuticle  occurs  at  the  wound  site. 
Figure  1  depicts  such  a  patch  of  pupal  cuticle.  It  stands  out  sharply  from  the  adult 
cuticle  which  surrounds  it.  It  is  scale-less,  brown,  rugose  and  typically  pupal  in 


FIGURE  1.     Thorax  of  adult  Polyphemus  with  a  pupal  patch  produced  by  the  wax  test. 

Scales  have  been  removed. 


532  HOWARD  A.  SCHNEIDERMAN  AND  LAWRENCE  I.   GILBERT 

TABLE  I 

Effects  of  serial  dilutions  of  crude  juvenile  hormone  extract  in  para(jin 

Concentration  of 
hormone  in  paraffin  Effect* 

0  (Peanut  oil)  000 

0  (Inactive  oils)  000 

0  (Paraffin)  000 

1:2000  +00 

1:512  +00 

1:256  +00 

1:128  +        +         0 

1:64  +        +         0 

1:32  +        +        + 

1:16  +        +        + 

1:8  +        +        + 

1:4  +        +        + 

1:1  +        +        + 

*  Each  symbol  represents  a  test  animal. 

most  other  respects ;  it  may  even  have  pupal  setae.  In  cross-section  it  appears  to 
be  three  to  four  times  as  thick  as  adult  thoracic  cuticle.  In  short,  it  is  essentially 
indistinguishable  from  ordinary  pupal  cuticle. 

This  "wax  test"  appears  to  be  far  more  sensitive  than  other  tests  for  juvenile 
hormone  activity,  as  the  following  experiment  reveals.  A  crude  ether  extract  of 
the  abdomens  of  male  Cecropia  moths  was  serially  diluted  with  peanut  oil  (up  to 
1/1000)  and  these  dilutions  dissolved  in  equal  parts  of  wax  and  applied  to  thoracic 
wounds  as  described  above.  The  results  recorded  in  Table  I  reveal  that  the  assay 

•> 

permits  detection  of  final  dilutions  of  hormone  of  1/2000.  That  is,  in  principle,  an 
extract  that  contained  only  1/1 000th  as  much  juvenile  hormone  activity  as  male 
Cecropia  extract  would  yield  a  positive  result.  In  the  1  2000  dilution  recorded  in 
Table  I,  the  wax  patch  weighed  about  6  milligrams  and,  therefore,  contained  only 
3  micrograms  of  crude  extract.  Hence  it  is  possible  with  this  test  to  assay  the 
juvenile  hormone  Content  of  minute  quantities  of  material  extracted  from  a  part 
of  a  single  insect. 

It  is  important  to  note  that,  so  far  as  we  can  ascertain,  the  wax  test  is  absolutely 
specific  for  juvenile  hormone  activity.  Thus  wax  alone,  or  mixtures  of  wax  with 
peanut  oil,  have  never  given  us  false  "positive  tests"  although  dozens  of  control 
tests  have  been  made.  Moreover,  when  the  active  principle  is  removed  from  the 
crude  extract  by  repeated  liquid-liquid  extractions,  the  oil  that  remains,  containing 
virtually  all  of  the  ether-extractable  material  in  the  original  extract,  is  also  inactive 
in  the  wax  test. 

RESULTS  AND  DISCUSSION 

Using  this  sensitive  test,  extracts  from  13  classes  of  invertebrates  representing 
most  of  the  major  phyla  were  examined.  The  results  presented  in  Table  II  reveal 
that  ether  extracts  of  a  truly  diverse  array  of  invertebrates  possess  at  least  some 
juvenile  hormone  activity.  It  is  not  too  surprising  to  find  juvenile  hormone  ac- 
tivity in  crustaceans  and  even  in  annelids,  but  surely  its  presence  in  hydroids  and 
sea  cucumbers  is  unexpected. 


JUVENILE  HORMONE  IN  INVERTEBRATES 


533 


TABLE  1 1 
Juvenile  hormone  activity  of  ether  extracts  of  various  invertebrates 


Phylum 

Porifera 
Cnidaria 

Rhynchocoela 

Annelida 


Class 

Demospongiae 
Hydrozoa 

. \nthozoa 
Anopla 

Polychaeta 
Oligochaeta 
I nsecta 


Species 


Wax  test 


Arthropoda 


Mollusca 

Echinodermata 

Enteropneusta 


Crustacea 
(Decapoda) 


Arachnida 

Gastropoda 
Holothuroidea 

Echinoidea 
Balanoglossida 


Mi-crociona  prolifera 
Cliona  celata 
Pennaria  tiarella 
Tubularia  crocea 
Metridium  dianthus 
Cerebratulus  sp. 

(bodies) 

(heads) 
Nereis  virens 

(bodies) 

(heads) 
Lumbricus  terrestris 

(bodies) 

(heads) 

Numerous  Lepidoptera 
Tenebrio  nwlitor  (Coleoptera) 

(larvae)* 

(adults) 
Sarcophaga  bullata  (Diptera) 

(larvae) 
Neodiprion  lecontei  (Hymenoptera) 

(diapausing  prepupae) 
Apis  mellifera  (Hymenoptera) 

(winter  workers) 
Uca  pugilator 
Orconectes  immunis 

(entire) 

(purified  extract) 
Ho  mar  us  americanus 

(eyestalks) 
Carcinides  maenas 

(fronts) 

(rears) 

Palaemonetes  vulgaris 
Limulus  polyphemus 

(fronts) 

(rears) 

(purified  sterols) 
Deroceras  (Agrioliinax)  agreste 

(heads) 

Thyone  briareus 
Leptosynapta  inhaerens 
Arbacia  punctulata 
Saccoglossus  kowalevsKii 

(entire) 

(less  collar  and  proboscis) 


0 
0 


0 
0 


0 
0 
0 
0 


0 
0 


0 
0 
0 

0 
0 
0 

0 
0 


*  Tested  by  injecting  extract. 

It  is  of  some  interest  that  the  most  potent  non-insect  extract  came  from  the 
eyestalks  of  lobsters.  The  occurrence  of  high  concentrations  of  substances  with 
juvenile  hormone  activity  in  the  eyestalk,  which  is  a  well-known  endocrine  center 


534  HOWARD  A.  SCHNEIDER  MAX  AND  LAWRENCE  I.   GILBERT 

in  crustaceans  (Knowles  and  Carlisle,  1956),  suggests  that  in  crustaceans  the  eye- 
stalk  may  contain  a  gland  which  produces  a  substance  chemically  similar  to  the 
juvenile  hormone  of  the  corpora  allata.  A  likely  site  is  a  part  of  the  X-organ 
which  is  not  neurosecretory  hut  appears  glandular  (i.e.,  the  secretory  cells  of  the 
sensory  papilla  X-organ  (Knowles  and  Carlisle.  1956)).  Whether  the  juvenile 
hormone  plays  a  role  in  crustacean  development  or  egg  maturation  remains  to  be 
proved,  but  it  appears  likely.  In  addition  to  these  results  we  have  also  recently 
found  juvenile  hormone  activity  in  the  adrenal  cortex  of  cattle  (Gilbert  and 
Schneiderman,  195Sb).  Hence,  it  seems  safe  to  conclude  that  substances  with 
juvenile  hormone  activity  are  widespread  in  the  animal  kingdom.  As  far  as  we 
are  aware,  the  only  other  animal  growth  hormones  of  such  wide  distribution  are 
the  estrogens  (Loewre  et  al,  1932;  Hagerman  ct  a/.,  1957). 

Whether  or  not  these  juvenile  hormone  substances  are  similar  chemically  to 
the  juvenile  hormone  of  insects  cannot  be  answered  until  the  structure  of  the 
juvenile  hormone  is  known,  nor  do  we  know  at  present  what  role  these  juvenile 
hormone  substances  play  in  groups  other  than  insects.  Nevertheless,  it  remains 
an  intriguing  fact  that  substances  that  act  as  a  growth  hormone  for  insects  occur 
in  both  hydroids  and  cattle.  It  supports  the  view  that  in  the  course  of  evolution 
there  have  not  been  a  great  number  of  innovations  at  the  level  of  small  molecules 
since  the  Cambrian  Era,  and  that  the  evolution  of  humoral  mechanisms  has  pro- 
ceeded by  particular  groups  of  animals  adapting  available  and  often  ubiquitous 
molecules  to  special  tasks. 

We  wish  to  thank  Dr.  Berta  Scharrer  for  critical  reading  of  the  manuscript  of 
the  present  paper.  Purified  Li  in  nl  us  sterols  were  generously  supplied  by  Dr. 
Werner  Bergmann. 

SUMMARY 

1.  A  new  assay  for  the  juvenile  hormone  of  insects  is  described  which  permits 
detection  of  very  small  amounts  of  hormone  activity. 

2.  Using  this  procedure  extracts  of  a  variety  of  invertebrates  were  assayed  for 
juvenile  hormone  activity. 

3.  Juvenile  hormone  activity  was  detected  in  Hydrozoa,  Polychaeta,  Oligochaeta, 
Lepidoptera,  Coleoptera,  Decapoda,  Holothuroidea,  and  Balanoglossida. 

4.  The  richest  source  of  juvenile  hormone  outside  of  insects  was  the  eyestalk 
of  Crustacea  and  it  is  suggested  that  the  juvenile  hormone  plays  a  role  in  crustacean 
physiology. 

5.  The  significance  of  these  findings  is  discussed  in  relation  to  the  evolution  of 
humoral  mechanisms. 

LITERATURE  CITED 

GILBERT,  L.  I.,  AND  H.  A.  SCHNEIDERMAN,  1958a.  Recent  studies  on  the  distribution  of  the  juve- 
nile hormone  of  insects.  Anat.  Rcc.,  13  :557. 

GILBERT,  L.  L,  AND  H.  A.  SCHNEIDERMAN,  1958b.  The  occurrence  of  substances  with  juvenile 
hormone  activity  in  the  adrenal  cortex  of  vertebrates.  Science,  128:844. 

HAGERMAN,  D.  D.,  F.  M.  WELLINGTON  AND  C.  A.  VILLEE,  1957.  Estrogens  in  marine  inverte- 
brates. Biol.  Bull..  112:  180-183. 

KNOWLES,  F.  G.  W.,  AND  D.  B.  CARLISLE,  1956.  Endocrine  control  in  the  Crustacea.  Biol 
Rev.,  31 :  396-473. 


JUVENILE  HORMONE  IN  INVERTEBRATES  535 

LOEWE,  S.,  W.  RAUDENBUSCH  AND  H.  E.  Voss,  1932.     Nachweis  der  Sexualhormon-Vorkom- 

mens  bei  Schmetterlingen.     Biochem.  Zcitschr.,  244:  347-356. 
PIEPHO,    H.,    1950.     tiber    die    Hemmung    der    Falterhautung    durch    Corpora    allata.     Unter- 

suchungen  an  der  Wachsmotte  Galleria  mellonella  L.     Biol.  Zcntralbl.,  69:   261-271.. 
PIEPHO,  H.,  1951.     Uber  die  Lenkung  der  Insektenmetamorphose  durch  Hormone.     Verh.  dtsch. 

sool.  Ges.  (Wilhelmshaven)  :  62-75. 
PIEPHO,  H.,  AND  A.  HEIMS,  1952.     Das  Kutikulamuster  der  Schmetterlingslarve  und  die  hor- 

monale  Grundlage  seiner  Entstehung.     Unter suchungen  an  der  Wachsmotte   Galleria 

mellonella  L.     Zcitsclir.  f.  Naturforschg.,  76:  231-237. 
SCHNEIDERMAN,  H.  A.,  AND  L.  I.  GILBERT,  1957.     The  distribution  and  chemical  properties  of 

the  juvenile  hormone  of  insects.     Anat.  Rcc.,  128:  618. 

WILLIAMS,  C.  M.,  1952.     Morphogenesis  and  the  metamorphosis  of  insects.     The  Harvey  Lec- 
tures, 47 :  126-155. 

WILLIAMS,  C.  M.,  1956.     The  juvenile  hormone  of  insects.     Nature,  178:  212-213. 
WIGGLES  WORTH,  V.  B.,  1957.     The  action  of  growth  hormones  in  insects.     S\tnpos.  Soc. 

Biol.,  11:  204-227. 


A  STUDY  OF  SOME  EFFECTS  OF  GAMMA  RADIATION  ON 
THE  ADULTS  AND  EGGS  OF  AEDES  AEGYPTI 

LEVON  A.  TERZIAN  AND  NATHAN  STABLER* 
Naval  Medical  Research  Institute,  Bcthcsda  14,  Maryland 

The  studies  to  he  reported  in  this  paper  were  undertaken  to  evaluate  some  of  the 
biological  effects  of  ionizing  radiation  on  the  mosquito  Acdes  aeyypti.  This  phase 
of  the  work  describes  in  particular  the  effects  produced  by  gamma  radiation  on  the 
fertility  and  reproductive  capacities  of  the  males  and  females  of  this  mosquito  species, 
detailing  as  well,  additional  studies  on  the  effects  of  radiation  on  the  viability  of 
eggs  in  various  stages  of  development  or  age.  It  may  be  of  interest  to  note  that 
although  there  is  an  extensive  literature  on  irradiation  of  insects,  and  in  particular 
on  irradiation  of  Drosophila,  nevertheless,  these  studies  have  been  concerned  in  the 
main  with  genetic  effects  rather  than  with  the  specific  biological  effects  reported 
here. 

MATERIALS  AND  METHODS 

The  strain  of  A.  aegypti  used  in  these  experiments  was  obtained  originally  from 
the  U.  S.  Bureau  of  Entomology  and  Plant  Quarantine  Laboratory  of  Beltsville, 
Maryland,  in  June,  1945.  It  has  been  maintained  since  that  time  in  this  laboratory 
in  wire  screened  cages,  measuring  27  :<  24  X24  inches,  at  a  constant  laboratory 
temperature  of  80°  F.  and  a  relative  humidity  of  75  per  cent.  To  maintain  egg 
production  the  females  have  been  allowed  to  gorge  on  guinea  pigs  once  a  week. 
The  resulting  eggs  have  been  collected  on  strips  of  filter  paper,  and  then  conditioned 
by  storing  the  wet  strips  in  closed  jars  for  a  period  of  three  days,  after  which  they 
are  dried  and  stored  at  room  temperature  for  future  use.  The  larvae  from  hatched 
eggs  have  been  reared  in  glass  jars  containing  approximately  2500  ml.  of  tap  water, 
and  have  been  fed  on  guinea  pig  pellets  added  in  appropriate  amounts  each  day. 
On  this  regimen,  it  usually  requires  8  days  for  newly  hatched  larvae  to  reach  the 
pupal  stage.  All  the  adults  and  eggs  exposed  to  gamma  radiation  were  derived 
from  this  colony. 

The  adults  used  for  experimental  purposes  were  kept  in  plastic  cylinders  of  3 
inches  height  and  4  inches  diameter,  in  groups  of  40  males  and/or  40  females, 
and  were  fed  during  the  course  of  the  experiments  on  4  per  cent  sugar  solutions. 
Experimental  groups  which  require  blood  were  fed  exclusively  on  chicks,  usually 
weighing  about  300  grams.  Adult  mosquitoes  were  kept  in  the  same  cylinders 
during  exposure  to  radiation,  and  it  was  possible  to  expose  6  cylinders  simultaneously 
in  the  cobalt  60  irradiator  used  for  these  experiments.  The  strips  of  paper  holding 
the  eggs  were  placed  in  Petri  dishes,  and  following  exposure  to  radiation  were  stored 

1  The  opinions  or  assertions  contained  herein  are  the  private  ones  of  the  writers  and  are 
not  to  he  construed  as  official  or  reflecting  the  views  of  the  Navy  Department  or  the  naval 
service  at  large. 

536 


EFFECTS  OF  GAMMA  RADIATION  ON  AEDES  537 

in  the  same  dishes.     To  insure  a  maximal  hatch,  irradiated  eggs  were  kept  immersed 
in  water  for  at  least  72  hours. 

The  mosquitoes  were  exposed  to  gamma  radiation  at  an  approximate  rate  of  450 
r  per  minute  in  air.  in  a  cobalt  60  irradiator  of  4  pi  geometry. 

RESULTS 
Irradiation  of  the  male 

In  the  first  series  of  experiments  designed  to  determine  the  effects  of  varying 
dosages  of  gamma  radiation  on  male  A.  aegypti,  40  male  and  40  female  mosquitoes 
were  allowed  to  emerge  separately  into  plastic  cylinders  and  on  the  fourth  day 
following  emergence,  the  males  were  exposed  to  gamma  radiation  in  the  cobalt 
irradiator.  On  either  the  first,  the  eighth,  or  the  fifteenth  day  following  exposure 
to  radiation,  the  males  were  then  placed  in  the  cylinders  containing  the  females, 
and  shortly  after  pairing  the  females  were  allowed  to  gorge  themselves  on  normal 
chicks.  The  paired  mosquitoes  then  remained  together  for  the  duration  of  the 
experiment.  Control  groups  were  handled  in  exactly  the  same  manner  except  that 
they,  of  course,  received  no  radiation. 

At  each  dosage  level,  two  to  four  trials  were  conducted  for  both  the  experimental 
and  control  groups,  and  the  number  of  eggs  laid  by  each  group  and  the  percentage 
of  the  eggs  which  hatched  were  estimated  as  accurately  as  possible.  Following 
hatching,  the  larvae  from  the  experimental  groups  were  grown  in  the  usual  manner 
to  determine  whether  or  not  they  would  develop  into  viable  adults.  If  adults 
finally  developed,  these,  in  turn,  were  given  a  blood  meal  and  allowed  to  mate  and 
the  resulting  eggs  were  immersed  in  water  as  a  final  test  of  parental  fertility. 
Finally,  many  of  the  male  mosquitoes  were  dissected  at  suitable  intervals  following 
exposure  to  radiation  in  order  to  determine  the  presence  and  condition  of  the 
spermatozoa  in  the  seminal  vesicles.  At  the  same  time,  non-irradiated  females 
which  had  been  paired  with  irradiated  males  were  also  dissected  to  determine  the 
presence  and  condition  of  spermatozoa  within  the  spermathecae. 

Table  I  summarizes  most  of  the  data  concerning  the  effects  of  gamma  radiation 
on  male  A.  aegypti.  In  dosages  ranging  from  1000  r  to  30,000  r,  exposure  to 
radiation  produced  no  significant  effect  on  the  number  of  eggs  laid  in  those  groups 
in  which  the  males  were  irradiated  one  day  before  they  were  mated  to  normal 
females.  Both  the  control,  or  non-irradiated  animals,  and  the  experimental  ones 
deposited  about  the  same  total  number  of  eggs.  In  the  groups  in  which  the  males 
were  irradiated  but  were  not  paired  and  offered  a  blood  meal  until  8  days  after 
radiation,  however,  there  was  evidence  of  some  reduction  in  the  number  of  eggs 
deposited  among  those  in  which  the  males  had  received  30,000  r,  while  among  the 
groups  paired  and  given  a  blood  meal  15  days  after  the  males  had  been  irradiated, 
it  required  only  20,000  r  to  produce  a  significant  reduction  in  the  number  of  eggs 
deposited  by  the  females.  In  these  groups  the  pattern  of  egg-laying  activity 
resembled  that  of  5-day-old  and  19-day-old  virgin  females,  allowed  to  take  a  blood 
meal.  Such  non-inseminated  females  produced  about  20  per  cent  less  the  number 
of  eggs  than  that  produced  by  inseminated  females  of  comparable  age,  and  in  addi- 
tion, the  oviposition  period  of  such  females  extended  over  significantly  longer  periods 
•  of  time. 


538 


LEVON  A.  TERZIAN  AND  NATHAN  STAHLER 


TABLE  I 

The  oviposit  ion  pattern  of  normal  female  Aedes  acgypti  mated  to  males  exposed  to 

varying  doses  of  gamma  radiation 


Days  post-irradiation,  mating  and  blood-meal 

1 

8 

15 

osage 

Length 

Length 

Length 

No. 
eggs 
laid 

oviposi- 
tion 
period 

Per  cent 
hatch 

No. 
eggs 
laid 

ovi  posi- 
tion 
period 

Per  cent 
hatch 

No. 
eggs 
laid 

oviposi- 
tion 
period 

Per  cent 
hatch 

(days! 

(days) 

(days) 

0 

1060 

11 

100 

1250 

13 

100 

1100 

16 

too 

1,000 

1440 

12 

84 

900 

10 

95 

950 

9 

98 

2,500 

940 

10 

64 

1150 

12 

85 

1400 

13 

46 

3,500 

880 

14 

43 

1000 

13 

46 

1200 

17 

38 

5,000 

1150 

12 

5 

1050 

13 

8 

1600 

26 

9 

7,500 

1.110 

9 

5* 

1230 

11 

6* 

1  400 

21 

2* 

10,000 

1530 

14 

1 

1000 

14 

2 

1150 

25 

2 

20,000 

900 

14 

0 

1100 

22 

0 

800 

41 

0 

30,000 

1000 

11 

0 

850 

40 

0 

750 

57 

0 

*  Fertile  F-l  progeny  reared. 

The  length  of  the  oviposition  period  was  markedly  affected  by  both  the  quantity 
of  radiation  applied  to  the  males  and  the  interval  between  exposure  to  radiation 
and  mating.  In  27  control  groups  given  a  blood  meal  and  allowed  to  mate  on 
the  fifth  day  following  emergence,  eggs  were  deposited  for  periods  ranging  from  8 
to  15  days,  averaging  11  days,  after  the  blood  meal.  In  27  corresponding  experi- 
mental groups,  in  which  males  exposed  to  doses  of  radiation  ranging  from  1000  r 
to  30.000  r  on  the  fourth  day  following  emergence  were  mated  on  the  next  day 
and  the  females  then  given  a  blood  meal,  the  length  of  the  oviposition  period 
differed  very  little  from  that  of  the  control  animals.  In  a  second  series,  in  which  the 
females  of  25  control  groups  were  given  a  blood  meal  and  allowed  to  mate  on  the 
twelfth  post-emergence  day,  eggs  were  deposited  for  periods  ranging  from  8  to 
16  days,  averaging  13  days,  after  the  blood  meal.  In  the  corresponding  experi- 
mental groups,  in  which  the  males  were  irradiated  on  the  fourth  post-emergence  day 
and  mated  8  days  later,  there  was  no  significant  difference  in  the  length  of  the 
oviposition  period  among  those  groups  in  which  the  males  received  radiation 
dosages  ranging  up  to  10,000  r.  At  dosages  of  20,000  r,  however,  eggs  were 
deposited  up  until  22  days  following  the  blood  meal,  and  in  the  groups  in  which 
the  males  had  received  30,000  r,  the  females  continued  to  deposit  eggs  for  periods 
as  long  as  40  days  after  the  blood  meal. 

Increases  in  length  of  the  oviposition  period  were  even  more  apparent  in  the 
third  set  of  experimental  groups,  in  which  the  males  and  females  were  paired  and 
the  females  then  given  a  blood  meal  14  days  after  the  males  had  been  exposed  to 
radiation.  In  this  series,  24  control  groups  in  which  non-irradiated  mosquitoes 
were  mated  and  given  a  blood  meal  on  the  nineteenth  day  after  emergence,  eggs 
were  deposited  for  periods  ranging  from  9  to  25  days,  averaging  16  days,  after  the 


EFFECTS  OF  GAMMA  RADIATION  ON  AEDES  539 

blood  meal.  In  the  experimental  groups  in  which  the  males  had  received  radiation 
dosages  up  to  3500  r  on  the  fourth  day  and  mated  15  days  later  with  normal  females, 
the  length  of  the  oviposition  period  was  similar  to  that  of  the  control  groups  cited 
above.  At  dosages  of  5000  r  to  10,000  r,  however,  eggs  were  deposited  for  periods 
which  averaged  24  days  after  the  blood  meal,  while  at  20,000  r  dosages,  this  period 
was  extended  to  an  average  of  41  days,  until  at  30,000  r  dosages  the  females 
continued  to  lay  eggs  for  periods  averaging  57  days.  It  is  of  interest  to  note  that 
along  with  the  increase  in  the  egg-laying  period,  there  was  a  corresponding  increase 
in  the  number  of  days  in  which  the  females  laid  eggs.  Thus,  in  the  series  in  which 
males  exposed  to  30,000  r  were  mated  to  normal  females  8  days  later,  and  in  which 
the  oviposition  period  was  extended  to  40  days,  eggs  were  actually  laid  during  this 
interval  on  27  different  days  as  compared  to  9  days  on  which  normal  females  mated 
to  normal  males  laid  eggs.  In  the  most  extreme  case,  in  which  males  exposed  to 
30,000  r  were  mated  15  days  later,  the  females  laid  eggs  on  33  different  days  as 
compared  to  10  days  for  the  control  females  in  that  series. 

In  view  of  these  results,  it  was  necessary  to  determine,  first,  the  effects  of  gamma 
radiation  on  the  spermatozoa  in  the  male  mosquito.  For  this  purpose,  males  were 
dissected  and  the  seminal  vesicles  examined  at  appropriate  intervals  following 
exposure  to  varying  dosages  of  radiation.  At  dosages  up  to  10,000  r,  there  was 
no  evident  effect  on  motility,  nor  any  evidence  of  morphological  damage  to  the 
spermatozoa  for  at  least  40  days  following  exposure  to  radiation.  Similarly,  at 
dosages  of  20,000  r  there  was  no  evidence  of  loss  of  motility  or  morphological  damage 
to  the  spermatozoa  for  a  period  of  25  days.  After  25  days,  however,  during  which 
period  the  mosquitoes  began  dying  from  the  effects  of  the  radiation,  the  spermatozoa 
were  found  to  be  in  various  stages  of  fragmentation  and  degeneration.  In  males 
exposed  to  30,000  r,  loss  of  motility  and  deterioration  of  the  spermatozoa  did  not 
occur  until  20  days  after  irradiation,  at  which  time  again  the  mosquitoes  began 
dying  from  the  effects  of  the  irradiation.  With  the  radiation  dosage  increased  to 
50,000  r,  spermatozoa  remained  motile  and  normal  in  appearance  for  only  about 
10  days,  at  which  time  deterioration  of  the  spermatozoa  and  death  of  the  adults 
began  to  occur  simultaneously  as  usual.  In  general,  therefore,  spermatozoa  re- 
mained normal  in  appearance  and  motile  for  about  as  long  as  the  mosquitoes  them- 
selves were  able  to  survive  the  various  doses  of  radiation. 

Secondly,  to  determine  whether  insemination  had  occurred,  and  to  observe  the  con- 
dition of  the  spermatozoa  in  inseminated  females,  the  spermathecae  of  normal  fe- 
males mated  to  irradiated  males  were  examined  at  appropriate  intervals  after  the 
animals  had  been  paired.  As  a  result,  it  was  found  that  all  the  females  paired  with 
males  one  day  after  the  males  had  been  exposed  to  20,000  r  contained  motile  sperm- 
atozoa normal  in  appearance  for  at  least  two  weeks  after  mating.  On  the  other  hand, 
only  about  one-half  the  females  mated  15  days  after  the  males  had  been  exposed  to 
20,000  r  contained  normal  spermatozoa  the  third  day  after  mating.  In  the  remainder, 
either  the  spermatozoa  were  in  fragments  or,  more  usually,  there  were  no  spermatozoa 
present.  And  again,  the  spermathecae  of  all  the  females  mated  a  day  after  the 
males  had  been  exposed  to  30,000  r  contained  normal  appearing  spermatozoa  for  at 
least  two  weeks  after  pairing.  If,  however,  pairing  was  delayed  until  15  days  after 
exposure  of  the  males  to  radiation,  none  of  the  spermathecae  of  the  25  specimens 
were  found  to  contain  spermatozoa  when  examined  the  day  after  mating.  Thus, 


540  LEVON  A.  TERZIAN  AND  NATHAN  STAHLER 

these  data  suggest  that  the  reduction  in  egg  production  and  increase  in  the  period 
of  oviposition  evidenced  when  mating  was  delayed  following  exposure  of  the  males 
to  radiation  was  due  simply  to  inability  of  more  and  more  of  the  deteriorating  males 
to  copulate  successfully  rather  than  to  any  lack  of  motile  spermatozoa. 

That  morphological  integrity  and  motility  of  the  spermatozoa,  or  significant 
changes  in  the  egg-laying  pattern  of  females  mated  to  irradiated  male  mosquitoes, 
are  inadequate  criteria  for  determining  the  biological  effects  of  gamma  radiation  on 
the  fertility  of  the  males,  is  shown  by  observations  on  the  hatching  of  eggs  laid  by 
normal  females  mated  to  males  exposed  to  varying  doses  of  radiation,  and  the 
viability  of  the  larvae  emerging  from  such  eggs.  Thus,  as  shown  in  Table  I, 
although  in  all  the  groups  mated  at  various  intervals  following  irradiation  of  the 
males,  the  females  continued  to  lay  eggs  in  considerable  numbers,  nevertheless,  the 
number  of  larvae  hatching  from  these  eggs  become  progressively  less  with  increasing 
exposure  to  irradiation  of  the  males. 

In  view  of  the  fact  that  even  under  well  controlled  conditions  of  temperature 
and  humidity,  there  was  considerable  variation  in  the  percentage  of  eggs  hatching 
from  eggs  produced  from  normal  matings,  it  was  necessary  for  purposes  of  com- 
parison, to  consider  the  hatch  from  a  given  number  of  normal,  control  eggs  as  a 
100  per  cent  hatch.  And  again  because  of  variations  in  the  hatch  from  a  given 
collection  of  eggs,  differences  in  the  degree  of  hatch  between  eggs  from  control  and 
experimental  were  not  too  apparent  until  the  disparity  in  hatching  between  the 
two  groups  became  quite  considerable. 

As  shown  in  Table  I,  in  all  three  groups  mated  at  various  intervals  following 
irradiation  of  the  males,  the  first  real  evidence  of  any  appreciable  reduction  in  the 
percentage  of  larvae  hatching  from  a  given  number  of  eggs  was  in  those  groups  in 
which  the  males  were  exposed  to  at  least  2500  r.  In  the  groups  in  which  the  males 
had  received  3500  r  there  was  little  further  reduction  in  hatch  except  in  the  group 
in  which  the  males  were  not  mated  with  normal  females  until  15  days  after  their 
exposure  to  radiation,  in  which  case  the  hatch  was  only  18.4  per  cent  of  that  in  the 
control  group.  There  was,  however,  a  sharp  decrease  in  the  hatch  from  eggs 
derived  from  groups  in  which  the  males  had  received  5000  r,  so  that  in  the  ones 
mated  1  and  8  days  following  exposure  to  radiation,  only  5.3  per  cent  and  7.6 
per  cent,  respectively,  of  the  eggs  hatched,  as  compared  to  the  controls.  On  the 
other  hand,  as  if  to  illustrate  again  the  variations  in  hatching  which  may  occur,  in 
the  third  remaining  group  which  had  been  mated  15  days  following  exposure  to 
radiation,  20.0  per  cent  of  the  eggs  produced  hatched  into  viable  larvae. 

There  was  little  further  reduction  in  the  proportion  of  eggs  hatching  among  the 
groups  in  which  the  males  had  received  7500  r.  In  the  groups  in  which  the  males 
had  been  exposed  to  10,000  r,  however,  only  slightly  over  1  per  cent  of  the  eggs 
hatched,  while  in  the  groups  in  which  the  males  had  received  20.000  r  and  30,000  r, 
no  larvae  ever  hatched  from  the  eggs  which  were  produced. 

The  fact  that  the  number  of  viable  eggs  produced  was  the  same,  at  any  given 
dosage,  in  the  groups  mated  15  days  after  irradiation  as  in  the  groups  mated  the 
day  following  exposure,  indicates  that  once  a  sperm  was  damaged  to  the  extent  that 
it  was  no  longer  capable  of  fertilizing  an  egg,  there  was  no  further  recovery  and  the 
injury  remained  permanent,  whereas  if  a  sperm  escaped  such  lethal  injury  initially 
there  was  no  further  physiological  deterioration  and  it  remained  uninjured  and 


EFFECTS  OF  GAMMA  RADIATION  ON  AEDES  541 

capable  of  fertilizing  an  egg.  Since  there  was  no  decrease  or  increase  in  the 
production  of  viable  eggs  to  indicate  either  deterioration  or  recovery,  neither  of 
which  process  could  be  expected  to  proceed  at  the  same  rate,  it  appears  reasonable 
to  assume  that  gamma  irradiation  has  a  kind  of  all-or-none  effect  on  the  spermatozoa 
of  A.  aegypti,  and  that  the  extent  of  this  effect  will  depend  upon  the  level  of 
radiation  that  has  been  administered.  Further,  the  data  cited  above  lend  further 
support  to  the  assumption  that  spermatogenesis  is  not  a  continuing  process  during 
the  adult  life  of  the  male  A.  aegypti. 

It  is  of  interest  to  note  that  the  larvae  hatching  from  the  matings  described  above 
could  be  reared  successfully  and  the  resulting  adults,  when  mated  themselves,  pro- 
duced fertile,  viable  eggs,  providing  there  were  sufficient  larvae  present  to  eliminate 
the  cultivation  problems  that  arise  when  larval  colonies  are  too  small.  Thus,  from 
matings  in  which  the  males  had  received  dosages  up  to  3500  r,  larvae  were  reared 
with  comparative  ease,  but  at  dosages  of  5000  r  and  7500  r,  when  only  very  few 
larvae  were  available,  rearing  them  to  adulthood  became  a  major  problem  of 
cultivation.  Even  at  these  dosages,  however,  the  fewr  larvae  that  were  finally 
grown  mated  successfully  as  adults  and  produced  viable  progeny.  Although  several 
attempts  to  rear  the  isolated  larvae  hatching  from  matings  in  which  the  males  had 
received  10,000  r  were  unsuccessful,  there  is  some  reason  to  believe  that  with  enough 
care  such  larvae  could  be  grown  to  adults,  and  in  such  a  case  the  adults  would  in  all 
likelihood  produce  viable  progeny. 

Irradiation  of  the  females 

In  these  experiments,  designed  to  measure  the  effects  of  varying  doses  of  gamma, 
radiation  on  the  oviposition  habits  of  female  A.  aegypti,  again  40  male  and  40  female 
mosquitoes  were  allowed  to  emerge  separately  into  plastic  cylinders  except  that  in 
this  case  on  the  fourth  day  following  emergence,  the  females  were  irradiated  and 
then  paired  with  normal  male  mosquitoes  on  the  first,  eighth,  or  fifteenth  day 
following  exposure  to  radiation  and  offered  a  blood  meal  soon  after  they  had  been 
mated.  The  resulting  eggs  were  collected,  counted,  then  incubated  as  usual  in  a 
saturated  atmosphere  for  five  days,  and  finally  hatched.  The  larvae  were  grown  to 
adults  and  mated,  and  the  resulting  eggs  were  then  collected  and  allowed  to  hatch 
as  evidence  of  fertility  of  the  F-l  adults.  It  may  be  noted  here  that  as  in  the 
case  of  eggs  produced  from  matings  in  which  the  males  were  irradiated,  if  the  eggs 
hatched  they  could  usually  be  grown  to  adults. 

As  shown  in  Table  II,  there  was  no  significant  reduction  in  the  number  of  eggs 
produced  by  females  exposed  to  1000  r,  or  2000  r,  and  mated  the  following  day. 
There  was,  howrever,  a  significant  reduction  in  the  number  of  eggs  produced  by 
females  exposed  to  2500  r,  while  the  females  exposed  to  doses  as  high  as  10,000  r 
produced  only  a  few  isolated  eggs.  On  the  other  hand,  in  the  groups  mated  8  and 
15  days  following  irradiation,  although  again  there  was  little  or  no  reduction  in  the 
number  of  eggs  produced  by  females  exposed  to  doses  up  to  2000  r,  egg  production 
dropped  off  sharply  in  females  exposed  to  2500  r.  while  females  exposed  to  5000  r 
laid  no  eggs  at  all.  In  general,  therefore,  the  longer  the  mating  of  females  exposed 
to  radiation  doses  above  2000  r  was  delayed,  the  fewer  the  eggs  they  produced,  and 
the  lower  the  dosage  required  to  eliminate  egg  production  entirely.  Similarly,  the 
fewer  the  eggs  that  were  produced,  the  less  the  proportion  of  them  that  eventually 


542 


LEVON  A.  TERZIAN  AND  NATHAN  STAHLER 


hatched  into  viable  larvae.  Thus,  whereas  64.9  per  cent  of  the  eggs  laid  by  females 
exposed  to  3000  r  and  mated  one  day  later  hatched,  only  25.6  per  cent  of  the  eggs 
laid  by  females  exposed  to  the  same  dosage  but  mated  15  days  later  hatched,  and 
similarly  while  67.0  per  cent  of  the  eggs  laid  by  females  exposed  to  3500  r  hatched 
when  they  were  mated  24  hours  later,  only  24.0  per  cent  of  the  eggs  hatched  when 
mating  was  delayed  15  days.  And,  finally,  although  a  small  percentage  of  the 
eggs  laid  by  females  exposed  to  5000  r  and  mated  one  day  later  hatched  into  viable 
larvae,  none  of  the  females  exposed  to  5000  r  or  higher  laid  any  eggs  at  all  when 
mated  8  or  15  days  following  exposure. 

These  data  indicate  that  in  female  A.  aeyypti  exposed  to  radiation  above  certain 
threshold  levels,  in  this  case  approximately  2000  r,  the  ovaries  are  not  only  incapable 
of  recovering  from  the  injury  produced  by  radiation,  but  rather  that  the  functional 
activity  of  the  ovary  becomes  progressively  further  impaired  during  the  interval 
following  exposure.  It  may  be  noted  in  passing  that  unlike  the  case  of  normal 
females  mated  to  males  exposed  to  radiation,  in  which  the  reduction  in  egg  pro- 
duction resulted  in  lengthening  of  the  period  of  oviposition,  in  this  case  the  reduced 
egg  production  of  irradiated  females  mated  to  normal  males  resulted  instead  in 
significant  lessening  of  the  oviposition  period,  indicating  again  the  loss  in  functional 
activity  of  the  impaired  ovaries. 

To  determine  whether  irradiated  females  could  mate  successfully  and  whether 
spermatozoa  could  survive  in  them,  groups  of  virgin  females  were  exposed  to  doses 
of  20,000  r  and  30.000  r,  paired  with  normal  males  either  1  or  15  days  later,  and 
then  dissected  at  suitable  intervals  following  mating.  The  spermathecae  of  all  the 
females  exposed  to  20,000  r,  and  paired  the  day  following  radiation,  contained  motile 
spermatozoa  for  a  period  of  two  weeks  after  mating  had  occurred.  However,  when 

TABLE  II 

The  oviposition  pattern  of  female  Aedes  acgypti  exposed  to  varying  doses  of  gamma  radiation  and. 
then  mated,  at  different  intervals  following  irradiation,  to  normal  males 


Dosage 

(r) 

Days  post-irradiation,  mating  and  blood  meal 

1 

8 

15 

No.  eggs 
laid 

Per  cent 
hatch 

No.  eggs 
laid 

Per  cent 
hatch 

No.  eggs 
laid 

Per  cent 
hatch 

0 

1130 

100 

1070 

100 

1100 

100 

1,000 

1050 

100 

1100 

100 

1000 

100 

1,500 

1350 

71 

1050 

90 

1000 

91 

2,000 

1000 

87 

1000 

90 

700 

78 

2,500 

700 

87 

830 

62 

650 

32 

3,000 

650 

65 

800 

42 

400 

26 

3,500 

550 

43 

600 

35* 

190 

24* 

5,000 

25 

13* 

0 

0 

7,500 

10 

0 

10,000 

10 

0 

20,000 

0 

*  Fertile  F-l  progeny  reared. 


EFFECTS  OF  GAMMA  RADIATION  ON  AEDES 


543 


mating  was  delayed  until  15  days  after  irradiation,  an  average  of  only  8  out  of  10 
females  were  found  to  contain  spermatozoa  during  the  ensuing  two  weeks. 
Similarly,  the  spermathecae  of  all  females  exposed  to  30,000  r  contained  sperma- 
tozoa for  at  least  2  weeks  if  pairing  took  place  the  day  after  irradiation,  but  if 
mating  was  delayed  until  15  clays  after  exposure  to  radiation,  then  only  an  average 
of  4  out  of  10  females  dissected  at  intervals  during  the  subsequent  2  weeks  contained 
spermatozoa.  From  these  results,  it  would  appear  as  if  females  exposed  to  high 
doses  of  radiation  are  capable  of  mating  shortly  after  exposure  and  that  spermatozoa 
will  apparently  survive  in  them  for  at  least  two  weeks,  but  if  mating  is  delayed  too 
long,  radiation  injury  to  the  mosquito  as  a  whole  progressively  reduces  the  chances 
of  successful  mating 

The  second  series  of  experiments  was  designed  to  ascertain  whether  viability 
or  production  of  eggs  was  influenced  by  insemination  of  the  females  prior  to  radia- 
tion rather  than  subsequent  to  radiation  as  in  the  previous  experiments.  Accord- 
ingly, to  assure  insemination  40  females  and  40  males  were  allowed  to  emerge  into 
the  same  cylinders  and  were  maintained  together  for  four  days  following  emergence. 
At  the  end  of  this  period  the  males  were  removed  and  the  females  were  exposed  to 
varying  doses  of  radiation.  One,  eight  and  fifteen  days  after  irradiation  these 
females  were  allowed  to  take  a  blood  meal  and  the  resulting  eggs  were  collected, 
incubated  and  hatched.  The  data  presented  in  Table  III  indicate  that  females 
inseminated  prior  to  irradiation  and  given  blood  at  various  subsequent  intervals 
laid  about  the  same  number  of  eggs  as  females  inseminated  at  similar  intervals 
following  exposure  to  radiation,  as  shown  previously  in  Table  II.  Similarly,  as 
in  the  previous  experiment,  the  number  of  eggs  produced  decreased  significantly  at 


TABLE  III 

The  oviposition  pattern  of  female  Aedes  aegypti  mated  first  to  normal  males,  exposed  to  varying 

doses  of  gamma  radiation  after  mating,  and  then  allowed  to  take  a  blood 

meal  at  different  intervals  following  irradiation 


Dosage 

(r) 

Days  post-irradiation,  blood  meal 

1 

8 

IS 

No.  eggs 
laid 

Per  c°nt 
hatch 

No.  eggs 
laid 

Per  cent 
hatch 

No.  eggs 
laid 

Per  cent 
hatch 

0 

1300 

100 

1280 

100 

1000 

100 

1,000 

1400 

75 

1300 

68 

1250 

86 

1,500 

1450 

55 

1220 

68 

1320 

60 

2,000 

1350 

67 

1150 

61 

900 

57 

2,500 

1180 

67 

910 

33 

630 

44 

3,000 

600 

34 

620 

15* 

670 

14 

3,500 

380 

3 

200 

0 

520 

7* 

5,000 

30 

2* 

0 

0 

7,500 

30 

0 

0 

0 

10,000 

40 

0 

20,000 

0 

Fertile  F-l  progeny  reared. 


544  LEVON  A.  TERZIAN  AND  NATHAN  STAHLER 

2500  r  to  3000  r,  and  except  for  those  females  which  received  a  blood  meal  one 
day  after  irradiation,  egg-laying  was  almost  entirely  inhibited  at  doses  5000  r  or 
above. 

On  the  other  hand,  the  number  of  eggs  which  hatched  from  among  those  laid  by 
females  inseminated  pi  ior  to  exposure  to  radiation  was  consistently  below  the  num- 
ber which  hatched  from  among  those  laid  by  females  inseminated  subsequent  to 
exposure  to  radiation.  Thus,  in  the  groups  in  which  the  females  were  mated  and 
then  exposed  to  as  little  as  1000  r  there  was  a  significant  reduction  in  the  number 
of  eggs  hatching  from  among  those  produced,  while  there  was  no  reduction  in  the 
number  of  eggs  that  hatched  of  those  produced  by  females  inseminated  following 
exposure  to  the  same  dosage,  whereas  43  per  cent  of  the  eggs  laid  by  females  in- 
seminated subsequent  to  exposure  to  3500  r  hatched  out,  only  3  per  cent  of  the 
eggs  produced  by  females  inseminated  prior  to  exposure  hatched  into  viable  larvae. 
Obviously,  insemination  of  females  prior  to  exposure  to  radiation  reduces  the  via- 
bility of  the  eggs  simply  by  introducing  an  additional  source  of  injury,  namely, 
injury  to  the  spermatozoa  in  addition  to  the  effect  on  the  ovaries. 

As  before,  it  was  possible  to  rear  fertile  F-l  progeny  whenever  enough  larvae- 
hatched  out  from  the  eggs.  Thus,  fertile  F-l  progeny  were  reared  from  females 
inseminated  either  before  or  after  exposure  to  doses  as  high  as  5000  r  and  then 
given  a  blood  meal  the  day  after  radiation.  At  dosages  above  5000  r  although  a 
few  eggs  were  produced,  as  shown  in  Tables  II  and  III,  none  hatched.  On  the 
other  hand,  no  eggs  were  produced  by  females  exposed  to  5000  r  but  not  allowed 
a  blood  meal  until  8  and  15  days  following  exposure  to  radiation,  and  fertile  progeny 
were  reared  only  from  females  receiving  3500  r. 

In  female  A.  acgypti  ovarian  activity  is  apparently  suspended  until  the  animal 
takes  a  blood  meal.  Following  a  blood  meal  development  of  the  egg  proceeds, 
fertilization  takes  place  providing  the  spermathecae  contain  spermatozoa,  and  finally 
oviposition  begins.  At  the  temperatures  maintained  in  this  laboratory,  female 
A.  aegypti  will  begin  to  produce  fertile  eggs  approximately  48  hours  after  a  blood 
meal  has  been  taken. 

The  next  series  of  experiments  was  designed  to  study  the  effects  of  radiation 
on  the  cycle  of  events  occurring  in  fertilized  females  between  the  time  blood  is  in- 
gested and  oviposition  begins.  Thus,  in  these  experiments  again  40  males  and  40  fe- 
males were  allowed  to  remain  together  for  4  days  following  emergence,  then  at  the 
conclusion  of  this  period,  the  males  were  removed  and  the  females  were  given  a 
blood  meal.  Subsequently,  at  intervals  of  4,  24  and  42  hours  after  the  blood  meal 
the  females  were  exposed  to  varying  doses  of  radiation. 

The  results  shown  in  Table  IV  indicate  that  the  various  doses  of  radiation  had 
their  greatest  effect  on  egg  production  in  those  groups  irradiated  4  hours  after  a 
blood  meal,  a  significantly  lesser  effect  in  the  groups  irradiated  20  hours  later,  and 
the  least  effect  in  the  groups  irradiated  42  hours  after  the  blood  meal.  Thus, 
whereas  the  group  exposed  to  radiation  4  hours  after  it  had  received  a  blood  meal 
laid  a  total  of  2305  eggs  and  no  eggs  were  produced  by  any  females  exposed  to 
more  than  10,000  r,  the  group  exposed  24  hours  after  it  had  engorged  laid  12,610 
eggs  and  a  few  eggs  were  produced  by  females  receiving  as  much  as  70,000  r. 
Finally,  in  the  group  irradiated  42  hours  after  the  blood  meal,  11,010  eggs  were 
laid  and  it  required  exposures  in  excess  of  100.000  r  to  inhibit  egg  production  com- 


EFFECTS  OF  GAMMA  RADIATION  ON  AEDES 


545 


TABLE  IV 

Oviposit  ion  and  egg  viability  of  inseminated  female  Aedes  aegypti  exposed,  to  gamma  radiation 

at  various  intervals  following  a  blood  meal 


Hours  post-blood-meal,  irradiation 

Dosage 

4 

24 

42 

(r) 

No.  eggs 

Per  cent 

No.  eggs 

Per  cent 

No.  eggs 

Per  cent 

laid 

hatch 

laid 

hatch 

laid 

hatch 

0 

1200 

100 

1400 

100 

1480 

100 

2,500 

1060 

69 

1380 

45 

1270 

29 

3.000 

600 

53 

1400 

38 

1400 

13 

3,500 

230 

31 

1660 

30 

1340 

7* 

5,000 

160 

7* 

1590 

10* 

1280 

1 

7,500 

110 

8 

1720 

6 

1480 

0 

10,000 

40 

0 

1970 

0 

1180 

0 

20,000 

0 

970 

0 

980 

0 

70,000 

10 

0 

310 

0 

80,000 

0 

130 

0 

100,000 

10 

0 

110,000 

0 

*  Fertile  F-l  progeny  reared. 

pletely.  It  would  appear  from  these  data  that  the  early  phases  of  the  complicated 
sequence  of  physiological  events  leading  to  egg  production  are  highly  sensitive  to 
radiation  injury  but  that  once  the  mechanism  has  been  established  and  has  proceeded 
to  some  specific  developmental  stage,  oviposition  will  take  place  in  spite  of  excessive 
radiation  injury. 

On  the  other  hand,  the  data  show  that  although  the  mechanisms  responsible  for 
egg  production  become  more  resistant  as  development  proceeds,  the  eggs  themselves 
become  more  sensitive  to  the  effects  of  radiation  as  they  mature  during  the  pre- 
oviposition  period.  Thus,  in  the  groups  irradiated  4  and  24  hours  following  the 
blood  meal,  about  8  and  6  per  cent,  respectively,  of  the  eggs  produced  by  the  females 
exposed  to  7500  r  hatched  into  viable  larvae  but  in  the  groups  irradiated  42  hours 
after  the  blood  meal,  only  about  1  per  cent  of  the  eggs  produced  by  females  exposed 
to  5000  r  hatched,  while  none  of  the  eggs  produced  by  females  exposed  to  7500  r 
proved  to  be  viable.  And  similarly,  in  the  lower  dosages,  the  hatch  from  eggs  pro- 
duced by  females  exposed  to  radiation  4  hours  after  the  blood  meal  was  far  more 
abundant  than  the  hatch  from  eggs  produced  by  females  exposed  42  hours  later. 

Viable,  fertile  F-l  progeny  were  obtained  from  the  eggs  produced  by  females 
exposed  to  5000  r  4  and  24  hours  after  the  blood  meal,  but  the  few  larvae  from 
eggs  of  females  exposed  to  6000  r  and  7500  r  died  shortly  before  the  pupal  stage. 
In  the  groups  irradiated  42  hours  following  the  blood  meal,  viable,  fertile  F-l 
progeny  were  reared  from  the  larvae  that  hatched  from  eggs  deposited  by  females 
exposed  to  3500  r,  but  again  the  few  larvae  hatching  from  eggs  deposited  by  females 
exposed  to  5000  r  died  during  cultivation.  There  is  reason  to  believe  that  with 
greater  numbers  of  larvae  available,  it  might  have  been  possible  to  rear  adults  from 


546 


LEVON  A.  TERZIAN  AND  NATHAN  STAHLER 


the  groups  in  this  experiment  in  which  the  larvae  died  during  cultivation.  Never- 
theless, as  before,  whenever  larvae  from  irradiated  parents  could  be  grown  to 
adulthood,  the  progeny  always  proved  to  be  fertile. 

Irradiation  of  eggs 

In  the  first  experiments  designed  to  determine  the  effects  of  ionizing  radia- 
tion on  the  eggs  of  A.  aegypti,  eggs  of  two  different  age  groups  were  selected 
for  study.  The  first  group  consisted  of  eggs  that  were  25  to  50  hours  old  in  which 
embryonation  had  not  been  completed,  while  the  second  group  consisted  of  eggs 
approximately  400  hours  old  in  which  such  development  had  long  been  completed 
so  that  the  eggs  would  normally  hatch  at  once  upon  immersion  in  water.  In  this 
laboratory,  properly  conditioned,  normal  eggs  usually  require  65  hours  from  the 
time  they  are  laid  until  they  hatch.  Since  the  dose  rate  was  about  450  r  per  minute, 
the  eggs  in  the  first  groups  were  in  the  cobalt  irradiator  for  periods  not  above  100 
minutes,  but  the  eggs  of  the  second  group,  which  required  extraordinarily  high 
doses  of  radiation  to  inhibit  hatching,  had  to  be  kept  in  the  cobalt  irradiator  for 
periods  up  to  about  23  hours.  Following  exposure  to  radiation,  the  eggs  were 
kept  at  insectary  temperatures  for  1,  8,  or  15  days  before  being  allowed  to  hatch. 
About  2000  eggs  were  used  for  each  exposure  and  there  were  from  4  to  8  different 
exposures  for  each  age  category.  To  assure  hatching,  if  hatching  was  to  occur  at 
all,  eggs  were  kept  immersed  for  72  hours  whenever  necessary. 

The  data  presented  in  Table  V  indicate  that  the  hatching  capabilities  of  the 
eggs  which  were  25  to  50  hours  old  when  irradiated,  and  which  have  been  desig- 
nated as  2  days  old,  were  far  more  susceptible  to  radiation  injury  than  the  eggs 
which  were  16  days  old  when  they  were  irradiated.  Thus,  whereas  in  the  former 
it  required  only  10,000  r  to  reduce  the  hatch  by  50  per  cent,  in  the  latter  it  re- 
quired dosages  ranging  from  20,000  r  to  100.000  r  to  reduce  hatching  to  the  same 
extent.  And  again,  while  a  radiation  dose  of  20,000  r  sufficed  to  inhibit  completely 

TABLE  V 

The  effect  of  gam  ma  radiation  on  the  hatch  of  2 -day-old  and  16-day-old  Aedcs  aegypti  embryos 


Dosage 

(r) 

2-day-old  eggs  (25-50  hrs.) 
Days  post-irradiation,  per  cent  hatcli 

16-day-old  eggs 
Days  post-irradiation,  per  cent  hatch 

1 

8 

15 

1 

8 

15 

2,500 

100 

100 

100 

100 

100 

100 

5,000 

100 

100 

80 

100 

100 

80 

7,500 

75 

75 

75 

100 

80 

80 

10,000 

50 

50 

50 

80 

75 

75 

15,000 

25 

25 

25 

80 

75 

75 

20,000 

0 

0 

0 

80 

75 

50 

30,000 

75 

50 

25 

100,000 

50 

20 

10 

150,000 

25 

0 

0 

200,000 

10 

500,000 

10 

550,000 

0 

EFFECTS  OF  GAMMA  RADIATION  ON  AEDES  547 

the  hatching  of  2-day-olcl  eggs,  which  were  being  exposed  while  embryonic  develop- 
ment was  still  in  progress,  it  required  doses  ranging  from  150,000  r  to  as  much  as 
550,000  to  eliminate  entirely  the  hatching  of  16-day-old  eggs  in  which,  of  course, 
embryonic  development  had  been  completed  prior  to  exposure.  In  addition,  as 
shown  in  Table  V,  it  was  found  that  storage  of  2-day-old  eggs  following  exposure 
to  radiation  produced  no  further  deterioration  in  the  ability  of  the  eggs  to  hatch 
but  that  storage  of  16-day-old  irradiated  eggs  resulted  in  marked  deterioration,  at 
the  various  dosage  levels,  in  the  ability  of  the  eggs  to  hatch  successfully.  Thus, 
in  2-day-old  eggs,  as  many  eggs  hatched  out  when  immersed  15  days  subsequent 
to  doses  of  10,000  r  and  20,000  r  as  had  hatched  following  immersion  only  one  day 
following  exposure,  but  with  16-day-old  eggs,  none  of  the  eggs  exposed  to  150,000  r 
hatched  out  at  the  end  of  15  clays  although  25  per  cent  of  them  had  hatched  when 
immersed  the  day  following  exposure.  And  again,  although  10  per  cent  of  the 
eggs  which  had  received  500,000  r  hatched  when  immersed  the  day  following  ex- 
posure, a  comparable  hatch  was  obtained  from  eggs  which  had  received  only 
100,000  r  when  immersion  was  delayed  15  days  following  exposure  to  radiation. 

It  is  of  interest  to  note  that  although  it  required  enormous  doses  of  radiation 
to  destroy  the  ability  of  the  larvae  to  hatch,  and  there  were  wide  differences  be- 
tween 2-day-old  and  16-day-old  eggs  in  the  amount  of  radiation  required  to  pro- 
duce this  effect,  nevertheless,  fertile  F-l  adults  could  not  be  produced  from  either 
2-day-old  or  16-day-old  eggs  exposed  to  more  than  2000  r.  whether  hatched  1,  8, 
or  15  days  following  exposure.  Thus,  only  about  50  per  cent  of  the  larvae  from 
either  2-day-old  or  16-day-old  eggs  exposed  to  1000  r  developed  into  adults,  almost 
all  of  which,  however,  were  able  to  mate  successfully.  From  eggs  exposed  to 
1500  r,  no  more  than  about  10  per  cent  of  the  larvae  developed  into  adults,  and 
of  these  probably  one-half  were  able  to  mate.  From  eggs  exposed  to  2000  r  only 
about  one  per  cent  of  the  larvae  developed  into  adults,  and  of  these  approximately 
one-third  were  able  to  mate  and  produce  viable  eggs.  The  remaining  adults  in 
these  groups  were  usually  too  feeble  even  to  feed  and  most  of  them  died  almost 
immediately  after  emergence.  However,  all  the  males  examined  in  such  cases 
were  found  to  contain  motile  spermatozoa.  It  may  be  noted,  too,  that  a  considerable 
proportion  of  the  mortality  in  these  groups  occurred  after  ecdysis  when  the  animals, 
apparently  too  weak  to  fly  off  properly,  simply  fell  back  into  the  water.  The  larvae 
developing  from  eggs  exposed  to  2500  r  died  during  either  the  fourth  larval  instar 
or  the  pupal  stage,  while  very  few  larvae  from  eggs  exposed  to  3000  r  survived 
beyond  the  third  instar,  but  of  those  that  did,  all  died  during  the  fourth  larval  stage. 

Finally,  experiments  were  designed  to  determine  the  effects  of  varying  doses 
of  radiation  on  the  hatching  ability  of  eggs  of  A.  aegypti  of  various  ages.  For  this 
purpose  the  eggs  were  kept  at  room  temperature  for  periods  ranging  from  less  than 
24  hours  up  to  180  days,  exposed  to  radiation  and  then  immersed  for  hatching 
4  days  after  having  been  irradiated.  Immersion  had  to  be  delayed  4  days  in  order 
to  insure  embryonation  of  the  eggs  which  were  irradiated  while  they  were  still 
less  than  65  hours  old.  About  2000  eggs  were  used  in  each  trial  and  every  group 
of  irradiated  eggs  was  matched  by  a  control,  or  non-radiated  group  of  eggs  from 
the  same  adult  colony.  The  experiment  was  terminated  with  ISO-day-old  eggs, 
since  it  was  found  that  the  hatching  of  control  eggs  was  so  poor  after  that  period 


548 


LEVON  A.  TERZIAN  AND  NATHAN  STAHLER 


that  it  was  almost  impossible  to  isolate  any  effect  of  radiation.     No  hatch  whatso- 
ever could  be  obtained  from  270-day-old  control  eggs. 

It  is  evident  from  the  data  presented  in  Figure  1  that  resistance  or  susceptibility 
to  radiation  was  related  to  the  age  of  the  egg.  During  the  first  24-hour  period  fol- 
lowing oviposition,  a  time  of  active  embryonic  development,  the  eggs  were  particu- 
larly susceptible  to  radiation  damage,  so  that  as  little  as  800  r  caused  a  50  per  cent 
reduction  in  hatch  while  6000  r  inhibited  hatching  entirely.  By  the  time  the  eggs 
were  48  hours  old  and  embryonic  development  was  nearly  complete,  however,  their 
resistance  had  increased  so  markedly  that  it  required  7500  r  to  produce  a  50  per 
cent  reduction  in  hatch  and  25.000  r  to  eliminate  hatching  entirely.  During  the 
period  in  which  hatching  would  have  normally  occurred  (third  day),  resistance 
was  still  further  increased  so  that  it  required  30,000  r  to  effect  a  50  per  cent  reduc- 
tion in  hatch.  Resistance  to  radiation  increased  to  maximal  dosage  levels  in  four- 
and  five-day-old  eggs  so  that  it  required  75,000  r  to  produce  a  50  per  cent  reduc- 
tion in  hatch,  and  at  least  130,000  r  to  inhibit  hatching  completely.  Further  aging 
gradually  lessened  resistance,  however,  until  by  the  180th  day  after  oviposition, 
administration  of  4000  r  reduced  the  hatch  by  50  per  cent  although  it  still  required 
some  75,000  r  to  eliminate  hatching  completely.  Progeny  were  reared  from  eggs 
of  various  ages  irradiated  at  dosages  ranging  from  1000  r  to  2500  r  and  there  was 
no  discernible  evidence  to  indicate  that  the  age  of  the  egg  influenced  in  any  way 


ro 
O 

130- 

X 

120- 

z 
o 

110- 

Q 
C£ 

100-  4 
90- 
80- 

1 

70- 

0 

60- 

O 

50- 

LJ 

«. 

CD 
O 
Q 

30- 

10  -I 

o  =  50%    HATCH 
•  =  NO   HATCH 


0     10    20    30    40    50   60  90  120  150  180 

AGE  OF  EGGS  (DAYS) 

FIGURE  1.     The  relation  between  the  radiation  dosage  required  to  inhibit  hatching  and 

the  age  of  the  eggs  of  Aedcs  aegypti. 


EFFECTS  OF  GAMMA  RADIATION  ON  AEDES  549s 

the  viability  of  the  F-l  progeny.  Attempts  to  rear  fertile  progeny  from  these 
irradiated  eggs  produced  results  almost  identical  to  the  results  obtained  with  2-day- 
old  and  16-day-old  eggs  except  that  in  this  case,  after  numerous  attempts,  a  few 
adults  were  finally  obtained  from  eggs  exposed  to  2500  r,  which  did  mate  and  lay 
fertile  eggs.  Thus,  again,  although  the  dosage  required  to  impair  the  impetus  to 
hatch  varied  with  the  age  of  the  egg,  the  dosage  required  to  destroy  the  viability 
of  the  egg  was  constant  regardless  of  age.  It  is  of  interest  to  note,  again,  that  the 
dosage  was  directly  related  to  the  length  of  time  larvae  developed  or  survived  fol- 
lowing exposure  to  radiation,  in  that  the  higher  the  dosage  the  quicker  larval  de- 
velopment was  arrested  and  the  larvae  died. 

SUMMARY 

1.  It  has  been  shown  that  when  normal  A.  aegypti  females  are  mated  and  given 
a  blood  meal  24  hours  after  exposure  of  the  males  to  gamma  radiation  in  doses 
up  to  30,000  r,  egg  production  is  not  significantly  affected.     If,  however,  mating  is 
delayed  8  or  15  days  following  irradiation  of  the  males,  egg  production  decreases 
and  the  period  of  oviposition  increases  apparently  because  fewer  males  are  able  to 
copulate  even  though  they  still  contain  motile  spermatozoa. 

2.  Although  eggs  continue  to  be  produced  in  some  quantity  whether  mating  is 
immediate  or  delayed,  fewer  larvae  hatch  from  eggs  produced  by  females  mated  to 
males  exposed  to  2500  r,  while  a  very  few  larvae  hatch  from  eggs  produced  from 
matings  in  which  the  males  received  10,000  r.     However,  it  was  possible  to  grow 
larvae  successfully  to  fertile  adults  capable  of  mating  and  producing  viable  eggs 
only  from  matings  in  which  the  males  had  received  a  maximum  of  7500  r. 

3.  It  has  been  shown,  too,  that  the  egg  production  of  female  mosquitoes,  ex- 
posed first  to  gamma  radiation  and  mated  24  hours  later  to  normal  males,  is  sig- 
nificantly reduced  among  those  receiving  2500  r,  and  almost  entirely  eliminated 
among  those  exposed  to  10,000  r.     However,  larvae  which  could  be  grown  to  fertile 
adults  were  obtained  only  from  eggs  produced  by  females  exposed  to  a  maximum 
of  5000  r.     When  mating  was  delayed,  no  eggs  were  produced  by  females  exposed 
to  doses  in  excess  of  3500  r,  although  viable  larvae  hatched  from  eggs  of  females 
exposed  to  3500  r. 

4.  Females  inseminated  prior  to  being  exposed  to  radiation  produced  approxi- 
mately the  same  number  of  eggs  at  the  various  dosage  levels  as  females  inseminated 
subsequent  to  exposure.     However,  significantly  fewer  larvae  hatched  from  these 
eggs  than  from  the  eggs  laid  by  females  inseminated  subsequent  to  exposure. 

5.  To  determine  the  effects  of  radiation  during  the  cycle  of  egg  development 
which  occurs  in  A.  aegypti  following  a  blood  meal,  inseminated  females  were  ex- 
posed to  gamma  radiation  at  various  intervals  following   engorgement.     It   was 
found  that  egg  production  was  almost  entirely  inhibited  in  females   exposed  to 
10,000  r  4  hours  after  the  blood  meal,  whereas  it  required  in  excess  of  100,000  r 
to  inhibit  egg  production  in  females  in  which  exposure  had  !;een  delayed  42  hours 
after  the  blood  meal.     On  the  other  hand,  although  it  required  higher  and  higher 
doses  of  radiation  to  inhibit  egg  production  the  longer  irradiation  was  delayed, 
nevertheless,  the  eggs  became  more  and  more  sensitive  to  radiation  as  they  matured 
within  the  body.     Thus,  whereas  viable  larvae  resulting  in  fertile  adults  developed 
from  eggs  produced  by  females  exposed  to  5000  r  4  or  24  hours  after  the  blood 


550  LEVON  A.  TERZIAN  AND  NATHAN  STAHLER 

meal,  viable  larvae  could  be  obtained  from  the  eggs  of  females  exposed  to  only 
3500  r  when  exposure  was  delayed  42  hours. 

6.  Finally,  it  has  been  shown  that  although  the  dosage  required  to  inhibit  hatch- 
ing of  the  eggs  of  A.  aegypti  exposed  to  gamma  radiation  varied  enormously  ac- 
cording to  the  age  of  the  egg,  nevertheless,  eggs  exposed  to  doses  in  excess  of 
2000  r,  regardless  of  age,  could  not  be  grown  to  adults.  Again,  however,  as  in 
the  experiments  in  which  either  males  or  females  were  exposed  to  radiation,  when- 
ever larvae  could  be  grown  successfully  to  adults,  the  resulting  adults  proved  to 
be  fertile  and  capable  of  producing  viable  eggs  if  they  were  physically  capable 
of  mating. 


QUATERNARY  AMMONIUM  BASES  IN  THE  COELENTERATES  l 

JOHN  H.  WELSH  AND  PEGGY  B.  PROCK 

Biological  Laboratories,  Harvard  University,  Cambridge  38,  Massachusetts 

The  stinging  organelles  or  nematocysts  of  coelenterates  appear  to  serve  two 
functions.  By  injecting  an  irritating  substance  they  serve  as  effective  weapons  of 
defense,  while  accompanying  paralyzing  action,  probably  by  a  different  agent  or 
combination  of  agents,  is  useful  in  quieting  prey  in  the  process  of  feeding.  In 
spite  of  a  considerable  amount  of  work  over  the  past  fifty  years,  the  chemical  nature 
of  the  nematocyst  toxin  is  still  unknown.  Some  of  the  earlier  literature  has  been 
summarized  elsewhere  along  with  an  account  of  some  recent  work  (Welsh,  1956). 

Aqueous  extracts  of  nematocyst-bearing  tentacles  of  representatives  of  each  of 
the  three  classes  of  coelenterates,  when  injected  in  crabs,  produce  a  preliminary 
excitation.  Spontaneous  autotomy  of  legs  may  accompany  the  excitation.  After 
a  time  the  crabs  become  paralyzed  and,  if  the  dose  is  sufficient,  they  fail  to  recover. 
An  injection  of  tetramethylammonium  chloride  mimics  the  paralyzing  action  of 
extracts,  while  an  earlier  or  simultaneous  injection  of  a  salt  of  tetraethylammonium 
antagonizes  the  paralyzing  actions  both  of  extracts  and  a  tetramethylammonium 
halide  (Welsh,  1956).  Since  Ackermann,  Holtz  and  Reinwein  (1923)  had  isolated 
tetramethylammonium  hydroxide  ("tetramine")  from  sea  anemones,  the  pos- 
sibility existed  that  this  substance,  or  some  derivative,  was  the  active  paralyzing 
principle. 

Largely  through  the  efforts  of  Ackermann  and  co-workers  several  other 
quaternary  ammonium  bases  have  been  isolated  from  sea  anemones  and  chemically 
identified.  Ackermann,  Holtz  and  Reinwein  (1924a)  isolated  and  identified  N- 
methylpyridinium  hydroxide  from  Actinia  equina,  along  with  a  compound  tentatively 
named  "actinin."  Later,  the  same  authors  (1924b)  presented  evidence  that  led 
them  to  suggest  that  actinin  was  probably  the  alkaloid  stachydrine.  Ackermann 
(1927),  however,  determined  actinin  to  be  y-butyrobetaine,  and  not  stachydrine. 
Recently  Ackermann  (1953)  found  homarine  and  trigonelline  in  extracts  of  the 
sea  anemone,  Anemonia  sulcata,  along  with  an  unidentified  base  which  he  first 
named  "anemonin,"  but  later  (1954)  changed  to  "zoo-anemonin."  Evidence  for 
the  occurrence  of  trigonelline  in  the  siphonophore,  Velella  spirans,  had  been  pre- 
sented earlier  by  Haurowitz  and  Waelsch  (1926).  Zoo-anemonin  was  identified 
as  the  dimethylbetaine  of  imidazole  acetic  acid  by  Ackermann  and  Janka  (1953), 
but  the  correctness  of  the  structural  formula  that  they  gave  will  be  discussed  later. 

The  present  study  began  as  an  attempt  to  determine  whether  or  not  tetramine 
was  generally  present  in  coelenterates.  Since  paper  chromatography  was  used, 
followed  by  reagents  that  help  in  the  visualization  of  quaternary  ammonium  bases, 
it  soon  became  obvious  that  several  such  compounds  were  present.  The  work  was 

1  This  investigation  was  supported  in  part  by  research  grant  B-623  from  the  National 
Institute  of  Neurological  Diseases  and  Blindness  of  the  National  Institutes  of  Health,  Public 
Health  Service. 

551 


552 


JOHN  H.  WELSH  AND  PEGGY  B.   PROCK 


extended  in  an  attempt  to  identify  these.  Unfortunately  certain  of  the  papers  of 
Ackermann  and  others  were  not  known  to  us  until  we  had  spent  considerable  time 
in  the  identification  of  those  bases.  We  have  now  identified,  with  reasonable 
certainty,  tetramethylammonium  (I),  homarine  (III),  trigonelline  (IV)  and  y- 
butyrobetaine  (V)  in  representatives  of  all  three  classes  of  coelenterates  (see  Fig. 
1).  We  find  what  probably  corresponds  with  Ackermann's  zoo-anemonin  (VI) 
in  a  horny  coral  and  in  two  species  of  sea  anemone.  With  the  method  used,  we 
have  been  unable  to  identify  N-methylpyridinium  (II)  in  any  coelenterate,  although 
using  the  same  method  we  can  demonstrate  its  presence  in  certain  molluscan  tissues. 
Some  spots  which  react  as  quaternary  ammonium  bases  have  not  been  identified. 
Preliminary  tests  of  the  toxicity  of  the  identified  bases  have  been  made. 


(CH3)4N 


-COO" 


III 


-COO' 


VI 

FIGTKE  1.  Structural  formulae  of  compounds  included  in  this  study.  I  =  tetramethylam- 
monium  ;  II  =  N-methylpyridinium  ;  III  =  homarine  ;  IV  —  trigonelline ;  V  --  7-butyrobetaine  ; 
VI  =  zoo-anemonin. 

MATERIALS  AND  METHODS 
1  extracts  were  made  from  the  following: 

Class  Hydrozoa 

Hydra  littoralis — some  supplied  by   Dr.  W.   F.   Loomis;   some  mass  cultured 
according  to  Loomis  and  Lenhoff  (1956)  ;  others  collected  locally. 
Physalia  physalis  L. — the  Portuguese  man-of-war,  fishing  filaments  only ;  col- 
lected in  Bermuda  and  Bimini,  B.  W.  I. 

Class  Scyphosoa 

Cyanea  capillata  (L.) — brown  or  red  jellyfish,  tentacles  only;  collected  in  Puget 
Sound,  Washington. 


QUATERNARY  BASES  IN  COELENTERATES  553 

Class  Anthozoa 

Plexaura  fle.vuosa- — a  horny  coral,  whole  animal;  collected  in  Bermuda. 
Metridium  dianthits  (Ellis) — sea  anemone,  whole  animal  or  tentacles;  collected 
at  Nahant  and  Rockport,  Massachusetts. 

Condylactis    gigantea    (Weiland) — pink-tipped    sea    anemone,    tentacles    only;, 
collected  in  Bermuda. 

Whole  animals  were  macerated  in  a  Waring  Blendor  and  4-5  volumes  of  acetone 
added.  Tentacles  were  cut  off  and  placed  in  4-5  volumes  of  acetone.  The  tissues 
in  acetone  were  stored  in  a  refrigerator.  When  needed,  a  given  volume  (20  or  25 
ml.)  of  the  acetone  extract  was  decanted,  filtered  and  the  acetone  removed  under 
reduced  pressure.  The  remaining  material  was  dried  and  washed  with  about 
10  ml.  of  petroleum  ether,  the  bases  were  then  taken  up  in  1  or  2  ml.  of  95% 
ethanol  for  chromatography.  No  attempt  was  made  to  secure  quantitative  yields 
but  the  results  give  a  good  idea  of  the  relative  amounts  of  different  bases  that  were 
extracted  from  a  given  species. 

Extracts  and  knowns  were  chromatographed  using  wide  strips  of  Whatman 
No.  3  MM  filter  paper.  After  trying  a  variety  of  acidic  and  basic  solvent  systems 
we  found  that  the  most  satisfactory  separation  was  obtained  with  a  mixture  of 
95  parts  of  95%  ethanol  and  5  parts  of  ammonium  hydroxide  (28%),  as 
recommended  by  Bregoff,  Roberts  and  Delwiche  (1953).  When  two-dimensional 
chromatograms  were  run,  the  second  solvent  system  was  n-butanol-acetic  acid-water 
(10:3:8-9).  The  jars  were  allowed  to  saturate  for  at  least  seven  hours  and  the 
papers  equilibrated  2-3  hours.  The  ascending  method  was  used.  Jars  were  kept 
in  a  chamb'-r  in  which  the  temperature  was  maintained  at  25°  C.  ±  1°.  The  most 
satisfactory  chromatograms  were  obtained  after  runs  of  9-10  hours.  After  drying, 
the  chromatograms  were  examined  under  ultraviolet  light  (short-wave  "Minera- 
light")  and  any  ultraviolet  absorbing  areas  outlined  with  pencil.  Of  the  several 
reagents  used  to  visualize  the  areas  occupied  by  quaternary  ammonium  compounds, 
the  most  generally  satisfactory  was  DragendorfFs  solution  (KBiI4  reagent)  as 
modified  and  used  by  Bregoff,  Roberts  and  Delwiche  (1953). 

To  identify  zoo-anemonin,  a  solvent  system  consisting  of  n-butanol-dioxane- 
water  in  the  proportions  of  4:1:5,  was  also  used  to  permit  comparison  of  the  Rf 
value  with  that  obtained  by  Ackermann  and  Janka  (1953). 

In  order  to  make  more  certain  the  identification  of  homarine  and  trigonelline, 
both  of  which  absorb  strongly  in  the  ultraviolet,  absorption  spectra  of  eluates  were 
compared  with  those  of  synthetic  compounds  using  the  Gary  recording  spectro- 
photometer.  Rather  large  amounts  of  extracts  were  placed  on  paper  and  run  with 
ethanol-ammonia  solvent.  Ultraviolet  absorbing  areas  were  outlined  and  a  strip 
was  cut  from  one  side  for  development  with  KBiI4.  The  desired  areas  were  cut 
out  and  eluted  with  distilled  water.  They  were  appropriately  diluted  and  absorption 
spectra  were  obtained.  We  are  greatly  indebted  to  Mr.  and  Mrs.  Paul  Brown  for 
their  cooperation  in  this  part  of  the  study. 

The  toxicities  of  tetramethylammonium  bromide,  N-methylpyridinium,  homarine, 
trigonelline,  y-butyrobetaine  and  N,N'-dimethylimidazole  acetic  acid  were  deter- 
mined on  the  fiddler  crab,  Uca  pugilator,  from  Florida.  Each  was  tested  on  one 
or  more  lots  of  5  crabs,  by  injecting  0.02  or  0.05  ml.  of  a  1%  solution  at  the 
base  of  one  of  the  walking  legs. 


554  JOHN  H.  WELSH  AND  PEGGY  B.  FROCK 

The  known  quaternary  ammonium  standards  used  in  this  study  were  from  the 
following  sources :  tetramethylammonium  bromide,  Eastman  Organic  Chemicals ; 
N-methylpyridinium  bromide,  kindness  of  Dr.  J.  A.  Aeschlimann,  Hoffmann-La 
Roche  Inc. ;  homarine,  kindness  of  Dr.  E.  L.  Gasteiger ;  trigonelline,  General 
Biochemicals  Inc. ;  while  y-butyrobetaine  was  prepared  from  y-carbomethoxypropyl- 
trimethylammonium  bromide  (generously  supplied  by  Dr.  R.  W.  Fleming,  Parke 
Davis  and  Co.)  after  the  method  suggested  by  Bregoff,  Roberts  and  Delwiche 
(1953).  A  sample  of  the  dimethylbetaine  of  imidazole  acetic  acid,  as  the  hydro- 
chloride  (C7H10O2N,-HCL-H,OJ,  was  kindly  furnished  by  Dr.  D.  Ackermann. 
A  second  sample  was  made  from  imidazole  acetic  acid  (supplied  by  Dr.  H.  Bauer, 
National  Institutes  of  Health)  in  the  laboratory  of  Dr.  R.  B.  Woodward.  The 
two  samples  had  similar  melting  points  and  similar  Rf  values.  Dr.  Woodward 
informs  us  that  the  structural  formula  for  anemonin  (zoo-anemonin)  as  given  by 
Ackermann  and  Janka  (1953)  is  in  error  and  that  the  correct  formula  is  as  given  in: 
the  series  of  structural  formulae.  The  more  descriptive  name  for  this  substance 
\vould,  therefore,  be  N,N'-dimethylbetaine  of  imidazole  acetic  acid. 

RESULTS 
Chromatognwis 

Extracts  of  tentacles  of  whole  animals  of  the  six  selected  species,  representing 
each  of  the  three  classes  of  coelenterates,  were  chromatographed  according  to  the 
procedure  outlined  in  the  section  on  Methods.  Each  extract  was  run  many  times 
along  with  one  or  more  samples  of  known  quaternary  ammonium  bases.  The  rela- 
tive Rf  values  of  these  bases  are  given  at  the  left  of  Figure  2.  All  results  are  for 
the  ethanol-ammonia  solvent  system.  It  may  be  seen  that  tetramethylammonium 
bromide  (I)  gave  an  Rf  value  of  0.75;  N-methylpyridinium  bromide  (II)  an  Rf 
of  0.64;  homarine  HC1  (III)  an  Rf  of  0.54;  trigonelline  (IV)  an  Rf  of  0.32;  and 
y-butyrobetaine  bromide  (V)  an  Rf  of  0.27.  For  each  species,  the  compounds 
found  and  identified  with  reasonable  certainty,  with  the  exception  of  N,N'-dimethyl- 
betaine  of  imidazole  acetic  acid  (VI),  are  represented  by  shaded  areas. 

Tetramine  was  present  in  each  of  the  species  examined,  being  the  only  base 
found  in  Hydra.  The  two  sea  anemones  yielded  smaller  amounts  than  the  other 
species  and  in  Mctndiinu  this  spot  was  most  distinct  when  an  extract  of  tentacles, 
rather  than  of  whole  animal,  was  used.  Extracts  of  the  gorgonian,  Plc.vaura 
flc.ruosa,  contained  relatively  large  amounts  of  tetramine,  as  suggested  by  the 
larger  shaded  area.  It  is  of  interest  to  note  that  separate  extracts  were  made  of 
purple  and  brown  varieties  of  colonies  of  Plc.vaura.  The  chromatograms  of  these 
extracts  were  so  similar  that  they  are  represented  by  the  one  set  of  spots  of  the  four 
bases  that  were  identified. 

In  none  of  the  species  examined  did  we  find  an  indication  of  the  presence  of 
N-methylpyridinium.  Since  the  methods  employed  have  enabled  us  to  identify 
this  substance  in  extracts  of  certain  molluscan  tissues,  we  believe  it  to  be  absent,  or 
present  in  very  small  amounts,  in  the  coelenterates  investigated.  Homarine  is  a 
compound  now  known  to  be  widely  distributed  among  marine  invertebrates  (Gas- 
teiger, Gergen  and  Haake,  1955).  We  found  it  in  all  five  marine  species  of  coelen- 
terates examined.  Although  present  in  relatively  large  amount  in  our  extracts  of 


QUATERNARY  BASES  IN  COELENTERATES  555 

Metridium,  it  was  determined  with  least  certainty  in  the  pink-tipped  sea  anemone, 
Condylactis. 

In  the  ethanol-ammonia  solvent,  the  Rf  values  of  trigonelline  and  y-butyrobetaine 
were  so  similar  that  the  spots  overlapped.  In  the  case  of  Plc.raura  extracts,  where 
a  relatively  large  amount  of  trigonelline  was  present,  two-dimensional  chromato- 
grams  were  run.  This  permitted  a  clear-cut  separation  of  trigonelline  and  y-butyr- 
obetaine. Trigonelline  was  not  found  in  our  extracts  of  Physalia,  although  it  was 
identified  with  reasonable  certainty  in  the  other  four  species.  Extracts  of  the  pink- 
tipped  sea  anemone,  Condylactis,  contained  large  amounts  of  y-butyrobetaine,  while 
extracts  of  Physalia  and  Metridium  appeared  to  lack  this  substance. 


i.o 


Solvent   Front 
.9  _ 


.7  I 


Ill 

Rf     .5   _ 
.4 


.3    _    IV 
.2  _ 


o  o  o  o  o  o  o 

Knowns  Hydra  Physalia  Cyanea  Metridium        Condylactis          Plexauro 


FIGURE  2.  Composite  of  chromatograms  giving  Rf  values  for  five  of  the  compounds  with 
which  this  study  was  concerned.  In  instances  where  identification  was  tentative,  the  spots  are 
shown  with  broken  boundary  lines.  I  =  tetramethylammonium  ;  II  =  N-methylpyridinium  ; 
III  —  homarine  ;  IV  —  trigonelline  ;  V  =  7-butyrobetaine.  Solvent  =  ethanol-ammonia. 

During  the  period  when  most  of  the  work  reported  here  was  in  progress  we  were 
not  aware  of  the  identification  of  zoo-anemonin  as  the  dimethylbetaine  of  imidazole 
acetic  acid.  Now  having  samples  of  the  synthesized  material  we  find  what  we 
believe  to  be  zoo-anemonin  in  Metridium,  Condylactis  and  Plexaura.  Unfortu- 
nately, when  ethanol-ammonia  is  used  as  a  solvent  system,  the  Rf  value  of  zoo- 
anemonin  is  between  0.2  and  0.3.  This  is  so  similar  to  that  for  y-butyrobetaine 
that  some  other  solvent  system  must  be  used  for  their  separation.  We  have  tried 
n-butanol-dioxane-water  (4:1:5)  as  used  by  Ackermann  and  Janka  (1953)  for 
zoo-anemonin.  With  this  they  obtained  an  Rf  of  0.17.  Extracts  of  Metridium  and 


556 


JOHN  H.  WELSH  AND  PEGGY  B.  PROCK 


P  lex  aura  run  with  this  solvent  give  a  relatively  large  spot  appearing  between  Rf  0.1 
and  0.2  and  probably  representing  zoo-anemonin. 

Identification  by  ultraviolet  absorption 

Spots  of  N-methylp)  ridinium,  trigonelline  and  homarine  are  readily  detected  as 
absorbing  areas  when  dried,  untreated  chromatograms  are  examined  with  short- 

i.O 


0.5 


0.0 


240 


260 


280 


300 


320 


mjj 


FIGURE  3.  Ultraviolet  absorption  curves  for  N-methylpyridinium  (II),  homarine  (III), 
trigonelline  (IV),  and  for  eluates  of  spots  from  chromatograms  of  Metridium  tentacle  extracts 
believed  to  represent  homarine  (dashed  line)  and  trigonelline  (dotted  line).  Ordinate  =  arbi- 
trary units  of  absorption. 


QUATERNARY  BASES  IN  COELENTERATES  557 

wave  ultraviolet  light.  This  was  very  helpful  in  the  location  of  regions  on  the 
paper  where  these  compounds  occurred.  Thus,  an  absorbing  area  was  never  found 
on  chromatograms  of  coelenterate  extracts  in  a  region  where  N-methylpyridinium 
should  have  occurred,  if  it  had  been  present.  It  should  be  noted,  however,  that 
N-methylpyridinium  bromide,  when  run  in  ethanol-ammonia,  gives  two  spots,  the 
lower  of  which  is  close  to  homarine.  This  could  give  rise  to  some  confusion  but 
we  believe,  in  this  instance,  that  it  has  not  done  so. 

The  ultraviolet-absorbing  characteristic  of  these  pyridine  derivatives  was  further 
used  in  the  identification  of  homarine  and  trigonelline.  Samples  of  crystalline  N- 
methylpyridinium,  trigonelline  and  homarine  were  run  in  a  Gary  recording 
spectrophotometer.  Tracings  of  their  absorption  curves  are  combined  in  Figure  3. 
Since  we  were  interested  mainly  in  the  wave-length  at  which  maximum  absorption 
occurred,  their  extinction  coefficients  were  not  determined.  N-methylpyridinium 
absorbed  maximally  at  258  m^,  trigonelline  at  264  m/x,  and  homarine  at  272-3  m/x. 

Extracts  of  Metridium  and  Physalia  were  streaked  on  5-inch-wide  strips  of 
paper.  After  running  in  ethanol-ammonia  the  papers  were  dried  and  the  areas 
believed  to  be  occupied  by  trigonelline  or  homarine  were  outlined  under  an  ultra- 
violet light  source.  These  areas  were  then  cut  out  and  eluted  with  distilled  water. 
After  appropriate  dilution  (determined  by  trial)  the  absorption  curves  for  these 
extracts  were  also  obtained.  Figure  3  shows  curves  for  eluates  of  the  areas 
of  Metridium  chromatograms  believed  to  represent  homarine  and  trigonelline. 
Maximum  absorption  of  the  eluate  supposed  to  contain  trigonelline  is  seen  to  cor- 
respond precisely  with  that  of  the  authentic  samples  of  trigonelline  (IV).  The 
absorption  curves  for  both  samples  have  similar  characteristic  shoulders. 

The  absorption  curve  of  a  Metridium  eluate,  believed  to  contain  homarine,  is 
identical  in  shape  with  that  for  synthetic  homarine.  In  a  similar  manner,  homarine 
was  identified  in  extracts  of  tentacles  of  Cyanea  and  Physalia.  The  procedure  was 
not  used  with  the  other  species. 

On  chromatograms  of  tentacle  extracts  of  Metridium  and  Physalia,  run  in 
ethanol-ammonia,  an  ultraviolet-absorbing  area  was  found  with  an  Rf  of  about  0.2. 
Eluates  of  this  area  gave  absorption  curves  with  a  maximum  absorption  at  248  m/A. 
The  substance  responsible  for  this  was  not  identified. 

The  action  of  quaternary  ammonium  bases  on  fiddler  crabs 

Aqueous  extracts  or  homogenates  of  tentacles  of  certain  coelenterates  have  been 
shown  to  influence  the  autotomy  reflex  of  crustaceans  (Welsh,  1956).  Likewise, 
certain  quaternary  ammonium  bases  were  found  to  facilitate  or  to  reduce  the 
tendency  to  autotomize  legs.  In  order  to  determine  whether  or  not  the  bases  under 
investigation  in  the  present  study  would  reproduce  the  actions  of  coelenterate 
extracts,  the  following  experiments  were  performed.  A  given  volume  of  Metridium 
tentacles  was  homogenized  with  an  equal  volume  of  sea  water.  After  centrifuging, 
0.05  ml.  of  the  clear  supernatant  was  injected  into  each  of  five  Uca  pug  Hat  or.  In 
three  minutes,  six  legs  had  spontaneously  autotomized  and  the  crabs  were  showing 
signs  of  severe  paralysis,  from  which  none  recovered.  The  original  extract  was 
diluted  1 : 10  with  sea  water  and  five  crabs  injected  with  0.05  ml.  each.  In  five 
minutes,  four  of  the  crabs  had  dropped  25  legs  (one  crab  failed  to  autotomize  any 
legs).  In  a  few  more  minutes,  they  were  completely  paralyzed  and  none  recovered. 


558  JOHN  H.  WELSH  AND  PEGGY  B.  PROCK 

A  dilution  of  1:100  with  sea  water  produced  12  autotomies  in  15  minutes.  After 
24  hours  two  crabs  were  dead  and  after  48  hours  two  additional  crabs.  Thus,  an 
aqueous  extract  of  Metridium  tentacles  contains  factors  which,  in  considerable 
dilution,  produce  spontaneous  autotomy  of  legs  followed  by  paralysis  and  death, 
in  this  species  of  crustacean. 

An  abundance  of  Hydro  littoralis,  from  mass  cultures,  made  it  convenient  to 
test  the  action  of  a  hydra  extract  on  a  crustacean.  Approximately  2000  hydra, 
unfed  for  one  week,  were  blotted  and  weighed.  The  wet  weight  was  410  mg. 
They  were  homogenized  and  one  nil.  of  distilled  water  added.  After  centrifuging, 
0.02  ml.  of  the  clear  supernatant  was  injected  into  Uca  pugilator.  The  following 
is  a  typical  record : 

1 :30  PM     Injected  0.02  ml.  at  base  of  second  left  walking  leg  of  a  specimen 

of  Uca  weighing  3.16  gm. 

1:31   PM     First  and  second  left  legs  ''paralyzed" 
1 :33  PM     Crab  cannot  right  itself  when  turned  on  back 
1 :34  PM     Only  slight  limb  movements 

1  :40  PM     All    spontaneous    movements    have    ceased    and    no    response    to 

stimulation 

2  :45  PM     No  indication  of  recovery ;  appears  dead,  but  on  removing  carapace 

the  heart  is  found  beating. 

This  extract  when  diluted  1:10  with  sea  water  was  almost  as  effective  in  causing 
paralysis  of  Uca  as  the  undiluted  extract.  However,  when  heated  at  100°  C.  for 
five  minutes  an  injection  of  0.02  ml.  was  entirely  without  effect  on  Uca. 

Next,  a  series  of  tests  was  made  to  determine  whether  or  not  any  one  of  the 
six  bases  used  in  this  study,  and  available  in  crystalline  form,  would  mimic  in  any 
respect  the  extract  of  Metridium  tentacles.  Each  base  was  made  up  as  a  one 
per  cent  solution  in  sea  water  and  0.05  ml.  injected  into  each  of  five  Uca  pugilator. 
Of  the  six  bases  only  tetramethylammonium  bromide  appeared  to  have  significant 
action.  This  substance  produced  a  type  of  paralysis  from  which  only  three  of  five 
crabs  recovered. 

It  seemed  possible  that  a  mixture  of  the  bases  in  question  might  have  an  action 
that  individual  members  lacked.  Therefore,  they  were  combined  and  injected. 
The  action  on  the  crabs  was  unspectacular  and  did  not  differ  from  that  produced 
by  an  equivalent  amount  of  a  tetramethylammonium  salt.  From  these  injection 
experiments  it  would  appear  that  the  toxic  action  of  an  aqueous  extract  of  Metridium 
tentacles,  or  whole  hydra,  on  the  fiddler  crab,  Uca  pugilator,  could  not  be  due  solely 
to  the  presence  of  the  quaternary  ammonium  bases  with  which  this  study  was 
chiefly  concerned. 

The  presence  of  a  tetramethylammonium  compound  in  all  species  of  coelenterates 
that  were  examined ;  its  occurrence  as  the  only  quaternary  base  identifiable  in 
Hydra  littoralis  (by  the  methods  used)  and  its  known  effects  on  crustaceans 
(Welsh,  1956)  would  all  appear  to  support  the  earlier  suggestion  of  Ackermann, 
Holtz  and  Reinwein  (1923)  that  tetramethylammonium  hydroxide  (tetramine) 
might  be  the  paralyzing  factor  in  nematocyst  toxin.  Two  observations  made  in 
the  present  study  make  this  suggestion  unlikely.  They  are  (1)  that  a  dose  of 
hydra  extract  calculated  to  contain  the  active  material  from  0.14  mg.  of  dry 


QUATERNARY  BASES  IN  COELENTERATES  559 

hydra  is  fatal  to  a  specimen  of  Uca,  while  0.5  mg.  of  crystalline  tetramethylam- 
monium  bromide  is  not,  and  (2)  that  heating  for  5  minutes  at  100°  C.  destroys  or 
greatly  lowers  the  activity  of  an  aqueous  hydra  extract.  This  should  have  little, 
if  any,  effect  on  a  tetramethylammonium  salt. 

DISCUSSION 

Studies  made  on  extracts  of  whole  coelenterates,  their  tentacles,  or  their  acontia, 
will  not  conclusively  identify  the  chemical  constituents  of  nematocyst  contents  and, 
therefore,  coelenterate  or  nematocyst  toxins,  as  was  recently  pointed  out  by  Phillips 
and  Abbott  (1957).  Such  studies  may,  however,  give  valuable  clues  to  the  nature 
of  the  toxic  substance,  and  toxic  components  of  extracts  of  tissues,  rich  in 
nematocysts,  may  then  be  sought  in  extracts  of  the  isolated  and  cleaned  stinging 
organelles.  Methods  for  isolating  undischarged  nematocysts  have  been  developed 
(Phillips,  1956;  Phillips  and  Abbott,  1957)  and  are  being  adopted  by  others  (e.g. 
Dodge  and  Lane,  1958;  Lane  and  Dodge,  1958). 

The  work  reported  here  was  an  attempt  to  learn  more  about  the  distribution  of 
tetramine  and  other  quaternary  ammonium  bases  in  representative  coelenterates. 
While  several  bases  were  found  in  marine  coelenterates,  only  tetramine  was  present 
in  the  fresh  water  hydra  in  sufficient  amounts  to  be  identified  with  the  methods 
employed.  This  finding,  and  the  observation  that  tetramine  was  the  only  base 
employed  in  this  study  that  had  significant  paralyzing  action  on  Uca,  provide 
further  evidence  that  this  substance  may  be  a  constituent  of  nematocyst  toxin. 
Almost  certainly  it  is  not  solely  responsible  for  the  paralyzing  effects  of  coelenterate 
stings.  One  or  more  proteins  could  be  additional  components.  This  is  suggested 
by  the  decreased  activity  of  hydra  extracts  that  have  been  heated  (see  above)  and 
by  the  loss  of  toxicitv  by  isolated  nematocysts  that  have  been  treated  with  ether, 
alcohol  or  drying  (Phillips  and  Abbott,  1957).  Against  the  view  that  proteins 
may  be  important  in  nematocyst  toxin  is  an  earlier  observation  that  deproteinization 
with  trichloracetic  acid  did  not  significantly  alter  the  toxicity  of  extracts  of  acontia 
of  Adamsia  palliata,  when  Carchuts  and  Astacns  were  used  as  test  animals 
(Cantacuzene  and  Damboviceanu,  1934a).  Further  observations  on  the  trichlora- 
cetic acid  extract  of  Adamsia  acontia  suggest  that  the  crustacean-paralyzing  factor 
is  a  relatively  small  and  quite  stable  molecule  (Cantacuzene  and  Damboviceanu, 
1934b). 

It  is  not  unreasonable  to  theorize  that  an  association  of  tetramine  with  a  protein 
might  produce  a  substance  more  toxic  than  tetramine  alone.  This  is  based  partly 
on  the  evidence  that  two  alkylated  tetracovalent  nitrogens,  properly  spaced  in  a 
molecule,  can  produce  highly  active  junctional  blocking  agents  such  as  curare  and 
the  many  synthetic,  curariform,  bis-quaternary  substances.  Protein  denaturation 
by  heat,  or  otherwise,  might  alter  the  spacing  of  the  tetramines  on  the  protein  or 
set  them  free  and  therein-  reduce,  but  not  abolish,  the  paralyzing  action  of  an 
extract.  In  support  of  such  a  suggestion  is  the  observation  that  nematocysts  have 
a  high  affinity  for  methylene  blue,  a  basic  dye  with  two  methylated  nitrogens  which, 
through  resonance,  may  become  tetracovalent.  This  implies  that  there  are 
molecules  within  the  nematocyst  (presumably  protein)  that  bind  methylene  blue 
and  that  might  bind  other  quaternary  ammonium  bases. 


560  JOHN  H.  WELSH  AND  PEGGY  B.   PROCK 

Although  certain  pyridinium  derivatives  have  a  weak  curariform  action  in 
vertebrates  (Craig,  1948)  those  studied  here,  as  well  as  the  other  betaines,  were 
characterized  by  their  lack  of  paralyzing  action  on  Uca.  Since  homarine,  one  of 
the  compounds  in  question,  occurs  widely  in  marine  invertebrates  but  not  in  those 
from  fresh  water  it  has  been  suggested  that  it  may  serve  an  osmoregulatory  function 
(Gasteiger  et  al.,  1955).  This  may  be  the  role  of  some  of  the  other  nitrogenous 
bases  of  marine  invertebrates. 

When  this  study  was  first  begun  we  tentatively  identified  one  of  the  bases  of 
Metridium  and  Physalia  extracts  as  urocanylcholine  (Welsh,  1956).  This  identi- 
fication was  based  partly  on  the  ultraviolet  absorption  of  eluates  of  chromatograms 
and  their  comparison  with  known  urocanylcholine.  Although  the  curves  and  peaks 
of  absorption  correspond  rather  precisely  at  a  certain  pH  value,  we  later  learned  that 
the  absorption  maximum  of  the  eluted  material  did  not  change  with  pH  as  does 
that  of  urocanylcholine  (Erspamer  and  Benati,  1953).  Later  we  learned  that  the 
suspected  urocanylcholine  was  actually  homarine. 

Although  we  do  not  yet  know  what  is  responsible  for  the  paralysis  produced 
by  the  nematocysts  of  coelenterates,  the  renewed  interest  in  this  question  should 
eventually  provide  an  answer. 

SUMMARY 

This  was  a  study  of  the  identification  and  distribution  of  quaternary  ammonium 
bases  in  representative  coelenterates.  The  purpose  was  to  determine  if  bases  were 
present  with  paralyzing  actions  greater  than  that  of  tetramethylammonium 
(tetramine)  which  was  found  to  occur  in  all  species  examined.  Four  other  bases 
(homarine,  trigonelline,  y-butyrobetaine  and  the  dimethylbetaine  of  imidazole  acetic 
acid)  were  found  in  some  species.  The  bases  other  than  tetramine  were  found  to 
have  no  observable  paralyzing  action  on  Uca  pugilator,  in  the  doses  employed. 
However,  it  is  not  possible  to  account  for  the  powerful  paralyzing  actions  of  cold, 
aqueous  extracts  of  Metridium  tentacles  or  whole  hydra  on  the  basis  of  their 
tetramine  content.  It  is  suggested  that  this  base,  in  conjunction  with  a  specific 
protein,  might  be  responsible  for  the  paralyzing  action  of  nematocysts. 

LITERATURE  CITED 

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Biol,  86:  199-202. 
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mittelung  des  Tetramins,  eines  Giftes  aus  Aktinia  equina.    Zeitschr.  f.  Biol.,  79  :  113-120. 
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equina.     Zeitschr.  f.  Biol.,  80:   131-136. 
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QUATERNARY  BASES  IN  COELENTERATES  561 

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136-138. 
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INDEX 


\  TP  as  energy  source  for  sperm  motility,  326. 

Abnormalities  in  chick  embryos  treated  with 
normal  sera  and  homologous  antisera,  239. 
Absorption  spectrum  of  Urechis  eggs,  136. 
Abstracts  of  papers  presented  at  the   Marine 

Biological  Laboratory,  319 
Accessory  electric  organs  of  Narcine,  126. 
Acclimation  of  Amphiprion  to  symbiosis  with 

Stoichactis,  397. 

Acetylcholine,  effects  of  on  mollusc  tissues,  471. 
Acid,     hydrochloric,     gustatory     response     of 

cockroach  to,  490. 

Acid  accumulation  in  Desmarestia,  101. 
Action  of  insulin  on  cells  and  protoplasm,  < 
Action  potentials,  nerve,  of  cockroach,  490. 
Activity  of  thyroid  in  salamanders,  411. 
Adrenaline,  effects  of  on  mollusc  tissues,  471. 
Adult  mosquitoes,  gamma  radiation  of,  536. 
Aedes  adults  and  eggs,  gamma  irradiation  of, 

536. 

Age  in  relation  to  form-stability  of  ciliates,  3 
Aggregates,  subnuclear,  in  protozoan  cells,  269. 
Alga,  marine,  sulfuric  acid  in,  101. 
Alga-salamander  embryo  symbiosis,  483. 
ALLEN,  R.  D.     Polarization  optical  studies  on 

amebae,  327. 

Alloxan  injection  of  toadfish,  357. 
AMATNIEK,    E.     See    R.     MATHEWSON,     126; 

M.  V.  L.  BENNETT,  331. 
Ameba,  effects  of  heparin  on,  459. 
Amino  acid   uptake   of   marine   invertebrates, 

341. 

Amino  acids  in  Physalia  nematocysts,  219. 
Ammonium  bases,  quaternary,  in  coelenterates, 

551. 
Amphibia,  effects  of  goitrogens  on  thyroids  ot, 

411. 

Amphibian  embryo,  localization  of  antigens  in, 

201. 
Amphibian  embryos,   symbiosis  of  with  alga, 

483. 
Amphiprion,  symbiosis  of  with  sea  anemone, 

397. 

Amylose  and  amylopectin  fractions,  320. 
Anatomy,  salt  and  water,  of  crabs,  180. 
Anatomy  of  gull  nasal  gland,  162. 
Anatomy  of  Narcine  electric  organs,  126. 
Anatomy  of  toadfish  swimbladder,  172. 
ANDERSON,  J.  M.,  AND  J.  C.  JOHANN.     Some 

aspects    of    reproductive    biology    in    the 


fresh-water     triclad     turbellarian,     Cura  , 

375. 

Anemone,  sea,  symbiosis  of  with  fish,  397. 

Annual  Report  of  the  Marine  Biological 
Laboratory,  1. 

Anolis,  male,  responses  of  to  changes  in  day- 
length,  427. 

Antennal  gland,  role  of  in  osmotic  regulation 
in  crabs,  180. 

Antherea,  use  of  for  assay  of  juvenile  hormone 
activity,  530. 

Anthopleura,  association  of  with  Amphiprion, 

397. 

Anthozoa,  nematocyst  toxins  of,  551. 
Antibacterial  activity  of  Limulus  blood,  341. 
Antibacterial  activity  of  Phascolosoma  blood, 

343. 

Antifertilizin  of  Mellita,  74. 
Antigens,  localization  of  in  frog  embryo,  2 
Antisera,     homologous,     effect     of     on     chirk 

embryo,  239. 

Antisera  to  frog  brain,  lens  and  cattle  lens,  2 
Aqueous  humor  of  dogfish,  335. 
Arachnid,  neuromuscular  transmission  in,  2 
Arbacia  eggs,  effects  of  insulin  on,  459. 
ARMSTRONG,  P.  B.     Retinal  development  and 
phototactic  responses  in  developing  Ameiu- 
rus  embryos,  332. 
Arthropod  chemoreceptors,  114. 
Arthropod  neuromuscular  transmission,  209. 
Ascidian,  regeneration  of  buds  in,  147. 
ASHTON,  F.  T.     See  L.  V.  Heilbrunn,  459. 
Assay  of  juvenile  hormone  activity,  530. 
Asteroid  coelomic  corpuscles,  53. 
Astropecten  coelomic  corpuscles,  53. 
AUCLAIR,  W.     Methyl  green  "vital  staining 

in  Arbacia  eggs,  342. 

AUCLAIR,    W.,    AND    D.    MARSLAND.     Form- 
stability  of  ciliates  in  relation  to  pressure 
and  temperature,  384. 
AUCLAIR,    W.     See    D.    MARSLAND,    3^6;    J. 

PADAWER,  359. 
Axoplasm,  lobster,  physical  properties  ot,  o 

BACTERIA,  photosynthetic  sulfur,  enrichment 
'   studies  on,  343. 

BANG,   F.   B.,  AND  S.   M.   KRASSNER.     Anti- 
bacterial activity  of  Phascolosoma  bio 
343. 

BANG,  F.  B.     See  M.  V.  SHIRODKAR,  341. 

562 


INDEX 


565 


BARNWELL,  F.  H.     See  F.  A.  BROWN,  JR.,  344. 

Bases,  quaternary  ammonium,  in  coelenterates, 
551. 

Bat,  echolocation  in,  107. 

BATTLEY,  E.  H.  Enrichment  studies  on  the 
photosynthetic  sulfur  bacteria,  343. 

BEDFORD,  B.     See  R.  F.  DOOLITTLE,  335. 

Behavioral  responses  of  cockroach,  490. 

BENNETT,  M.  V.  L.,  R.  D.  KEYNES  AND  H. 
GRUNDFEST.  Electric  organ  electrogenesis 
in  Malapterurus,  330. 

BENNETT,  M.  V.  L.,  M.  WURZEL,  E.  AMATNIEK 
AND  H.  GRUNDFEST.  Electroplaque  ac- 
tivity in  marine  electric  fishes,  331. 

BENSAM,  A.     See  W.  S.  VINCENT,  342. 

BENSAM,  B.     See  W.  S.  VINCENT,  342. 

Bermuda  crab,  osmotic  regulation  in,  180. 

BERNATOWICZ,  A.  J.  A  Bermudian  marine 
Vaucheria  at  Cape  Cod,  344. 

BERNATOWICZ,  A.  J.  Ecological  isolation  and 
independent  speciation  of  the  alternate 
generations  of  plants,  323. 

BETTELHEIM,  F.  A.  The  nature  of  chroma- 
tographic  amylose  and  amylopectin  frac- 
tions, 320. 

BETTELHEIM,  F.  A.,  AND  D.  E.  PHILPOTT. 
Electron  microscopic  investigation  of  the 
structure  of  hyaluronic  acid  gels  and 
hyaluronic  acid-protein  complexes,  333. 

Biological  rhythms,  440. 

Biological  rhythms,  exogenous  reference-clock 
for,  81. 

Biology  of  reproduction  in  Cura,  375. 

Bioluminescence  of  Gonyaulax,  440. 

Bioluminescence  of  Mnemiopsis,  336. 

Bird,  marine,  salt  gland  of,  162. 

Birds,  marine,  parasites  of,  276. 

BISHOP,  D.  W.  Sperm  cell  models  and  the 
question  of  ATP-induced  rhythmic  mo- 
tility,  326. 

BLACK,  R.  E.,  S.  EPSTEIN  AND  A.  TYLER.  The 
oxidation  of  carbon  monoxide  by  fertilized 
eggs  of  Urechis,  shown  by  use  of  a  C-13 
label,  153. 

Blepharisma,  form-stability  of,  384. 

Blood,  role  of  in  transportation  of  strontium 
and  yttrium  in  teleosts,  64. 

Blood  pigment  of  hagfish,  227. 

Blood  specific  gravity  of  crabs,  180. 

Bloom,  diatom,  dynamics  of,  257. 

Bohr  effect  of  hagfish  hemoglobin,  227. 

BOOLOOTIAN,  R.  A. ,  AND  A.  C.  GIESE  .  Coelomic 
corpuscles  of  echinoderms,  53. 

BORGESE,  T.  A.     See  J.  W.  GREEN,  352. 

BOROUGHS,  H.,  AND  D.  F.  REID.  The  role  of 
the  blood  in  the  transportation  of  stron- 
tium-90  and  yttrium-90  in  teleost  fish,  64. 

Botryllus,  bud  regeneration  in,  147. 

BOVELL,  C.  R.     See  R.  W.  EPPLEY,  101. 


Brachyuran  crabs,  osmotic  regulation  in,   180. 

BRAVERMAN,  M.  H.  An  inhibitory  extract 
of  chick  tissues,  344. 

BRAVERMAN,  M.  Neural  and  mesodermal 
hierarchies  in  chick  development,  319. 

Breeding  season  in  relation  to  response  of  male 
lizard  reproductive  system  to  changes  in 
day-length,  427. 

BRETT,  W.  J.     See  F.  A.  BROWN,  JR.,  345. 

BROCK,  L.  G.     See  R.  M.  ECCLES,  330. 

BROOKBANK,  J.  W.  Dispersal  of  the  gelatinous 
coat  material  of  Mellita  eggs  by  homologous 
sperm  and  sperm  extracts,  74. 

BROWN,  F.  A.,  JR.  An  exogenous  reference- 
clock  for  persistent,  temperature-inde- 
pendent, labile,  biological  rhythms,  81. 

BROWN,  F.  A.,  JR.,  AND  F.  H.  BARNWELL. 
The  explanation  of  the  two-day  physio- 
logical anticipation  of  barometric  pressure 
changes,  344. 

BROWN,  F.  A.,  JR.,  W.  J.  BRETT  AND  H.  M. 
WEBB.  The  rhythmic  nature  of  metabo- 
lism in  Ilyanassa  in  constant  conditions, 
345. 

BROWN,  F.  A.,  JR.,  H.  M.  WEBB  AND  W.  J. 
BRETT.  Correlation  between  oxygen  con- 
sumption in  Fucus  in  constant  conditions, 
including  pressure,  and  specific  baro- 
metric pressure  parameters,  345. 

BROWN,  F.  A.,  JR.     See  H.  M.  WEBB,  303. 

BROWNELL,  K.  A.,  AND  F.  A.  HARTMAN.  The 
interrenal  of  the  sting  ray,  345. 

BRYANT,  S.  H.  Aspects  of  synaptic  trans- 
mission in  the  squid  stellate  ganglion,  331. 

BUCK,  J.     See].  CASE,  346. 

Bud  regeneration  in  Botryllus,  147. 

BURBANCK,  W.  D.,  AND  M.  P.  BURBANCK. 

Chromosomes    of     the    estuarine    isopod 

Cyathura,  346. 
Busycon   tissues,  effects  of   neurohumors  and 

drugs  on,  471. 

Butterflies,  chemoreceptors  of,  1 14. 
Butyrobetaine    in    coelenterate    nematocysts, 

551. 

QAGLE,  J.     See  A.  K.  PARPART,  340. 

Calcium,  possible  role  of  in  action  of  insulin, 

459. 

Cambarus,  chemoreceptors  of,  114. 
CARANASOS,  G.     See  J.  E.  SCHUH,  363. 
Carbamylcholine,  effects  of  on  mollusc  tissues, 

471. 
Carbon-13,  use  of  in  demonstrating  oxidation 

of  CO  by  Urechis  eggs,  153. 
Carbon    dioxide   formation   by   Arbacia   eggs, 

effect    of    substituted    phenols    on,    350. 
Carbon  monoxide,  effect  of  on  respiration  of 

Urechis  eggs,  136. 


564 


INDEX 


Carbon  production  by  diatoms,  257. 
CARDELL,   R.   R.,  JR.,  AND   D.   E.   PHILPOTT. 
Observations    on    the    structure    of    the 
cercaria  of  Himasthla,  346. 
Carotenoid  pigments  in  phytoplankton,  257. 
Carrot,  biological  rhythms  of,  81. 
CASCARANO,  J.     See  E.  K-VY- ROSENBERG,  354. 
CASE,  J.,  AND  J.  BUCK.     Regulation  of  flashing 

in  the  firefly,  346. 

CASPARI,  S.  B.     See  P.  W.  WHITING,  321. 
Cells,  action  of  insulin  on,  459. 
Centrifuging  of  pressure-treated  ciliates,   384. 
Cercaria  of  Himasthla,  346. 
CHAET,  A.  B.,  AND  S.  I.  COHEN.     A  source  of 

the  toxic  factor (s)  in  scalded  starfish,  347. 
Chaetopterus  eggs,  effects  of  insulin  on,  459. 
Chaos,  effects  of  heparin  on,  459. 
Chemoreception  in  cockroach,  490. 
Chemoreceptors  of  arthropods,  114. 
CHESBOROUGH,  C.     See  R.  F.  DOOLITTLE,  335. 
Chick    embryo,    effect    of    normal    sera    and 

homologous  antisera  on,  239. 
CHILD,  F.   M.     The  isolation  and  analysis  of 

cilia,  327. 

Chloride  concentration  in  crab  blood,  180. 
Chloride  excretion,  role  of  salt  gland  in,   162. 
Chlorophyll  a  in  phytoplankton,  257. 
Choline  esters,  mode  of  action  of,  322. 
Chromatin  extrusion  in  Tetrahymena,  269. 
Chromatography   of  coelenterate   nematocyst 

toxins,  551. 
Chromatophores,  squid,  action  of  chemicals  on, 

340. 

Chromatophorotropic  principle  from  Uca,  367. 
Chromatophorotropins  of   Palaemonetes,   351, 

352. 

Chromosomes  of  Cyathura,  346. 
Cilia,  isolation  and  analysis  of,  327. 
Ciliary  motion  in  oyster  gills,  320. 
Ciliates,  form-stability  of,  384. 
Ciliates,  subnuclear  aggregates  in,  269. 
CLAFF,  C.  L.,  F.  N.  SUDAK  AND  V.  MOLONEY. 

Survival  of  Uca  in  sand,  water  and  vegeta- 
tion contaminated  with  2,  4-D,  347. 
CLAFF,  C.  L.     See  F.  N.  SUDAK,  368. 
Clam,  parasites  of,  276. 
Clam    heart,    effect    of    Physalia    nematocyst 

toxin  on,  219. 
Cleavage,  behavior  of  metachromatic  granules 

during,  in  Spisula,  325. 
Cleavage  of  Arbacia,  effects  of  pentahalophenols 

on,  354. 
Cleavage  furrows  in  Arbacia  egg,  experimental 

induction  of,  356. 

Cleavage  inhibition  by  urethan,  363. 
"Clock"  mechanism  in  Gonyaulax,  440. 
•"Clock"    mechanism   as   related   to   biological 

rhythms,  81. 


CLOWES,   G.   H.   A.     See  R.   K.   CRANE,   350; 

A.  K.  KELTCH,  354. 

CO  oxidation  by  fertilized   Urechis  eggs,   153. 
Cobalt-60  irradiation  of  Aedes,  536. 
Cockroach,  corpus  allatum  cells  of,  508,  521. 
Cockroach  gustatory  responses,  490. 
Coelenterate,  symbiosis  of  with  fish,  397. 
Coelenterate  nematocysts,  219. 
Coelenterates,    quaternary    ammonium    bases 

in,  551. 

Coelomic  corpuscles  of  echinoderms,  53. 
COHEN,  J.     See  M.  ROCKSTEIN,  361. 
COHEN,  S.  I.     See  A.  B.  CHAET,  347. 
Coleoptera,  juvenile  hormone  substance  from, 

530. 
COLLIER,  J.   R.     A  study  of  ribonucleic  acid 

during  the  development  of  Ilyanassa,  348. 
Colloidal    changes    in    protoplasm,    effects    of 

insulin  on,  459. 

Colonial  ascidian,  regeneration  in,  147. 
COLWIN,  A.   L.,   AND  L.   H.   COLWIN.     Some 

characterization    of    the    egg    membrane 

lytic  agent   derived   from   sperm  extracts 

of  Hydroides,  348. 
COLWIN,  A.  L.,  AND  L.  H.  COLWIN.     Effects 

of  sperm  extract  and  other  agents  on  the 

egg    membranes    in    relation     to    sperm 

entry  in  Hydroides,  324. 
COLWIN,    L.    H.,   AND   A.    L.    COLWIN.     The 

effects  of  certain  enzymes  and  other  sub- 
stances on  the  egg  membranes  of  Hydroides, 

349. 
Commensalism  of  polychaetes  and  crustaceans, 

method  for  study  of,  323. 
Comparative    study    of    cockroach    gustatory 

responses,  490. 

Comparison  of  effects   of  goitrogens  on   sala- 
manders, 411. 
Complement,  role  of  in  toxicity  of  rabbit  serum 

to  chick  embryo,  239. 
Conduction     in     dogfish     and     Phascolosoma 

muscles,  366. 
Conduction  in  Phascolosoma  fusiform  muscle, 

360. 

Condylactis,  nematocyst  toxins  of,  551. 
Constancy  of  salt  levels  in  crabs,  180. 
CONWAY,  D.  M.,  AND  A.  I.  CsAPO.     Potassium 

contracture  in  a  variety  of  conditions,  333. 
Copepod  from  gills  of  conger  eel,  370. 
Corpus  allatum  of  Leucophaea,  508,  521. 
Corpuscles,  coelomic,  of  echinoderms,  53. 
Correlation  of  anatomical  and  electrophysio- 

logical     properties     of     Narcine     electric 

organs,  126. 
COSTELLO,    D.    P.     Membrane   removal   from 

the  egg  of  the  annelid,  Hydroides,  349. 
COUTINHO,   E.   M.,  AND  A.   I.   CSAPO.     Cal- 

cium,  oxytocin  and  the  regulation  of  the 

myometrium,  334. 


INDEX 


565 


'Crabs,  salt  and  water  anatomy  of,  180. 
CRAIG,  L.  C.     See  S.  P.  MARFEY,  339. 
CRANE,  R.  K.,  A.  K.  KELTCH,  C.  P.  WALTERS 

AND  G.    H.   A.   CLOWES.     The  action  of 

substituted  phenols  on  the  conversion  of 

glucose- 1-CU  and  glucose-6-C14  to  carbon 

dioxide  by  the  eggs  of  Arbacia,  350. 
CRANE,  R.  K.     See  S.  M.  KRANE,  355. 
Crayfish,  chemoreceptors  of,  114. 
Crinoid  coelomic  corpuscles,  53. 
CROWELL,  S.     Tail  regeneration  in  lengthened 

and  shortened  earthworms,  321. 
Crustacea,  juvenile  hormone  substances  from, 

530. 

Crustacea,  osmotic  regulation  in,  180. 
Crustacean,    effects    of    Physalia    nematocyst 

toxin  on,  219,  551. 

Crustacean,    patterns    in    respiration    of,    303. 
CSAPO,  A.  I.     See  D.  M.  CON  WAY,  333;  E.  M. 

COUTINHO,  334;  B.  A.  CURTIS,  334;   M. 

GOTO,  335;  T.  SAKAI,  341. 
Culture  methods  for  ciliates,  384. 
Culture  methods  for  Cura,  375. 
Cura,  reproduction  in,  375. 
CURTIS,  B.  A.,  AND  A.  I.  CSAPO.     Iodide  con- 

tracture  in  potassium-treated  muscle,  334. 
Curtisia  (Cura),  reproduction  in,  375. 
Cyanea,  nematocyst  toxins  of,  551. 
Cyanide,   effect   of   on   respiration   of   Urechis 

eggs,  136. 

Cycles  in  potatoes  and  carrots,  81. 
Cycles  of  luminescence  in  Gonyaulax,  440. 
Cyclic  fluctuations  in  histology  of  male  lizard 

reproductive  system,  427. 
Cyclostome,  hemoglobin  of,  227. 
Cytochrome  system  in  Urechis,  136. 
Cytology  of  Leucophaea  corpus  allatuni,  508, 

521. 

Cytology  of  Tetrahymena,  269. 
Cytolysis  in  pressure-treated  ciliates,  384. 
Cytoplasm  of  sand  dollar  eggs,  329. 
Cytoplasmic    content    of    Leucophaea    corpus 

allatum  cells,  508,  521. 

2,4-D,  effects  of  on  Uca,  347. 

2,4-D,  physiological  properties  of,  368. 

DNA  in  Tetrahymena,  269. 

Daily  cycles  in  potatoes  and  carrots,  81. 

Damselfish-anemone  symbiosis,  397. 

Dark,  effect  of  on  luminescence  of  Gonyaulax, 

440. 
Darkness,    effect    of    on    reproductive    system 

of  male  lizards,  427. 
Daucus,  biological  rhythms  of,  81. 
DAVENPORT,    D.     A  technique  for  the  study 

of    the    effects    of    "host-factor"    on    the 

behavior  of  commensal   polychaetes  and 

Crustacea,  323. 


DAVENPORT,  D.,  AND  K.  S.  NORRIS.     Observa- 
tions on  the  symbiosis  of  the  sea  anemone 

Stoichactis     and     the     pomacentrid     fish 

Amphiprion,  397. 

Day-length,  effect  of  on  male  lizards,  427. 
Day-length,  relation  of  to  cycles  in  potatoes 

and  carrots,  81. 
Decapoda,  juvenile  hormone   substance  from, 

530. 

Decompression  effects  on  ciliates,  384. 
Dendraster  coelomic  corpuscles,  53. 
DENT,  J.  N.,  AND  W.  G.  LYNN.     A  comparison 

of    the   effects    of   goitrogens   on    thyroid 

activity    in  Triturus  and   Desmognathus, 

411. 

Desmarestia,  sulfuric  acid  accumulation  in,  101. 
Desmognathus,  effects  of  goitrogens  on  thyroid 

of,  411. 

Desoxyribonucleic  acid  in  Tetrahymena,  269. 
DESSAUER,  H.  C.     See  W.  Fox,  427. 
Development     of     alga-infected     Ambystoma 

embryos,  483. 
Development  of  Arbacia,  effect  of  triphenyl- 

ethanol  derivative  on,  364. 
Development  of  chick,  effect   of  normal  sera 

and  homologous  antisera  on,  239. 
Development  of  frog  embryos,   in  relation  to 

localization  of  antigens,  201. 
Development  of  reproductive  system  in  Cura, 

375. 
Development  of  retina  in  Ameiurus  embryos, 

332. 
Development  of  Urechis  eggs,  as  affected  by 

cyanide  and  carbon  monoxide,   136,   153. 
Diatom  bloom,  dynamics  of,  257. 
Dictyoneurum,  analysis  of  cytoplasm  of,   101. 
Diemyctylus,  effects  of  goitrogens  on  thyroid 

of,  411. 

Differentiation  of  Botryllus  buds,  147. 
Differentiation  in  frog  embryo,  in  relation  to 

antigen  localization,  201. 
Digenetic  trematodes,  276. 
DINGLE,  A.  D.     See  P.  F.  NACE,  357. 
Dinoflagellate,    persistent    diurnal    rhythm    of 

luminescence  in,  440. 
Diphosphopyridine     nucleotides     of     Arbacia 

eggs,  355. 

Discharge  of  nematocysts  by  Stoichactis,  397. 
Dispersal  of  Mellita  egg  jelly-coat  by  sperm,  74. 
Diurnal  rhythm,  melanophore,  of  Uca,  effect 

of  population  size  on,  368. 
Diurnal  rhythm  of  luminescence  in  Gonyaulax, 

440. 

Diurnal  rhythms  in  respiration  of  Uca,  303. 
Division  of  Tetrahymena,  269. 
DODGE,  E.     See  C.  E.  LANE,  219. 
DOOLITTLE,    R.    F.,    B.    BEDFORD,    C.    CHES- 

BOROUGH,  C.  THOMAS  AND  W.  STONE,  JR. 

Some   aspects   of   the   chemical   composi- 


566 


INDEX 


tion   of   the   aqueous   humor  and   plasma 

of  the  smooth  dogfish,  335. 
Drugs,  effects  of  on  mollusc  tissues,  471. 
DUBNAU,  D.     Substrate  induction  of  adenosine 

deaminase   activity    in   Arbacia    embryos, 

350. 
Dynamics  of  a  diatom  bloc-m,  257. 

]}CCLES,   R.   M.,  AND  L.   G.   BROCK.     The 

membrane     potentials     during     rest     and 

activity  of  the  electroplate  of  Raia,  330. 
Echinochrome  granules  of  Mellita  egg,  74. 
Echinoderm  coelomic  corpuscles,  53. 
Echinoderm  egg  gelatinous  coat  dispersal,  74. 
Echinoid  coelomic  corpuscles,  53. 
Echolocation  in  the  fruit  bat,  107. 
Ecological    isolation   and  speciation  of  plants, 

323. 

Ecological  study  of  phytoplankton,  257. 
Ecology  of  hagfish,   in   relation   to   properties 

of  its  hemoglobin,  227. 
Effects  of  gamma  radiation  on  Aedes  adults 

and  eggs,  536. 

Effects  of  goitrogens  on  salamanders,  411. 
Effects  of  neurohumors  and  drugs  on  mollusc 

tissues,  471. 
Effects  of  normal  sera  and  homologous  antisera 

on  chick  embryos,  239. 
Egg-deposition  by  Cura,  375. 
Egg  development  in  starved  Leucophaea,  521. 
Egg  gelatinous  coat  material,  dispersal  of,  74. 
Eggs,   Ambystoma,   effects  of   symbiotic   alga 

on,  483. 

Eggs,  effects  of  insulin  on,  459. 
Eggs,  mosquito,  gamma  irradiation  of,  536. 
Eggs,  Urechis,  oxidation  of  CO  by,  153. 
Eggs,  Urechis,  oxidative  metabolism  of,  136. 
Egregia,  analysis  of  cytoplasm  of,  101. 
Electric  organs  of  Narcine,  126. 
Electrical    activity    of    Limulus    nerve-muscle 

preparations,  209. 
Electrogenesis  of  electric  organ  of  Malapteru- 

rus,  330. 

Electrolyte  relations  in  crabs,  180. 
Electron     micrographs     of     Narcine     electric 

organs,  126. 
Electron    microscopes,    method   for   correcting 

astigmatism  in,  360. 
Electrophysiological     properties     of     Narcine 

electric  organs,  126. 

Electrophysiological  studies  of  arthropods,  114. 
Electrophysiological  studies  on  cockroach,  490. 
Electrophysiology  of  lobster  muscle  fibers,  332. 
Electrophysiology  of  Raia,  330. 
Electrophysiology  of  Romalea,  329,  356. 
Electroplaque  activity  of  marine  fishes,  331. 
Embryo,    chick,    effect    of    normal    sera    and 

homologous  antisera  on,  239. 
Embryo,  frog,  localization  of  antigens  in,  201. 


Embryology  of  Urechis  as  affected  by  cyanide 
and  carbon  monoxide,  136,  153. 

Embryos  of  Ambystoma,  symbiosis  of  with 
Oophila,  483. 

Embryos  of  Arbacia,  enzyme  activity  in,  350. 

Embryos  of  Narcine,  electric  organs  of,  126. 

Embryos  of  Spisula,  dehydrogenase  activity  in,. 
354. 

Endocrine  activity  of  substances  from  inver- 
tebrates, 530. 

Endocrinology  of  salamanders,  411. 

Endogenous  rhythm  of  luminescence  in 
Gonyaulax,  440. 

Endothelial  elements  of  Rana,  phagocytic 
behavior  of,  357. 

Environment,  importance  of  in  biological 
rhythms,  81. 

EPPLEY,  R.  W.,  AND  C.  R.  BOVELL.  Sulfuric 
acid  in  Desmarestia,  101. 

EPSTEIN,  S.     See  R.  E.  BLACK,  153. 

Erythrocytes  of  hagfish,  227. 

Eserine,  effects  of  on  mollusc  tissues,  471. 

Estrogens,  effects  of  on  Arbacia  development, 
364. 

Estuarine  flora,  bloom  of,  257. 

Euphysoclist  swimbladder,  172. 

Evolution  of  hemoglobin,  227. 

Excitation-contraction  coupling  in  Limulus, 
209. 

Excretion  of  radioactive  elements  by  fish,  64. 

Exogenous  reference-clock  for  biological 
rhythms,  81. 

Eyestalk  of  lobsters,  juvenile  hormone  sub- 
stance from,  530. 

pANGE,  R.,  AND  J.  B.  WITTENBERG.     The 

swimbladder  of  the  toadfish,  Opsanus,  172. 
FANGE,    R.,    K.    SCHMIDT-NIELSEN    AND    H. 

OSAKI.     The    salt    gland    of    the    herring 

gull,  162. 

FAUST,  R.  G.,  AND  A.  K.  PARPART.     Perme- 
ability studies  on  Arbacia  eggs,  350. 
Feeding,  role  of  in  gamma  radiation  effects  on 

mosquitoes,  536. 
Feeding  in  Physalia,  338. 
FELDHERR,  C.     Physical  properties  of  lobster 

nerve  axoplasm,  328. 

FELDHERR,  C.     See  L.  V.  HEILBRUNN,  459. 
Female  Leucophaea  corpus  allatum  cells,  508, 

521. 
Fertility    of    mosquitoes,    effects    of    gamma 

radiation  on,  536. 

Fertilization,  changes  in  flux  of  K  upon,  339. 
Fertilization,  importance  of  gelatinous  coat  of 

egg  in,  74. 
Fertilization  and  agglutination  inhibitors  from 

Arbacia,  325. 

Fertilized  Urechis  eggs,  metabolism  of,  136. 
Fertilized  Urechis  eggs,  oxidation  of  CO  by,  153. 


INDEX 


567 


Fertilizin  from  Arbacia  eggs,  36Q. 

Fertilizin    of   Arbacia,    splitting-off   of   sulfate 

from,  337. 

Fertilizin  of  Mellita  egg,  74. 
Fiddler  crab,   effects   of   Physalia   nematocyst 

toxin  on,  219,  551. 

Fiddler  crab,   patterns  of  respiration   in,   303. 
FIGGE,  F.  H.  J.     See  R.  WICHTERMAN,  369. 

FlNGERMAN,      M.,      M.      E.     LOWE     AND     B.      I. 

SUNDARARAJ.  Direct  evidence  for  a  distal 
retinal  pigment  dark-adapting  hormone 
in  Palaemonetes,  351. 

FlNGERMAN,    M.,    M.    I.    SANDEEN    AND    M.    E. 

LOWE.  Influence  of  long-term  back- 
ground adaptation  on  the  lability  of 
chromatophores  and  the  sources  of  chroma- 
tophorotropins  in  Palaemonetes,  351. 

FlNGERMAN,   M.,   B.    I.   SUNDARARAJ  AND   M.    I. 

SANDEEN,  Further  studies  on  the  chro- 
matophorotropins  of  Palaemonetes,  352. 

Fish,  effects  of  Physalia  nematocyst  toxin  on, 
219. 

Fish,  marine,  electric  organs  of,  126. 

Fish,  symbiosis  of  with  sea  anemone,  397. 

Fish,  transportation  of  radioactive  elements 
in  blood  of,  64. 

Fission  of  Tetrahymena,  269. 

FITTON  JACKSON,  S.  Some  aspects  of  morpho- 
genesis in  ascidians,  335. 

Flatworm,  reproduction  in,  375. 

Flatworms,  studies  on,  276. 

FLEMISTER,  L.  J.  Salt  and  water  anatomy, 
constancy  and  regulation  in  related  crabs 
from  marine  and  terrestrial  habitats,  180. 

FLICKINGER,  R.  A.  Regional  localization  of 
neural  and  lens  antigens  in  the  frog  em- 
bryo in  relation  to  induction,  201. 

Flies,  chemoreceptors  of,  114. 

Food,  role  of  in  maintenance  of  normal  mor- 
phology of  Leucophaea  corpus  allatum 
cells,  521. 

Form-stability  of  ciliates,  384. 

Formation  of  subnuclear  aggregates  in  pro- 
tozoan cells,  269. 

Fox,  \V.,  AND  H.  C.  DESSAUER.  Response 
of  the  male  reproductive  system  of  lizards 
(Anolis)  to  unnatural  day-lengths  in 
different  seasons,  427. 

Fresh-water  turbellarian,  reproduction  in,  375. 

Frog,  effect  of  Physalia  nematocyst  toxin  on, 
219. 

Frog  embryo,  localization  of  antigens  in,  201. 

Fruit  bat,  echolocation  in,  107. 

Fundulus,  effects  of  Stoichactis  tentacles  on, 
397. 


QALTSOFF,  P.  S.  Coordination  of  ciliary 
motion  and  muscular  contractions  in  the 
gills  of  Crassostrea,  320. 


Gamma   irradiation  of  Aedes  adults  and  eggs, 

536. 

Gas  secretion  in  toadfish  swimbladder,  172. 
Gas  uptake  of  fertilized  Urechis  eggs,  153. 
Gastropod  tissues,  effects  of  neurohumors  and 

drugs  on,  471. 

Gecarcinus,  osmotic  regulation  in,  180. 
Gel  changes  in   pressure-treated   ciliates,   384. 
Gelatinous  coat  dispersal  by  sperm,  74. 
Gephyrean    worm    eggs,    metabolism    of,    136, 

153. 

Ghost  crab,  osmotic  regulation  in,  180. 
GIESE,  A.  C.     See  R.  A.  BOOLOOTIAN,  53. 
GILBERT,    L.    I.     See    H.    A.    SCHNEIDERMAN, 

530. 

Gland,  salt,  of  the  gull,  162. 
Glucagon  and  blood  glucose  in  Lophius,  371. 
Glucose  permeability  in  relation  to  action   of 

insulin,  459. 

Goitrogens,    effects    of    on    salamanders,    411. 
Gonads  of  male  lizards  exposed  to  changes  in 

day-length,  427. 

Goniopsis,  osmotic  regulation  in,  180. 
Gonyaulax,     persistent     diurnal     rhythm     of 

luminescence  in,  440. 

Gorgonocephalus  coelomic  corpuscles,  53. 
GOTO,    M.,   AND  A.    I.   CSAPO.     The  effect   of 

ovarian  steroids  on  the  membrane  poten- 
tial of  the  uterus,  335. 

GREEN,  J.  P.     See  G.  C.  STEPHENS,  367,  368. 
GREEN,  J.  W.,  AND  T.  A.  BORGESE.     Sodium 

and    potassium   exchanges   in   photosensi- 
tized fish  red  cells,  352. 
GREENBERG,  A.     See  F.  N.  SUDAK,  368. 
GRIFFIN,   D.   R.,  A.   NOVICK  AND  M.   KORN- 

FIELD.     The    sensitivity    of    echolocation 

in  the  fruit  bat,  Rousettus,  107. 
Gross  anatomy  of  gull  salt  gland,  162. 
Growth    of   Ambystoma    embryos    in    relation 

to  presence  of  symbiotic  alga,  483. 
Growth  control  in  tadpoles,  320. 
Growth  of  Leucophaea  corpora  allata,  508,  521. 
GRUNDFEST,   H.     Graded  electrical  responses, 

329. 
GRUNDFEST,   H.,  J.   P.   REUBEN  AND   \Y.    H. 

RICKLES,     JR.         Electrophysiology     and 

pharmacology  of  lobster  muscle  fibers,  332. 
GRUNDFEST,    H.     See    R.    MATHEWSON,    126; 

M.  V.  L.  BENNETT,  330,  331;  F.  V.  Mc- 

CANN,  356. 

GRUPP,  E.     See  R.  RUGH,  362. 
Guinea  pig  serum,  effect  of  on  chick  embryo, 

239. 

Gull,  salt  gland  of,  162. 
Gustatory  stimuli,  responses  of  cockroach  to, 

490. 

GUTTMAN,  B.     See  G.  C.  STEPHENS,  367,  368. 
Gymnophallus,  studies  on,  276. 


568 


INDEX 


"LJABITAT  in  relation  to  salt  and  water 
anatomy  of  crabs,  180. 

Hagfish,  respiratory  properties  of  hemoglobin 
of,  227. 

HAMMEN,  C.  S.     See  V.  H.  HUTCHISON,  483. 

HANEDA,  Y.,  F.  H.  JOHNSON  AND  E.  H.-C.  SIE. 
Luciferin  and  luciferase  extracts  of  a 
fish,  Apogon,  and  their  luminescent  cross- 
reactions  with  those  of  a  crustacean, 
Cypridina,  336. 

HARDING,  C.  V.  The  osmotic  behavior  of 
marine  oocyte  nuclei,  371. 

HARDING,  C.  V.,  AND  W.  L.  HUGHES.  Uptake 
of  tritium-labelled  thymidine  by  Arbacia 
eggs  and  embryos,  372. 

VON  HARNACK,  M.  Histophysiological  studies 
on  the  corpus  allatum  of  Leucophaea. 
II.,  521. 

VON  HARNACK,   M.     See  B.   SCHARRER,   508. 

HARTMAN,  F.  A.     See  K.  A.  BROWNELL,  345. 

HARVEY,  E.  B.  A  crescent-shaped  figure  in 
the  hyaline  layer  of  the  Arbacia  egg,  352. 

HARVEY,  E.  N.,  AND  S.  P.  MARFEY.  Fluo- 
rescence, phosphorescence  and  biolumines- 
cence  in  the  ctenophore  Mnemiopsis,  336. 

HARVEY,  E.  N.     See  S.  P.  MARFEY,  339. 

HASTINGS,  J.  \V.,  AND  B.  M.  SWEENEY.  A 
persistent  diurnal  rhythm  of  luminescence 
in  Gonyaulax,  440. 

Hatching  capacity  of  mosquito  eggs  after 
gamma  irradiation,  536. 

HATHAWAY,  R.,  AND  A.  TYLER.  Evidence 
for  the  splitting-off  of  S35-labelled  sulfate 
from  the  fertilizin  of  Arbacia  eggs  upon 
the  spontaneous  reversal  of  sperm  aggluti- 
nation, 337. 

HATHAWAY,  R.  R.     See  A.  TYLER,  369. 

HAUSMAN,  S.  A.     See  M.  ROCKSTEIN,  361. 

Heart-beat  of  molluscs,  471. 

Heat-lability  of  gelatinous  coat-dispersal  in 
Mellita,  74. 

Heat-stable  and  -labile  factors  in  rabbit  serum, 
239. 

Heat-treatment  of  Tetrahymena,  269. 

HEILBRUNN,  L.  V.,  AND  W.  L.  WILSON.  A 
physical  study  of  the  ground  substance  of 
the  Spisula  egg,  328. 

HEILBRUNN,  L.  V.,  F.  T.  ASHTON,  C.  FELD- 
HERR  AND  W.  L.  WILSON.  The  action  of 
insulin  on  cells  and  protoplasm,  459. 

Heliometra  coelomic  corpuscles,  53. 

Hemoglobin,  evolution  of,  227. 

HENLEY,  C.  Studies  on  membrane  elevation 
in  the  eggs  of  Chaetopterus  and  Nereis,  353. 

Herring  gull,  salt  gland  of,  162. 

Hiatella,  parasites  of,  276. 

HIATT,  H.  H.     See  A.  K.     KELTCH,  354. 

High  temperature,  effect  of  on  Tetrahymena, 
269. 


HILL,    R.    B.     The   effects    of   certain    neuro- 

humors  and  of  other  drugs  on  the  ventricle 

and  radula  protractor  of  Busycon  and  on 

the  ventricle  of  Strombus,  471. 
Histology  of  Cura,  375. 
Histology  of  gull  nasal  gland,  162. 
Histology  of  salamander  thyroid,  411. 
Histology  of  toadfish  swimbladder,  172. 
Histophysiological     studies     on     Leucophaea 

corpus  allatum,  508,  521. 
HODGSON,   E.  S.     Electrophysiological  studies 

of  arthropod  chemoreception.  III.  Chemo- 

receptors    of    terrestrial    and    fresh-water 

arthropods,  114. 

Holothuroid  coelomic  corpuscles,  53. 
Holothuroids,     juvenile     hormone     substance 

from,  530. 

HOLTZER,  H.     See  }.  W.  LASH,  322. 
HOLZ,     G.     G.,     JR.     Mercaptoethanol     and 

Tetrahymena,  354. 

Homarine  in  coelenterate  nematocysts,  551. 
Homologous    antisera,     effects    of     on     chick. 

embryo,  239. 
Homologous  sperm  extracts,  use  of  in  dispersal. 

of  Mellita  gelatinous  egg  coat,  74. 
Hormone,  dark-adapting,  from  Palaemonetes, 

351. 

Hormone,  possible  mode  of  action  of,  459. 
Hormone  activity   of   materials  from   inverte- 
brates, 530. 
Horseshoe    crab,    neuromuscular    transmission' 

in,  209. 

HOYLE,  G.     Studies  on  neuromuscular  trans-- 
mission in  Limulus,  209. 
HUGHES,  W.  L.     See  C.  V.  HARDING,  372. 
HULBURT,  E.  M.     See  J.  H.  RYTHER,  257. 
Humoral  mechanisms,  evolution  of,  338,  530. 
HUTCHISON,    V.    H.,    AND    C.    S.    HAMMEN. 

Oxygen    utilization    in    the    symbiosis    of 

embryos   of   the   salamander  Ambystoma 

and  the  alga  Oophila,  483. 
Hyaline  layer  of  Arbacia  egg,  crescent-shaped 

figure  in,  352. 
Hyaline    layers    of    Arbacia    egg,    action    of 

enzymes  on,  340. 
Hyaluronic  acid,   electron   microscope    studies. 

on,  333. 

Hydra,  nematocyst  toxins  of,  551. 
Hydrogen     ion     concentration,     effect    of    on. 

oxygen  equilibria  of  hagfish  blood,  227. 
Hydrogen   ion   concentration   of   Desmarestia, 

101. 

Hydrostatic  pressure  studies  with  ciliates,  384. 
Hydroxytryptamine,     effects    of    on     mollusc 

hearts,  471. 
Hydrozoa,  juvenile   hormone  substance  from... 

530. 
Hydrozoa,  nematocyst  toxins  of,  551. 


INDEX 


569 


TMMUNOLOGICAL  studies  on  rhick  em- 
bryo, 239. 

Induction  in  frog  embryo,  relation  of  to 
localization  of  antigens,  201 

Inhibition  of  tissue  formation  in  rhick  develop- 
ment, 319. 

Inhibitory  extract  of  chick  tissues,  preparation 
of,  344. 

Innervation  of  Narcine  electric  organs,  126. 

Insect,  corpus  allatum  cells  of,  508,  521. 

Insect  adults  and  eggs,  gamma  irradiation  of, 
536. 

Insect  chemoreception,  114,  490. 

Insect  juvenile  hormone  material  from  in- 
vertebrates, 530. 

Insulin,  action  of  on  cells  and  protoplasm,  459. 

Interrenal  of  sting  ray,  345. 

Tnulin  concentration  in  crab  blood,  180. 

Invertebrates,  juvenile  hormone  substance 
from,  530. 

lodine-131,  uptake  of  by  salamander  thyroids, 
411. 

Ion  exchanges  in  photosensitized  fish  red  cells, 
352. 

Ionic  regulation  in  spider  crab,  362. 

Irradiation  of  Aedes  adults  and  eggs,  536. 

Isotopes,  radioactive,  use  of  in  study  of 
salamander  thyroid  activity,  411. 

Isotopes,  transportation  of  in  fish  blood,  64. 

|  AH,\T,  T.  L.     See  O.  H.  SCHERBAUM,  269. 

JOHANX,  J.  C.     See  J.  M.  ANDERSON,  375. 
JOHNSON,  F.  H.     See  Y.  HANEDA,  336. 
Junctional  potentials  in  Limulus,  209. 
Juvenile  hormone  activity  of  substances  from 
invertebrates,  530. 

T7"AGEY,  K.  S.  See  E.  Kivv-RosENBERG, 
-  354. 

KELTCH,  A.  K.,  H.  H.  HIATT,  C.  P.  WALTERS 
\\D  G.  H.  A.  CLOWES.  The  action  of 
pentahalophenols  on  oxygen  consumption 
and  cell  division  and  on  the  glucose-6- 
phosphate  dehydrogenase  of  the  eggs 
of  Arbacia,  354. 

KELTCH,  A.  K.     See  R.  K.  CRANE,  350. 

KENNEDY,  I).  Neural  photosensitivity  in 
Mactra,  338. 

KEYNES,  R.  D.     See  M.  Y.  L.  BENNETT,  330. 

KlVY-ROSENBERG,     E.,     K.     S.     IvAGEY     AND     J- 

CASCARANO.  Dehydrogenase  activity  in 
developmental  stages  of  Spisula  as  meas- 
ured with  a  tetrazolium  salt,  354. 

KORNFIELD,  M.     See  D.  R.  GRIFFIN,  107. 

KRANE,  S.  M.,  AND  R.  K.  CRANE.  Changes 
in  the  levels  of  triphosphopyridine  nticleo- 


tide    in    the   eggs   of    Arbacia    subsequent 
to  fertilization,  355. 
KRASSNER,  S.  M.     See  F.  B.  BANG,  343. 

T  ABELLED  carbon,  use  of  in  demonstrating 
oxidation  of  CO  by  Urechis  eggs,  153. 

Labile  biological  rhythms,  81. 

Land  crab,  osmotic  regulation  in,  180. 

LANE,  C.  E.,  AND  E.  DODGE.  The  toxicity 
of  Physalia  nematocysts,  219. 

Larus,  salt  gland  of,  162. 

Larvae,  frog,  antigen  preparations  from,   201. 

LASH,  J.  YV.,  AND  H.  HOLTZER.  The  uptake 
of  radiosulfur  during  the  in  vitro  induction 
of  cartilage,  322. 

LENHOFF,  H.  M.  The  biological  and  chemical 
mechanisms  of  protein  utilization  by 
Hydra,  356. 

LENHOFF,  H.  M.,  AND  H.  A.  SCHNEIDERMAN. 
The  humoral  control  of  feeding  in  Physalia 
and  its  evolutionary  significance,  338. 

Lens  antigen,  localization  of  in  frog  embryo, 
201. 

Lepidoptera,  juvenile  hormone  substance  from, 
530. 

Leucophaea,  histophysiological  studies  on 
corpus  allatum  of,  508,  521. 

LEWIN,  R.  A.  Genes  controlling  the  move- 
ment of  flagella  in  Chlamydomonas,  339. 

Life-cycle  of  Leucophaea  corpus  allatum  cells, 
508. 

Light,  effect  of  on  oxygen  consumption  of 
Ambystoma-alga  symbioses,  483. 

Light,  effect  of  on  reproductive  system  of  male 
lizards,  427. 

Light,  effect  of  on  Urechis  egg  respiration,  153. 

Light,  effects  of  on  respiration  of  CO-treated 
Urechis  eggs,  136. 

Light  emission  by  Gonyaulax,  440. 

Light  penetration,  relation  of  to  diatom  bloom, 
257. 

Limulus,  neuromuscular  transmission  in,  209. 

Lizards,  male,  responses  of  to  changes  in  day- 
length,  427. 

Lobsters,  juvenile  hormone  substance  from,  530. 

Localization  of  antigens  in  frog  embryo,  201. 

LOUDERBACK,  A.     See  O.  H.  SCHERBAUM,  269. 

LOWE,  M.  E.     See  M.  FINGERMAN,  351. 

Luciferin,  Cypridina,  fractionation  of,  339. 

Luciferin-luciferase  cross-reactions,  336. 

Luminescence  of  firefly,  346. 

Luminescence  in  Gonyaulax,  440. 

Lunar-day  rhythm  in  Uca,  303. 

LYNN,  W.  G.     See  J.  N.  DENT,  411. 

Lytic  agent  from  Hydroides  sperm  extract,  348. 

\/f  ACRONUCLEAR     volume     in     Tetrahy- 

mena,  269. 
Mactra  (Spisula)  eggs,  effects  of  insulin  on,  459. 


570 


INDEX 


Male  Leucophaea  corpus  allatum  cells,  508,  521. 

Male  lizards,  response  of  to  changes  in  day- 
length,  427. 

Mangrove  crab,  osmotic  regulation  in,  180. 

MANWELL,  C.  On  the  evolution  of  hemo- 
globin. Respiratory  properties  of  the 
hemoglobin  of  the  California  hagfish 
Polistotrema,  227. 

MARFEY,  S.  P.,  L.  C.  CRAIG  AND  E.  N.  HARVEY. 
Fractionation  of  Cypridina  luciferin  and 
its  benzoyl  derivative,  339. 

MARFEY,  S.  P.     See  E.  N.  HARVEY,  336. 

Marine  bird,  salt  gland  of,  162. 

Marine  crabs,  osmotic  relations  in,  180. 

Marine  dinoflagellate,  persistent  rhythm  of 
luminescence  in,  440. 

Marine  eggs,  effects  of  insulin  on,  459. 

Marine  fish,  electric  organs  of,  126. 

MARSLAND,  D.,  AND  W.  AUCLAIR.  Experi- 
mental induction  of  clevage  furrows  in  the 
Arbacia  egg,  356. 

MARSLAND,  D.  See  J.  PADAWER,  359;  \V. 
AUCLAIR,  384. 

Mast  cells  of  rat,  centrifugally-deformed,  359. 

MATHEWSON,  R.,  A.  MAURO,  E.  AMATNIEK 
AND  H.  GRUNDFEST.  Morphology  of  main 
and  accessory  electric  organs  of  Narcine 
and  some  correlations  with  their  electro- 
physiological  properties,  126. 

Mating  in  Cura,  375. 

MAURO,  A.     See  R.  MATHEWSON,  126. 

McCANN,  F.  V.,  R.  WERMAN  AND  H.  GRUND- 
FEST. Graded  and  all-or-none  activity 
in  insect  muscle  fibers,  356. 

Mediaster  coelomic  corpuscles,  53. 

Mellita  egg  gelatinous  coat,  74. 

Membrane  elevation  in  Chaetopterus  and 
Nereis  eggs,  353. 

Membrane  permeability,  in  relation  to  action 
of  insulin,  459. 

Membrane  potential  of  uterus,  effect  of  ovarian 
steroids  on,  335. 

Membrane  removal  from  Hydroides  egg,  349. 

Membranes  of  Hydroides  egg,  effects  of  various 
agents  on,  349. 

Menidia,  hybridization  in,  361. 

Mercaptoethanol  treatment  of  Tetrahymena, 
354. 

MERRIAM  R.  \V.  The  nuclear  envelope  as  a 
possible  agent  in  specific  synthetic  events 
in  the  cytoplasm  of  sand  dollar  eggs,  329. 

Metabolism,  explanation  for  correlation  of, 
with  barometric  pressure  change,  344. 

Metabolism  of  Ambystoma  embryos,  483. 

Metabolism  of  Urechis  eggs,  136,  153. 

Metacercariae  of  trematodes,  276. 

Metachromatic  reaction  inhibition  by  insulin, 
459. 

Metamorphosis  of  Botryllus,  147. 


Methyl  green  staining  of  Arbacia  eggs,  342. 

Metridium,  nematocyst  toxins  of,  551. 

METZ,  C.   B.     Fertilization  and  agglutination 

inhibitors  from  Arbacia,  325. 
Mice,  effects  of  Physalia  nematocyst  toxin  on, 

219. 

Millipede,  chemoreceptors  of,  114. 
Mitotic  activity  of  Leucophaea  corpus  allatum 

cells,  508. 

Molecular  weight  of  hagfish  hemoglobin,  227. 
Molluscan  heart,  effects  of  Physalia  nematocyst 

toxin  on,  219. 
Mollusc   tissues,   effects   of    neurohiunors   and 

drugs  on,  471. 
Molluscs,  parasites  of,  276. 
MOLONEY,  V.     See  C.  L.  CLAFF,  347. 
MONROY,    A.,    AND    A.    TYLER.     Changes    in 

efflux     and     influx     of     potassium     upon 

fertilization  in  eggs  of  Arbacia,  measured 

by  use  of  K-42,  339. 
Mormoniella,  factors  and  genes  in,  321. 
Morphogenesis  of  ascidians,  335. 
Morphogenesis    of    chick    embryo,    effect    of 

normal  sera  and  homologous  antisera  on, 

239. 

Morphology  of  gull  nasal  gland,  162. 
Morphology    of    Leucophaea    corpus    allatum 

cells,  508,  521. 

Morphology  of  Narcine  electric  organs,  126. 
Morphology  of  toadfish  swimbladder,  172. 
Morphology  of  trematodes,  276. 
Mosquito  adults  and  eggs,  gamma  irradiation 

of,  536. 
Movement,     amoeboid,     polarization     optical 

study  of,  327. 

Movement  of  flagella  in  Chlamydomonas,  339. 
MUN,    A.    M.     Toxic   effects   of    normal    sera 

and    homologous    antisera    on    the    chick 

embryo,  239. 

MURRELL,  L.  R.     See  P.  F.  NACE,  357. 
Muscle,  Busycon,  effects  of  neurohumors  and 

drugs  on,  471. 

Muscle,  Limulus,  A  band  of,  325. 
Muscle,  potassium  contracture  in,  333,  334. 
Muscle    contraction    without    membrane    po- 
tential change,  341. 

Muscle-nerve  physiology  of  Limulus,  209. 
Mussel,  parasites  of,  276. 
Mya,  parasites  of,  276. 
Myogenic  molluscan  hearts,  471. 
Myometrium,  calcium,  oxytocin  and  regulation 

of,  334. 
Mytilus,  parasites  from,  276. 

VTACE,  P.  F.,  J.  E.  SCHUH,  L.  R.  MURRELL 
AND  A.  D.  DINGLE.  Hyperglycemia  and 
islet  damage  after  intracardiac  injection 
of  alloxan  in  toadfish,  357. 


INDEX 


571 


NAGLER,  A.  L.,  AND  B.  W.  ZWEIFACH.  The 
effect  of  bacterial  endotoxins  and  bio- 
genie  amines  on  the  phagocytic  behavior 
of  endothelial  elements  in  the  frog,  Rana, 
357. 

Narcine,  electric  organs  of,  126. 

Nasal  gland  of  gull,  162. 

NELSON,  L.  ATP — an  energy  source  for 
sperm  motility,  326. 

Nematocyst  discharge  by  Stoichactis,  397. 

Nematocysts,  Physalia,  toxicity  of,  219. 

Nematocysts  of  coelenterates,  chemical  nature 
of  toxin  in,  551. 

Nerve  tissues  of  cockroach,  gustatory  responses 
of,  490. 

Neural  antigens,  localization  of  in  frog  em- 
bryo, 201. 

Neurohumors,  effects  of  on  mollusc  tissues,  471. 

Neuromuscular  transmission  in  Limulus,  209. 

Neurophysiological  studies  on  cockroach,  490. 

Newts,  effects  of  goitrogens  on  thyroids  of,  411. 

Nitrogen  utilization  by  Oophila  symbionts  of 
Ambystoma  embryos,  483. 

Noise,  extraneous,  role  of  in  echolocation  of 
fruit  bat,  107. 

Noradrenaline,  effects  of  on  mollusc  tissues,  471. 

Normal  cell  life-cycle  in  Leucophaea  corpus 
allatum,  508. 

Normal  sera,  effects  of  on  chick  embryo,  239. 

NORRIS,  K.  S.     See  D.  DAVENPORT,  397. 

NOVICK,  A.     See  D.  R.  GRIFFIN,  107. 

Nuclear  processes  in  Tetrahymena,  269. 

Nuclear-cytoplasmic  ratio  in  Leucophaea  cor- 
pus allatum  cells,  508,  521. 

Nutrients  giving  rise  to  diatom  bloom,  257. 

Nutrition,  role  of  in  gamma  radiation  effects  on 
mosquitoes,  536. 

Nutrition,  role  of  in  morphology  of  Leucophaea 
corpus  allatum  cells,  521. 

QCYPODE,  osmotic  regulation  in,  180. 

Oligochaete,  juvenile  hormone  substance  from, 
530. 

Oophila,  symbiosis  of  with  Ambystoma  em- 
bryos, 483. 

Ophiuroid  coelomic  corpuscles,  53. 

Opsanus,  swimbladder  of,  172. 

Organ-specific  frog  sera,  201. 

Orientation  of  fruit  bat,  107. 

Orthopteran  chemoreceptors,  114. 

OSAKI,  H.     See  R.  FANGE,  162. 

Osmotic  behavior  of  marine  oocyte  nuclei,  371. 

Osmotic  effects  of  insulin  on  marine  eggs,  459. 

Osmotic    function    of    gull    salt    gland,     162. 

Osmotic  regulation  in  crabs,  180. 

OSTERHOUT,  W.  J.  V.  Changes  in  behavior 
of  the  cell  wall  and  cytoplasm  due  to 
injuries  in  Nitella,  358. 


OSTERHOUT,  W.  J.  V.  Changes  in  permeabil- 
ity to  an  acid  dye  due  to  protoplasmic 
lesions  in  Nitella,  358. 

OSTERHOUT,  W.  J.  V.  Inhibitory  effect  of 
electrolytes  on  the  penetration  of  organic 
molecules  into  Nitella,  359. 

Ova,  Ambystoma,  effects  of  symbiotic  alga  on, 
483. 

Ova,  dispersal  of  gelatinous  coat  of,  74. 

Ova,  effects  of  insulin  on,  459. 

Ova,  mosquito,  gamma  irradiation  of,  536. 

Ova,  Urechis,  metabolism  of,  136. 

Ova,  Urechis,  oxidation  of  CO  by,  153. 

Ovarian  activity  of  starved  Leucophaea,  521. 

Oviposition  of  mosquitoes  after  gamma  irradia- 
tion, 536. 

Oxidation  of  CO  by  fertilized  Urechis  eggs,  153. 

Oxidative   metabolism   of    Urechis   eggs,    136. 

Oxygen  consumption  of  Arbacia  eggs,  effects 
of  pentahalophenols  on,  354. 

Oxygen  consumption  of  diatoms,  257. 

Oxygen  consumption  of  Fucus,  345. 

Oxygen  consumption  of  potatoes  and  carrots, 
81. 

Oxygen  consumption  of  Uca,  303. 

Oxygen  content  of  toadfish  swimbladder,  172. 

Oxygen  dissociation  curve  for  hagfish  hemo- 
globin, 227. 

Oxygen  transport  in  fish  swimbladder,  372. 

Oxygen  uptake  of  Urechis  eggs,  136,  153. 

Oxygen  utilization  of  Ambystoma  embryos, 
483. 

pADAWER,    J.,    D.    MARSLAND    AND    W. 

AUCLAIR.     Rate    of    recovery   of  centrif- 

ugally-deformed  mast  cells  as  a  function 

of  age  in  the  rat,  359. 
Paramecium,  form-stability  of,  384. 
Parasitic  worms,  studies  on,  276. 
PARPART,   A.    K.,   J.    CAGLE   AND   L.    WOOD. 

Action  of  enzymes  on  the  hyalines  of  the 

Arbacia  egg,  340. 

PARPART,  A.  K.     See  R.  G.  FAUST,  350. 
Parvatrema,  studies  on,  276. 
Patiria  coelomic  corpuscles,  53. 
Pattern,  repetition  of,  in  respiration  of  LTca, 

303. 
Perchlorate,  effects  of  on  salamander  thyroids, 

411. 

Periplaneta,  gustatory  responses  of,  490. 
Perivisceral  fluid  of  echinoderms,  53. 
Permeability  of  Arbacia  egg,  350. 
Permeability  of  Desmarestia,  101. 
Permeability  of  Nitella,  358,  359. 
Permeability  theory  of  insulin  action,  459. 
Persistent  biological  rhythms,  81. 
Persistent  diurnal  rhythm  of  luminescence  in 

Gonyaulax,  440. 


572 


INDEX 


pH,  effect  of  on  oxygen  equilibria  of  hagfish 
blood,  227. 

pH  of  Desmarestia  cytoplasm,  101. 

Pharmacology  of  Busycon  and  Strombus,  471. 

Philippine  fish,  symbiosis  of  with  sea  anemone, 
397. 

PHILPOTT,  D.  E.  A  ncvel  method  for  cor- 
recting astigmatism  in  electron  microscopes, 
360. 

PHILPOTT,  D.  E.  See  G.  \V.  DE  VILLAFRANCA, 
325;  F.  A.  BETTELHEIM,  333;  R.  R.  CAR- 
DELL,  JR.,  346. 

Photoperiod  in  relation  to  sex  characteristics 
of  male  lizards,  427. 

Photo-reversal  of  respiration  of  CO-treated 
Urechis  eggs,  136. 

Photosensitive  pigment  from  Asterias  skin  and 
eyespots,  361. 

Photosensitivity  in  Mactra,  338. 

Photosynthesis  in  relation  to  luminescence  of 
Gonyaulax,  440. 

Photosynthesis  in  relation  to  oxygen  produc- 
tion by  Oophila,  483. 

Photosynthetic  activity  of  diatoms,  257. 

Phylogenetic  significance  of  hemoglobin,   227. 

Physalia  float,  CO  in,  370. 

Physalia   nematocysts,    toxicity   of,    219,    551. 

Physical  chemistry  of  haghsh  blood,  227. 

Physiology  of  action  of  insulin,  459. 

Physiology  of  a  diatom  bloom,  257. 

Phytoplankton  bloom,  dynamics  of,  257. 

Pigment  concentration  in  diatom  bloom,  257. 

Pisaster  coelomic  corpuscles,  53. 

Planarian,  reproduction  in,  375. 

Plasma,  role  of  in  transportation  of  radioactive 
elements  in  fish  blood,  64. 

Plasmagel  changes  in  pressure-treated  ciliates, 
384. 

Platyhelminth,  reproduction  in,  375. 

Platyhelminthes,  parasitic,  276. 

Plexaura,  nematocyst  toxins  of,  551. 

Polistotrema,  respiratory  properties  of  hemo- 
globin of,  227. 

Polychaetes,  juvenile  hormone  substance  from, 
530. 

Polyphemus,  use  of  for  assay  of  juvenile 
hormone  activity,  530. 

Pomacentrid  fish,  symbiosis  of  with  sea 
anemone,  397. 

Population  bloom  in  diatoms,  257. 

Poraniopsis,  coelomic  corpuscles  of,  53. 

Portuguese  man-of-war  nematocysts,  219. 

Potassium  perchlorate,  effects  of  on  salamander 
thyroids,  411. 

Potatoes,  biological  rhythms  of,  81. 

Potentials,  nerve  action,  of  cockroach,  490. 

Precipitin  reactions  with  frog  sera,  201. 

Pressure,  relation  of  to  form-stability  of  ciliates, 
384. 


PROCK,  P.  B.     See  J.  H.  WELSH,  551. 
Productivity  of  diatoms,  257. 
Properdin,  effect  of  on  chick  embryo,  239. 
PROSSER,  C.  L.     See.  C.  L.   RALPH,  360;  \Y.. 

W.  STEINBERGER,  366. 
Protein  utilization  by  Hydra,  356. 
Proteins  of  chick  embryo,  239. 
Protochordate,  regeneration  of  buds  in,  147. 
Protoplasm,  action  of  insulin  on,  459. 
Protozoa,  formation  of  subnuclear  aggregates 

in,  269. 

Protozoa,  form-stability  of,  384. 
Protozoan,  effects  of  heparin  on,  459. 
Protractor  muscle  of  Busycon,  effect  of  neuro- 

humors  on,  471. 

Pseudopolydesmus,  chemoreceptors  of,  114. 
Pycnopodia  coelomic  corpuscles,  53. 

QUATERNARY  bases  in  coelenterates,  551. 

Quinine,  gustatory  response  of  cockroach  to, 
490. 

D  ABBIT  serum,  effects  of  on  chick  embryo, 

1X  239. 

Radiation  effects  on  Aedes,  536. 

Radioactive  strontium  and  yttrium,  transpor- 
tation of  in  teleost  blood,  64. 

Radioiodine,  uptake  of  by  salamander  thyroids, 
411. 

Radiosulfur,  uptake  of  during  in  vitro  induc- 
tion of  cartilage,  322. 

Radula  protractor  of  Busycon,  effects  of 
neurohumors  on,  471. 

RALPH,  C.  L.,  AND  C.  L.  PROSSER.  Conduc- 
tion in  Phascolosoma  fusiform  muscle,  360. 

Rana  embryos,  localization  of  antigens  in,  201. 

REBHUN,  L.  I.  Behavior  of  metachromatic 
granules  during  cleavage  in  Spisula,  325 

Receptors,  taste,  of  cockroach,  490. 

Reconstitution  in  Cordylophora,  319. 

Red  blood  cell  counts  of  fish,  64. 

Reference-clock  for  biological  rhythms,  81. 

Regeneration  inhibitor  in  Tubularia,  369. 

Regeneration  of  buds  in  Botryllus,  147. 

Regeneration  of  tail  in  shortened  and  length- 
end  earthworms,  321. 

Regional  localization  of  antigens  in  frog  em- 
bryo, 201. 

Regulation  of  salt  levels  in  crabs,  180. 

Regulation  of  size  in  Botryllus  regenerates,  147.. 

REID,  D.  F.     See  H.  F.  BOROUGHS,  64. 

Relation  of  ciliate  form-stability  to  pressure- 
and  temperature,  384. 

Repetition  of  pattern  in  respiration  of  Uca,. 
303. 

Reproductive  biology  of  Cura,  375. 

Reproductive  system  of  male  lizards,  response- 
of  to  changes  in  day-length,  427. 


INDEX 


573 


Reptile,  male,  effect  of  day-length  on  reproduc- 
tive system  of,  427. 

Respiration  of  Ambystoma  embryos,  483. 

Respiration  of  diatoms,  257. 

Respiration  of  potatoes  and  carrots,  81. 

Respiration  of  Uca,  303. 

Respiration  of  Urechis  eggs,  136,  153. 

Respiratory  properties  of  hagfish  hemoglobin, 
227. 

Response  of  male  Anolis  to  changes  in  day- 
length,  427. 

Responses  of  cockroach  tissues  to  gustatory 
stimuli,  490. 

Retia  mirabilia  of  toadfish  swimbladder,  172. 

REUBEN,  J.  P.     See  H.  GRUNDFEST,  332. 

Rhythm  of  luminescence  in  Gonyaulax,  440. 

Rhythmic  nature  of  metabolism  in  Ilyanassa, 
345. 

Rhythms,    exogenous    reference-clock   for,    81. 

Rhythms  of  male  lizard  reproductive  system, 
427. 

Rhythms  in  respiration  of  Uca,  303. 

Ribonucleic  acid  content  of  developing  Ilyan- 
assa embryos,  348. 

Ribonucleic  acid  synthesis  by  nucleoli,  342. 

RICKLES,  W.  H.,  JR.     See  H.  GRUNDFEST,  332. 

ROCKSTEIN,  M.,  J.  COHEN  AND  S.  A.  HAUSMAN. 
A  photosensitive  pigment  from  the  dorsal 
skin  and  eyespots  of  the  starfish  Asterias, 
361. 

Role  of  blood  in  strontium-yttrium  trans- 
portation of  fish,  64. 

ROSE,  S.  M.  A  feed-back  mechanism  of 
growth  control  in  tadpoles,  320. 

ROSENBLUM,   W.,   AND   B.   W.   ZWEIFACH.       The 

action   of   certain   chemical    agents    upon 

squid  chromatophores,  340. 
ROTHSCHILD,     LORD,    AND    A.     TYLER.     The 

oxidation  metabolism  of  eggs  of  Urechis, 

136. 

Rousettus,  echolocation  in,  107. 
ROYS,    C.    C.     A    comparison    between    taste 

receptors  and  other  nerve  tissues  of  the 

cockroach  in  their  responses  to  gustatory 

stimuli,  490. 

RUBINOFF,  I.,  AND  E.  SHAW.  Artificial  hy- 
bridization between  two  species  of  Menidia, 

361. 
RUGH,  R.,  AND  E.  GRUPP.     The  effect  of  x- 

irradiation  of  the  early  fish  embryo,  362. 
RYTHER,  J.  H.,  C.  S.  YENTSCH,  E.  M.  HULBURT 

AND  R.  F.  VACCARO.     The  dynamics  of  a 

diatom  bloom,  257. 

QACCOGLOSSUS,     juvenile     hormone     sub- 
stance from,  530. 

SAKAI,  T.,  AND  A.  I.  CSAPO.  Contraction 
without  membrane  potential  change,  341. 


Salamander  embryos,  symbiosis  of  with  alga, 

483. 
Salamanders,  effects  of  goitrogens  on  thyroids 

of,  411. 

Salinity  in  relation  to  diatom  bloom,  257. 
Salinity  relations  in  crabs,  180. 
Salt  gland  of  the  gull,  162. 
Salt  and  water  anatomy  of  crabs,  180. 
SANBORN,  R.  C.     Ionic  regulation  in  a  spider 

crab,  362. 

Sand  dollar  egg  gelatinous  coat  dispersal,   74. 
SANDEEN,  M.  I.     See  M.  FINGERMAN,  351,  352. 

SCHARRER,  B.,  AND  M.  VON  HARNACK.       HistO- 

physiological  studies  on  the  corpus  allatum 

of  Leucophaea.      I.,  508. 
SCHECHTER,  V.     Water  relations  of  the  Spisula 

egg,  362. 
SCHEINBLUM,    T.    S.     See   G.    \Y.    DE    VILLA- 

FRANCA,  325. 
SCHERBAUM,  O.   H.,  A.  L.  LOUDERBACK  AND 

T.  L.  JAHN.     The  formation  of  subnuclear 

aggregates    in    normal    and    synchronized 

protozoan  cells,  269. 
SCHINSKE,  R.  A.     See  G.  C.  STEPHENS,  341, 

368. 
SCHMIDT-NIELSEN,  K.     See  R.  FANGE,  162. 

SCHNEIDERMAN,     H.    A.,    AND    L.     I.     GILBERT. 

Substances  with  juvenile  hormone  activity 
in  Crustacea  and  other  invertebrates,  530. 

SCHNEIDERMAN,  H.  A.  See  H.  M.  LENHOFF, 
338. 

Schooling  behavior  of  Menidia,  324,  365. 

SCHUEL,  H.  Urethan  inhibition  of  cleavage 
in  the  Chaetopterus  egg  and  its  antagonism 
by  various  substances,  363. 

SCHUH,  J.  E.,  AND  G.  CARANASOS.  Additional 
evidence  for  somatic  reduction  in  the 
metamorphosis  of  the  ileum  of  the  mos- 
quitoes by  the  use  of  tritiated  thymidine, 
363. 

SCHUH,  J.  E.     See  P.  F.  NACE,  357. 

SCOTT,  SISTER  FLORENCE  M.  Affinity  of 
tissues  in  reconstituting  tunicates,  363. 

Scyphozoa,  nematocyst  toxins  of,  551. 

Sea  anemone,  symbiosis  of  with  fish,  397. 

Seasonal  variation  in  biological  rhythms  of 
carrots  and  potatoes,  81. 

Seasonal  variations  in  response  of  male  lizards 
to  changes  in  day-length,  427. 

Secretory  activity  of  Leucophaea  corpus  allatum 
cells,  508,  521. 

Secretory  function  of  gull  salt  gland,  162. 

SEGAL,  S.  J.,  AND  A.  TYLER.  Inhibiting  action 
of  a  triphenylethanol  derivative  on  the 
development  of  eggs  of  Arbacia  and  on 
the  fertilizing  capacity  of  the  sperm,  364. 

SEGAL,  S.  J.,  AND  A.  TYLER.  Structure- 
activity-relationships  concerning  the  in- 
hibitory activity  of  synthetic  estrogens 


574 


INDEX 


and  some  triphenylethanol  derivatives  on 
developing  eggs  of  Arbacia,  364. 

Self-fertilization  in  Cura,  375. 

Semi-lunar  rhythms  in  Uca,  303. 

Sensitivity  of  echolocation   in   fruit   bat,    107. 

Sensitivity  of  mosquitoes  to  gamma  radiation, 
536.  ' 

Sera,  normal,  effects  of  on  chick  embryo,  239. 

Serological  studies  of  frog  embryos,  201. 

Serology  of  chick  embryo,  239. 

Sex  characteristics  of  male  lizards,  changes 
in  after  alteration  of  day-length,  427. 

Sex  differences  among  mosquitoes  in  radiation 
effects,  536. 

Sex  differences  in  Leucophaea  corpora  allata, 
508. 

Sex  differences  in  reactivity  of  frog  serum,  201. 

Sexual  reproduction  in  Cura,  375. 

Shape  changes  in  ciliates,  384. 

SHAW,  E.  The  development  of  schooling 
behavior  in  the  genus  Menidia,  324. 

SHAW,  E.  A  study  of  current  orientation  as  a 
stimulus  to  schooling  behavior  in  Menidia, 
365. 

SHAW,  E.  A  study  of  visual  attraction  as  a 
stimulus  to  schooling  behavior  in  Menidia, 
365. 

SHAW,  E.     See  I.  RUBINOFF,  361. 

SHIRODKAR,  M.  V.,  F.  B.  BANG  AND  A.  WAR- 
WICK. Antibacterial  action  of  Limulus 
blood  on  an  in  vitro  system,  341. 

SIE,  E.  H.-C.     See  Y.  HANEDA,  336. 

Silkworm,  use  of  for  assay  of  juvenile  hormone 
activity,  530. 

Siphonophore  nematocyst  toxicity,  219. 

Size  regulation  in  regeneration  of  Botryllus, 
147. 

Sodium  chloride,  gustatory  response  of  cock- 
roach to,  490. 

Solanus,  biological  rhythms  of,  81. 

Solar  day  in  relation  to  rhythms  of  luminescence 
in  Gonyaulax,  440. 

Solational  changes  in  pressure-treated  ciliates, 
384. 

SOLOMON,  H.     See.  R.  WICHTERMAN,  369. 

Somatic  reduction  in  ileum  of  mosquitoes,  363. 

Sound  production  by  toadfish  swimbladder, 
172. 

SPEIDEL,  C.  C.  Motion  pictures  of  some 
changes  in  cells  induced  by  x-ray  treat- 
ments of  tadpoles  and  tetrahymenae,  322. 

SPEIDEL,  C.  C.  The  occurrence  of  amicronu- 
cleate  tetrahymenae  as  facultative  para- 
sites in  embryos  of  the  catfish,  Ameiurus, 
366. 

SPEIDEL,  C.  C.  Radiation-induced  variation 
in  the  micronucleus  of  Tetrahymena,  366. 

Sperm,  mosquito,  effects  of  gamma  radiation 
on,  536. 


Sperm  entry  in  Hydroides,  324. 

Sperm  extracts,  use  of  in  dispersal  of  Mellita 
egg  gelatinous  coat,  74. 

Sperm  motility,  ATP-induced,  326. 

Spermatogenesis  in  lizards,  427. 

Spisula  egg,  water  relations  in,  362. 

Spisula  eggs,  effects  of  insulin  on,  459. 

Stability  of  ciliate  morphology,  384. 

STAHLER,  N.     See  L.  A.  TERZIAN,  536. 

Starvation,  effect  of  on  Leucophaea  corpus 
allatum  cells,  521. 

STEINBERGER,  W.  W.,  AND  C.  L.  PROSSER. 
Conduction  in  dogfish  spiral-valve  retrac- 
tor and  Phascolosoma  proboscis  retractor 
muscles,  366. 

STEPHENS,  G.  C.,  AND  J.  P.  GREEN.  Enzymatic 
inactivation  of  chromatophorotropic  prin- 
ciples from  the  fiddler  crab,  Uca,  367. 

STEPHENS,  G.  C.,  J.  P.  GREEN,  B.  GUTTMAN 
AND  R.  A.  SCHINSKE.  Studies  on  the 
effect  of  population  size  on  the  diurnal 
melanophore  rhythm  of  the  fiddler  crab, 
Uca,  368. 

STEPHENS,  G.  C.,  B.  GUTTMAN  AND  J.  P. 
GREEN.  Chromatophorotropic  principles 
of  the  green  gland  of  the  fiddler  crab, 
Uca,  367. 

STEPHENS,  G.  C.,  AND  R.  A.  SCHINSKE.     Amino 
acid  uptake  in  marine  invertebrates,  341. 

Stichopus  coelomic  corpuscles,  53. 

Stimuli,  gustatory,  responses  of  cockroach  to, 
490. 

Stinging  organelles  of  coelenterates,  toxins  in, 
551. 

Stoichactis,  symbiosis  of  with  fish,  397. 

STONE,  W.,  JR.     See  R.  F.  DOOLITTLE,  335. 

Strombus  ventricle,  effects  of  neurohumors 
and  drugs  on,  471. 

Strongylocentrotus  coelomic  corpuscles,  53. 

Strontium-90,  transportation  of  in  fish  blood, 
64. 

STUNKARD,  H.  W.,  AND  J.  R.  UZMANN.  Studies 
on  digenetic  trematodes  of  the  genera 
Gymnophallus  and  Parvatrema,  276. 

Subnuclear  aggregates  in  protozoan  cells,  269 

Substances  with  juvenile  hormone  activity 
from  invertebrates,  530. 

Sucrose,  gustatory  response  of  cockroach  to, 
490. 

SUDAK,  F.  N.,  C.  L.  CLAFF  AND  A.  GREENBERG. 
Relation  of  halogen  position  to  physio- 
logical properties  of  mono-,  di-,  and  tri- 
chlorophenoxyacetic  acid,  368. 

SUDAK,  F.  N.     See  C.  L.  CLAFF,  347. 

Sulfuric  acid  accumulation  in  Desmarestia,  101. 

SUNDARARAJ,    B.    I.       See   M.    FlNGERMAN,    351, 

352. 

SWEENEY,  B.  M.     See  J.  W.  HASTINGS,  440. 
Swimbladder  of  toadfish,  172. 


INDEX 


575 


Symbiosis  of  Ambystoma  and  Go  phi  la,  483. 

Symbiosis  of  sea  anemone  and  fish,  397. 

Synaptic  transmission  in  squid  stellate  gan- 
glion, 331. 

Synchronized  protozoan  cultures,  formation  of 
subnuclear  aggregates  in,  269. 

-yADPOLES  of  Botryllus,  147. 

Taste  perception  in  cockroach,  490. 

Taxonomy  of  trematodes,  276. 

Teleost  fish,  swimbladder  of,  172. 

Teleost   fish,   symbiosis  of  with  sea  anemone, 

397. 
Teleost     fish,     transportation     of     radioactive 

elements  in  blood  of,  64. 
Temperature,  effect  of  on   circulation   in   Cis- 

tenides,  370. 
Temperature,   effect   of  on   light   emission   by 

Gonyaulax,  440. 
Temperature,  effect  of  on  hagfish  hemoglobin 

oxygen  equilibria,  227. 
Temperature-independent   biological   rhythms, 

81. 

Temperature  and  pressure  in  relation  to  form- 
stability  of  ciliates,  384. 

Terrestrial  arthropod,  chemoreceptors  of,  114. 
Terrestrial  crabs,  osmotic  relations  in,  180. 
TERZIAN,  L.  A.,  AND  N.  STAHLER.     A  study 

of  some  effects  of  gamma  radiation  on  the 

adults  and  eggs  of  Aedes,  536. 
Testis  weight  of  male  lizards  exposed  to  changes 

in  day-length,  427. 
Tetrahymena,    amicronucleate,    as   facultative 

parasite  of  catfish,  366. 

Tetrahymena,  formation  of  subnuclear  aggre- 
gates in,  269. 
Tetramethylammonium  chloride  in  nematocysts, 

551. 

Tetramine  in  coelenterate  nematocysts,  551. 
Thiocyanate  concentration  in  crab  blood,  180. 
Thiourea,   effects  of  on   salamander   thyroids, 

411. 

THOMAS,  C.     See  R.  F.  DOOLITTLE,  335. 
Thymidine,  uptake  of  by  Arbacia  embryos,  372. 
Thyroid  activity  of  salamanders,  411. 
Tilapia,  transportation  of  radioactive  elements 

in  blood  of,  64. 

Tissue  affinity  in  reconstituting  tunicates,  363. 
Toadfish,  swimbladder  of,  172. 
Tonotropic  effects  of  drugs  on  mollusc  tissues, 

471. 
Toxic  effects  of  normal  sera  and  antisera  on 

chick  embryo,  239. 

Toxic  factor  from  scalded  starfish,  347. 
Toxicity  of  Physalia  nematocysts,  219. 
Toxins  of  coelenterate  nematocysts,  551. 
Transmission,  neuromuscular,  in  Limulus,  209. 


Transportation    of    radioactive    substances    in 

fish  blood,  64. 

Triclad  turbellarian,  reproduction  in,  375. 
Trigonelline  in  coelenterate  nematocysts,  551. 
Triturus,  effects  of  goitrogens  on  thyroid  of, 

411. 

Turbellarian,  triclad,  reproduction  in,  375. 
TWEEDELL,    K.    S.     A    bacteria-free    inhibitor 

of  regeneration  in  Tubularia,  369. 
TYLER,  A.,  AND  R.   R.   HATHAWAY.     Produc- 
tion  of   S35-labelled    fertilizin    in    eggs   of 

Arbacia,  369. 
TYLER,  A.     See  LORD  ROTHSCHILD,  136;  R.  E. 

BLACK,    153;   R.   R.   HATHAWAY,  337;  A. 

MONROY,  339;  S.  J.  SEGAL,  364. 


in, 


1  ]  CA,   repetition   of  respiration   patterns   i 

303. 
Uca,  use  of  in  assay  of  coelenterate  nematocyst 

toxins,  219,  551. 

Urechis  eggs,  oxidation  of  CO  by,  153. 
Urechis  eggs,  oxidative  metabolism  of,  136. 
Urine  chloride  levels  in  crabs,  180. 
Urodeles,  effects  of  goitrogens  on  thyroids  of,. 

411. 
UZMANN,  J.  R.     See  H.  W.  STUNKARD,  276. 

yACCARO,  R.  F.     See  J.  H.  RYTHER,  257. 
Vacuoles    as    site    of    acid    accumulation    in 

Desmarestia,  101. 

Vascular  supply  of  toadfish  swimbladder,  172. 
Vaucheria,  Bermudian,  at  Cape  Cod,  344. 
Ventricle  of  molluscs,  effects  of  neurohumors 

on,  471. 
Venus   heart,   effects   of   Physalia   nematocyst 

toxin  on,  219. 
Viability    of    mosquito    larvae    after    gamma 

irradiation,  536. 

DE  VlLLAFRANCA,  G.  W.,  T.  S.  SCHEINBLUM 

AND    D.    E.    PHILPOTT.     The  A   band  of 

muscle  from  Limulus,  325. 
VINCENT,  W.  S.,  B.  BENSAM  AND  A.  BENSAM. 

Synthesis  of  ribonucleic  acid  by  nucleoli, 

342. 
Viscosity  of  Spisula  ground  substance,  328. 

YV ALTERS,  C.  P.     See  R.  K.  CRANE,  350; 

A.  K.  KELTCH,  354. 

WARWICK,  A.     See  M.  V.  SHIRODKAR,  341. 
Water  and  salt  anatomy  of  crabs,  180. 
WATKINS,    M.    J.     Regeneration    of    buds    in 

Botryllus,  147. 
"Wax    test"    for    assay    of    juvenile    hormone 

activity,  530. 
WEBB,  H.  M.,  AND  F.  A.  BROWN,  JR.     The 

repetition  of  pattern  in  the  respiration  of 

Uca,  303. 


576 


INDEX 


WEBB,  H.  M.     See  F.  A.  BROWN,  JR.,  345. 
WELSH,  J.  H.,  AND  P.  B.  PROCK.     Quaternary 

ammonium  bases  in  the  coelenterates,  551. 
WERMAN,  R.     See  F.  V.  McCAxx,  356. 
WHITING,  P.  W.,  AND  S.  B.  CASPARI.     Factors 

and  genes  in  Mormoniella,  321. 

WlCHTERMAN,    R.,    H.    SOLOMON   AND    F.    H.    J. 

FIGGE.  The  influence  of  protoporphyrin- 
nitroresorcinol  and  other  phenols  on  x- 
radiation  sensitivity  of  Paramecium,  369. 

WILBER,  C.  G.  Effect  of  temperature  on 
circulation  in  Cistenides,  370. 

WILLEY,  C.  H.  The  morphology  of  the  cope- 
pod  Congericola  from  the  gills  of  Conger 
taken  at  Woods  Hole,  370. 

WILSON,  W.  L.  See  L.  V.  HEILBRUNX,  328, 
459. 

WITTENBERG,  J.  B.  Active  transport  of 
oxygen,  372. 

WITTENBERG,  J.  B.  Carbon  monoxide  in  the 
float  of  Physalia,  371. 

WITTENBERG,  J.  B.     See  R.  FANGE,  172. 

WOOD,  L.     See  A.  K.  PARPART,  340. 

Worm  eggs,  oxidation  of  CO  by,  153. 


Worm  eggs,  oxidative  metabolism  of,  136. 
WRIGHT,  P.  A.     Glucagon  and  blood  glucose 

in  Lophius,  371. 
WURZEL,  M.     Mode  of  action  of  choline  esters. 

Substrate    specificity    of    their    "receptor- 

protein,"  322. 
WURZEL,  M.     See  M.  V.  L.  BENNETT,  331. 


X    of    Fundulus    embryos, 


362. 


X-irradiation  of  Paramecium,  369. 
X-irradiation   of   tadpoles   and    Tetrahymena, 

322. 
X-irradiation  of  Tetrahymena,  366. 

yENTSCH,  C.  S.     See  J.  H.  RYTHER,  257. 
Yttrium-90,  transportation  of  in  fish  blood,  64. 

'TOO-anemonin   in  coelenterate  nematocysts, 

'   551. 
ZWEIFACH,  B.     See  W.   ROSENBLUM,  340;  A. 

NAGLER,  357. 
ZWILLING,    E.     Complete  reconstitution   from 

ectoderm  in  Cordylophora,  319. 


CT 
LiSRARV 


VU1UU1C 


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CONTENTS 


Page 

Annual  Report  of  the  Marine  Biological  Laboratory 1 

BOOLOOTIAN,  RICHARD  A.,  AND  ARTHUR  C.  GIESE 

Coelomic  corpuscles  of  echinoderms 53 

BOROUGHS,  HOWARD,  AND  DELIA  F.  REID 

The  role  of  the  blood  in  the  transportation  of  strontium90- 
yttrium90  in  teleost  fish 64 

BROOKBANK,  JOHN  W. 

Dispersal  of  the  gelatinous  coat  material  of  Mellita  quinquies- 
perf  orata  eggs  by  homologous  sperm  and  sperm  extracts ....  74 

BROWN,  FRANK  A.,  JR. 

An  exogenous  reference-clock  for  persistent,  temperature- 
independent,  labile,  biological  rhythms 81 

EPPLEY,  RICHARD  W.,  AND  CARLTON  R.  BOVELL 

Sulfuric  acid  in  Desmarestia 101 

GRIFFIN,  D.  R.,  A.  NOVICK  AND  M.  KORNFIELD 

The  sensitivity  of  echolocation  in  the  fruit  bat,  Rousettus ...   107 

HODGSON,  EDWARD  S. 

Electrophysiological  studies  of  arthropod  chemoreception. 
III.  Chemoreceptors  of  terrestrial  and  fresh-water  arthropods  114 

MATHEWSON,  ROBERT,  ALEXANDER  MAURO,  ERNEST  AMATNIEK 
AND  HARRY  GRUNDFEST 

Morphology  of  main  and  accessory  electric  organs  of  Narcine 
brasiliensis  (Olfers)  and  some  correlations  with  their  electro- 
physiological  properties 126 

\ 

ROTHSCHILD,  LORD,  AND  ALBERT  TYLER 

The  oxidative  metabolism  of  eggs  of  Urechis  caupo 136 

WATKINS,  MARGARET  J. 

Regeneration  of  buds  in  Botryllus 147 


i  I  II  i