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THE 

BIOLOGICAL  BULLETIN 


PUBLISHED  BY 

THE  MARINE  BIOLOGICAL  LABORATORY 


Editorial  Board 

T.  H.  BULLOCK,  University  of  California,  E.  T.  MOUL,  Rutgers  University 

Los  Angeles  ARTHUR  W.  POLLISTER,  Columbia  University 

E.  G.  BUTLER,  Princeton  University  MARY  E  RAWLES>  johns  Hopkins  University 

K.  W.  COOPER,  University  of  Rochester  BERTA  STARRER,  Albert  Einstein  College 
L.  V.  HEILBRUNN,  University  of  Pennsylvania  of  Medicine 

M.  E.  KRAHL,  University  of  Chicago  J.  H.  WELSH,  Harvard  University 

J.  H.  LOCHHEAD,  University  of  Vermont  A.  R.  WHITING,  University  of  Pennsylvania 

DONALD  P.  COSTELLO,  University  of  North  Carolina 
Managing  Editor 


VOLUME  111 

AUGUST  TO  DECEMBER,  1956 


Printed  and  Issued  by 

LANCASTER  PRESS,  Inc. 

PRINCE  &  LEMON  STS. 

LANCASTER,  PA. 


II 


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,  Department  of  Zoology,  University  of  North 
Carolina,  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,  1956  PAGE 

Annual  Report  of  the  Marine  Biological  Laboratory 1 

CAIN,  GERTRUDE  L. 

Studies  on  cross-fertilization  and  self-fertilization  in  Lymnaea  stagnalis 
appressa  Say 45 

DEHNEL,  PAUL  A.,  AND  EARL  SEGAL 

Acclimation  of  oxygen  consumption  to  temperature  in  the  American 
cockroach  (Periplaneta  americana) 53 

DURAND,  JAMES  B. 

Neurosecretory  cell  types  and  their  secretory  activity  in  the  crayfish .  .      62 

FRASER,  RONALD  C. 

The   presence   and   significance   of   respiratory   metabolism   in   streak- 
forming  chick  blastoderms 77 

FRINGS,  HUBERT,  AND  MABLE  FRINGS 

The  location  of  contact  chemoreceptors  sensitive  to  sucrose  solutions  in 
adult  Trichoptera 92 

HASTINGS,  J.  WOODLAND,  AND  JOHN  BUCK 

The  firefly  pseudoflash  in  relation  to  photogenic  control 101 

LAVOIE,  MARCEL  E. 

How  sea  stars  open  bivalves 114 

ROGICK,  MARY 

Studies  on  marine  bryozoa.     VIII.   Exochella  longirostris  Jullien  1888.  .    123 

SEGAL,  EARL 

Microgeographic    variation    as    thermal    acclimation    in    an    intertidal 
mollusc 129 

TYLER,  ALBERT,  ALBERTO  MONROY,  C.  Y.  KAO  AND  HARRY  GRUNDFEST 

Membrane  potential  and  resistance  of  the  starfish  egg  before  and  after 
fertilization 153 

No.  2.  OCTOBER,  1956 

ANDERSON,  JANE  COLLIER 

Relations  between  metabolism  and  morphogenesis  during  regeneration 

in  Tubifex  tubifex.     II 179 

BILEAU,  SISTER  M.  CLAIRE  OF  THE  SAVIOR 

The  uptake  of  I131  by  the  thyroid  gland  of  turtles  after  treatment  with 
thiourea.  190 


in 


1 88ft 


iv  CONTENTS 

DETHIER,  V.  G.,  D.  R.  EVANS  AND  M.  V.  RHOADES 

Some  factors  controlling  the  ingestion  of  carbohydrates  by  the  blowfiy 

GIBOR,  AARON 

The  culture  of  brine  algae. 

GIBOR,  AARON 

Some  ecological  relationships  between  phyto-  and  zooplankton . 

GOLDSMITH,  TIMOTHY  H.,  AND  DONALD  R.  GRIFFIN 
Further  observations  of  homing  terns.  . 

PARK,  HELEN  D. 

Modification   of  x-ray   injury   to  Hydra   littorahs  by   post-irradiation 
treatment  with  magnesium  sulfate  and  glutathione. 

STUNKARD,  HORACE  W. 

The  morphology  and  life-history  of  the  digenetic  trematode,  Azygia 

sebago  Ward,  1910... 

TURNER,  C.  L. 

Twinning  and  reproduction  of  twins  in  Pelmatohydra  ohgactis.  . 

TWAROG,  B.  M.,  AND  K.  D.   ROEDER 

Properties  of  the  connective  tissue  sheath  of  the  cockroach  abdominal 

9  78 
nerve  cord .... 

Abstracts  of  papers  presented  at  the  Marine  Biological  Laboratory: 

Tuesday  Evening  Seminars 

General  Meetings.  .  .  . 

Lalor  Fellowship  Reports 


No.  3.  DECEMBER,  1956 

AIRTH,  R.  L.,  AND  L.  R.  BLINKS 

A  new  phycoery thrin  from  Porphyra  naiadum 321 

BOOLOOTIAN,  R.  A.,  AND  A.   R.  MOORE 

Hermaphroditism  in  echinoids 

BOROUGHS,  H.,  SIDNEY  J.  TOWNSLEY  AND  ROBERT  W.  HIATT 

The  metabolism  of  radionuclides  by  marine  organisms.  I.  The  uptake, 
accumulation,  and  loss  of  strontium89  by  fishes 336 

BOROUGHS,  H.,  SIDNEY  J.  TOWNSLEY  AND  ROBERT  W.  HIATT 

The  metabolism  of  radionuclides  by  marine  organisms.  1 1 .  The  uptake, 
accumulation,  and  loss  of  yttrium91  by  marine  fish,  and  the  importance 
of  short-lived  radionuclides  in  the  sea 352 

FINGER,  IRVING 

Immobilizing  and  precipitating  antigens  of  Paramecium 358 

Hsu,  W.  SIAN<; 

Oogenesis  in  Habrotrocha  tridens  (Milne) 364 

KENNEDY,  DONALD,  AND  ROGER  D.  MILKMAN 

Selective  light  absorption  by  the  lenses  of  lower  vertebrates,  and  its 
influence  on  spectral  sensitivity 375 


CONTENTS  v 

LpJDSANOFF,  V.  L.,  AND  C.  A.  NOMEJKO 

Relative  intensity  of  oyster  setting  in  different  years  in  the  same  areas 

of  Long  Island  Sound 387 

MOULTON,  JAMES  M. 

Influencing  the  calling  of  sea  robins  (Prionotus  spp.)  with  sound 393 

RASQUIN,  PRISCILLA 

Cytological  evidence  for  a  role  of  the  corpuscles  of  Stannius  in  the 
osmoregulation  of  teleosts 399 

YISHNIAC,  HELEN  S. 

On  the  ecology  of  the  lower  marine  fungi 410 

WILSON,  \YILBOR  O.,  ALLEN  E.  WOODARD  AND  HANS  ABPLANALP 

The  effect  and  after-effect  of  varied  exposure  to  light  on  chicken  de- 
velopment     415 


Vol.  Ill,  No.  1 


August,  1956 


THE 

BIOLOGICAL 


PUBLISHED  BY  THE   MARINE  BIOLOGICAL  LABORATORY 


THE  MARINE  BIOLOGICAL  LABORATORY 

FIFTY-EIGHTH  REPORT,  FOR  THE  YEAR  1955 — SIXTY-EIGHTH  YEAR 

I.     TRUSTEES  AND  EXECUTIVE  COMMITTEE  (AS  OF  AUGUST  12,  1955)    .  .  . 

STANDING  COMMITTEES 
II.     ACT  OF  INCORPORATION   

III.  BY-LAWS  OF  THE  CORPORATION  

IV.  REPORT  OF  THE  DIRECTOR 

Statement   

Addenda : 

1.  The  Staff 

2.  Investigators  and  Students   

3.  The  Lalor  Fellows 

4.  Tabular  View  of  Attendance,  1951-1955  

5.  Subscribing  and  Cooperating  Institutions 

6.  Evening  Lectures 

7.  Shorter  Scientific  Papers  (Seminars)   

8.  Members  of  the  Corporation 

V.     REPORT  OF  THE  LIBRARIAN  

VI.     REPORT  OF  THE  TREASURER 


3 
4 
6 

7 

8 
11 
18 
19 
19 
20 
21 
22 

39 
40 


I.     TRUSTEES 

EX    OFFICIO 

GERARD  SWOPE,  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 

ROBERT  CHAMBERS,  425  Riverside  Drive,  New  York  City 

1 


MARINE  BIOLOGICAL  LABORATORY 

W.  C.  CURTIS,  University  of  Missouri 

B.  M.  DUGGAR,  University  of  Wisconsin 
Ross  G.  HARRISON,  Yale  University 

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

F.  P.  KNOWLTON,  Syracuse  University 

A.  P.  MATHEWS,  University  of  Cincinnati 
W.  J.  V.  OSTERHOUT,  Rockefeller  Institute 
CHARLES  PACKARD,  Woods  Hole,  Mass. 

G.  H.  PARKER,  Harvard  University 
LAWRASON  RIGGS,  120  Broadway,  New  York  City 

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,  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 

E.  B.  HARVEY,  Princeton  University 
ALBERT  I.  LANSING,  Emory  University 
DANIEL  MAZIA,  University  of  California 
S.  MERYL  ROSE,  University  of  Illinois 

ALBERT  TYLER,  California  Institute  of  Technology 

TO   SERVE   UNTIL    1957 

E.  G.  BALL,  Harvard  University  Medical  School 

F.  A.  BROWN,  JR.,  Northwestern  University 

P.  S.  GALTSOFF,  Woods  Hole  Bureau  of  Fisheries 
E.  N.  HARVEY,  Princeton  University 
L.  H.  KLEINHOLZ,  Reed  College 

C.  L.  PROSSER,  University  of  Illinois 

A.  E.  SZENT-GYORGYI,  Institute  for  Muscle  Research 
WM.  RANDOLPH  TAYLOR,  University  of  Michigan 

TO   SERVE   UNTIL   1956 

E.  S.  G.  BARRON,  University  of  Chicago 

D.  W.  BRONK,  Rockefeller  Institute 

G.  FAILLA,  College  of  Physicians  and  Surgeons 
L.  V.  HEILBRUNN,  University  of  Pennsylvania 
R.  T.  KEMPTON,  Vassar  College 

S.  KUFFLER,  Johns  Hopkins  Hospital 

A.  H.  STURTEVANT.  California  Institute  of  Technology 

GEORGE  WALD,  Harvard  University 


TRUSTEES 


EXECUTIVE  COMMITTEE  OF  THE  BOARD  OF  TRUSTEES 


GERARD  SWOPE,  JR.,  Chairman 
A.  K.  PARPART 

J.    H.    WlCKERSHAM 

P.  B.  ARMSTRONG 
F.  A.  BROWN,  JR. 


R.  T.  KEMPTON 

C.  L.  PROSSER 

D.  MAZIA 

D.  P.  COSTELLO 
H.  B.  STEINBACH 


THE  LIBRARY  COMMITTEE 


MARY  SEARS,  Chairman 
HAROLD  F.  BLUM 


E.  G.  BUTLER 
T.  P.  TRINKAUS 


THE  APPARATUS  COMMITTEE 


C.  LLOYD  CLAFF,  Chairman 
T.  H.  BULLOCK 


ALBERT  I.  LANSING 


THE  SUPPLY  DEPARTMENT  COMMITTEE 


RUDOLF  KEMPTON,  Chairman 
C.  B.  METZ 


ROBERT  DAY  ALLEN 
L.  V.  HEILBRUNN 


THE  EVENING  LECTURE  COMMITTEE 


P.  B.  ARMSTRONG,  Chairman 

E.  G.  BALL 

E.  G.  BOETTIGER 


L.  V.  HEILBRUNN 
MAC  V.  EDDS 


THE  INSTRUCTION  COMMITTEE 


S.  MERYL  ROSE,  Chairman 
L.  H.  KLEINHOLZ 


C.  L.  PROSSER 
I.  M.  KLOTZ 


THE  BUILDINGS  AND  GROUNDS  COMMITTEE 


EDGAR  ZWILLING,  Chairman 
RALPH  WICHTERMAN 


C.  B.  METZ 
SEARS  CROWELL 


THE  RADIATION  COMMITTEE 


G.   FAILLA,   Chairman 
CLAUDE  VILLEE 


RAYMOND  ZIRKLE 
ROBERTS  RUGH 


No.  3170 


II.     ACT  OF  INCORPORATION 


COMMONWEALTH  OF  MASSACHUSETTS 


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


MARINE  BIOLOGICAL  LABORATORY 

scientific  study  and  investigation,  and  a  school  for  instruction  in  biology  and  natural  his- 
tory, and  have  complied  with  the  provisions  of  the  statutes  of  this  Commonwealth  in  such 
case  made  and  provided,  as  appears  from  the  certificate  of  the  President,  Treasurer,  and 
Trustees  of  said  Corporation,  duly  approved  by  the  Commissioner  of  Corporations,  and 
recorded  in  this  office; 

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

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

.[SEAL]  HENRY  B.  PIERCE, 

Secretary  of  the  Commonwealth. 


III.     BY-LAWS  OF  THE  CORPORATION   OF  THF   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  by  any  seven  Trustees,  to  be  held  at 
such  time  and  place  as  may  be  designated,  and  the  Secretary  shall  give  notice  thereof  by 
written  or  printed  notice,  mailed  to  each  Trustee  at  his  address  as  shown  on  the  records 
of  the  Corporation,  at  least  one  (1)  week  before  the  meeting.     At  such  special  meeting 


ACT  OF  INCORPORATION  5 

only  matters  stated  in  the  notice  shall  lie  considered.     Seven  Trustees  of  those  eligihle  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  \vho  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  Com- 
mittee 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  shalJ  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  Bi- 
ological Laboratory.     In  case  of  dissolution,  the  property  shall  be  disposed  of  in  such 
manner  and  upon  such  terms  as  shall  be  determined  by  the  affirmative  vote  of  two-thirds 
of  the  Board  of  Trustees. 

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

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


6  MARINE  BIOLOGICAL  LABORATORY 

IV.     REPORT  OF  THE  DIRECTOR 

To  THE  TRUSTEES  OF  THE  MARINE  BIOLOGICAL  LABORATORY  : 
Gentlemen : 

I  submit  herewith  the  report  of  the  sixty-eighth  session  of  the  Laboratory. 

Except  during  some  of  the  World  War  II  years,  the  Laboratory  has  always 
operated  at  capacity  in  the  summer  months.  However,  there  have  always  been 
more  applicants  for  research  space  than  could  be  accommodated.  The  total  num- 
ber of  investigators  at  the  Laboratory  for  the  past  few  years  has  not  been  as  great 
as  immediately  after  the  war.  This  has  been  due  in  part  to  the  diversion  of  some 
laboratories  from  research  to  special  uses  such  as  radioisotope  laboratories,  electron- 
microscopy,  and  for  special  equipment,  in  all,  some  six  laboratories.  Preference  is 
given  to  those  investigators  who  can  devote  the  major  share  of  the  summer  to 
their  projects  and  who  use  the  marine  biological  materials  available  in  the  Woods 
Hole  area.  However,  there  is  not  a  rigid  adherence  to  these  two  factors.  The 
Laboratory  is  primarily  interested  in  fostering  and  promoting  outstanding  biologi- 
cal research,  in  its  broadest  terms,  and  in  developing  scientists.  The  allotment  of 
space,  however,  is  becoming  more  difficult  due  to  an  increasing  number  of  appli- 
cations for  research  space.  For  this  year  an  increased  number  of  well  qualified 
investigators  cannot  be  accommodated. 

1.  Plant  Changes  and  Improvements 

Through  a  grant  of  $50,000  from  the  National  Science  Foundation  the  extensive 
repairs  of  the  hurricane  damage  of  1954  were  completed  and  some  modifications 
were  made  in  the  Main  Building  and  Pump  House  to  prevent  a  recurrence  of  the 
flooding  of  these  buildings  such  as  occurred  in  the  1954  hurricanes.  Included  were 
the  bricking  up  of  some  of  the  basement  windows  in  the  Main  Building  and  in  the 
rump  House  as  well  as  other  stand-by  measures  to  be  used  in  an  emergency.  It 
was  necessary  to  replace  and  relocate  considerable  equipment  which  was  irrepara- 
bly damaged  by  salt  water. 

2.  Pension  Plan 

During  the  past  year  a  study  was  made  of  a  number  of  pension  plans  in  force 
in  other  organizations  with  a  view  to  developing  a  pension  plan  for  the  full-time 
employees  of  the  Laboratory.  A  plan  was  developed  and  became  effective  Sep- 
tember 1,  1955.  The  employees  of  the  Laboratory  were  already  under  Social 
Security.  The  Laboratory's  pension  plan  will  supplement  Social  Security  benefits. 
The  entire  cost  of  the  pension  plan  will  be  borne  by  the  Laboratory.  For  the 
past  26  years  the  Laboratory  has  put  aside  annually  amounts  up  to  10%  of  the 
payroll  for  pension  purposes  so  there  have  accumulated  funds  which  are  adequate 
to  carry  the  plan. 

3.  Grants,  Contracts  and  Contributions 

The  total  income  from  these  sources  of  support  amounted  to  $192,555.94  in 
1955.  This  represents  39%  of  the  total  Laboratory  budget  and  consists  of  the 
following  accounts : 


REPORT  OF  THE  DIRECTOR  7 

Amer.  Cancer  Soc. — 026 — Function  of  Nuclei  and  Nucleic  Acids  ....   $  13,500.00 

Amer.  Cancer  Soc. — R-7F — Fundamental  Studies  in  Radiobiology  .  .  6,600.00 
A.   E.   C.-M.   B.   L.— At  30-1-1343— Program  of   Research   on   the 

Physiology  of  Marine  Organisms  Using  Radioisotopes   7,674.00 

N.  I.  H. — -B6430 — Encephalization  in  Embryonic  Development 1,998.00 

N.  I.  H. — SA43PH  423 — Investigations  of  the  Microscopic  Physiol- 
ogy of  Various  Forms  of  Living  Marine  Life 1,350.00 

N.  I.  H.—B799— Electrical  and  Mechanical  Changes  in  Muscle 864.00 

N.  I.  H. — RG4359 — Biological  Research  on  the  Morphology,  Ecology, 

Physiology,  Biochemistry  and  Biophysics  of  Marine  Organisms  . .  40,000.00 
National    Sci.    Found. — G1469 — Provision    of    Funds    for    Scientific 

Equipment  and  Facilities  for  Biological  Research 50,000.00 

National  Sci.  Found.— G1807— Mechano-Chemical  Coupling  in  Muscle  11,500.00 
National     Sci.     Found. — G1395 — Osmoregulation     of     Excretion     in 

Tunicates    1,708.04 

O.  N.  R.—RG4359— Studies  in  Marine  Biology 15,000.00 

O.  N.  R.— 09701— Studies  on  Isolated  Nerve  Fibers 6,359.34 

O.  N.  R.— 09702 — Investigation  of  Environmental  Factors  Influenc- 
ing Certain  Marine  Biological  Populations  in  the  Woods  Hole  Area  4,437.56 

American  Philosophical  Soc 2,500.00 

M.  B.  L.  Associates   2,940.00 

Eli  Lilly  Co 5,000.00 

Rockefeller  Found 20,000.00 

Other  .  1,125.00 


$192,555.94 


4.  Instruction 


Dr.  Daniel  Mazia  completed  five  years  as  head  of  the  course  in  Physiology  in 
1955  and  will  be  succeeded  by  Dr.  Stephen  Kurfier.  Dr.  S.  Meryl  Rose  also  com- 
pleted a  five-year  term  as  head  of  the  course  in  Embryology  and  will  be  succeeded 
by  Dr.  Mac  V.  Edds.  The  success  of  these  courses  is  attested  by  the  large  number 
of  applicants  for  admission  to  both  of  the  courses.  The  Laboratory  is  to  be  con- 
gratulated in  having  such  excellent  leadership  in  its  various  courses. 

5.  Fclloivships  and  Scholarships 

Under  a  new  plan  all  of  the  Lalor  Fellows  will  be  selected  by  a  panel  set  up  by 
the  Lalor  Foundation.  The  successful  applicants  who  elect  to  work  at  the  Marine 
Biological  Laboratory  will  be  permitted  to  do  so.  It  is  hoped  that  under  this  new 
plan  the  Laboratory  will  continue  to  have  a  fair  number  of  Lalor  Fellows.  The 
Laboratory  has  much  to  offer  these  young  postdoctoral  fellows  and  would  not 
realize  its  full  educational  potentialities  without  them.  Also  these  fellowships  have 
been  of  real  value  to  the  Laboratory,  resulting  in  the  recruitment  of  highly  quali- 
fied investigators  in  subsequent  years. 


MARINE  BIOLOGICAL  LABORATORY 

6.  Losses  by  Death 

Since  the  last  report  the  Laboratory  has  lost  through  death  two  eminent  scien- 
tists who  served  for  many  years  on  the  Board  of  Trustees,  Professor  George 
Howard  Parker  and  Professor  Warder  Clyde  Alice.  Both  of  these  scientists  con- 
tributed in  an  effective  way  to  the  prestige  and  development  of  the  Laboratory. 

Respectfully  submitted, 

PHILIP  B.  ARMSTRONG, 

Director 

1.  THE  STAFF,  1955 

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

SENIOR  STAFF  OF  INVESTIGATION 

A.  P.  MATHEWS,  Professor  of  Biochemistry,  Emeritus,  University  of  Cincinnati 
G.  H.  PARKER,  Professor  of  Zoology,  Emeritus,  Harvard  University 

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,  Associate  Professor  of  Zoology,  University  of  California,  Los 

Angeles,  in  charge  of  course 

JOHN  H.  LOCHHEAD,  Associate  Professor  of  Zoology,  University  of  Vermont 
NORMAN  A.  MEINKOTH,  Associate  Professor  of  Zoology,  Swarthmore  College 
GROVER  STEPHENS,  Assistant  Professor  of  Zoology,  University  of  Minnesota 
JOHN  M.  ANDERSON,  Associate  Professor  of  Zoology,  Cornell  University 
MURIEL  SANDEEN,  Department  of  Zoology,  Duke  University 
L.  M.  PASSANO,  Department  of  Zoology,  University  of  Washington,  Seattle 
MORRIS  ROCKSTEIN,  Department  of  Physiology,  New  York  University,  Bellevue  Medical 

Center 

III.     LABORATORY  ASSISTANTS 

ALLISON  BURNETT,  Ithaca,  New  York 
DOROTHY  M.  SKINNER,  Tufts  College 

EMBRYOLOGY 

I.     INSTRUCTORS 

S.  MERYL  ROSE,  Associate  Professor  of  Zoology,  University  of  Illinois,  in  charge  of  course 

CLIFFORD  GROBSTEIN,  National  Cancer  Institute 

MAC  V.  EDDS,  JR.,  Associate  Professor  of  Biology,  Brown  University 

NELSON  T.  SPRATT,  JR.,  Professor  of  Zoology,  University  of  Minnesota 

J.  P.  TRINKAUS,  Assistant  Professor  of  Zoology,  Yale  University 

EDGAR  ZWILLING,  Associate  Professor  of  Genetics,  University  of  Connecticut 


REPORT  OF  THE  DIRECTOR  () 

II.  LABORATORY  ASSISTANTS 

JOAN  K.  ERICKSEN,  Radcliffe  College 
ROGER  D.  MILKMAN,  Harvard  University 

PHYSIOLOGY 

I.  CONSULTANTS 

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

OTTO  LOEWI,  Professor  of  Pharmacology,  New  York  University,  School  of  Medicine 

ARTHUR  K.  PARPART,  Professor  of  Biology,  Princeton  University 

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

E.  S.  GUZMAN  BARRON,  Associate  Professor  of  Biochemistry,  University  of  Chicago 

II.  INSTRUCTORS 

DANIEL  MAZIA,  Associate  Professor  of  Zoology,  University  of  California,  in  charge  of 
course 

HERMAN  M.  KALCKAR,  National  Institutes  of  Health 

STEPHEN  KUFFLER,  Associate  Professor  of  Ophthalmology,  Wilmer  Institute,  Johns  Hop- 
kins University  Medical  School 

MAX  A.  LAUFFER,  Professor  and  Head  of  Dept.  of  Biophysics,  University  of  Pittsburgh 

GEORGE  WALD,  Professor  of  Biology,  Harvard  University 

ANDREW  SZENT-GYORGYI,  Independent  Investigator,  The  Institute  for  Muscle  Research 

III.  LABORATORY  ASSISTANT 
PAUL  BERNSTEIN,  Department  of  Zoology,  Columbia  University 

BOTANY 

I.  CONSULTANTS 

MAXWELL  S.  DOTY,  Associate  Professor  of  Botany,  University  of  Hawaii 
WM.  RANDOLPH  TAYLOR,  Professor  of  Botany,  University  of  Michigan 

II.  INSTRUCTORS 

HAROLD  C.  BOLD,  Professor  of  Biology,  Vanderbilt  University,  in  charge  of  course 

ROBERT  W.  KRAUSS,  University  of  Maryland 

RICHARD  C.  STARR,  Instructor  in  Botany,  University  of  Indiana 

III.    LABORATORY  ASSISTANTS 
FRANCIS  R.  TRAINOR,  Vanderbilt  University 

IV.     COLLECTOR 
ANN  ALLEN,  University  of  Indiana 

MARINE  ECOLOGY 

I.     CONSULTANTS 
ALFRED  C.  REDFIELD,  Woods  Hole  Oceanographic  Institution 


10  MARINE  BIOLOGICAL  LABORATORY 

II.  INSTRUCTORS 

BOSTWICK  H.  KETCHUM,  Marine  Microbiologist,  Woods  Hole   Oceanographic  Institu- 
tion, in  charge  of  course 

EDWIN  T.  MOUL,  Assistant  Professor  of  Botany,  Rutgers  University 
CHARLES  JENNER,  Associate  Professor  of  Zoology,  University  of  North  Carolina 

III.  ASSISTANT 
RUDOLF  SCHELTEMA,  George  Washington  University 

THE  LABORATORY  STAFF,  1955 
HOMER  P.  SMITH,  General  Manager 

MRS.  DEBORAH  LAWRENCE  HARLOW,  ROBERT  KAHLER.  Superintendent, 

Librarian  Buildings  and  Grounds 

JAMES  MC!NNIS,  Manager  of  Supply  ROBERT  B.  MILLS,  Manager,  De- 
Department  partment  of  Research  Service 

GENERAL  OFFICE 

IRVINE  L.  BROADBENT 

POLLY  L.  CROWELL  NANCY  SHAVE 

MRS.  LILA  MYERS  ELIZABETH  CORRELLUS 

LIBRARY 

MARY  E.  CASTELLANO,  Assistant  Librarian 
MARY  A.  ROHAN  NAOMI  BOTELHO 

ALBERT  NEAL 

MAINTENANCE  OF  BUILDINGS  AND  GROUNDS 

ROBERT  ADAMS  JOHN  HEAD 

EDMOND  BOTELHO  GEORGE  A.  KAHLER 

ARTHUR  CALLAHAN  DONALD  B.  LEHY 

ROBERT  GUNNING  ALTON  J.  PIERCE 

JAMES  S.  THAYER 

DEPARTMENT  OF  RESEARCH  SERVICE 

GAIL  M.  CAVANAUGH  SEAVER  HARLOW 

JOHN  P.  HARLOW  PATRICIA  PHILPOTT 

SUPPLY  DEPARTMENT 

RUTH  S.  CROWELL  GEOFFREY  LEHY 

MILTON  B.  GRAY  ROBERT  O.  LEHY 

WALTER  E.  KAHLER  CARL  SCHWEIDENBACK 

ROBERT  PERRY  BRUNO  TRAPASSO 

PATRICIA  M.  CONWAY  JAMES  P.  WHITCOMB 

H.  S.  WAGSTAFF 


REPORT  OF  THE  DIRECTOR 


11 


BIOLOGICAL  BULLETIN 

DONALD  P.  COSTELLO,  Managing  Editor 

University  of  North  Carolina,  Dept.  of  Zoology 
Chapel  Hill,  North  Carolina 

CATHERINE  HENLEY,  Assistant  to  the  Editor 

2.  INVESTIGATORS  AND  STUDENTS 
Independent  Investigators,  1955 

ABBOTT,  ROBINSON  SHEWELL,  Assistant  Professor  of  Botany,  Cornell  University 

AGNEW',  L.  R.  C.,  Research  Fellow,  Department  of  Nutrition,  Harvard  University 

ALLEN,  ROBERT  DAY,  Instructor  in  Zoology,  University  of  Michigan 

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

ARMSTRONG,"  PHILIP  B.,  Professor  of  Anatomy,  State  Univ.  of  New  York,  College  of  Medicine 

ARNOLD,  WILLIAM  A.,  Scientific  Investigator,  Oak  Ridge  National  Laboratory 

BARTON,  JAY,  Assistant  Professor  of  Zoology,  Columbia  University 

BENNETT,  MIRIAM  F.,  Instructor  in  Biology,  Sweet  Briar  College 

BERGER,  CHARLES  A.,  Chairman,  Department  of  Biology,  Fordham  University 

BLUM,  HAROLD  E.,  Physiologist,  Princeton  University 

BOETTIGER,  EDWARD  G.,  Associate  Professor,  University  of  Connecticut 

BOLD,  HAROLD  C.,  Vanderbilt  University 

BRIDGMAN,  JOSEPHINE,  Professor  of  Biology,  Agnes  Scott  College 

BROWN,  FRANK  A.,  JR.,  Chairman,  Dept.  of  Biological  Sciences,  Northwestern  University 

BRUMMETT,  ANNA  RUTH,  Instructor  in  Biology,  Carleton  College 

BULLOCK,  THEODORE  H.,  Associate  Professor  of  Zoology,  Univ.  of  California,  Los  Angeles 

BURBANCK,  W.  D.,  Department  of  Biology,  Emory  University 

BUTLER,  ELMER  G.,  Professor  of  Zoology,  Princeton  University 

CHAET,  A.  B.,  Instructor  in  Zoology,  University  of  Maine 

CHANG,  C.  Y.,  Research  Associate,  State  University  of  Iowa 

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

CHENEY,  RALPH  HOLT,  Professor  of  Biology,  Brooklyn  College 

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

CLARK,  ELLIOT  R.,  Professor  Emeritus  of  Anatomy,  Univ.  of  Pennsylvania  School  of  Medicine 

CLAYTON,  RODERICK  K.,  Associate  Professor  of  Physics,  U.  S.  Naval  P.  G.  School 

CLOWES,  G.  H.  A.,  Research  Director  Emeritus,  Eli  Lilly  and  Company 

COLE,  KENNETH  S.,  Chief,  Laboratory  of  Biophysics,  National  Institutes  of  Health 

COLWIN,  ARTHUR  L.,  Associate  Professor  and  Lecturer,  Queens  College 

COOPERSTEIN,  SHERWIN  J.,  Assistant  Professor  of  Anatomy,  Western  Reserve  Univ.  School  of 

Medicine 

CORLISS,  CLARK  E.,  Instructor  in  Anatomy,  University  of  Tennessee 
COSTELLO,  DONALD  P.,  Kenan  Professor  of  Zoology,  Univ.  of  North  Carolina 
CROWELL,  SEARS,  Assistant  Professor  of  Zoology,  Indiana  University 
CSAPO,  A.,  Carnegie  Institution  of  Washington 

DWYER,  JOHN  D.,  Director,  Dept.  of  Biology,  St.  Louis  University 
EDDS,  M.  V.,  JR.,  Associate  Professor  of  Biology,  Brown  University 
ELLIOTT,  ALFRED  M.,  Associate  Professor  of  Zoology,  University  of  Michigan 
FAILLA,  G.,  Professor,  Columbia  University 

FiTzHuGH,  RICHARD,  Instructor  in  Physiological  Optics,  Johns  Hopkins  University 
FLAVIN,  MARTIN,  JR.,  Postdoctoral  Fellow,  New  York  University 
FREYGANG,  WALTER  H.,  JR.,  S.  A.  Surg.  (R)  U.  S.  Public  Health  Service 
GALL,  JOSEPH  G.,  Instructor  in  Zoology,  University  of  Minnesota 
GAMOW,  GEORGE,  Professor,  George  Washington  University 

GASTEIGER,  EDGAR  L.,  Assistant  Professor  of  Physiology,  Harvard  Medical  School 
GILMAN,  LAUREN  C.,  Associate  Professor  of  Zoology,  University  of  Miami 
GREEN,  JAMES  W.,  Assistant  Professor  of  Physiology,  Rutgers  University 


' 


12  MARINE  BIOLOGICAL  LABORATORY 

GREEN,  MAURICE,  Instructor  of  Biochemistry,  Childrens  Hospital,  University  of  Pennsylvania 

GREGG,  JAMES  H.,  Assistant  Professor  of  Biology,  University  of  Florida 

GROBSTEIN,  CLIFFORD,  Biologist,  National  Cancer  Institute 

GROSCH,  DANIEL  S.,  Associate  Professor  of  Genetics,  North  Carolina  State  College 

GRUNDFEST,  HARRY,  Associate  Professor  of  Neurology,  College  of  Physicians  and  Surgeons 

GUTTMAN,  RITA,  Assistant  Professor  of  Biology,  Brooklyn  College 

HAGIWARA,  SUSUMU,  Research  Associate,  University  of  California,  Los  Angeles 

HARVEY,  ETHEL  BROWNE,  Independent  Investigator,  Biology  Department,  Princeton  University 

HARVEY,  E.  NEWTON,  Professor  of  Physiology,  Princeton,  New  Jersey 

HAY,  ELIZABETH  D.,  Instructor  in  Anatomy,  Johns  Hopkins  University  School  of  Medicine 

HAYWOOD,  CHARLOTTE,  Professor  of  Physiology,  Mount  Holyoke  College 

HEILBRUNN,  L.  V.,  Zoological  Laboratory,  University  of  Pennsylvania 

HERVEY,  JOHN  P.,  Electronic  Engineer,  Rockefeller  Institute  for  Medical  Research 

HOLZ,  GEORGE  G.,  JR.,  Assistant  Professor  of  Zoology,  Syracuse  University 

HOWARD,  ROBERT  STEARNS,  Assistant  Professor  of  Biology,  University  of  Delaware 

HYDE,  BEAL  B.,  Assistant  Professor  of  Plant  Sciences,  University  of  Oklahoma 

JACOBS,  M.  H.,  Professor  of  General   Physiology,  Medical  School,  University  of  Pennsylvania 

JENNER,  CHARLES  E.,  Associate  Professor  of  Zoology,  University  of  North  Carolina 

JENKINS,  GEORGE  B.,  Emeritus  Professor  of  Anatomy,  George  Washington  University 

KALCKAR,  HERMAN  M.,  Visiting  Scientist,  National  Institutes  of  Health 

KAVANAU,  J.  LEE,  Assistant,  Rockefeller  Institute  for  Medical  Research 

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

KEOSIAN,  JOHN,  Professor  of  Biology,  Rutgers  University 

KIND,  C.  ALBERT,  Assistant  Professor  of  Zoology,  University  of  Connecticut 

KING,  JOHN  W.,  Professor  of  Biology,  Morgan  State  College 

KLEINHOLZ,  L.  H.,  Professor  of  Biology,  Reed  College 

KLOTZ,  IRVING  M.,  Professor  of  Chemistry,  Northwestern  University 

KRAHL,  MAURICE  E..  Professor  of  Physiology,  University  of  Chicago 

KUFFLER,  STEPHEN  W.,  Associate  Professor  of  Ophthalmology,  Johns  Hopkins  Hospital 

KUNKEL,  HENRY  G.,  Rockefeller  Institute 

KUNTZ,  ELOISE,  Assistant  Professor,  Vassar  College 

LANSING,  ALBERT  L,  Professor  of  Anatomy,  Emory  University 

LAUFFER,  MAX  A.,  Professor  and  Head  of  Dept.  of  Biophysics,  University  of  Pittsburgh 

LAZAROW,  ARNOLD,  Professor  and  Head  of  Dept.  of  Anatomy,  University  of  Minnesota 

LEVINE,  ROBERT  P.,  Assistant  Professor,  Harvard  University 

LEWIN,  RALPH  A.,  National  Research  Council,  Maritime  Regional  Laboratory,  Halifax,  N.  S. 

LEVY,  MILTON,  Professor,  New  York  University,  Bellevue  Medical  Center 

LLOYD,  DAVID  P.  C.,  Rockefeller  Institute  for  Medical  Research 

LOCHHEAD,  JOHN  H.,  Professor  of  Zoology,  University  of  Vermont 

LOVE,  WARNER  E.,  Associate,  Johnson  Foundation,  Maloney  Clinic 

LYNCH,  WILLIAM  F.,  Professor  of  Biology,  St.  Ambrose  College 

MCCLEMENT,  PATRICIA,  Research  Scientist,  Columbia  University 

MARSLAND,  DOUGLAS,  Professor  of  Biology,  New  York  University,  Washington  Square  College 

MACCHI,  ITALO  A.,  Assistant  Professor  of  Physiology,  Clark  University 

MAZIA,  DANIEL,  Professor  of  Zoology,  University  of  California 

MEINKOTH,  NORMAN  A.,  Associate  Professor  of  Biology,  Swarthmore  College 

MENKIN,  VALY,  Head  of  Experimental  Pathology,  Temple  University  School  of  Medicine 

METZ,  CHARLES  B.,  Associate  Professor  of  Zoology,  Florida  State  University 

MILKMAN,  ROGER,  Teaching  Fellow,  Harvard  University 

MILLER,  JAMES  A.,  Professor  of  Anatomy,  Emory  University 

MOORE,  JOHN  W.,  Biophysicist,  National  Institutes  of  Health 

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

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

NACE,  PAUL  FOLEY,  Associate  Professor  of  Anatomy,  New  York  Medical  College 

NELSON,  LEONARD,  Assistant  Professor  of  Physiology,  LIniversity  of  Nebraska 

O'MALLEY,  BENEDICT  B.,  160  West  88  Street,  New  York  City  24,  New  York 

OOMURA,  YUTAKA,  Research  Associate,  Neuropsychiatric  Institute,  University  of  Illinois 

OSTERHOUT,  W.  J.  V.,  Member  Emeritus,  Rockefeller  Institute  for  Medical  Research 


REPORT  OF  THE  DIRECTOR  13 

PARKER,  JOHNSON,  Assistant  Professor  of  Botany,  University  of  Idaho 
PASSANO,  LEONARD  M.,  Instructor,  University  of  Washington 
PIERCE,  MADELENE  E.,  Professor  of  Zoology,  Vassar  College 
PLOUGH,  HAROLD  H.,  Professor  of  Biology,  Amherst  College 
PROSSER,  C.  LADD,  Professor  of  Physiology,  University  of  Illinois 
RAY,  CHARLES,  JR.,  Assistant  Professor  of  Biology,  Emory  University 
RAY,  DAVID  T.,  Instructor  of  Zoology,  Howard  University 

ROCKSTEIN,  MORRIS,  Assistant  Professor  of  Physiology,  New  York  University,  Bellevue  Medi- 
cal Center 

ROGERS,  K.  T.,  Assistant  Professor  of  Zoology,  Oberlin  College 
ROYS,  CHESTER,  Research  Associate,  Tufts  College 
RUBEN,  LAURENS  NORMAN,  Princeton  University 

Rur.it,  ROBERTS,  Associate  Professor  of  Radiology,  Columbia  University 
SANDEEN,  MURIEL  I.,  Assistant  Professor  of  Zoology,  Duke  University 
SCHECHTER,  VICTOR,  Associate  Professor  of  Biology,  City  College  of  New  York 
SCOTT,  ALLAN,  Professor  of  Biology,  Colby  College 

SCOTT,  SISTER  FLORENCE  MARIE,  Professor  of  Biology,  Seton  Hill  College 
SCOTT,  GEORGE  T.,  Professor  of  Zoology,  Oberlin  College 

SEAMAN,  GERALD  R.,  Associate  Professor  of  Physiology,  University  of  Texas  Medical  Branch 
SHEIILOVSKY,  THEODORE,  Rockefeller  Institute  for  Medical  Research 
SLIFER,  ELEANOR  H.,  Associate  Professor  of  Zoology,  State  University  of  Iowa 
SMALL,  JEAN  E.,  Graduate  Student,  Brown  University 

SOLOMON,  SIDNEY,  Assistant  Professor  of  Physiology,  Medical  College  of  Virginia 
SPIEGEL,  MELVIN,  Research  Fellow,  California  Institute  of  Technology 
SPEIDEL,  CARL  C.,  Professor  and  Chairman,  Dept.  of  Anatomy,  University  of  Virginia 
SPYROPOULOS,  CONSTANTINE,  Assistant  Scientist,  National  Institutes  of  Health 
STARR,  RICHARD  C.,  Assistant  Professor  of  Botany,  Indiana  University 
STEELE,  R.  H.,  Research  Fellow,  Muscular  Dystrophy  Association  of  America 
STEINBACH,  H.  B.,  Professor  of  Zoology,  University  of  Minnesota 
STEINBERG,  MALCOM  S.,  Graduate  Student,  University  of  Minnesota 
STEINHARDT,    JACINTO,    Director,    Operations    Evaluation    Group,    Massachusetts    Institute    of 

Technology 

STEPHENS,  GROVER  C.,  Instructor  in  Zoology,  University  of  Minnesota 
STEPHENSON,  WILLIAM  K.,  Assistant  Professor  of  Biology,  Earlham  College 
STOREY,  ALMA  G.,  Emeritus  Professor  of  Plant  Science,  Mount  Holyoke  College 
STUNKARD,  HORACE  W.,  Professor  Emeritus  of  Biology,  New  York  University 
SZENT-GYORGYI,  A.  E.,  Institute  for  Muscle  Research  at  Marine  Biological  Laboratory 
TAKAGI,  SADAYUKI,  Research  Associate,  Neuropsychiatric  Institute,  University  of  Illinois 
TASAKI,  ICHIJI,  Visiting  Scientist,  National  Institutes  of  Health 
TAYLOR,  WM.  RANDOLPH,  Professor  of  Botany,  University  of  Michigan 
TRAUTWEIN,  WOLFGANG,  Fellow,  Johns  Hopkins  University  Medical  School 
TRINKAUS,  J.  P.,  Assistant  Professor  of  Zoology,  Osborn  Zoological  Laboratory,  Yale  University 
TYLER,  ALBERT,  Professor  of  Embryology,  California  Institute  of  Technology 
URETZ,  ROBERT  B.,  Instructor  in  Biophysics,  University  of  Chicago 

VINCENT,  WALTER  S.,  Instructor  in  Anatomy,  State  University  of  New  York  College  of  Medicine 
WARNER,  ROBERT  C.,  Associate  Professor,  New  York  University-Bellevue  Medical  Center 
WEBB,  MARGUERITE,  Assistant  Professor  of  Physiology,  Goucher  College 
WEISS,  PAUL,  Rockefeller  Institute  for  Medical  Research 
WEISZ,  PAUL  B.,  Associate  Professor  of  Biology,  Brown  University 
WHITING,  P.  W.,  Professor  of  Zoology,  University  of  Pennsylvania 
WICHTERMAN,  RALPH,  Professor  of  Biology,  Temple  University 

WILBER,  CHARLES  G.,  Chief,  Animal  Ecology  Branch,  Chemical  Corps  Medical  Laboratories 
WILCZYNSKI,   J.,    Professor    of   General    Biology   and    Genetics,    Lebanese    University,    Beirut, 

Lebanon 

WILBRANDT,  WALTER,  Head  of  Dept.  of  Pharmacology,  University  of  Berne,  Switzerland 
WILLEY,  CHARLES  H.,  Professor  of  Biology,  New  York  University,  Heights 
WILSON,  WALTER   L.,   Assistant   Professor   of   Physiology,   University  of  Vermont   College   of 
Medicine 


14  MARINE  BIOLOGICAL  LABORATORY 

YNTEMA,  CHESTER  L.,  Professor  of  Anatomy,  State  University  of  New  York  Upstate  Medical 

Center 

YOUNG,  R.  T.,  University  of  Maryland 

ZIRKLE,  RAYMOND  E.,  Professor  of  Radiobiology,  University  of  Chicago 
ZWEIFACH,   BENJAMIN   W.,   Associate    Prof,   of   Biology,   New   York   University,    Washington 

Square  College 
ZWILLING,  EDGAR,  Associate  Professor,  University  of  Connecticut 

Beginning  Investigators,  1955 

AIELLO,  EDWARD,  Assistant  in  Zoology,  Columbia  University 

BRADFORD,  WILLIAM  DALTON,  Medical  Student,  Western  Reserve  University  School  of  Medicine 

DRAKE,  JOHN  W.,  Graduate  Assistant,  California  Institute  of  Technology 

GEIGER,  H.  JACK,  Student,  Western  Reserve  University 

KAYE,  ALVIN  M.,  Assistant  Instructor,  University  of  Pennsylvania 

LARIS,  PHILIP  C,  Graduate  Student,  Princeton  University 

LAVOIE,  MARCEL  E.,  Syracuse  University 

McKiNNELL,  ROBERT  GILMORE,  University  of  Minnesota 

MORRILL,  JOHN  B.,  Florida  State  University 

Research  Assistants,  1955 

AOTO,  TOMOJI,  Research  Assistant,  State  University  of  lm\u 

ADAMS,  TERRY,  Massachusetts  Horticultural  Society 

ALLEN,  M.  ANN,  Indiana  University 

ALLERAND,  C.,  Albany  Medical  College 

BALABANIS,  REBECCA,  South  Milwaukee,  Wisconsin 

BASCH,  PAUL  FREDERICK,  University  of  Michigan 

BIRSKY,  BILL,  Indiana  University 

BROWN,  ROBERT  A.,  Northwestern  University 

BOWLING,  JOHN  ALAN,  Harvard  College 

ELLIS,  GORDON  W.,  University  of  California 

ERICKSON,  JOAN,  Radcliffe  College 

FRIZ,  CARL  T.,  University  of  Minnesota 

KAHN,  KENNETH,  University  of  Pennsylvania 

LACHANCE,  LEO  E.,  North  Carolina  State  College 

LEFKOWITZ,  LEWIS  B.,  Southwestern  Medical  School 

NATHANSON,  DONALD  L.,  Amherst  College 

OBERLANDER,  MARCIA  L,  State  University  of  New  York  College  of  Medicine  at  Syracuse 

RAFFERTY,  KEEN  A.,  JR.,  University  of  Illinois 

REGEHR,  HULDA,  University  of  Minnesota 

SHELBURNE,  JAMES  CHRISTIE,  Emory  University 

SKINNER,  DOROTHY  M.,  Radcliffe  College 

WATT,  DONALD,  Columbia  University  College  of  Physicians  and  Surgeons 

WILT,  FRED,  Indiana  University 

ZIMMERMAN,  ARTHUR  M.,  New  York  University,  Washington  Square  College 

Library  Readers,  1955 

BECK,  LYLE  V.,  Associate  Professor  of  Physiology,  University  of  Pittsburgh  School  of  Medicine 

BEUTNER,  REIN  HARD  H.,  Des  Moines  Still  College  of  Osteopathy 

BODANSKY,  OSCAR,  Attending  Clinical  Biochemist,  Sloan-Kettering  Institute 

CROUSE,  HELEN  V.,  Associate  Professor,  Goucher  College 

DEAN,  HELEN  WENDLER,  Cambridge,  Mass. 

DuBois,  EUGENE,  Emeritus  Professor  of  Physiology,  Cornell  University  Medical  College 

EICHEL,  HERBERT  J.,  Research  Associate  in  Biological  Chemistry,  Hahnemann  Medical  College 

FREUND,  JULES,  Public  Health  Research  Institute  of  the  City  of  New  York 

GABRIEL,  MORDECAI,  Assistant  Professor,  Brooklyn  College 


REPORT  OF  THE  DIRECTOR  15 

GAFFRON,  HANS,  Professor  of  Biochemistry,  University  of  Chicago 

GINSBERG,  HAROLD  S.,  Associate  Professor  of  Preventive  Medicine,  Western  Reserve  Univer- 
sity School  of  Medicine 

GLASS,  H.  BENTLEY,  Professor  of  Biology,  Johns  Hopkins  University 

GOLDTHWAIT,  DAVID,  Associate  Member,  Dept.  of  Biochemistry,  Western  Reserve  University 
GRANT,  PHILIP,  Research  Associate,  Institute  for  Cancer  Research 

GREIF,  ROGER  L.,  Associate  Professor  of  Physiology,  Cornell  University  Medical  College 
GUDERNATSCH,  FREDERICK,  Cornell  University  Medical  College 
JOHANSSON,  ARNE,  Foreign  Operations  Administration,  University  of  Colorado 
JOHNSON,  THOMAS  N.,  Assistant  Professor  of  Anatomy,  George  Washington  University  Medi- 
cal School 

JONES,  SARAH  R.,  Instructor  in  Zoology,  Connecticut  College 

KABAT,  ELVIN  A.,  Professor  of  Microbiology,  College  of  Physicians  and  Surgeons 
KARUSH,   FRED,   Associate   Professor   of   Immunology,   University   of   Pennsylvania    School   of 

Medicine 

KATZ,  Louis  NELSON,  Professorial  Lecturer  in  Physiology,  University  of  Chicago 
KERSCHNER,  JEAN,  Assistant  Professor,  Western  Maryland  College 
KINDRED,  JAMES  E.,  Professor  of  Anatomy,  University  of  Virginia  School  of  Medicine 
KINERSLY,  THORN,  Research  Fellow,  Yale  University  School  of  Medicine 
KLEINFELD,  RUTH  G.,  Postdoctoral  Fellow,  National  Cancer  Institute,  Ohio  State  University 
KOLIN,  ALEXANDER,  Associate  Professor  of  Physics,  University  of  Chicago 
LEVINE,  RACHMIEL,  Chairman,  Dept.  of  Medicine,  Michael  Reese  Hospital 
LIPPMAN,  HEINZ  I.,  Assistant  Clinical  Professor,  Albert  Einstein  College  of  Medicine 
LOEWI,  OTTO,  Research  Professor  of  Pharmacology,  N.  Y.  U.,  College  of  Medicine 
LOVE,  Lois  H.,  Research  Associate,  National  Research  Council 
LOEWENFELD,  IRENE  E.,  Research  Technician,  Columbia  University 
LOWENSTEIN,  OTTO,  Research  Associate,  Columbia  University 
MCDONALD,  SISTER  ELIZABETH  SETON,  College  of  Mount  St.  Joseph  on  the  Ohio 
MILSTEIN,  SEYMOUR  W.,  Research  Associate,  Hahnemann  Medical  College 
NACHMANSOHN,  DAVID,  Professor  of  Biochemistry,  Columbia  University 
PEQUEGNAT,  WILLIS,  Professor  of  Zoology,  Pomona  College 

PICK,  JOSEPH,  Associate  Professor  of  Anatomy,  N.  Y.  U.  Bellevue  Medical  Center 
RAVIN,  ARNOLD  W.,  Assistant  Professor  of  Biology,  University  of  Rochester 
ROBERT,  NAN  L.,  Instructor  in  Biological  Sciences,  Hunter  College 
ROOT,  WALTER  S.,  Professor  of  Physiology,  College  of  Physicians  and  Surgeons 
ROTH  STEIN,  FRED,  Hahnemann  Medical  College 
SCHLESINGER,  R.  WALTER,  Associate  Member,  The   Public   Health  Research   Institute  of  New 

York 

SCHNEYER,  LEON  H.,  Associate  Professor  of  Clinical  Dentistry,  College  of  Alabama 
SCHWABE,  LOUISE  A.,  Science  Teacher,  Kenmore  Senior  High  School 
SMELSER,  GEORGE  K.,  Professor  of  Anatomy,  Columbia  University 
SULKIN,  S.  EDWARD,  Professor  of  Microbiology,  Southwestern  Medical  School 
TAUBER,  HANS-LUKAS,  Assistant  Professor,  New  York  University  College  of  Medicine 
WAINIO,  WALTER  W.,  Associate  Professor  of  Biochemistry,  Rutgers  University 
WAKSMAN,  BRYON  H.,  Associate  in  Bacteriology,  Harvard  University 
WEIDMAN,  S.,  State  University  of  New  York  College  of  Medicine  at  Brooklyn 
WHEELER,  GEORGE  E.,  Instructor  in  Biology,  Brooklyn  College 

Students,  1955 
BOTANY 

AHMADJIAN,  VERNON,  Clark  University 

ARCE,  GINA,  Vanderbilt  University 

CASHMAN,  MARJEAN  L.,  University  of  Maryland 

COURTENAY,  WALTER  ROWE,  JR.,  Vanderbilt  University 

Cox,  SAMUEL  F.,  Vanderbilt  University 

CROSS,  CAROLINE  B.,  Acadia  University 


16  MARINE  BIOLOGICAL  LABORATORY 

EIGER,  JOAN  V.,  City  College  of  New  York 

FREUDENTHAL,  HUGO  D.,  Columbia  University,  College  of  Pharmacy 

GALLOWAY,  RAYMOND  A.,  University  of  Maryland 

GATES,  JOHN,  Cornell  University 

GREEN,  PAUL  B.,  Princeton  University 

HILFERTY,  FRANK,  State  Teachers  College 

HOFFER,  JOSEPH  L.,  Fordham  University 

LAMB,  IVAN  MACKENZIE,  Harvard  University 

OVERSTREET,  ROSE  ALICE,  Indiana  University 

POKORNY,  FRANK  J.,  St.  John's  University 

RADER,  PHILIP  SCOTT,  Middle  Tennessee  State  College 

SCHELTEMA,  RUDOLF  S.,  Harvard  University 

WILSON,  VANNIE  WILLIAM,  Morgan  State  College 

EMBRYOLOGY 

BAGNARA,  JOSEPH  T.,  State  University  of  Iowa 

BEARD,  ROBERT  GORDON,  Indiana  University 

BORODACH,  GEROLD  N.,  Brown  University 

BOURKE,  ROBERT  SAMUEL,  Harvard  University 

CAREY,  FRANCIS  GERALD,  Harvard  University 
"  DAVIS,  ROWLAND  HALLOWELL,  Harvard  University 

DE  LA  PAZ,  JUSTO,  Cornell  University 

DE  TERRA,  NOEL,  Barnard  College 

GOLDSTEIN,  JOEL  B.,  Haverford  College 

GREENLESS,  JANET  LUCILE,  University  of  Wisconsin 
*  HARRIS,  PATRICIA  J.,  Yale  University 

HICKSON,  ELIZABETH,  Brown  University 

KNEPTON,  JAMES  C,  JR.,  Duke  University 

LAUFER,  WILA  P.,  Tufts  University 

LYSER,  KATHERINE  MAY,  Oberlin  College 

McARDLE,  EUGENE  WILLIAM,  Marquette  University 

MATHESON,  GAIL  E.,  Wheaton  College 

MENDELSOHN,  EVERETT,  Harvard  University 

NATHANSON,  DONALD  LAWRENCE,  Amherst  College 

NEU,  HAROLD  C.,  Creighton  University 

ORELUP,  ALETHEA  ANN,  University  of  Illinois 

PIERCE,  PETER  G.,  Colby  College 

RAFFERTY,  NANCY  S.,  University  of  Illinois 

SAGE,  JANET  KATHLEEN,  DePauw  University 

SCHULTES,  SANDRA  JEAN,  Goucher  College 

SHOGER,  Ross  L.,  University  of  Minnesota 

SKINNER,  DOROTHY  M.,  Radcliffe  College 

SPENCER,  CHARLES  DAVID,  Wesleyan  University 

TULL,  DADE  LOUISE,  Vassar  College 

YOUNG,  ROBERT  RICE,  Yale  University 

PHYSIOLOGY 

BAKER,  K.  FRANCE,  Columbia  University 

CAZORLA,  F.  ALBERTO,  Institute  NCNAL  de  Enferme-Dades  Neoplasicas,  Lima,  Peru 

CORDEAU,  JEAN  PIERRE,  Universite  de  Montreal 

CROCKER,  CHARITY  S.,  Institute  de  Biofisica,  Rio  de  Janeiro,  Brazil 

CURRY,  GEORGE  MONTGOMERY,  Harvard  University 


REPORT  OF  THE  DIRECTOR  17 


EKBERG,  DONALD  ROY,  University  of  Illinois 

FERNANDES,  JOSE  FERREIRA,  Medical  Faculty,  Sao  Paulo,  Brazil 

FLEISHER,  JOSEPH  H.,  Chemical  Corps,  Medical  Laboratories 

FLEMING,  WILLIAM  WRIGHT,  Harvard  University 

GOE,  DON  RICHARD,  University  of  Southern  California 

GOLDSMITH,  TIMOTHY  H.,  Harvard  University 

GUCCIONE,  IGNATIUS,  New  York  University 

HERRANEN,  AILENE  M.,  University  of  Wisconsin 

HURWITZ,  CHARLES,  V.  A.  Hospital,  Albany,  New  York 

KEPCHAR,  JOHN  HOWARD,  University  of  North  Carolina 

LEFKOWITZ,  LEWIS  B.,  JR.,  Southwestern  Medical  School 

LEVINE,  LAURENCE,  Wayne  University 

MACHATTIE,  LORNE  ALLISTER,  University  of  Buffalo 

MAUZERALL,  DAVID  C.,  Rockefeller  Institute  for  Medical  Research 

MENDELSOHN,  MARY  L.,  Radcliffe  College 

MESSINEO,  LUIGI,  Institute  of  Zoology  of  Palermo,  Italy 

NEUHAUS,  FRANCIS  C.,  Duke  University 

RHODES,  WILLIAM  C.,  Johns  Hopkins  University 

ROBERTS,  JANE  C.,  University  of  California,  Los  Angeles 

ROGERS,  PALMER,  Johns  Hopkins  University 

SMALL,  ARLENE  MAY,  Mount  Holyoke  College 

STEVENS,  CARL  MANTLE,  State  College  of  Washington 

URBAN,  THEODORE  J.,  Creighton  University 

WAHBA,  ALBERT  J.,  Cornell  University 

WOHLHIETER,  JOHN  ANDREW,  LTniversity  of  Pittsburgh 

ZOOLOGY 

ABBOTT,  JOAN,  Washington  University 

ADAM,  BETTY  ROSE,  De  Paul  University 

BABCOCK,  RICHARD  G.,  University  of  Michigan 

BATES,  GRIFFIN  MILLER,  Hamilton  College 

BEARD,  ROBERT  G.,  Indiana  University 

BISHOP,  JANE  ELLEN,  Oberlin  College 

BROWN,  EDWARD  R.,  University  of  Cincinnati 

BRUENING,  BETTY  L.,  Goucher  College 

BUTZ,  ANDREW,  Fordham  University 

CALI,  CARMEN  T.,  Fordham  University 

CAMOUGIS,  GEORGE,  Harvard  University 

CAREY,  FRANCIS  GERALD,  Harvard  University 

CHRISTENSEN,  ALBERT  KENT,  Harvard  University 

CHURCH,  CHARLES  HENRY,  JR.,  Wesleyan  University 

CRANSTON,  MARGARET  B.,  Mount  Holyoke  College 

DAVIS,  PETER  WRIGHT,  Bowdoin  College 

DICKINSON,  WINIFRED,  Pennsylvania  College  for  Women 

DRURY,  GEORGE  L.,  S.  J.,  Boston  College 

DUNGAN,  SHIRLEY  R.,  DePauw  University 

EAKIN,  EDWIN  LLOYD,  Kenyon  College 

FABINY,  ROBERT  JOHN,  Marquette  University 

FASSULIOTIS,  GEORGE,  New  York  University 

FEELEY,  EDWARD  J.,  Fordham  University 

FINLAY,  PETER  S.,  Syracuse  University 

GATES,  JOHN  OTIS,  Cornell  University 

GOLDEN,  HAROLD  J.  J.,  Saint  Louis  University 

GROSS,  CHARLES,  Harvard  College 


18  MARINE  BIOLOGICAL  LABORATORY 

HALL,  WILLIAM  H.,  University  of  Virginia 

HOFSTETTER,  SISTER  ADRIAN  MARIE,  Notre  Dame  University 

HORRELL,  HARRY  CUNNINGHAM,  University  of  Chicago 

JOSEPHSON,  ROBERT  KARL,  Tufts  University 

KINYON,  NANCY,  Northwestern  University 

LISA,  JOSEPH  D.,  Fordham  University 

MACHATTIE,  LORNE  A.,  University  of  Buffalo 

MARKHAM,  ALICE  ELINOR,  Mount  Holyoke  College 

MAURIELLO,  GEORGE  E.,  New  York  University 

MENGES,  ELIZABETH  V.,  Smith  College 

ORR,  ANTOINETTE  M.,  Marlboro  College 

OZBURN,  GEORGE  W.,  Ontario  College 

PANUSKA,  JOSEPH  ALLEN,  St.  Louis  University 

PARSONS,  JOHN  A.,  Pennsylvania  State  University 

PASCALE,  JANE  FAY,  University  of  Chicago 

PEACOCK,  RONNIE,  Earlham  College 

PICCIANO,  ANTOINETTE  A.,  Northwestern  University 

Ross,  DONALD  J.,  Fordham  University 

ROUSALL,  PAUL  G.,  S.  J.,  Fordham  University 

SALYERDS,  ANNE  MARTHA,  Agnes  Scott  College 

SCOTT,  DIANA  F.,  Swarthmore  College 

SMITH,  SALLY,  Vassar  College 

SPANO,  REV.  ANTHONY  A.,  Fordham  University 

SWADER,  LAURA  LYNN,  Drew  University 

TAYLOR,  PETER  B.,  Cornell  University 

TRUONG,  REV.  HOANG,  Northwestern  University 

WHITIN,  NAVAMONIE,  Wellesley  College 

WISCHNITZER,  SAUL,  University  of  Notre  Dame 

WOODS,  JAMES  E.,  DePaul  University 

ECOLOGY 

BING,  PETER  S.,  Los  Angeles  24,  California 

CAREY,  ANDREW  G.,  Princeton  University 

COHEN,  MATANAH,  University  of  Colorado 

DAVIS,  ROGER  E.,  University  of  Wisconsin 

DIAL,  NORMAN  A.,  University  of  Illinois 

FELITTI,  VINCENT  JUSTUS,  Dartmouth  College 

FREUDENTHAL,  ANITA  R.,  New  York  University 

FREUDENTHAL,  HUGO  D.,  Columbia  University 

HAWS,  CLAYTON,  Drew  University 

MCLAUGHLIN,  JOHN  J.,  Haskins  Laboratories,  New  York  University 

MORRILL,  JOHN  B.,  JR.,  Florida  State  University 

NAGLE,  MARY  ELIZABETH,  Clark  University 

WRIGHT,  THEODORE,  Yale  University 

3.  LALOR  FELLOWS,  1955 

CLAYTON,  RODERICK  K.,  U.  S.  Naval  P.  G.  School 

GEST,  H.,  Western  Reserve  University 

GREEN,  MAURICE,  Childrens  Hospital,  University  of  Pennsylvania 

LEWIN,  RALPH,  National  Research  Council,  Maritime  Regional  Laboratory,  Halifax,  N.  S. 

MACCHI,  ITALO,  Clark  University 

ROTH,  JAY,  Hahnemann  Medical  College 

WILBRANDT,  W.,  University  of  Berne,  Switzerland 


REPORT  OF  THE  DIRECTOR 


19 


4.  TABULAR  VIEW  OF  ATTENDANCE,  1951-1955 

1951  1952        1953  1954  1955 

INVESTIGATORS— TOTAL 303  306  310  298  250 

Independent     186  172  176  180  162 

Under  Instruction   28  38  37  20  9 

Library  Readers    37  49  46  54 

Research  Assistants    52  47  51  46  25 

STUDENTS— TOTAL  124  123  136  134  148 

Zoology    55  55  55  56  56 

Embryology 27  23  30  29  30 

Physiology 29  27  31  28  30 

Botany    13  11  11  12  19 

Ecology    : 7  9  9  13 

TOTAL  ATTENDANCE   427  429  446  432  398 

Less  persons  represented  as  both  students  and  investi- 
gators      2 

427  427  446  427  398 

INSTITUTIONS   REPRESENTED — TOTAL    158  149  155  136  129 

By  Investigators   115  92  90  104  95 

By  Students    43  57  65  32  34 

SCHOOLS  AND  ACADEMIES  REPRESENTED 

By  Investigators    1  1  2  3 

By  Students    3  1  1  2 

FOREIGN  INSTITUTIONS  REPRESENTED 

By  Investigators    8  7  15  11  8 

By  Students    3  2  6  13  6 

5.  COOPERATING  AND  SUBSCRIBING  INSTITUTIONS,  1955 
Cooperating  Institutions 


Amherst  College 

American  Cancer  Society 

American  Philosophical  Society 

Brooklyn  College 

Brown  University 

Bryn  Mawr  College 

California  Institute  of  Technology 

Children's  Hospital  of  Philadelphia 

City  College  of  New  York 

Colby  College 

College  of  Mt.   St.  Joseph  on  the  Ohio 

Columbia  University 

Columbia    University,    College    of    Physicians 

and  Surgeons 
Cornell  University 
Cornell  University  Medical  School 
Duke  University 
Elmira  College 
Emory  University 
Florida  State  University 
Fordham  University 
George  Washington  University  Medical 

School 

Grass  Foundation 
Hahnemann  Medical  College 
Harvard  University 


Harvard  University  Medical  School 

Institute  for  Cancer  Research 

Institute  for  Muscle  Research 

Johns  Hopkins  University 

Johns  Hopkins  University  Medical  School 

Lalor  Foundation 

Eli  Lilly    and  Company 

Morgan  State  College 

Mount  Holyoke  College 

National  Institutes  of  Health 

National  Science  Foundation 

New  York  University,  College  of  Medicine 

New  York  University- — Heights 

New    York    University — Washington    Square 

College 

North  Carolina  State  College 
Northwestern  University 
Oberlin  College 
Office  of  Naval  Research 
Princeton  University 
Public  Health  Institute  of  New  York- 
Rockefeller  Foundation 
Rockefeller  Institute  for  Medical  Research 
Rutgers  University 
Saint  Louis  University 
Sloan-Kettering  Institute 


20 


MARINE  BIOLOGICAL  LABORATORY 


Southwestern  Medical  College 

State  University  of  Iowa 

State    University   of    New    York,    College    of 

Medicine,  at  Syracuse 
Syracuse  University 
Temple  University 
Tufts  College 
University  of  Chicago 
University  of  Connecticut 
University  of  Delaware 
University  of  Illinois 

University  of  Maryland  School  of  Medicine 
University  of  Michigan 
University  of  Minnesota 


University  of  Nebraska 

University  of  North  Carolina 

University  of  Pennsylvania 

University  of  Pennsylvania  Medical  School 

University  of  Rochester 

University  of  Virginia,  School  of  Medicine 

University  of  Wisconsin 

Yassar  College 

Washington  University 

Washington  and  Jefferson  College 

Wesllesley  College 

Wesleyan  University 

Western  Reserve  University 

Yale  University 


Subscribing  Institutions 


Acadia  University 

Boston  College 

Ethicon  Corporation 

Goucher  College 

Guggenheim  Foundation 

Hamilton  College 

Indiana  University 

Marquette  University 

Pennsylvania  College  for  Women 

Purdue  University 


Radcliffe  College 

Saint  Ambrose  College 

State  University  of  New  York  at  Brooklyn 

University  Center  of  Georgia 

University  of  Alabama  School  of  Dentistry 

University  of  Illinois,  College  of  Medicine 

University  of  Maine 

University  of  Oklahoma 

University  of  Texas  Medical  School 

Yale  University  School  of  Medicine 


6.  EVENING  LECTURES,  1955 

June  24 

PAUL  WEISS    "Some  thoughts   and   experiments   on   mor- 
phogenesis" 
July  1 

GEORGE  WALD   "Tin-  origin  of  life — some  special  problems" 

July  8 

F.    SJOSTRAND    "L'ltr  (structural   organization   of  retinal   re- 
ceptor cells" 
July  15 

BRADLEY  M.  PATTEN   "Micmmoving    picture    studies    of   the    first 

heart   beats   in   the  beginning  of  the  em- 
bryonic circulation" 
July  22 

F.  O.  SCHMITT   "Chemical    and    structural    studies    of   nerve 

fibers" 

July  29 

WALTER  WILBRANDT   "Carrier  transport  systems  and  their  kinetics" 

August  5 

F.  A.   BROWN,   JR "The  rhythmic  nature  of  life" 

August  12 

ARNOLD   LAZAROW    "Diabetic  toadfish ;   their   use   in   studies  on 

the  etiology  of  diabetes  mellitus" 
August  19 

KENNETH  S.  COLE  "Squid  axon  excitation" 

August  26 

GEORGE  KIDDER   "Metabolic      studies      on      animal      micro- 


REPORT OF  THE  DIRECTOR  21 

7.  TUESDAY  EVENING  SEMINARS,  1955 

July  5 

R    P.   LEVINE    "Cations   i  n    chromosome    structure :    their 

relation    to    the    mechanism    of    crossing 
over" 
DANIEL  MAZIA  and  "Distribution  of  parental  material  in  chro- 

WALTER  S.  PLAUT  mosome  reproduction" 

MAX  A.  LAUFFER  and  "The  effects  of  ionizing  radiations  on  cock- 

HERMAN  CEMBER  roach  embryos" 

July  12 
J.  P.  TRINKAUS  and  "Differentiation  of  mixed  aggregates  of  dis- 

PEGGY  W.  GROVES   sociated  tissue  cells" 

R.  E.  ZIRKLE,  W.  BLOOM  and  "Chromosome  movements   and  cell  division 

R.   B.  URETZ   after  spindle  destruction  by  irradiation  of 

cytoplasm" 

R.  B.  URETZ,  W.  BLOOM  and  "Changes   in  refractive  index  of  irradiated 

R.  E.  ZIRKLE chromosome  segments" 

July  19 

GERALD  R.  SEAMAN  "Purification  and  properties  of  an  enzyme 

system  which  reversibly  cleaves  succinate 
to  join  two  molecules  of  acetyl  coenzyme 
A" 

JAY  S.  ROTH   "Ribonuclease  inhibition" 

ELIZABETH  ANDERSON  "Metabolism  of  uridinediphosphoglycosyl 

compounds" 

ALEXANDER  KOLIN  "Some  recent  experiments  on  electrokinetic 

separation  of  proteins  and  of  micro- 
organisms" 

July  26 

R.  K.  CLAYTON "Tactic  responses  of  purple  bacteria" 

\\~ARNER  E.  LOVE "The   molecular   weight   of   hemerythrin   of 

Phascolosoma  gouldi  by  x-ray  diffraction" 

BARBARA  E.  WRIGHT "Pteridine      coenzymes      in      one      carbon 

metabolism" 

I'.  F.  SCHOLANDER  "Secretion  of  inert  gases  and  oxygen  by  the 

swim-bladder  of  fishes" 
August  2 

CARL  C.   SPEIDEL   "Motion  pictures  of  cellular  changes  in  tad- 
poles following  x-ray  irradiation" 

ROBERT  ALLEN    "Protoplasmic      streaming      and     amoeboid 

movement" 

S.  G.  A.  ALIVISATOS   "Enzymic  synthesis  of  new  dinucleotides  by 

a  novel  method  of  biosynthesis" 

PAUL  S.  GALTSOFF "Structure  and   function  of  the  ligament  of 

Pelecypoda" 
August  9 

E.  G.  BUTLER  and  "Effects   of  ultraviolet   on   regenerative  ac- 

H.  F.  BLUM    tivity  in  urodeles" 

S.  HAGIWARA  and  "Study   of   intracellular   potentials   in   pace- 

T.  H.  BITLLOCK maker  and  integrative  neurons  of  the  lob- 
ster cardiac  ganglion" 
T.  H.  BULLOCK  and  "Further  study  of  the  giant  synapse  in  the 

S.  HAGIWARA   stellate  ganglion  of  squid" 

CHARLES  G.  WILBER "Electrocardiogram  of  the  alligator" 


22  MARINE  BIOLOGICAL  LABORATORY 

August  16 
ANNA  R.  WHITING  and  "Differences    in    response    of   x-rayed    eggs 

WILLIAM   E.   MURPHY    and     spermatozoa     of     Habrobracon     to 

anoxia" 

RALPH  A.  LEWIN    "Paralysis    in    double-mutants    of    Chlamy- 

domonas" 
H.  H.  PLOUGH  and  "High  frequency  of  transduction  of  genes  by 

MARGARET  ROBERTS   bacteriophage  in  Salmonella" 

JOYCE  C.  LEWIN  "Physiological  races  of  the  diatom,  Navicnla 

pelliculosa" 

August  23 

PHILIP  GRANT    "Some  observations  on  the  incorporation  of 

glycine    C-14    into    amphibian    embryos" 

MAURICE  GREEN       "Fucose   metabolism    in   Eschcrichia   coli" 

JAMES  A.  MILLER,  JR "The  potentiation  by  narcosis  of  the  bene- 
ficial effects  of  hypothermia  in  asphyxia 
of  the  neonatal  guinea  pig" 

W.  WILBRANDT  and  "Action  of  a  thrombocyte  protein  on  capil- 

P.    LUESCHER    lary  permeability" 

8.  MEMBERS  OF  THE  CORPORATION.   1955 
1.    LIFE  MEMBERS  OF  THE  CORPORATION 

BILLINGS,  MR.  R.  C.,  66  Franklin  Street,  Boston,  Massachusetts 

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

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

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

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

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

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

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

JACKSON,  MR.  CHARLES  C.,  24  Congress  Street,  Boston,  Massachusetts 

JACKSON,  Miss  M.  C.,  88  Marlboro  Street,  Boston,  Massachusetts 

KING,  MR.  CHARLES  A. 

LEWIS,  PROF.  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,  PROF.  E.  F.,  6610  North  llth  Street,  Philadelphia  26,  Pennsylvania 

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

MOORE,  DR.  GEORGE  T.,  Missouri  Botanical  Gardens,  St.  Louis,  Missouri 

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

NOYES,  Miss  EVA  J., 

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

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

RIGGS,  MR.  LAWRASON,  120  Broadway,  New  York  City,  New  York 

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

SEARS,  DR.  HENRY  F.,  86  Beacon  Street,  Boston,  Massachusetts 

SHEDD.  MR.  E.  A. 

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


REPORT  OF  THE  DIRECTOR 

WALLACE,  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 

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  Den- 
tistry, Rochester,  New  York 
ALBAUM,  DR.  HARRY  G.,  Biology  Department,  Brooklyn  College,  Brooklyn,  New 

York 

ALBERT,  DR.  ALEXANDER,  Mayo  Clinic,  Rochester,  Minnesota 
ALLEN,  DR.  M.  JEAN,  Dept.  of  Zoology,  University  of  New  Hampshire,  Durham, 

New  Hampshire 

ALLEN,  DR.  ROBERT  D.,  University  of  Michigan,  Ann  Arbor,  Michigan 
ALSCHER,  DR.  RUTH,  Dept.  of  Physiology,  Manhattanville  College  of  the  Sacred 

Heart,  Purchase,  New  York 
AMBERSON,  DR.  WILLIAM  R.,  Dept.  of  Physiology,  University  of  Maryland  School 

of  Medicine,  Baltimore,  Maryland 

ANDERSON,  DR.  J.  M.,  Dept.  of  Zoology,  Cornell  University,  Ithaca,  New  York 
ANDERSON,  DR.  RUBERT  S.,  Medical  Laboratories,  Army  Chemical  Center,  Maryland 
ANDERSON,  DR.  T.  F.,  University  of  Pennsylvania,  Philadelphia,  Pennsylvania 
ARMSTRONG,  DR.  PHILIP  B.,  State  University  of  New  York  College  of  Medicine, 

Syracuse  10,  New  York 

ATWOOD,  DR.  KIMBALL  C.,  681/2  Outer  Drive,  Oak  Ridge,  Tennessee 
AUSTIN,  DR.  MARY  L.,  Wellesley  College,  Wellesley,  Massachusetts 
AYERS,  DR.  JOHN  C.,  Dept.  of  Oceanography,  Cornell  University,  Ithaca,  New  York 
BAITSELL,  DR.  GEORGE  A.,  Osborn  Zoological  Laboratory,  Yale  University,  New 

Haven,  Connecticut 

BAKER,  DR.  H.  B.,  Zoological  Laboratory,  University  of  Pennsylvania,  Philadel- 
phia, Pennsylvania 
BALL,  DR.  ERIC  G.,  Dept.  of  Biological  Chemistry,  Harvard  University  Medical 

School,  Boston  15,  Mass. 

BANG,  DR.  F.  B.,  Dept.  of  Parasitology,  Johns  Hopkins  University  School  of  Hy- 
giene, Baltimore  5,  Maryland 

BALLARD,  DR.  WILLIAM  W.,  Dartmouth  College,  Hanover,  New  Hampshire 
BARD,    PROF.    PHILIP,    Johns    Hopkins    University    Medical    School,    Baltimore, 

Maryland 

BARRON,  DR.  E.  S.  G.,  Dept.  of  Medicine,  University  of  Chicago,  Chicago,  111. 
BARTII,  DR.  L.  G.,  Dept.  of  Zoology,  Columbia  University,  New  York  City,  New 

York 

BARTLETT,  DR.  JAMES  H.,  Dept.  of  Physics,  University  of  Illinois,  Urbana,  Illinois 
BEAMS,  DR.  HAROLD  W.,  Dept.  of  Zoology,  State  University  of  Iowa,  Iowa  City, 

Iowa 

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


24  MARINE  BIOLOGICAL  LABORATORY 

BEERS,  DR.  C.  D.,  University  of  North  Carolina,  Chapel  Hill,  North  Carolina 
BEHRE,  DR.  ELINOR  H.,  Louisiana  State  University,  Baton  Rouge,  Louisiana 
BERNSTEIN,  DR.  MAURICE,  Virus  Lab.,  University  of  California,  Berkeley  4, 

California 

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

City,  New  York 
BIGELOW,  DR.  HENRY  B.,  Museum  of  Comparative  Zoology,  Harvard  University, 

Cambridge,  Mass. 

BIGELOW,  PROF.  ROBERT  P.,  72  Blake  Road,  Brookline  46,  Massachusetts 
BISHOP,  DR.  DAVID  W.,  Dept.  of  Embryology,  Carnegie  Institute  of  Washington, 

Baltimore  5,  Maryland 
BLANCHARD,  DR.  K.   C,  Johns  Hopkins  University   Medical   School,   Baltimore, 

Maryland 
BLOCK,  DR.  ROBERT,  Dept.  of  Botany,  University  of  Pennsylvania,  Philadelphia, 

Pennsylvania 
BLUM,  DR.  HAROLD  F.,  Dept.  of  Biology,  Princeton  University,  Princeton,  New 

Jersey 
BODANSKY,  DR.  OSCAR,  Dept.  of  Biochemistry,  Sloan-Kettering  Division,  Cornell 

University  Medical  College,  New  York  City,  New  York 
BODIAN,  DR.  DAVID,  Dept.  of  Epidemiology,  Johns  Hopkins  University,  Baltimore 

5,  Maryland 

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

Connecticut 

BOLD,  DR.  H.  C.,  Dept.  of  Botany,  Vanderbilt  University,  Nashville,  Tennessee 
BOREI,  DR.   HANS,   Dept.   of  Zoology.   University  of  Pennsylvania,   Philadelphia, 

Pennsylvania 

BRADLEY,  PROF.  HAROLD  C.,  2639  Durant  Avenue,  Berkeley  4,  California 
BRONK,  DR.  DETLEV  W.,  Rockefeller  Institute,  66th  St.  and  York  Avenue,  New 

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

4,  California 
BROWN,  DR.  FRANK  A.,  JR.,  Dept.  of  Biological  Sciences,  Northwestern  University, 

Evanston,  Illinois 
BROWN,  DR.  DUGALD  E.  S.,  Dept.  of  Zoology,  University  of  Michigan,  Ann  Arbor, 

Michigan 

BROWNELL,  DR.  KATIIERINE  A.,  Ohio  State  University,  Columbus,  Ohio 
BUCK,  DR.  JOHN  B.,  Laboratory  of  Physical  Biology,  National  Institutes  of  Health, 

Bethesda,  Maryland 

BULLINGTON,  DR.  W.  E.,  Randolph-Macon  College,  Ashland,  Virginia 
BULLOCK,  DR.  T.  H.,  Dept.  of  Zoology,  University  of  California,  Los  Angeles  24, 

California 

BURBANCK,  DR.  WILLIAM  D.,  Box  834,  Emory  University,  Georgia 
BURDICK,  DR.  C.  LALOR,  The  Lalor  Foundation,  Lancaster  Pike  and  Old  Baltimore 

Road,  Wilmington,  Delaware 


REPORT  OF  THE  DIRECTOR 

BURKENROAD,  DR.  M.  D.,  c/o  Lab.  Nal.  de  Pesca,  Apartado  3318,  Estofeta  No.  1, 

Olindania,  Republic  de  Panama 

BUTLER,  DR.  E.  G.,  Dept.  of  Biology,  Princeton  University,  Princeton,  New  Jersey 
CAMERON,  DR.  J.  A.,  Baylor  College  of  Dentistry,  Dallas,  Texas 
CANTOXI,  DR.  GIULIO,  National  Institutes  of  Health,  Mental  Health,  Bethesda  14, 

Maryland 
CARLSON,  PROF.  A.  J..  Dept.  of  Physiology,  University  of  Chicago,  Chicago  37, 

Illinois 

CAROTHERS,  DR.  E.  ELEANOR,  9  Gladys  Sparr,  Murdock,  Kansas 
CARPENTER,  DR.  RUSSELL  L.,  Tufts  College,  Medford  55,  Massachusetts 
CARSON,  Miss  RACHEL,  204  Williamsburg  Drive,  Silver  Spring,  Maryland 
CATTELL,  DR.  McKEENE,  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.,  University  of  Maine,  Orono,  Maine 
CHAMBERS,  DR.  EDWARD,  Dept.  of  Physiology,  University  of  Miami  Medical  School, 

Coral  Gables,  Florida 

CHAMBERS,  DR.  ROBERT,  425  Riverside  Drive,  New  York  City,  New  York 
CHARLES,  DR.  DONALD  R.,  Dept.  of  Zoology,  Division  of  Biological  Sciences,  Uni- 
versity of  Rochester,  Rochester  3,  New  York 
CHASE,  DR.  AURIN  M.,  Dept.  of  Biology,  Princeton  University.  Princeton,  New 

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

New  York 
CHURNEY,  DR.  LEON,  Dept.  of  Physiology,  Louisiana  State  University  School  of 

Medicine,  New  Orleans,  Louisiana 

CLAFF,  MR.  C.  LLOYD,  5  Van  Beal  Road.  Randolph,  Massachusetts 
CLARK,  DR.  A.  M.,  Dept.  of  Biology,  University  of  Delaware,  Newark,  Delaware 
CLARK,  PROF.  E.  R.,  The  Wistar  Institute,  Woodland  Avenue  and  36th  St.,  Phila- 
delphia 4,  Pennsylvania 

CLARK,  DR.  LEONARD  B.,  Dept.  of  Biology,  Union  College,  Schenectady,  New  York 
CLARKE,  DR.  GEORGE  L.,  Harvard  University,  Biological  Laboratories,  Cambridge 

38,  Massachusetts 

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

COLE,  DR.  ELBERT  C.,  Dept.  of  Biology,  Williams  College,  Williamstown,  Massa- 
chusetts 
COLE,  DR.  KENNETH  S..  National  Institutes  of  Health   (NINDB),  Bethesda  14, 

Maryland 

COLLETT,  DR.  MARY  E.,  34  Weston  Road,  Wellesley  81,  Massachusetts 
COLTON,  PROF.  H.  S.,  Box  601,  Flagstaff,  Arizona 

COLWIN,  DR.  ARTHUR  L.,  Dept.  of  Biology,  Queens  College,  Flushing,  New  York 
COLVVIN,  DR.  LAURA  H.,  Dept.  of  Biology,  Queens  College,  Flushing,  New  York 


26  MARINE  BIOLOGICAL  LABORATORY 

COOPER,  DR.  KENNETH  W.,  Dept.  of  Biology,  University  of  Rochester,  Rochester 

3,  New  York 
COOPERSTEIN,  DR.  SHERWIN  J.,  Dept.  of  Anatomy,  Western  Reserve  University 

Medical  School,  Cleveland,  Ohio 

COPELAND,  DR.  D.  E.,  1027  N.  Manchester  Street,  Arlington  5,  Virginia 
COPELAND,  PROF.  MANTON,  Boudoin  College,  Brunswick,  Maine 
COPLEY,  DR.  ALFRED  L.,  Charge  de  Recherches,  Laboratories  de  Recherches,  Centre 

International  de  L'Enfance,  Chateau  de  Longchamp,  Bois  de  Bologne,  Paris  16, 

France 

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

Hill,  North  Carolina 
COSTELLO,  DR.  HELEN  MILLER,  Dept.  of  Zoology,  University  of  North  Carolina, 

Chapel  Hill,  North  Carolina 
CRAMPTON,  PROF.  H.  E.,  American  Museum  of  Natural  History,  New  York  City, 

New  York 

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

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

Indiana 

CURTIS,  DR.  MAYNIE  R.,  University  of  Miami,  Box  1015,  South  Miami,  Florida 
CURTIS,  PROF.  W.  C,  University  of  Missouri,  Columbia,  Missouri 
DAN,  DR.  KATSUMA,  Misaki  Biological  Station,  Misaki,  Japan 
DANIELLI,  DR.  JAMES  F.,  Dept.  of  Zoology,  King's  College,  London,  England 
DAWSON,  DR.  A.  B.,  Harvard  University,  Cambridge  38,  Massachusetts 
DAWSON,  DR.  J.  A.,  College  of  the  City  of  New  York,  New  York  City,  New  York 
DILLER,  DR.  IRENE  C.,  Institute  for  Cancer  Research,  Philadelphia,  Pennsylvania 
DILLER,  DR.  WILLIAM  F.,  2417  Fairhill  Avenue,  Glenside,  Pennsylvania 
DODDS,  PROF.  G.  S.,  School  of  Medicine.  West  Virginia  University,  Morgantown, 

West  Virginia 

DOLLEY,  PROF.  WILLIAM  L.,  University  of  Buffalo,  Buffalo  14,  New  York 
DONALDSON,  DR.  JOHN   C.,  University  of  Pittsburgh  School  of  Medicine,   Pitts- 
burgh, Pennsylvania 

DOTY,  DR.  MAXWELL  S.,  Dept.  of  Biology,  University  of  Hawaii,  Honolulu,  T.  H. 
DRINKER,  DR.  CECIL  K.,  Box  502,  Falmouth,  Massachusetts 
DuBois,  DR.  EUGENE  F.,  200  East  End  Avenue,  New  York  28,  New  York 
DUGGAR,  DR.  BENJAMIN  M.,  Lederle  Laboratories  Inc.,  Pearl  River,  New  York 
DURYEE,  DR.  WILLIAM  R.,  George  Washington  University  School  of  Medicine, 

Dept.  of  Physiology,  Washington  5,  D.  C. 
EDDS,  DR.  MAC  V.,  JR.,  Dept.  of  Biology,  Brown  University,  Providence,  Rhode 

Island 

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.,  Dept.  of  Zoology,  University  of  Michigan,  Ann  Arbor, 

Michigan 


REPORT  OF  THE  DIRECTOR  27 

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,  PROF.  EMMANUEL,  College  de  France,  Paris,  France 
FERGUSON,  DR.  F.  P.,  Dept.  of  Physiology,  University  of  Maryland  Medical  School, 

Baltimore  1,  Maryland 

FERGUSON,  DR.  JAMES  K.  W.,  Connough  Laboratories,  University  of  Toronto,  On- 
tario, Canada 
FIGGE,  DR.  F.  H.  J.,  University  of  Maryland  Medical  School,  Lombard  &  Green 

Sts.,  Baltimore  1,  Maryland 
FINGERMAN,  DR.  MILTON,  Dept.  of  Zoology,  Newcomb  College,  Tulane  University, 

New  Orleans  18,  Louisiana 
FISCHER,  DR.  ERNST,  Dept.  of  Physiology,  Medical  College  of  Virginia,  Richmond 

19,  Virginia 
FISHER,  DR.  JEANNE  M.,  Dept.  of  Biochemistry,  University  of  Toronto,  Toronto, 

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

Canada 
FORBES,  DR.  ALEXANDER,  Biological  Laboratories,  Harvard  University,  Cambridge 

38,  Massachusetts 
FRAENKEL,  DR.  GOTTFRIED  S.,  Dept.  of  Entomology,  University  of  Illinois,  Urbana, 

Illinois 
FRIES,  DR.  ERIK  F.  B.,  Dept.  of  Biology,  City  College  of  New  York,  New  York 

City,  New  York 

FRISCH,  DR.  JOHN  A.,  Canisius  College,  Buffalo,  New  York 
FURTH,  DR.  JACOB,  18  Springdale  Road,  Wellesley  Farms,  Massachusetts 
GABRIEL,  DR.  MORDECAI,  Dept.  of  Biology,  Brooklyn  College,  Brooklyn,  New7  York 
GAFFRON,  DR.  HANS,  Research  Insts.,  University  of  Chicago,  5650  Ellis  Ave.,  Chi- 
cago 37,  Illinois 
GALL,  DR.  JOSEPH  G.,  Dept.  of  Zoology,  University  of  Minnesota,  Minneapolis  14, 

Minnesota 

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

GASSER,  DR.  HERBERT  S.,  Director,  Rockefeller  Institute,  New  York  21,  New  York 
GATES,  DR.  REGINALD  R.,  Dept.  of  Anthropology,  Harvard  University,  Peabody 

Museum,  Cambridge,  Massachusetts 

GEISER,  DR.  S.  W.,  Southern  Methodist  University,  Dallas,  Texas 
GERARD,  PROF.  R.  W.,  Illinois  Neuropsychiatric  Institute,  Chicago  12,  Illinois 
GILMAN,  DR.  LAUREN  C.,  Dept.  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.,  Dept.  of  Biology,  Emory  University,  Emory  Uni- 
versity, 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.,  Medical  Sciences  Information  Exchange,  1113  Dupont  Circle 
Bldg.,  Washington,  D.  C. 


MARINE  BIOLOGICAL  LABORATORY 

GRAHAM,  DR.  HERBERT,  Director,  Woods  Hole  Lab.,  Fish  and  Wildlife  Service, 

Woods  Hole,  Massachusetts 
GRAND,  MR.  C.  G.,  Dade  County  Cancer  Inst..   1155  N.W.   15th  Street,  Miami, 

Florida 

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

New  Jersey 

GREGG,  DR.  JAMES  H.,  University  of  Florida,  Gainesville,  Florida 
GREGG,  DR.  J.  P.,  Dept.  of  Zoology,  Columbia  University,  New  York  27,  New  York 
GROSCH,  DR.  DANIEL  S.,  North  Carolina  State  College,  Raleigh,  North  Carolina 
GRUNDFEST,  DR.  HARRY,  Columbia  University  College  of  Physicians  and  Surgeons, 

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,  Dept.  of  Physiology,  Brooklyn  College,  Brooklyn,  New  York 
GUYER,  PROF.  MICHAEL  F.,  University  of  Wisconsin,  Madison,  Wisconsin 
HAGUE,  DR.  FLORENCE,  Sweet  Briar  College,  Sweet  Briar,  Virginia 
HAJDU,  DR.  STEPHEN,  U.  S.  Public  Health  Institute,  Bethesda,  Maryland 
HALL,  PROF.  FRANK  G.,  Duke  University,  Durham,  North  Carolina 
HAMBURGER,  DR.  VIKTOR,  Dept.  of  Zoology,  Washington  University,  St.  Louis, 

Missouri 

HAMILTON,  DR.  HOWARD  L.,  Iowa  State  College,  Ames,  Iowa 
HANCE,  DR.  ROBERT  T.,  Box  108,  R.  R.  No.  3,  Loveland,  Ohio 
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,  PROF.  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.,  Div.  of  Marine  Botany,  Scripps  Institution  of  Oceanog- 
raphy, University  of  California,  La  Jolla,  California 
HAYASHI,  DR.  TERU,  Dept.  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.,  Dept.  of  Zoology,  University  of  Pennsylvania,  Philadel- 
phia, Pennsylvania 

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


REPORT  OF  THE  DIRECTOR 

HENLEY,  DR.  CATHERINE,  Dept.  of  Zoology,  University  of  North  Carolina.  Chapel 

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

York 

HESS,  PROF.  WALTER  N.,  Hamilton  College,  Clinton,  New  York 
HIBBARD,  DR.  HOPE,  Dept.  of  Zoology,  Oberlin  College,  Oberlin,  Ohio 
HILL,  DR.  SAMUEL  E.,  135  Brunswick  Road,  Troy,  New  York 
HINRICHS,  DR.  MARIE,  Bd.  of  Education,  Bureau  of  Health  Service,  228  N.  LaSalle 

St.,  Chicago,  Illinois 

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

Massachusetts 

HODES,  DR.  ROBERT,  Nuffield  Dept.  of  Orthopaedic  Surgery,  Oxford,  England 
HODGE,  DR.  CHARLES,  IV,  Temple  University,  Dept.  of  Zoology,   Philadelphia, 

Pennsylvania 
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.,  Institute  for  T.B.  Research,  Rm.  201,  1835  W.  Har- 
rison St.,  Chicago  12,  Illinois 
HUTCHENS,  DR.  JOHN  O.,  Dept.  of  Physiology,  University  of  Chicago,  Chicago  37, 

Illinois 
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,  PROF.  M.  H.,  School  of  Medicine,  University  of  Pennsylvania,  Philadel- 
phia, Pennsylvania 

JENKINS,  DR.  GEORGE  B.,  5339  42nd  Street  N.W.,  Washington  15,  D.  C. 
JENNER,  DR.  CHARLES  E.,  Dept.  of  Zoology,  University  of  North  Carolina,  Chapel 

Hill,  North  Carolina 
JONES,  DR.  E.  RUFFIN,  JR.,  Biological  Dept.,  University  of  Florida,  Gainesville, 

Florida 
KAAN,  DR.  HELEN  W.,  National  Heart  Institute,  National  Institutes  of  Health, 

Bethesda  14,  Maryland 
KABAT,  DR.  E.  A.,  Neurological  Institute,  College  of  Physicians  and  Surgeons,  New 

York  City,  New  York 
KARUSH,  DR.  FRED,  Dept.  of  Pediatrics,  University  of  Pennsylvania,  Philadelphia, 

Pennsylvania 
KAUFMAN,  PROF.  B.  P.,  Carnegie  Institute,  Cold  Spring  Harbor,  Long  Island,  New 

York 

KEMPTON,  PROF.  RUDOLF  T.,  Vassar  College,  Poughkeepsie,  New  York 
KETCHUM,  DR.  BOSTWICK,  Woods  Hole  Oceanographic  Institution,  Woods  Hole, 

Massachusetts 


30  MARINE  BIOLOGICAL  LABORATORY 

KILLE,  DR.  FRANK  R.,  Carleton  College,  Northfield,  Minnesota 

KIND,   DR.   C.   ALBERT,   Dept.   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 

KNOWLTON,  PROF.  F.  P.,  1356  Westmoreland  Avenue,  Syracuse,  New  York 
KOLIN,  DR.  ALEXANDER,  Division  of  Biol.  Sciences,  University  of  Chicago,  Chicago, 

Illinois 
KOPAC,  DR.  M.  J.,  New  York  University,  Washington  Square  College,  New  York 

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

Missouri 

KRAHL,  DR.  M.  E.,  Dept.  of  Physiology,  University  of  Chicago,  Chicago  37,  Illinois 
KREIG,  DR.  WENDALL  J.  S.,  303  East  Chicago  Avenue,  Chicago,  Illinois 
KUNITZ,  DR.  MOSES,  Rockefeller  Institute,  66th  St.  and  York  Ave.,  New  York  21, 

New  York 

KUFFLER,  DR.  STEPHEN,  Dept.  of  Ophthalmology,  Johns  Hopkins  Hospital,  Balti- 
more 5,  Maryland 
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.,  Dept.  of  Anatomy,  Emory  University,  Emory,  Georgia 
LAUFFER,  DR.  MAX  A.,  Dept.  of  Biophysics,  University  of  Pittsburgh,  Pittsburgh, 

Pennsylvania 

LAVIN,  DR.  GEORGE  I.,  3714  Springdale  Avenue,  Baltimore,  Maryland 
LAZAROW,   DR.    ARNOLD,    Dept.    of   Anatomy,    University   of   Minnesota    Medical 

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

New  York 
LEFEVRE,  DR.  PAUL  G.,  Division  of  Biology  and  Medicine,  U.  S.  Atomic  Energy 

Commission,  Washington  25,  D.  C. 
LESSLER,  DR.  MILTON  A.,  Dept.  of  Physiology,  Ohio  State  University,  Columbus, 

Ohio 

LEVINE,  DR.  RACHMIEL,  Michael  Reese  Hospital,  Chicago  16,  Illinois 
LEVY,  DR.  MILTON,  Chemistry  Dept.,  New  York  University  School  of  Medicine, 

New  York  City,  New  York 
LEWIS,  PROF.  I.  F.,  1110  Rugby  Road,  Charlottesville,  Virginia 


REPORT  OF  THE  DIRECTOR  31 

LITTLE,  DR.  E.  P.,  150  Causeway  St.,  Anderson  Nichols  and  Co.,  Boston  24, 
Massachusetts 

LOCHHEAD,  DR.  JOHN  H.,  Dept.  of  Zoology,  University  of  Vermont,  Burlington, 
Vermont 

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

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

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

LOR  AND,  DR.  LASZLO,  Dept.  of  Chemistry,  College  of  Liberal  Arts,  Northwestern 
University,  Evanston,  Illinois 

LOVE,  DR.  Lois  H.,  4253  Regent  Street,  Philadelphia  4,  Pennsylvania 

LOVE,  DR.  WARNER  E.,  Johnson  Foundation,  University  of  Pennsylvania,  Phila- 
delphia, Pennsylvania 

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

LYNCH,  DR.  RUTH  STOCKING,  Dept.  of  Botany,  University  of  California,  Los  An- 
geles 24,  California 

LYNN,  DR.  WILLIAM  G.,  Dept.  of  Biology,  Catholic  University  of  America,  Wash- 
ington, D.  C. 

MACDOUGAL,  DR.  MARY  S.,  Mt.  Vernon  Apts.,  423  Clairmont  Avenue,  Decatur, 
Georgia 

McCoucn,  DR.  MARGARET  SUMWALT,  University  of  Pennsylvania  Medical  School, 
Philadelphia,  Pennsylvania 

MCDONALD,  SR.  ELIZABETH  SETON,  Dept.  of  Biology,  College  of  Mt.  St.  Joseph, 
Mt.  St.  Joseph,  Ohio 

MCDONALD,  DR.  MARGARET  H.,  Carnegie  Institute  of  Washington,  Cold  Spring 
Harbor,  Long  Island,  New  York 

MACKLIN,  DR.  CHARLES  C.,  37  Gerard  Street,  London,  Ontario 

MAGRUDER,  DR.  SAMUEL  R.,  Dept.  of  Anatomy,  Tufts  Medical  School,  136  Harri- 
son Avenue.,  Boston,  Massachusetts 

MANWELL,  DR.  REGINALD  D.,  Syracuse  University,  Syracuse,  New  York 

MARSHAK,  DR.  ALFRED,  Woods  Hole,  Massachusetts 

MARSLAND,  DR.  DOUGLAS  A.,  New  York  University,  Washington  Square  College, 
New  York  City,  New  York 

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

MATHEWS,  PROF.  A.  P.,  Woods  Hole,  Massachusetts 

MATTHEWS,  DR.  SAMUEL  A.,  Thompson  Biological  Lab.,  Williams  College,  Wil- 
liamstown,  Massachusetts 

MAVOR,  PROF.  JAMES  W.,  Greenwood  Park,  Cambridge  58,  Massachusetts 

MAZIA,  DR.  DANIEL,  University  of  California,  Department  of  Zoology,  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.,  Dept.  of  Biology,  Swarthmore  College,  Swarthmore, 
Pennsylvania 

MEM  HARD,  MR.  A.  R.,  Riverside,  Connecticut 


32  MARINE  BIOLOGICAL  LABORATORY 

MENKIN,  DR.  VALY,  Temple  University  Medical  School,  Philadelphia,  Pennsylvania 
METZ,  DR.  C.  B.,  Dept.  of  Zoology,  Florida  State  University,  Tallahassee,  Florida 
METZ,  PROF.  CHARLES  W.,  University  of  Pennsylvania,  Philadelphia,  Pennsylvania 
MILLER,  DR.  J.  A.,  Basic  Science  Building,  Emory  University,  Georgia 
MILNE,  DR.  LORUS  J.,  Department  of  Zoology,  University  of  New  Hampshire,  Dur- 
ham, New  Hampshire 
MINNICH,  PROF.  D.  E.,  Dept.  of  Zoology,  University  of  Minnesota,  Minneapolis 

14,  Minnesota 
MOE,  MR.  HENRY  A.,  Secretary  General,  Guggenheim  Memorial  Fund,  551  Fifth 

Ave.,  New  York  17,  New  York 

MONROY,  DR.  ALBERTO,  Inst.  Compar.  Anatomy,  University  of  Palermo,  Italy 
MOORE,  DR.  CARL  R.,  University  of  Chicago,  Chicago  37,  Illinois 
MOORE,  DR.  GEORGE  M.,  Dept.  of  Zoology,  University  of  New  Hampshire,  Durham, 

New  Hampshire 
MOORE,  DR.  JOHN  W.,  Lab.  of  Biophysics,  NINDR,  National  Institutes  of  Health, 

Bethesda  14,  Maryland 
MOUL,  DR.  E.  T.,  Dept.  of  Botany,  Rutgers  University,  New  Brunswick,  New 

Jersey 

MOUNTAIN,  MRS.  J.  D.,  9  Coolidge  Avenue,  White  Plains,  New  York 
MULLER,  PROF.  H.  J.,  Dept.  of  Zoology,  Indiana  University,  Bloomington,  Indiana 
NABRIT,  DR.  S.  M.,  Texas  Southern  University,  3201  Wheeler  Avenue,  Houston  4, 

Texas 
NACE,  DR.  PAUL  FOLEY,  Dept.  of  Anatomy,  New  York  Medical  College,  New 

York  City,  New  York 

NACHMANSOHN,  DR.  DAVID,  College  of  Physicians  and  Surgeons,  Columbia  Uni- 
versity, New  York  City,  New  York 

NAVEZ,  DR.  ALBERT  E.,  206  Churchill's  Lane,  Milton  86,  Massachusetts 
NELSON,  DR.  LEONARD,  Dept.  of  Physiology,  University  of  Nebraska,  Lincoln, 

Nebraska 
NEUBERG,  DR.  CARL,  New  York  Medical  College,  5th  Avenue  at  106th  Street,  New 

York  29,  New  York 
NEURATH,  DR.  H.,  Dept.  of  Biochemistry,  University  of  Washington,  Seattle  5, 

Washington 

NEWMAN,  PROF.  H.  H.,  173  Devon  Drive,  Clearwater,  Florida 
NICOLL,  DR.    PAUL  A.,   Dept.   of   Physiology,   Indiana   University,   Bloomington, 

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

New  York 
OPPENHEIMER,  DR.  JANE  M.,  Dept.  of  Biology,  Bryn  Mawr  College,  Bryn  Mawr, 

Pennsylvania 
OSBORN,  PROF.  R.  C.,  Botany-Ecology  Bldg.,  Ohio  State  University,  Columbus  10, 

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

Maryland 
OSTERHOUT,  PROF.  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 


REPORT  OF  THE  DIRECTOR  33 

PACKARD,  DR.  CHARLES.  Woods  Hole,  Massachusetts 
PAGE,  DR.  IRVINE  H.,  Cleveland  Clinic,  Cleveland,  Ohio 

PARMENTER,  DR.  CHARLES  L.,  Dept.  of  Zoology,  University  of  Pennsylvania,  Phila- 
delphia, Pennsylvania 
PARPART,  DR.  ARTHUR  K.,  Dept.  of  Biology,  Princeton  University,  Princeton,  New 

Jersey 
PASSANO,  DR.  LEONARD  M.,  Osborn  Zoological  Laboratory,  Yale  University,  New 

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

Michigan 

PEEBLES,  PROF.  FLORENCE,  380  Rosemont  Avenue,  Pasadena  3,  California 
PERKINS,  DR.  JOHN  F.,  JR..  Dept.  of  Physiology,  University  of  Chicago,  Chicago 

37,  Illinois 
PETTIBONE,  DR.  MARIAN  H.,  Dept.  of  Zoology,  University  of  New  Hampshire, 

Durham,  New  Hampshire 

PIERCE,  DR.  MADELENE  E.,  Vassar  College,  Poughkeepsie,  New  York 
PLOUGH,  PROF.  HAROLD  H.,  Amherst  College,  Amherst,  Massachusetts 
POLLISTER,  DR.  A.  W.,  Columbia  University,  New  York  City,  New  York 
POND,  DR.  SAMUEL  E.,  53  Alexander  Street,  Manchester,  Connecticut 
PRATT,  DR.  FREDERICK  H..  105  Hundreds  Road,  Wellesley  Hills  82,  Massachusetts 
PROCTOR,  DR.  NATHANIEL,  Dept.  of  Biology,  Morgan  State  College,  Baltimore  12. 

Maryland 
PROSSER,  DR.  C.  LADD,  401  Natural  History  Bldg.,  University  of  Illinois,  Urbana, 

Illinois 
PROVASOLI,  DR.  LUIGI,  Dept.  of  Biology,  Haskins  Lab.,  305  E.  43rd  Street,  New 

York  17,  New  York 

QUASTEL,  DR.  JUDA  H.,  Dept.  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.,   Dept.    of   Zoology,   University  of   Connecticut,    Storrs, 

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

New  York  9,  New  York 

RAY,  DR.  CHARLES,  JR.,  Dept.  of  Biology,  Emory  University,  Emory,  Georgia 
REDFIELD,  DR.  ALFRED  C.,  Woods  Hole,  Massachusetts 
REID,  DR.  W.  M.,  Monmouth  College,  Monmouth,  Illinois 
REINER,  DR.  J.  M.,  Columbia-Presbyterian  Medical  Center,  622  W.  168  St.,  New 

York  32,  New  York 
RENN,  DR.  CHARLES  E.,  200  Whitehead  Hall,  Johns  Hopkins  University,  Baltimore 

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

New  York  City,  New  York 

RICE,  PROF.  E.  L.,  2241  Seneca  Avenue,  Alliance,  Ohio 
RICHARDS,  PROF.  A.,  2950E  Mabel  Street,  Tucson,  Arizona 
RICHARDS,  DR.  A.  GLENN,  Entomology  Dept.,  University  of  Minnesota,  St.  Paul, 

Minnesota 


34  MARINE  BIOLOGICAL  LABORATORY 

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

RIESER,  DR.  PETER,  Dept.  of  Zoology,  University  of  Pennsylvania,  Philadelphia  4, 
Pennsylvania. 

ROCKSTEIN,  DR.  MORRIS,  Dept.  of  Physiology,  N.  Y.  U.  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 

RONKIX,  DR.  RAPHAEL  R.,  Dept.  of  Physiology,  University  of  Delaware,  Newark, 
Delaware 

ROOT,  DR.  R.  W.,  Dept.  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,  Dept. 
of  Physiology,  New  York  City,  New  York 

ROSE,  DR.  S.  MERYL,  Dept.  of  Zoology,  University  of  Illinois,  Champaign,  Illinois 

ROSENTHAL,  DR.  THEODORE  B.,  Dept.  of  Anatomy,  Emory  University  Medical 
School,  Emory  University,  Georgia 

ROSSIE,  DR.  HAROLD  H.,  Dept.  of  Radiology.  Columbia  University,  New  York  32, 
New  York 

ROTH,  DR.  JAY  S.,  Dept.  of  Biochemistry,  Hahnemann  Medical  College,  Philadel- 
phia 2,  Pennsylvania 

ROTHENBERG,  DR.  M.  A.,  Chief,  Chemical  Labs.,  Duguay  Proving  Ground,  Duguay, 
Utah 

RUGH,  DR.  ROBERTS,  Radiological  Research  Lab.,  College  of  Physicians  and  Sur- 
geons, New  York  City,  New  York 

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

RUT  MAN,  DR.  ROBERT  J.,  Dept.  of  Zoology,  University  of  Pennsylvania,  Philadel- 
phia, Pennsylvania. 

RYAN,  DR.  FRANCIS  J.,  Columbia  University,  New  York  City,  New  York 

SAMPSON,  DR.  MYRA  M.,  Smith  College,  Northampton,  Massachusetts 

SANDEEN,  DR.  MURIEL  I.,  Dept.  of  Zoology,  Duke  University,  Durham,  North 
Carolina 

SAUNDERS,  MR.  LAWRENCE,  R.  D.  7,  Bryn  Mawr,  Pennsylvania 

SCHAEFFER,  DR.  ASA  A.,  Dept.  of  Biology,  Temple  University,  Philadelphia, 
Pennsylvania 

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

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

SCHLESSINGER,  DR.  R.  WALTER,  Public  Health  Research  Institute,  New  York  9, 
New  York 

SCHMIDT,  DR.  L.  H.,  Christ  Hospital,  Cincinnati,  Ohio 

SCHMITT,  PROF.  FRANCIS  O.,  Dept.  of  Biology,  Massachusetts  lust,  of  Technology, 
Cambridge,  Massachusetts 

SCHMITT,  DR.  O.  H.,  Dept.  of  Physics,  University  of  Minnesota,  Minneapolis  14, 
Minnesota 


REPORT  OF  THE  DIRECTOR 

SCHOLANDER,  DR.  P.  F.,  Inst.  Zoophysiology,  University  of  Oslo,  Oslo,  Norway 
SCHOTTE,  DR.  OSCAR  E.,  Dept.  of  Biology,  Amherst  College,  Amherst,   Massa- 
chusetts 
SCHRADER,  DR.  FRANZ,  Dept.  of  Zoology,  Columbia  University,  New  York  City, 

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

York  City,  New  York 

SCHRAMM,  PROF.  J.  R.,  University  of  Pennsylvania,  Philadelphia,  Pennsylvania 
SCOTT,  DR.  ALLAN  C,  Colby  College,  Waterville,  Maine 
SCOTT,  SISTER  FLORENCE  M.,  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.,  Dept.  of  Anatomy,  College  of  Physicians  and  Sur- 
geons, New  York  City.  New  York 
SHANES,  DR.  ABRAHAM  M.,  Experimental  Biology  and  Medicine  Inst.,  National 

Institutes  of  Health,  Bethesda  14,  Maryland 

SHAPIRO,  DR.  HERBERT,  5800  No.  Camac  Street,  Philadelphia  41,   Pennsylvania 
SHAVER,  DR.  JOHN  R.,  Research  Fellow,  California  Institute  of  Technology,  Pasa- 
dena 4,  California 
SHEDLOVSKY,  DR.  THEODORE,  Rockefeller  Institute,  66th   St.  and  York  Avenue, 

New  York  21,  New  York 

SHUMWAY,  DR.  WALDO,  Stevens  Institute  of  Technology,  Hoboken,  New  Jersey 
SICHEL,  DR.  FERDINAND  J.  M.,  University  of  Vermont,  Burlington,  Vermont 
SICHEL,  MRS.  F.  J.  M.,  35  Henderson  Terrace,  Burlington,  Vermont 
SILVA,  DR.  PAUL,  Dept.  of  Botany,  University  of  Illinois,  Urbana,  Illinois 
SLIFER,  DR.  ELEANOR  H.,  Dept.  of  Zoology,  State  University  of  Iowa,  Iowa  City, 

Iowa 
SMITH,  DR.  DIETRICH  C.,  Dept.  of  Physiology,  University  of  Maryland  School  of 

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

Massachusetts 

SMITH,  MR.  HOMER  P.,  Marine  Biological  Laboratory,  Woods  Hole,  Massachusetts 
SMITH,  DR.  RALPH   I.,  Dept.   of  Zoology,  University  of  California,   Berkeley  4, 

California 
SONNEBORN,    DR.    T.    M.,    Dept.   of   Zoology,    Indiana    University,    Bloomington, 

Indiana 

SONNENBLICK,  DR.  B.  P.,  40  Rector  Street,  Newark  2,  New  Jersey 
SPEIDEL,  DR.  CARL  C.,  University  of  Virginia,  University,  Virginia 
SPIEGEL,  DR.  MELVIN,  Kerckhoff  Labs.,  California  Institute  of  Technology,  Pasa- 
dena 4,  California 

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

STARR,  DR.  RICHARD  C,  Dept.  of  Botany,  Indiana  University,  Bloomington,  Indiana 
STEINBACH,  DR.  HENRY  BURR,  Dept.  of  Zoology,  University  of  Minnesota,  Minne- 
apolis 14,  Minnesota 

STEPHENS,  DR.  GROVER  C.,  Dept.  of  Zoology,  University  of  Minnesota,  Minne- 
apolis 14,  Minnesota 


36  MARINE  BIOLOGICAL  LABORATORY 

STERN,  DR.  KURT  G.,  Polytechnic  Institute,  Dept.  of  Chemistry,  84  Livingston 

St.,  Brooklyn,  New  York 

STEWART,  DR.  DOROTHY,  Rockford  College,  Rockford,  Illinois 
STOKEY,  DR.  ALMA  G.,  Dept.  of  Botany,  Mt.  Holyoke  College,  South  Hadley, 

Massachusetts 

STRAUSS,  DR.  W.  L.,  JR.,  Johns  Hopkins  University,  Baltimore  18,  Maryland 
STUNKARD,  DR.  HORACE  W.,  New  York  University,  New  York  City,  New  York 
STURTEVANT,  DR.  ALFRED  H.,  California   Institute  of  Technology,   Pasadena  4, 

California 
SULKIN,  DR.  S.  EDWARD,  Dept.  of  Bacteriology,  Southwestern  Medical  School, 

University  of  Texas,  Dallas,  Texas 

SWOPE,  MR.  GERARD,  JR.,  570  Lexington  Avenue,  New  York  22,  New  York 
SZENT-GYORGYI,  DR.  A.  E.,  Woods  Hole,  Massachusetts 
SZENT-GYORGYI,  DR.  ANDREW  G.,  Institute  for  Muscle  Research,  MBL,  Woods 

Hole,  Massachusetts 

TASHIRO,  DR.  SHIRO,  University  of  Cincinnati  Medical  College,  Cincinnati,  Ohio 
TAYLOR,  DR.  WM.  RANDOLPH,  University  of  Michigan,  Ann  Arbor,  Michigan 
TEWINKEL,  DR.  Lois  E.,  Dept.  of  Zoology,  Smith  College,  Northampton,  Massa- 
chusetts 
TRACY,  DR.  HENRY  C,  Dept.  of  Anatomy,  University  Medical  Center,  Jackson, 

Mississippi 
TRACER,  DR.  WILLIAM,  Rockefeller   Institute,  66th   St.  and  York  Avenue,  New 

York  21,  New  York 
TRINKAUS,  DR.  J.  PHILIP,  Osborn  Zoological  Laboratory,  Yale  University,  New 

Haven,  Connecticut 

TURNER,  PROF.  C.  L.,  Northwestern  University,  Illinois 

TYLER,  DR.  ALBERT,  California  Institute  of  Technology,  Pasadena  4,  California 
UHLENHUTH,  DR.  EDWARD,  University  of  Maryland,  School  of  Medicine,  Balti- 
more, Maryland 
DEViLLAFRANCA,  DR.  GEORGE  W.,  Marine  Biological  Laboratory,  Woods  Hole, 

Massachusetts 

VILLEE,  DR.  CLAUDE  A.,  Harvard  Medical  School,  Boston  15,  Massachusetts 
VINCENT,  DR.  WALTER  S.,  Dept.  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  Laboratories,  Harvard  University,  Cambridge  38, 

Massachusetts 

WARBASSE,  DR.  JAMES  P.,  Woods  Hole,  Massachusetts 
WARNER,  DR.  ROBERT  C.,  Dept.  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,  Dept.  of  Physiology  &  Bacteriology,  Goucher  College, 

Towson,  Maryland 
WEISS,  DR.  PAUL  A.,  Lab.  of  Developmental  Biology,  Rockefeller  Institute,  New 

York  21,  New  York 


REPORT  OF  THE  DIRECTOR 

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  Dept,  Temple  University,  Philadelphia, 
Pennsylvania 

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

WILBER,  DR.  C.  G.,  Medical  Labs.,  Applied  Physiology  Branch,  Army  Chemical 
Center,  Maryland 

WILLIER,  DR.  B.  H.,  Dept.  of  Biology,  Johns  Hopkins  University,  Baltimore, 
Maryland 

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

WILSON,  DR.  WALTER  L.,  Dept.  of  Physiology,  University  of  Vermont  College  of 
Medicine,  Burlington,  Vermont 

WITSCHI,  PROF.  EMIL,  Dept.  of  Zoology,  State  University  of  Iowa,  Iowa  City, 
Iowa 

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

WOODWARD,  DR.  ARTHUR  A.,  Army  Medical  Center,  Maryland  (Applied  Physi- 
ology Branch,  Army  Chemical  Corps  Med.  Lab.) 

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

WRINCH,  DR.  DOROTHY,  Dept.  of  Physics,  Smith  College,  Northampton,  Massa- 
chusetts 

YNTEMA,  DR.  C.  L.,  Dept.  of  Anatomy,  University  of  New  York  College  of  Medi- 
cine, Syracuse  10,  New  York 

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

ZINN,  DR.  DONALD  J.,  Zoology  Dept.,  University  of  Rhode  Island,  Kingston,  Rhode 
Island 

ZORZOLI,  DR.  ANITA,  Dept.  of  Physiology,  Southern  Illinois  University,  Carbon- 
dale,  Illinois 

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

3.  ASSOCIATE  MEMBERS,  1955 

ALDRICH,  Miss  AMEY  OWEN  BRADLEY,  MR.  ALBERT  L. 

ALTON,  DR.  AND  MRS.  BENJAMIN  H.  BRADLEY,  MRS.  CHARLES  CRANE 

ANTHONY,  MR.  RICHARD  A.  BROWN,  MRS.  THORNTON 

ARMSTRONG,  DR.  AND  MRS.  P.  B.  BURDICK,  MR.  CHARLES  L. 

BARBOUR,  MR.  Lucius  CAHOON,  MRS.  SAMUEL 

BARTOW,  MR.  AND  MRS.  CLARENCE  CALKINS,  MR.  G.  NATHAN,  JR. 

BARTOW,  MRS.  FRANCIS  D.  CALKINS,  MRS.  GARY  N. 

BARTOW,  MR.  AND  MRS.  PHILIP  CALKINS,  MR.  SAMUEL 

BELL,  MRS.  ARTHUR  CARLETON,  MRS.  WINSLOW 


38 


MARINE  BIOLOGICAL  LABORATORY 


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 

CROSSLEY,  MR.  AND  MRS.  ARCHIBALD  M. 

CROWELL,  MR.  PRINCE  S. 

DANIELS,  MR.  AND  MRS.  F.  HAROLD 

DAY,  MR.  AND  MRS.  POMEROY 

DRAPER,  MRS.  MARY  C. 

ELSMITH,  MRS.  DOROTHY 

ENDERS,  MR.  FREDERICK 

EWING,  MR.  FREDERICK 

FAY,  MR.  AND  MRS.  HENRY  H. 

FISHER,  MRS.  BRUCE  CRANE 

GALTSOFF,  MRS.  EUGENIA 

GlFFORD,  MR.  AND  MRS.  JOHN  A. 

GlLCHRIST,  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. 
HOUSTON,  MR.  AND  MRS.  HOWARD  E. 
HOWE,  MRS.  HARRISON  E. 
JANNEY,  MRS.  WALTER  C. 
JEWETT,  MR.  AND  MRS.  GEORGE  F. 
KEITH,  MR.  AND  MRS.  HAROLD  C. 
KIDDER,  MRS.  HENRY  M. 
KING,  MR.  FRANKLIN 
KOLLER,  MRS.  LEWIS 
LAWRENCE,  MR.  MILFORD 
LEMANN,  MRS.  SOLEN  B. 
LOBB,  MRS.  JOHN 
McCLINTIC,  MRS.  GUTHRIE 
MARVIN,  MRS.  WALTER  T. 
MAST,  MRS.  S.  O. 
MEIGS,  MRS.  EDWARD  B. 
MEIGS,  DR.  AND  MRS.   ].  WISTER 


MEIGS,  Miss  MARY  ROBERTS 
MELLON,  MRS.  RICHARD  K. 
MISKELL,  MR.  JOSEPH  B. 
MITCHELL,  MRS.  JAMES  McC. 
MIXTER,  MRS.  JASON 
MONTGOMERY,  MRS.  T.  H. 
MOORE,  MRS.  WILLIAM  A. 
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. 
PECK,  MR.  AND  MRS.  SAMUEL  A. 
PENNINGTON,  Miss  ANNE  H. 
REDFIELD,  MRS.  ALFRED 
REZNIKOFF,  DR.  PAUL 
RIGGS,  MRS.  LAWRASON 
RIYINUS,  MR.  AND  MRS.  F.  MARKOE 
RODES,  MRS.  BOYLE 
ROOT,  MRS.  WALTER 
RUDD,  MRS.  H.  W.  DWIGHT 
SANDS,  Miss  ADELAIDE  G. 
SAUNDERS,  MRS.  LAWRENCE 
SINCLAIR,  MR.  W.  R. 
SMITH,  MRS.  EDWARD  H. 
STANWOOD,  MRS.  F.  A. 
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. 
VANNEMAN,  DR.  AND  MRS.  JOSEPH 
WAKSMAN,  MRS.  SELMAN  A. 
WARBASSE,  DR.  JAMES  P. 
WEBSTER,  MRS.  EDWIN  S. 
WHITELY,  MR.  AND  MRS.  G.  W.,  JR. 
WHITELY,  Miss  MABEL  W. 

WlCKERSHAM,  MR.  AND  MRS.  JAMES  H. 

WILLISTON,  Miss  EMILY 
WILLISTON,  PROF.  SAMUEL 
WILSON,  MRS.  EDMUND  B. 

WOLFINSOHN,    MRS.    WOLFE 


REPORT  OF  THE  LIBRARIAN  39 

V.  REPORT  OF  THE  LIBRARIAN 

In  1955,  the  number  of  currently-received  journals  totalled  1554  (51  new). 
Of  these  titles,  there  were  467  (5  new)  Marine  Biological  Laboratory  subscrip- 
tions; 604  (10  new)  exchanges  and  179  (13  new)  gifts;  74  (2  new)  were  Woods 
Hole  Oceanographic  Institution  subscriptions;  183  (12  new)  were  exchanges  and 
47  (9  new)  were  gifts. 

The  Laboratory  purchased  56  books,  received  84  complimentary  copies  (10  from 
authors  and  74  from  publishers)  and  60  miscellaneous  presentations.  The  Insti- 
tution purchased  28  titles.  The  total  number  of  new  books  accessioned  amounted 
to  228. 

By  purchase,  the  Laboratory  completed  6  journal  sets  and  partially  completed 
13  sets.  The  Institution  completed  two  sets  and  partially  completed  two  sets. 
Volumes  and  numbers  received  by  gift  and  by  exchange  completed  13  sets  and  par- 
tially completed  10  sets. 

There  were  3891  reprints  added  to  the  collection,  of  which  2317  were  of  current 
issue. 

The  Library  sent  out  on  inter-library  loan  190  volumes  and  borrowed  57  for  the 
convenience  of  the  investigators.  A  sum  of  $246.43  was  realized  from  the  sale  of 
duplicate  material. 

At  the  end  of  the  year,  Dr.  E.  Newton  Harvey  presented  to  the  Laboratory  his 
large  and  valuable  collection  of  12,000  reprints.  Of  these,  2500  were  added  to  the 
shelves,  many  of  which  filled  in  sets,  making  them  of  far  greater  value  than  hereto- 
fore. This  gift  is  greatly  appreciated  and  grateful  acknowledgment  is  herewith 
conveyed  to  the  donor.  The  duplicates  of  the  reprints  already  in  the  Library's 
possession  will  be  sent  to  another  library  of  Dr.  Harvey's  choosing. 

Other  gifts  of  reprints  and  books  were  received  from  Drs.  Wm.  R.  Amberson, 
Roberts  Rugh,  Kurt  G.  Stern  and  Ralph  A.  Lewin.  Without  these  generous  con- 
tributions, the  Library  would  be  minus  man}-  worthwhile  acquisitions. 

At  the  end  of  the  year,  the  Library  contained  65,463  bound  volumes  and  196,089 
reprints. 

During  the  years  1914-1923,  Dr.  R.  P.  Bigelow  served  as  Librarian.  In  the 
years  following,  he  did  not  lose  interest  in  the  growth  and  development  of  the 
Library  and  throughout  the  past  ten  years,  he  made  many  generous  and  valuable 
contributions.  In  keeping  with  his  thoughtfulness,  Mrs.  Bigelow  presented  four 
fine  portraits  which  had  hung  in  her  husband's  study,  namely,  those  of  Brooks, 
Darwin,  Huxley  and  Lamarck.  Through  Dr.  Bigelow's  death,  the  Library  has  lost 
a  great  friend. 

Respectfully  submitted, 

DEBORAH  L.  HARLOW, 

Librarian 


40  MARINE  BIOLOGICAL  LABORATORY 

VI.  REPORT  OF  THE  TREASURER 

MARINE  BIOLOGICAL  LABORATORY 

BALANCE  SHEET 
December  31,  1955 


Investments 
Investments  held  by  Trustee: 

Securities,  at  cost  (approximate  market  quotation  $1,500,773) $    979,202 

Cash.  1,441 


980,643 

Investments  of  other  endowment  and  unrestricted  funds: 

Pooled  Investments,  at  cost  (approximate  market  quotation  $230,368) 213,784 

Other  investments  (note  B) 53,076 

Cash.  9.155 


276,015 


Plant  Assets 

Land,  buildings,  library  and  equipment  (note  A) 2,406,077 

Less  allowance  for  depreciation  (note  A) 955,753 


1,450,324 


Current  Assets 

Cash 36,857 

Accounts  receivable  ($3,596  from  U.  S.  Government) 34,754 

Inventories  of  specimens  and  Bulletins 61,158 

Prepaid  insurance  and  other 8,432 


$2,848,183 


Notes : 


A — 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  1%  to  5% 
of  the  original  cost  of  the  assets. 

B — The  Laboratory  has  guaranteed  a  note  of  approximately  $4,000  of  the  M.  B.  L. 
Club  and  has  pledged  as  security,  therefore,  bonds  with  an  original  cost  of  $7,900, 
included  in  other  investments. 


REPORT  OF  THE  TREASURER  41 


MARINE  BIOLOGICAL  LABORATORY 

BALANCE  SHEET 
December  31,  1955 

Endowment  Funds 

Endowment  funds  given  in  trust  for  the  benefit  of  the  Marine  Biological 

Laboratory $    980,643 

Endowment  funds  for  awards  and  scholarships: 

Principal 31,738 

Unexpended  income 1,377          33,115 

Unrestricted  funds  functioning  as  endowment 206,378 

Retirement  fund 35,408 

Pooled  investments — accumulated  gain 1,114 

276,015 


Plant  Liability  and  Funds 

Mortgage  payable  on  demand,  5% 5,000 

Funds  expended  for  plant,  less  retirements 2,401,077 

Less  allowance  for  depreciation  charged  thereto 955,753     1,445,324 


1,450,324 


Current  Liabilities  and  Funds 


Accounts  payable 5,767 

Unexpended  balances  of  gifts  for  designated  purposes 8,229 

Advance  payments  on  research  contracts 7,646 

Current  fund.  119,559 


$2,848,183 


42  MARINE  BIOLOGICAL  LABORATORY 

MARINE  BIOLOGICAL  LABORATORY 

STATEMENT  OF  OPERATING  EXPENDITURES  AND  INCOME 

Year  Ended  December  31,  1955 

Operating  Expenditures 
Direct  expenditures  of  departments: 

Research  and  accessory  services $145,306 

Instruction 23,967 

Library,  including  book  purchases 30,389 

Biological  Bulletin 12,684 


212,346 

Administration  and  general 46,988 

Plant  operation  and  maintenance 68,974 

Hurricane  emergency  repairs 22,059 

Dormitories  and  dining  services 128,460 

Equipment  purchased  from  current  funds 1,712 


480,539 
Less  depreciation  included  in  plant  operation  and  auxiliary  activities  above 

but  charged  to  plant  funds 36,429 


444,110 

Income 
Direct  income  of  departments: 

Research  fees 40, 194 

Accessory  services  (including  sales  of  biological  specimens,  $67,436) 93,230 

Instruction  fees 17,930 

Library  fees  and  income 6,537 

Biological  Bulletin,  subscriptions  and  sales 15,920 


173,811 

Allowance  for  indirect  costs  on  research  contracts 15,747 

Dormitories  and  dining  services  income 100,768 


290,326 

Investment  income 73,581 

Gifts  for  current  use 121,053 

Sundrv  income.  394 


Total  current  income 485,354 


Excess  of  income $  4 1 ,244 


Direct    costs    on    research    contracts    and    reimbursement    therefor, 
$41,311,  are  not  included  in  operating  expenditures  or  income. 


REPORT  OF  THE  TREASURER 


43 


MARINE  BIOLOGICAL  LABORATORY 
STATEMENT  OF  CURRENT  FUND 
Year  Ended  December  31,  1955 

Balance  January  1,  1955 $  84,586 

Less: 

Amount  transferred  to  unrestricted  funds  functioning  as  endowment  as  of  July  15, 

6,843 


1955. 


Add: 


77,743 


Provision  for  uncompleted  repairs  included  in  operating  expenditures 572 

Excess  of  income  over  operating  expenditures,  1955 41,244 


Balance  December  31,  1955 $119,559 


MARINE  BIOLOGICAL  LABORATORY 
SUMMARY  OF  INVESTMENTS 


December  31,  1955 


Cost 


%of 
Total 


Approximate  Investment 

Market          %  of        Income 
Quotations       Total          1955 


Securities  held  by  Trustee: 
General  endowment  fund : 


U.  S.  Government  bonds $184,206 

Other  bonds.  285,429 


469,635 

Preferred  stocks 85,788 

Common  stocks 265,663 


22.4 
34.8 

$  178,437 
288,716 

14.4 
23.3 

$  4,337 
9,079 

57.2 
10.4 
32.4 

467,153 
80,688 
691,929 

37.7 
6.5 
55.8 

13,416 
3,370 
25,148 

100.0 

1,239,770 

100.0 

41,934 

General  Education  Board  endow- 
ment fund : 


Other  bonds 


Preferred  stocks 
Common  stocks 


Total  securities  held  by 
Trustee. . 


iment  bonds  48  139 

304 

46  633 

17  9 

1  113 

30,637 

194 

30425 

11  6 

997 

78,776 
cks              27,281 
cks              52,059 

49.8 
17.3 
32.9 

77,058 
26,637 
157,308 

29.5 
10.2 
60.3 

2,110 
1,130 
5,350 

158,116 

100.0 

261,003 

100.0 

8,590 

$979.202 


$1,500,773 


$50,524 


44 


MARINE  BIOLOGICAL  LABORATORY 


MARINE  BIOLOGICAL  LABORATORY 

SUMMARY  OF  INVESTMENTS — Continued 

December  31,  1955 


Cost 


70  O 
Total 


Approximate  Investment 

Market          %  of        Income 
Quotations      Total          1955 


Investments  of  other  endowment  and 
unrestricted  funds: 
Pooled  investments: 


U   S  Government  bond^ 

$  40  340 

18.9 

$      38,231 

16.6 

$      952 

Other  bonds 

82  428 

38.5 

81,455 

35.4 

2,007 

Common  stocks  

122,777 
91,007 

57.4 
42.6 

119,686 
110,682 

52.0 
48.0 

2,959 

4,382 

213,784 

100.0 

230,368 

100.0 

7,341 

Other  investments: 


7,920 


Bonds 

Investment  in  General  Bio- 
logical Supply  House,  Inc.         12,700 
Real  estate  and  mortgage 32,456 


330 
17,780 

18,110 

$25,451 

Total  investment  income  received 75,975 

Custodian's  fees  charged  thereto (254) 


53,076 

Total  investments  of  other  en- 
dowment and  unrestricted 
funds.  .  $266,860 


Investment  income .  .  $75,72 1 


STUDIES  ON  CROSS-FERTILIZATION  AND   SELF-FERTILIZATION 
IN  LYMNAEA  STAGNALIS  APPRESSA  SAY1 

GERTRUDE  L.  CAIN  -' 
Department  of  Zoology,  University  of  Wisconsin,  Madison,  Wisconsin 

In  1817  Oken  obtained  fertile  eggs  from  L\innaca  anricularis  which  were  reared 
in  isolation  during  their  entire  reproductive  period.  Baudelot  (1863)  reported 
both  self-fertilization  and  cross-fertilization  in  Lynmaca.  Pelseneer  (1920)  saw 
only  one  polar  body  extruded  from  the  eggs  of  Lynmaca  (three  species),  and  con- 
cluded that  reproduction  in  isolated  snails  was  parthenogenetic.  However,  Colton, 
(1918)  in  L.  cohnnclla  and  Crabb  (1927a)  in  L.  stagnates  observed  two  polar 
bodies  and  on  the  basis  of  their  observations  concluded  that  parthenogenesis  did  not 
occur.  Colton  further  reported  that,  although  self-fertilization  did  occur,  cross- 
fertilization  was  the  rule;  Crabb  reported  that  cross-fertilization  was  mechanically 
impossible  (1927b).  Seshaiya  (1927)  concluded  from  a  study  of  breeding  habits 
of  L.  Inicola  that  both  cross-  and  self-fertilization  occurred  in  this  species. 

Lang  in  1900  claimed  that  self-fertilization  could  occur  without  self-copulation, 
while  Kunkel  (1908)  believed  that  self-copulation  was  indispensable  to  self-fertiliza- 
tion, basing  his  opinion  in  part  on  the  observation  of  self-copulation  in  L.  anricularis 
by  Von  Baer  in  1835.  Colton  and  Pennypacker  (1934)  reported  that  self-fertili- 
zation in  L.  colnmclla  for  93  generations  did  not  decrease  the  viability  of  the  strain. 
Boettger  (1944),  in  his  survey  of  the  Basommatophora,  concluded  that  self-  and 
cross-fertilization  were  both  common  in  this  order.  DeWitt  (1954)  found  the  per- 
centage of  hatching  less  in  self-fertilized  eggs  of  Physa  g \rina  than  in  cross-fertilized 
eggs. 

The  first  genetic  proof  that  both  self-  and  cross-fertilization  occur  in  snails  was 
supplied  by  Diver,  Boycott  and  Garstang  (1925)  in  a  study  of  the  inheritance  of 
inverse  symmetry  in  L.  pcrcgra.  Further  proof  was  obtained  in  the  study  of  the 
inheritance  of  albinism  in  this  snail  (Boycott  and  Diver,  1927).  Ikeda  and  Mura 
(1934),  using  shell  color  as  a  genetic  marker,  demonstrated  that  both  self-  and 
cross-fertilization  occurred  in  the  land  snail,  Bradybacna  siinilaris. 

Bretschneider  (1948a,  1948b)  investigated  the  mechanism  of  insemination  and 
oviposition  in  L.  stagnalis.  He  reported  that  he  had  seen  sperm  balls  leaving  the 
seminal  vesicle  and  being  swept  up  the  female  tract  to  the  hermaphroditic  duct, 
where  he  assumed  fertilization  occurred.  As  additional  evidence  he  reported  see- 
ing a  complete  spermatozoon  inside  the  cytoplasm  of  an  egg  still  in  the  duct. 

1  Revised  from  a  dissertation  presented  to  the  graduate  School  of  the  University  of  Wiscon- 
sin in  partial  fulfillment  of  requirements  for  the  degree  of  Doctor  of  Philosophy.     The  author 
wishes  to  express  gratitude  to  Professor  L.  E.  Noland  for  his  kindness  and  assistance  during 
the  course  of  this  study.     She  is  also  grateful  to  the  General  Education  Board  and  the  National 
Medical  Foundation  Inc.   (funds  contributed  by  the  National  Foundation  of  Infantile  Paralysis) 
for  financial  assistance  during  the  time  this  study  was  being  pursued. 

2  Present  address :  West  Virginia  State  College ;   Institute,  West  Virginia. 

45 


46  GERTRUDE  L.  CAIN 

Holm  (1946),  in  micro-anatomical  studies  on  the  reproductive  tract  of  L.  st  agnails, 
found  a  "fertilization  pocket"  homologous  to  that  found  by  Meisenheimer  (1912)  in 
Helix  pomatia,  but  saw  no  eggs  or  spermatozoa  in  it.  Perrot  (1940)  reported  a 
similar  structure  in  Lima*  maximus.  Abdel-Malek  (1954a,  1954b)  saw  ova  in  the 
corresponding  pockets  in  Helisoma  trivolvis  and  Biomphalaria  biossyi. 

The  present  study  was  undertaken  to  determine :  ( 1 )  the  mode  of  inheritance  of 
albinism  in  Lymnaea  st  agnails;  (2)  the  relative  frequency  of  self-fertilization  as 
compared  with  cross-fertilization  in  this  species;  (3)  the  survival  time  of  spermato- 
zoa after  transfer  from  one  snail  to  another;  and  (4)  the  possible  location  of 
fertilization  of  the  eggs  in  this  snail. 

MATERIALS  AND  METHODS 

The  snails  used  in  this  study  were  obtained  from  strains  that  had  been  main- 
tained in  laboratory  culture  at  the  University  of  Wisconsin  for  over  ten  years. 
Culture  methods  were  those  of  Noland  and  Carriker  (1946). 

MODE  OF  INHERITANCE  OF  ALBINISM 

Although  albinism  has  been  found  to  be  inherited  as  a  simple  Mendelian  reces- 
sive in  several  other  gastropods  (Boycott  and  Diver,  1927,  in  L.  pcrci/ra ;  Ikeda, 
1937,  in  Philomycus  bilincatus),  it  was  necessary  to  verify  this  for  L.  staynalis  be- 
fore albinism  could  be  used  as  a  genetic  marker  in  this  study. 

Accordingly  two  snails,  one  from  the  albino  culture  and  one  from  the  pigmented 
culture,  were  isolated  until  each  had  deposited  at  least  one  egg  mass.  The  offspring 
from  the  eggs  of  the  pigmented  snail  were  all  pigmented  and  those  from  the  albino 
snail  were  all  albinos.  The  two  parent  snails  were  then  paired  for  42  days.  Dur- 
ing this  time  one  copulation  was  observed  with  the  pigmented  snail  serving  as  the 
male.  Presumably  other  such  copulations  occurred  when  the  snails  were  not  under 
observation. 

After  42  days  the  two  snails  were  separated.  The  albino  was  kept  in  isolation 
culture,  and  its  egg  masses  were  collected.  The  offspring  resulting  from  these  egg 
masses  were  examined  under  a  binocular  microscope  six  days  after  hatching.  By 
this  time  pigment  had  developed  along  the  mantle  collar.  Any  young  not  showing 
pigment  were  re-examined  after  another  five  or  six  days.  Of  885  offspring  grown 
from  28  egg  masses  laid  by  the  albino  snail,  43  were  albinos,  resulting  presumably 
either  from  self-fertilization  or  from  previous  copulations  with  other  albinos  in  the 
original  stock  culture.  The  842  pigmented  offspring  of  the  albino  parent  were 
clearly  the  result  of  fertilization  of  the  eggs  of  the  albino  by  spermatozoa  from  its 
pigmented  mate. 

Six  of  these  pigmented  heterozygotes  were  selected  and  isolated  before  sexual 
maturity.  Each  was  maintained  in  solitary  culture  to  insure  that  only  self-fertiliza- 
tion would  occur.  This  self-fertilization  is  obviously  the  equivalent  of  crossing  two 
F!  heterozygotes.  Five  or  more  egg  masses  were  saved  from  each  snail,  and  the 
progeny  therefrom  were  grown  to  the  age  of  "pigment-testing."  Of  a  total  of  4909 
eggs  (49  egg  masses)  from  the  six  heterozygous  snails,  64.7%  hatched,  and  of  those 
that  hatched  92.9%  survived  to  be  examined  for  the  presence  of  pigment.  Of  2949 
thus  surviving,  2193  were  pigmented  and  765  were  albinos.  On  a  3  :  1  basis  the 


CROSS-FERTILIZATION  IN  LYMNAEA  47 

expected  ratio  would  have  been  2212:737.  The  agreement  is  close  and,  on  the 
basis  of  chi  square  tests,  the  difference  between  the  expected  and  the  observed  ra- 
tio was  not  significant.  It  may  therefore  be  concluded  with  confidence  that  albinism 
in  L.  stagnalis  is  inherited  as  a  simple  Mendelian  recessive,  as  in  other  gastropods. 

PREVALENCE  OF  SELF-FERTILIZATION 

Eighteen  albino  snails  (15  with  pedigreed  albino  parentage  and  3  from  ex- 
clusively albino  stock  cultures)  were  paired,  each  with  a  homozygous  pigmented 
snail  taken  from  the  stock  culture  which  for  ten  years  had  shown  no  albinos.  Each 
of  these  pairs  was  kept  in  a  separate  dish  for  varying  lengths  of  time  (from  20  to 
187  days,  depending  on  the  pair).  The  albino  partners  were  thereafter  maintained 
in  isolation  culture.  The  eggs  produced  by  these  albinos  were  saved  until  the 
hatching  snails  reached  the  "pigment-testing"  age  to  determine  the  relative  num- 
bers of  pigmented  and  albino  progeny.  It  was  assumed  that  the  albino  progeny 
resulted  from  self-fertilization  and  the  pigmented  offspring  from  cross-fertilization. 
There  was  a  slight  possibility  that  the  three  snails,  taken  as  adults  from  albino 
cultures,  might  have  cross-copulated  with  other  albinos  before  isolation.  Two  of 
these  three  snails  showed  100%  pigmented  offspring  in  their  first  egg  mass.  The 
third  \vas  never  seen  to  copulate  with  its  pigmented  partner,  and  produced  only 
albino  offspring  throughout  its  life.  Since  all  other  snails  were  young  (less  than 
130  days)  and  since  no  copulations  had  been  observed  in  the  cultures  from  which 
they  were  taken,  the  possibility  that  they  had  already  cross-copulated  with  other 
albinos  is  extremely  small. 

Of  the  18  albino  snails  paired  with  pigmented  mates,  15  of  them  after  separation 
produced  mainly  pigmented  offspring  during  the  first  month,  while  three  gave  only 
albino  progeny.  From  this  it  is  clear  that,  when  albino  and  pigmented  snails  are 
paired,  not  only  does  cross-fertilization  occur,  but,  contrary  to  the  opinion  of  Crabb 
(1927b),  it  is  the  predominating  process. 

Ten  of  the  15  albino  snails  that  produced  pigmented  offspring  after  separation 
from  their  pigmented  mate  gave  100%  pigmented  young  in  at  least  one  of  their 
egg  masses.  In  five  of  these  it  was  the  first  egg  mass  laid  after  isolation  that  gave 
only  pigmented  progeny.  One  showed  only  albinos  in  its  first  egg  mass,  but  by  its 
third  egg  mass  was  producing  100%  pigmented  young.  Of  the  ten  snails  that  gave 
100r;  pigmented  offspring  in  at  least  one  egg  mass,  four  were  producing  albinos 
exclusively  by  the  end  of  their  lives.  Three  others,  however,  were  still  producing 
100%  pigmented  progeny  in  the  last  egg  mass  laid  before  they  died.  Noland  and 
Carriker  (1946)  have  shown  that  snails  maintained  in  solitary  culture  their  entire 
lives  frequently  will  produce  more  fertile  eggs  than  snails  allowed  to  cross-copulate. 
It  is  therefore  unlikely  that  the  continued  production  of  pigmented  offspring  by  the 
three  snails  mentioned  above  could  have  been  due  to  any  lack  of  fertilizing  ability  on 
the  part  of  the  animal's  own  sperm. 

While  cytological  tests  were  not  made  to  eliminate  the  possibility  of  partheno- 
genesis in  the  case  of  isolated  snails,  this  seems  unlikely  because  of  the  almost  exact 
3  :  1  ratio  obtained  in  the  offspring  of  the  isolated  heterozygous  snails  mentioned 
earlier  in  this  paper.  Had  haploid  parthenogenesis  occurred,  a  ratio  nearer  to  1  :  1 
would  have  been  expected.  If  diploid  parthenogenesis  had  occurred  exclusively, 
only  pigmented  offspring  would  have  been  expected.  Moreover,  the  work  of  Col- 


48 


GERTRUDE  L.  CAIN 


ton  (1918)  and  Crabb  (1927b,  1928)  indicated  that  two  polar  bodies  are  extruded 
by  the  eggs  in  Lymnaca. 

Each  of  the  18  albino  snails  mentioned  above  was  kept  in  isolation  culture  until 
its  death,  with  one  exception.  This  snail  was  discarded  after  producing  nothing 
but  albino  offspring  in  its  first  five  egg  masses.  The  ages  of  the  snails  at  death,  in 
the  14  cases  where  it  was  known  exactly,  varied  from  128  to  465  days.  The  latter 
figure  represents  the  oldest  snail  ever  reared  in  this  laboratory. 

LONGEVITY  OF  TRANSFERRED  SPERM 

The  time  elapsing  between  the  separation  of  an  albino  snail  from  its  pigmented 
mate  and  the  laying  of  its  last  "pigment-producing"  egg  gives  an  approximate  figure 
for  the  survival  time  of  transferred  sperm  in  the  recipient  snail.  The  maximum 
time  found  in  this  study  was  116  days.  To  get  some  idea  about  how  fast  the  ferti- 
lizing power  of  transferred  sperm  is  lost,  the  data  obtained  from  13  of  the  18  snails 
referred  to  earlier  were  combined.  Of  the  five  snails  not  used  in  the  calculations, 
three  (as  mentioned  above)  had  not  received  sperm  from  their  pigmented  mate, 
and  two  others  died  too  early  to  give  significant  data. 


10  Q^ 


40 


60  SO 

after    I5oid.ti.on 


too 


120 


FIGURE  1.  Graph  showing  rate  of  decrease  in  percentage  of  pigmented  individuals  in  the 
offspring  of  albino  snails  that  were  isolated  after  receiving  sperm  from  pigmented  snails.  (Data 
obtained  from  egg  masses  deposited  by  the  albino  after  the  102nd  day  of  isolation  could  not  be 
treated  in  this  graph.) 


CROSS-FERTILIZATION  IN  LYMNAEA  49 

The  data  from  the  13  remaining  snails  represented  260  egg  masses,  containing 
15,545  eggs.  Of  these  72.5%  hatched;  and  of  those  that  hatched,  87.8%  survived 
to  the  "pigment-producing"  age.  Figure  1  presents  a  curve  showing  the  percentage 
of  pigmented  individuals  in  the  progeny  of  the  13  snails  plotted  against  time  elapsed 
since  separation  from  their  pigmented  mates.  The  curve  represents  a  moving 
average,  smoothed  as  follows  :  each  point  on  the  curve  represents  the  total  pigmented 
offspring  developing  from  eggs  laid  in  the  10  days  just  preceding  a  chosen  point, 
divided  by  the  total  offspring  produced  from  all  the  eggs  laid  during  the  same  period. 
The  curve  thus  represents  substantially  the  percentage  of  cross-fertilization  on  suc- 
cessive days  following  separation. 

Examination  of  the  curve  shows  that  in  the  first  50  days  cross-fertilization  was 
very  high  (over  80^)-  after  which  time  it  gradually  fell,  dropping  rather  suddenly 
near  the  100th  day.  As  stated  earlier  the  maximum  figure  obtained  was  116  days. 
Whether  this  figure  really  represents  the  maximum  survival  period  of  the  sperm 
or  merely  the  time  at  which  the  supply  of  transferred  sperm  was  all  used  up,  it  is 
impossible  to  say. 

To  arrive  at  a  more  exact  figure  for  sperm  survival  after  transfer,  it  would  be 
necessary  (1)  to  add  to  the  figure  obtained  (116  days)  the  time  elapsing  between 
the  last  copulation  and  the  separation  of  the  two  partners,  and  (2)  to  subtract  from 
the  figure  the  time  elapsing  between  actual  fertilization  and  the  laying  of  the  egg. 
These  corrections  cannot  be  made  from  the  data  here  obtained. 

LOCATION  OF  FERTILIZATION 

The  observations  of  Meisenheimer  (1912),  Holm  (1946)  and  Abdel-Malek 
(1954a,  1954b)  suggest  that  the  sperm  probably  enters  the  egg  in  or  near  the  "ferti- 
lization pocket."  Bretschneider  (1948a).  however,  thinks  that  fertilization  may 
occur  as  high  up  in  the  reproductive  tract  as  the  hermaphroditic  duct.  (The  anat- 
omy of  the  reproductive  system  of  Lymnaca  stagnalis  is  shown  in  Figure  2.) 

If  foreign  sperm  after  copulation  actually  travel  up  the  female  tract  as  far  as  the 
hermaphroditic  duct,  as  Bretschneider  implies,  it  would  seem  likely  that  they 
would  mix  with  the  sperm  of  the  recipient  snail.  Then  if  such  a  mixture  of  sperm 
were  later  transferred  in  copulation,  it  is  conceivable  that  some  of  the  foreign  sperm 
might  be  passed  along  to  a  third  snail.  This  possibility  was  tested  as  described 
below. 

Ten  albino  snails  that  had  never  been  with  pigmented  snails  were  paired  with 
pigmented  mates  until  the  albinos  were  seen  to  function  as  females  in  copulation 
with  those  mates.  Each  of  these  albinos  was  then  marked  with  finger  nail  polish  on 
the  tip  of  the  shell  and  placed  with  another  albino  which  had  never  been  with  a 
pigmented  snail.  The  pairs  were  maintained  until  the  marked  albino  was  observed 
functioning  as  a  male  in  copulation  with  the  second  albino.  Eggs  were  saved  from 
the  second  albino  after  isolation,  and  young  snails  grown  from  them.  In  no  case 
were  any  pigmented  offspring  obtained. 

This  negative  result  indicates  either  ( 1 )  that  the  transferred  sperm  did  not 
reach  the  level  of  the  hermaphroditic  duct  in  any  significant  number,  or  (2)  that 
foreign  sperm  cannot  survive  a  second  passage  through  the  reproductive  tract  in 
the  process  of  copulation  and  later  movement  up  the  female  tract.  Since  the  foreign 
sperm  had  already  made  such  a  passage  once,  it  seems  a  bit  unlikely  that  they  could 


50 


GERTRUDE  L.  CAIN 


ovotestis 


hermaphroditic     duct 


le 


seminal 


albumen 

•fertilization      pocket 
bifurcation    of    hermaphroditic 
duct 

uterus 

muciparous    gjand 
upper     prostate 


oothecal 


tacle 


L|      receptac 


-lower    prostate 
vas    deferens 

Seminal     receptacle     duct 


FIGURE  2.  Dorsal  view  of  the  reproductive  system  of  Lymnaea  stagnalis  apprcssa  (X  40). 
The  vas  deferens  has  been  cut  away  just  beyond  the  point  where  it  joins  with  the  lower  prostate 
gland.  The  copulatory  apparatus  is  not  shown. 


CROSS-FERTILIZATION  IN  LYMNAEA  51 

not  do  it  again  without  injury,  though  they  would  undoubtedly  be  greatly  diluted  by 
the  other  sperm  with  which  they  were  transferred.  These  results  therefore  sug- 
gest that  foreign  sperm  probably  do  not  travel  up  the  female  tract  as  far  as  the 
hermaphroditic  duct. 

If,  as  this  suggests,  fertilization  occurs  below  the  bifurcation  of  the  hermaphro- 
ditic duct  in  the  oviduct,  it  must  occur  very  high  up  in  this  latter  structure,  since  the 
albumen  and  egg  shell  is  laid  down  around  the  egg  very  soon  after  the  egg  enters 
the  oviduct  (according  to  Holm  and  Bretschneider),  and  no  micropyle  has  ever  been 
found  in  the  snail's  egg  shell. 

If  fertilization  does  not  occur  above  the  point  of  bifurcation  of  the  hermaphroditic 
duct,  self-fertilization  could  result  only  after  the  transfer  of  sperm  by  self-copula- 
tion. That  self-copulation  actually  does  occur  has  been  observed  by  many  workers. 
Experiments  were  made  to  test  this  possibility. 

Even  though  snails  are  extremely  difficult  subjects  for  surgical  experimentation, 
the  intromittent  organ  was  successfully  removed  in  9  out  of  14  cases.  These  snails 
continued  to  lay  eggs  after  self-copulation  was  no  longer  possible.  The  obvious 
possibility  of  prior  self-copulation  could  not  be  excluded.  In  several  snails  in 
which  a  section  of  the  vas  deferens  was  experimentally  removed  without  subsequent 
death  of  the  snail,  regeneration  re-established  a  connection.  The  question,  there- 
fore, remains  unsettled  as  to  whether  prevention  of  self-copulation  will  also  prevent 
self-fertilization. 

The  possibility  that  the  seminal  receptacle  might  serve  as  an  activating  organ 
for  the  sperm  was  excluded  by  examination  of  seminal  receptacles  removed  from 
snails  at  different  intervals  following  copulation.  Only  in  those  removed  within 
30  minutes  after  copulation  were  motile  sperm  found,  and  the  motility  was  less  than 
that  of  sperm  taken  from  the  vas  deferens  or  ovotestis.  The  problems  of  the  loca- 
tion of  fertilization  and  the  function  of  the  seminal  receptacle  still  remain  unsolved. 

SUMMARY 

1.  Albinism  in  Lyuinaca  stognalis  appressa  Say  is  inherited  as  a  simple  Men- 
delian  recessive. 

2.  Cross-fertilization  greatly  exceeds  self-fertilization  in  snails  allowed  to  cross- 
copulate. 

3.  Transferred  sperm  may  remain  viable  in  the  body  of  the  recipient  snail  for  as. 
long  as  116  days. 

4.  It  is  unlikely  that  foreign  sperm  are  stored  as  high  up  in  the  reproductive  tract 
as  the  seminal  vesicles,  since  albino  snails  previously  impregnated  by  pigmented 
snails  and  later  mated  to  virgin  albinos  engender  no  pigmented  offspring  in  the 
latter. 

LITERATURE  CITED 

ABDEL-MALEK,  E.,  1954a.     The  genital  organs  of  Biomphalaria  boissyi  (Subfamily:  Planorhinae, 

H.  A.  Pilsbry,  1934).    'Trans.  Amcr.  Micros.  Soc..  73:  285-296. 
ABDEL-MALEK,  E.,  1954b.     Genital  organs  of  Helisnma  trirnlvis  (Say).     Trans.  Amcr.  Micros. 

Soc..  73  :  103-124. 
BAUDELOT,    M.,    1863.     Recherches    sur    1'appareil    generateur    des    mollusques    gasteropodes. 

Ann.  dcs  Sci.  Nat.,  Ser.  4,  Zool.  19:  135-222. 
BOETTGER,  C.  R.,  1944.     Basommatophora.     Die  Tier-welt  dcr  Nord-  nnd  Ostscc.     Leipzig,  Akad. 

Verlags-Gesellsch.  35 :  241-478. 


52  GERTRUDE  L.  CAIN 

BOYCOTT,  A.  E.,  AND  C.  DIVER,   1927.     The  origin  of  an  albino  mutation  in  Limnaca  peregra. 

Nature,  119:  9. 
BRETSCHNEIDER,    L.    H.,    1948a.     Insemination    in    Limnaca    stagnalis    L.    Proc.    Kon.    Ncdcrl. 

Akad.  v.  Wetenschappcn.,  51 :  358-363. 
BRETSCHNEIDER,   L.    H.,    1948b.     The   mechanism   of   oviposition   in    Limnaca   stagnalis.     Proc. 

Kon.  Ncdcrl.  Akad.  v.  Wetenschappcn.,  51  :  2-12. 

COLTON,  H.  S.,  1918.     Self-fertilization  in  the  air  breathing  pond  snail.     Biol.  Bull..  35:  48—49. 
COLTON,  H.  S.,  AND  M.  PENNYPACKER,  1934.     The  results  of  twenty  years  of  self-fertilization 

in  the  pond  snail,  Lymnaca  columclla  Say.     Atncr.  Nat.,  68:   129-136. 
CRABB,  E.  D.,  1927a.     Anatomy  and  function  of  the  reproductive  system  in  the  snail,  Lymnaca 

stagnalis  apprcssa.     Biol.  Bull.,  53  :  55-56. 
CRABB,  E.  D.,   1927b.     The  fertilization  process  in  the  snail,  Lymnaca  stagnalis  apprcssa  Say. 

Biol.  Bull,  53  :  67-108. 

CRABB,  E.  D.,  1928.     Self-fertilization  in  the  pond  snail,  L\mnaca  palustris.     Trans.  Anicr.  Mi- 
cros. Soc.,  47  :  82-88. 
DE\VITT,  R.  M.,   1954.     Reproduction,  embryonic  development  and  growth  in  the  pond  snail, 

Physa  gyrina  Say.     Trans.  Amcr.  Micros.  Soc.,  73:   124-137. 
DIVER,  C.,  A.  E.  BOYCOTT  AND  S.  GARSTANG,   1925.     The  inheritance  of  inverse   symmetry  in 

Limnaca  peregra.     J.  Genetics,  15:  113-200. 
HOLM,  L.  W.,  1946.     Histological  and  functional  studies  on  the  genital  tract  of  Lymnaca  stau- 

nalis  apprcssa  Say.     Trans.  Amcr.  Micros.  Soc.,  65:  45-68. 
IKEDA,  K.,  1937.     Cytogenetic  studies  on  the  self-fertilization  of  Philomycus  bilincatns  Bensen. 

/.  Sci.  Hiroshima  Univ.  (B)  Zoo!..  5:  67-123. 
IKEDA,   K.,   AND  S.   E.   MVRA,    1934.     On  the  possibility  of   self-fertilization   and   longevity   of 

spermatozoa  in  the  receptaculum  seminis  in  the  land  snail,  Bradybaena  similaris  stimp- 

soni.     I'cnus.  4:  208-224. 
K.UNKEL,  K.,  1908.     Vermehrung  und  Lebensdauer  der  Limnaca  stat/nalis  L.     Nachbl.  deiitsch. 

malak.  Gesell.,  40 :  70-77. 

LANG,  ARNOLD,   1900.     Lehrbuch   der  vergleichenden  Anatomie  der   wirbellosen   Thiere.     Jena. 
MEISENHEIMER,   J.,    1912.     Die   Weinbergschnecke,   Helix   pomatia.     Leipzig,    Verlag    von   Dr. 

Werner  Klinkhart. 
NOLAND,    L.    E.,    AND    M.    R.    CARRIKER,    1946.     Observations    on    the    biology    of    the    snail, 

Lymnaca  stat/nalis  apprcssa,  during  twenty  generations  in  laboratory  culture.     Amcr. 

Mid.  Nat.,  36 :  467^93. 
J'ELSENEER,   PAUL,    1920.     Les   variations   et   leur   heredite   chez   les   mollusques.     Mem.   Acad. 

Sci.  Bchi.    (2  scr.),  5:   1-314. 

PERROT,  J.  L.,  1940.     Le  fecondation  chez  Liinax  maximiis  L.     Rev.  Suisse  Zoo/.,  47:  371-380. 
SESHAIYA,  R.  V.,   1927.     On  the  breeding  habits  and  fecundity  of  the  snail,   Limnaca   lutcola. 

J.  Bombay  Nat.  His.  Soc.,  32:  154-162. 


ACCLIMATION  OF  OXYGEN  CONSUMPTION  TO  TEMPERATURE 
IN  THE  AMERICAN  COCKROACH   (PERIPLANETA 

AMERICANA)  * 

PAUL  A.  DEHNEL  =  AND  EARL  SEGAL  a 
Department  of  Zonlof/y.  ['nii'crsity  of  California,  J.ns  .lu<iclcs.  24,  California 

The  extensive  literature  that  shows  metabolic  compensation  to  temperature 
among  marine  poikilotherms  (see  Dehnel,  1955;  Segal,  1955;  for  reviews)  has 
led  us  to  question  the  generally  reported  inability  to  compensate  among  insects. 
Scholander,  Flagg,  Walters  and  Irving  (1953),  having  compared  certain  arctic  and 
tropical  insects,  find  no  significant  metabolic  adaptation  to  temperature.  Edwards 
(1953)  generalizes:  he  proposes  that  the  metabolic  response  of  insects  to  tempera- 
ture can  be  expressed  by  a  single  metabolism/temperature  curve.  Although  several 
examples  of  insect  acclimation  are  cited  by  Bullock  (1955)  he  states  (p.  320)  that 
"in  spite  of  these  cases,  it  is  believed  that  insects  may  be  relatively  poor  in  ability 
to  compensate." 

Of  the  well  documented  cases  of  insect  acclimation  to  temperature,  four  are  either 
overwintering  or  in  summer  sleep  (Liihmann  and  Drees,  1952;  Marzusch,  1952)  ; 
one  is  aquatic  (Sayle,  1928),  and  one  is  in  pupa  (Heller,  1930).  Only  a  single 
example  concerns  an  active  isolated  insect  (Parhon,  1909). 4 

We  have  investigated  the  American  cockroach,  Pcriplancta  amcricana,  an  insect 
that  is  active  all  year,  to  see  whether  or  not  it  behaves  similarly  to  marine  poikilo- 
therms with  respect  to  temperature  adaptation  of  the  oxygen  consumption. 

MATKKIAL  AND  METHODS 

A  culture  of  nymphal  and  adult  cockroaches  was  obtained  from  the  Riverside 
campus  of  the  University  of  California.  This  culture  had  been  maintained  at  27° 
C.  for  a  minimum  of  three  generations.  Neither  molting  individuals  nor  adult  fe- 
males nor  individuals  under  approximately  0.3  gram  were  used  in  these  experi- 
ments. The  animals  were  divided  into  two  groups,  nymphs  and  adults,  the  former 
being  represented  by  a  wide  range  of  instars.  Each  of  the  above  two  groups  was 
subdivided  into  three  groups  of  randomly  selected  individuals.  There  were  fifty 
animals  in  each  of  the  six  groups.  One  group  of  nymphs  and  one  group  of  adults 
were  placed  at  10°  C.  Similar  groups  of  nymphs  and  adults  were  placed  at  16° 

1  This  study  was  conducted  at  the  University  of   California,   Los   Angeles.     We   wish  to 
thank  Dr.  T.  H.  Bullock  for  making  the  equipment  and  space  available  to  us. 

2  Present  address :  Department  of  Zoology,  University  of  British  Columbia,  Vancouver  8, 
Canada. 

3  Present    address :    Department    of    Biology,    Kansas    State    Teachers    College,    Emporia, 
Kansas. 

4  Parker    (1930)    shows  acclimation   of   growth   to   temperature   in   two   species   of  insects 
{Melanoplus  mc.ricanus  and   Camnula   pellitcida).     Thompson    (1937)    finds   that  embryos   of 
l\Iclanoplus  differentials  and  Melanoplus  femur-rubrum  kept  at  lower  temperatures    (20°  C.) 
show  a  more  rapid  heart  rate  than  embryos  incubated  at  higher  temperatures   (30°  and  35°  C.) 
when  measured  at  a  series  of  temperatures  from  20°  to  35°  C. 

S3 


54  PAUL  A.  DEHNEL  AND  EARL  SEGAL 

and  26°  C.  Cockroaches  placed  at  the  latter  temperature  were  at  essentially  the 
same  temperature  as  the  original  stock  (26° -27°  C.)  ;  this  permitted  them  to  be 
used  as  controls. 

In  order  to  eliminate  the  reported  light-controlled  diurnal  activity  rhythm 
(Cloudsley-Thompson,  1953),  the  animals  were  maintained  in  constant  darkness. 
The  cockroaches  were  weighed  after  each  experiment  was  completed,  as  it  wras  felt 
that  the  added  handling  of  weighing  would  stimulate  the  animals  to  increased 
activity. 

Each  of  the  six  groups  of  animals  was  given  an  initial  supply  of  food,  and 
fresh  water  was  added  every  other  day.  It  was  noticed  that  the  cockroaches  at 
10°  C.  did  not  feed.  In  order  to  determine  whether  non-feeding  had  any  effect  on 
the  results,  a  fresh  culture  was  obtained  and  the  experiment  was  repeated;  this 
time  none  of  the  animals  was  given  food. 

Oxygen  consumption  was  measured  with  the  use  of  Wennesland-Scholander 
microrespirometers  which  were  submerged  in  a  constant  temperature  bath  con- 
trolled to  ±0.5°  C.  The  cockroaches  were  kept  at  the  acclimation  (10°  and  16° 
C.)  and  control  (26°  C.)  temperatures  for  three  \veeks.  At  the  end  of  each  week 
measurements  were  made  on  a  nymphal  sample  from  each  of  the  three  temperature 
groups.  At  the  end  of  the  first  and  third  weeks  similar  measurements  were  made 
on  adult  samples  from  each  of  the  three  temperature  groups.  All  the  above  oxygen 
consumption  measurements  were  made  at  20°  C.  Those  cockroaches  from  16°  and 
26°  C.  were  kept  in  the  20°  C.  bath  for  one  hour  before  measurements  were  made. 
Animals  from  10°  C.  w-ere  kept  at  15°  C.  for  one  hour;  the  temperature  of  the  bath 
was  then  raised  to  20°  C.  After  one  hour  at  the  latter  temperature,  their  oxygen 
consumption  was  measured. 

In  addition  to  the  above  experiments,  two  samples  of  nymphs  were  separated 
from  the  original  culture  (26°  C.).  The  oxygen  consumption  of  those  from  the 
first  sample  was  measured  over  a  descending  series  of  temperatures  (30°,  25°,  20°, 
15°  and  10°  C.).  The  first  sample  was  then  placed  at  26°  C.  and  the  other  at  10° 
C.  At  the  end  of  three  weeks  the  oxygen  consumption  of  animals  from  the  first 
sample  was  measured  over  the  same  descending  series  of  temperatures.  The  oxy- 
gen consumption  of  animals  from  the  second  sample  was  measured  over  an  ascend- 
ing series  of  temperatures  (10°,  15°,  20°,  25°  and  30°  C.). 

Measurements  for  all  experiments  were  made  at  fifteen-minute  intervals  for  a 
period  of  one  and  one-half  to  three  hours.  On  all  figures  each  point  represents  one 
animal.  The  coordinates  are  log-log  and  the  curves  are  eye-fitted. 

RESULTS 

Nymphs.  When  the  oxygen  consumption  of  equal  weight  animals  kept  at  10°, 
16°  and  26°  C.  is  measured  at  20°  C.,  it  is  found  that  those  animals  maintained  at 
the  lower  temperatures  show  the  higher  consumption  (Fig.  1).  The  increase  in 
oxygen  consumption  of  animals  kept  at  10°  and  at  16°  C.  occurs  within  the  first 
week.  For  the  duration  of  the  experiment  no  further  increases  were  observed. 
Under  the  conditions  of  this  experiment  it  is  impossible  to  compare  the  time  re- 
quired for  acclimation  in  the  two  groups ;  it  can  be  said  only  that  in  both  it  is  com- 
plete within  one  week.  Because  there  is  no  difference  in  the  weekly  oxygen  con- 
sumption values  for  each  group,  they  are  combined  for  the  regression  curves  in 
Figure  1. 


ACCLIMATION  IN  THE  COCKROACH 


55 


For  purposes  of  comparison  within  the  three  groups  of  cockroaches,  animals 
with  an  average  weight  of  0.6  gram  were  chosen  from  the  regression  curves.  This 
weight  was  chosen  because  it  falls  approximately  within  the  center  of  the  weight 
range  on  each  of  the  regression  curves.  When  the  oxygen  consumption  of  this 
0.6-gram  animal  is  read  directly  from  the  graph  it  is  noted  that  (1)  this  weight 
animal  acclimated  to  10°  C.  consumes  67  mm.3/gm./hr.  (57%)  more  oxygen  than 
his  counterpart  acclimated  to  26°  C.,  (2)  this  weight  animal  acclimated  to  16°  C. 
consumes  42  mm.3/grn./hr.  (36%)  more  oxygen  than  his  counterpart  from  26°  C. 
and  (3)  this  weight  animal  from  10°  C.  consumes  25  mm.3/gm./hr.  (16%)  more 
oxygen  than  his  counterpart  from  16°  C.  It  is  apparent  that  the  animals  acclimated 


300 


o 

o 

o 

C\J 

(—    200 


cr 

D 
O 
I 


cr 


CO 

O 


90 
80 

70 
60 


50 


OO 


CD          o 


Y*  O  0    0 


yy 


,6°C. 


26°C. 


0.2 


0.3 


0.4 


0-5        0.6      0.7    0-8  0-9    1.0 


.5 


BODY   WEIGHT    IN   GRAMS 

FIGURE  1.  Weight-specific  oxygen  consumption  as  a  function  of  weight  in  nymphal 
Periplaneta  amcricana.  Animals  were  kept  at  10°,  16°  and  26°  C.  for  one  to  three  weeks,  and 
the  measurements  were  made  at  20°  C.  In  all  figures  each  point  represents  the  average  oxy- 
gen consumption  for  one  animal  over  a  period  of  one  and  one-half  to  three  hours.  Open  circles 
represent  10°  C.  animals,  crosses,  16°  C.  animals  and  closed  circles,  26°  C.  ones.  The  coordi- 
nates are  logarithmic,  and  all  curves  are  eye  fitted.  Results  from  feeding  and  non-feeding  ex- 
periments are  combined. 


56 


PAUL  A.  DEHNEL  AND  EARL  SEGAL 


700 

500 
400 

300 


D 

O  200 

I 


cr 
O 

<M 
O 


100 

70 

50 

40 

30 

20 


0.2      0.3    0.4  0.5    0.7      1.0  2.0 

BODY   WEIGHT    IN  GRAMS 


3.0 


FIGURE  2.  Weight-specific  oxygen  consumption  as  a  function  of  weight  in  nymphal 
Pcriplancta  amcricana  measured  over  a  series  of  temperatures.  Open  circles  represent  cold- 
adapted  animals  (10°  C.)  ;  closed  circles  represent  warm-adapted  animals  (26°  C. ). 


ACCLIMATION  IN  THE  COCKROACH 


57 


to  10°  C.  are  responding  to  the  increased  distance  (°  C.)  from  the  control  tempera- 
ture (26°  C.)  with  a  further  increase  in  oxygen  consumption.  The  values  indicate 
that  a  linear  relation  exists  between  the  increase  in  oxygen  consumption  and  the  de- 
crease in  acclimation  temperature  (4.2  mm.3  O2/gi~n-/hr.  increase  per  degree  centi- 
grade drop  in  temperature). 


400 


U 

o 

o 

C\J 


300 


o: 

Z) 

o 

I 


-200 


cr 


OJ 

O 


100 


o°c 


I6°C. 
26°C 


0.5        0-6      0.7 


1.0 


2.0 


BODY    WEIGHT    IN  GRAMS 


FIGURE  3.  Weight-specific  oxygen  consumption  as  a  function  of  weight  in  adult  Peri- 
plancta  amcricana.  Animals  were  kept  at  10°,  16°  and  26°  C.  for  one  to  three  weeks,  and  the 
measurements  were  made  at  20°  C. 

Nymphs,  rate /temperature  experiment.  The  results  of  the  rate/temperature 
experiment  are  presented  in  Figure  2.  The  rate  values  obtained  at  15°  and  25°  C. 
are  omitted  to  make  the  graph  easier  to  read.  From  10°  to  25°  C.  cold-acclimated 
nymphs  (10°  C.)  consume  more  oxygen  per  gram  per  hour  than  equal  weight 
warm-acclimated  nymphs  (26°  C.).  The  oxygen  consumption  of  the  cold-accli- 
mated nymphs  is  depressed  at  30°  C.,  i.e.,  less  oxygen  is  consumed  at  30°  C.  than  at 
20°  C.  Since  the  curves  at  30°  and  at  20°  C.  are  parallel,  the  oxygen  consumption 
of  large  and  small  nymphs  is  depressed  equally. 

At  all  temperatures  the  regression  lines  for  the  warm-acclimated  nymphs  are 


58  PAUL  A.  DEHNEL  AND  EARL  SEGAL 

steeper ;  the  regression  lines  at  the  different  temperatures  for  either  the  warm-  or 
cold-acclimated  animals  are  essentially  parallel. 

If  the  curves  (Figs.  1  and  2)  representing  nymphs  acclimated  to  10°  C.  and 
measured  at  20°  C.  are  compared,  it  is  seen  that  the  slopes  and  positions  of  the 
curves  are  the  same.  However,  if  a  similar  comparison  is  made  for  the  warm-ac- 
climated nymphs,  it  is  found  that  the  oxygen  consumption  is  constantly  lower  for 
these  animals  in  the  rate/temperature  experiment.  These  animals  spent  approxi- 
mately four  hours  at  30°  C..  before  they  were  measured  at  20°  C.  It  is  possible  that 
four  hours  is  sufficient  time  for  the  acclimation  process  to  have  begun.  Therefore, 
the  oxygen  consumption  at  20°  C.  is  lower  than  it  is  for  the  animals  brought  directly 
from  26°  to  20°  C. 

Adults.  Adult  cockroaches,  like  nymphal  cockroaches,  show  acclimation  of  their 
oxygen  consumption  to  temperature.  Those  adults  kept  at  10°  and  16°  C.  con- 
sume more  oxygen  per  gram  than  do  equal  weight  adults  kept  at  26°  C.  when  all 
are  measured  at  20°  C.  (Fig.  3).  As  with  the  nymphs  no  change  was  found  in  the 
weekly  (first  and  third)  oxygen  consumption  values  for  each  temperature  group. 
Therefore,  these  values  are  combined  for  each  of  the  regression  curves  in  Figure  3. 

For  the  adult  cockroaches,  animals  with  an  average  weight  of  0.9  gram  were 
chosen.  When  the  oxygen  consumption  of  this  0.9-gram  adult  is  read  from  the 
graph  it  is  noted  that  (1)  this  weight  animal  acclimated  to  10°  C.  consumes  55 
mm.3/gm./hr.  (40%)  more  oxygen  than  his  counterpart  acclimated  to  26°  C.,  (2) 
this  weight  animal  acclimated  to  16°  C.  consumes  25  mm.3/gm./hr.  (18%)  more 
oxygen  than  his  counterpart  from  26°  C.  and  (3)  this  weight  animal  from  10°  C. 
consumes  30  mm.3/gm./hr.  (18%)  more  oxygen  than  his  counterpart  from  16°  C. 
These  values  suggest  that  in  contrast  to  the  nymphs,  a  non-linear  relationship  exists 
between  the  increase  in  oxygen  consumption  and  the  decrease  in  acclimation  tem- 
perature (2.5  mm.3  O2/gm./hr.  increase  per  degree  centigrade  drop  in  temperature 
from  26°  to  16°  C. ;  5.0  mm.3  O.,/gm./hr.  increase  per  degree  centigrade  drop  in 
temperature  from  16°  to  10°  C.). 

Comparison  of  nyinpJis  and  adults.  Comparison  of  the  oxygen  consumption  of 
nymphal  and  adult  cockroaches  (Figs.  1  and  3)  that  have  been  acclimated  to  and 
measured  at  the  same  temperatures  shows  that  adult  cockroaches  consume  more  oxy- 
gen per  gram  than  equal  weight  nymphs.  Although  the  adult  curve  representing 
the  control  animals  (26°  C.)  is  displaced  above  the  curve  for  the  control  nymphs 
(26°  C.),  the  slopes  are  essentially  parallel.  With  acclimation  to  16°  and  to  10°  C. 
small  and  large  nymphs  respond  in  a  like  manner  and  these  curves  have  approxi- 
mately the  same  slopes  as  the  26°  C.  curve.  On  the  contrary,  small  and  large  adults 
show  a  differential  response  to  the  temperatures  of  acclimation  (with  extrapolation, 
the  curves  in  Fig.  3  would  intersect  to  the  right).  Small  adults  are  responding  to 
the  decreased  temperatures  of  acclimation  with  a  greater  increase  in  their  weight- 
specific  oxygen  consumption  than  are  large  adults.  Small  adults  are  therefore  do- 
ing a  better  job  of  acclimating  than  large  adults  and  all  sizes  of  nymphs  are  'doing  a 
better  job  than  all  sizes  of  adults. 

DISCUSSION 

The  object  of  this  investigation  was  to  see  if  the  insect  Pcriplancta  americana 
could  acclimate  its  metabolic  activity  to  temperature.  However,  we  would  first  like 


ACCLIMATION  IN  THE  COCKROACH  59 

to  discuss  an  additional  observation.  We  have  found  that  adult  cockroaches  are 
living  at  a  faster  metabolic  pace  than  are  nymphs  of  approximately  the  same  weight 
(see  Figs.  1  and  3).  Batelli  and  Stern  (1913)  showed  that  at  all  temperatures 
from  20°  to  40°  C.  fly  imagines  consume  more  oxygen  per  unit  body  weight  than  did 
larvae.  Similarly,  it  was  found  by  Ludwig  (1931)  that  the  weight-specific  oxy- 
gen consumption  of  adult  Japanese  beetles,  Popillia  japonica,  was  greater  than  that 
of  the  larvae.  Referring  to  the  Holometabola,  Wigglesworth  (1950,  p.  413)  has 
stated  that  "metabolism  at  a  given  temperature  is  generally  much  higher  in  the  adult 
than  in  the  larva  and  higher  in  the  larva  than  in  the  pupa."  Wigglesworth  at- 
tributes this  difference  to  the  increased  activity  metabolism  of  the  adult.  Many  in- 
vestigators will  speak  of  the  typical  "U-shaped"  respiratory  curve  during  the 
metamorphosis  of  holometabolus  insects  (see  Edwards,  1953,  for  references). 

The  available  data  for  the  Hemimetabola  (the  insects  in  which  there  is  little 
or  no  change  in  shape  during  ontogeny)  are  much  less  than  for  the  Holometabola. 
Edwards  (1953)  presents  a  curve  showing  the  change  in  weight-specific  oxygen 
consumption  from  egg  deposition  through  early  adulthood  of  the  milkweed  bug 
Oncopeltns  jasciatus.  Within  a  few  days  after  the  last  molt,  adults  consume 
more  oxygen  per  gram  per  hour  than  do  last  instar  nymphs.  If  the  oxygen  con- 
sumption during  molt  is  ignored,  then  the  curve  resembles  a  flattened  "U-shape." 
In  this  study  early  adult  and  late  nymphal  Periplaneta  americana  show  a  similar 
relationship.  With  increasing  size  of  the  adult,  the  characteristic  fall  in  weight- 
specific  oxygen  consumption  is  observed. 

We  do  not  know  what  makes  possible  the  elevated  metabolic  activity  of  the  early 
adult  cockroaches ;  we  have  not  observed  a  difference  in  locomotor  activity  that 
would  account  for  it.  Perhaps  the  elevation  in  rate  is  a  consequence  of  the  meta- 
morphosis from  nymph  to  adult.  A  similar  suggestion  was  offered  by  Groebbels 
(1925)  to  account  for  the  increase  in  metabolic  rate  found  during  metamorphosis  of 
Rana  tadpoles. 

Contrary  to  the  generalized  statement  of  Edwards  (1953)  that  insects  do  not 
compensate  metabolically  to  temperature,  both  nymphs  and  adults  of  Periplaneta 
americana  adapted  to  10°  C.  consume  more  oxygen,  per  animal  and  per  gram,  than 
equal  weight  control  animals  adapted  to  26°  C.  when  measured  at  the  same  tempera- 
ture. 

Liihmann  and  Drees  (1952)  and  Marzusch  (1952)  show  temperature  adaptation 
in  four  species  of  insects,  two  of  which  are  overwintering  (the  potato  beetle, 
Leptinotarsa  decemlineata,  and  the  leaf  beetle,  Phytodccta  rufipes}  and  two  in  sum- 
mer sleep  (the  potato  beetle,  Melasoma  populi,  and  the  leaf  beetle,  Galcrnca  tana- 
ccti).  These  investigators  are  unable  to  show  temperature  adaptation  during  the 
active  feeding  period.  Liihmann  and  Drees  have  suggested  that  the  compensatory 
response  is  masked  by  the  high  metabolic  activity  associated  with  feeding.  We  be- 
lieve that  if  such  a  response  can  be  demonstrated  at  any  given  time,  it  does  seem 
reasonable  to  expect  this  ability  to  be  present  at  all  times.  This  expectation  is  borne 
out  by  Periplaneta  americana,  which  is  active  and  feeds  all  year.  Therefore,  it  is 
difficult  to  understand  why  this  compensatory  response  appears  only  in  these  in- 
sects under  conditions  of  winter  and  summer  sleep.  It  would  be  well  to  note  that 
one  species  of  leaf  beetle  (Chrysomela  haemoptera)  shows  no  adaptation  even 
though  its  metabolic  level  was  depressed  during  the  summer  sleep  (Liihmann  and 
Drees.  1952). 


60  PAUL  A.  DEHNEL  AND  EARL  SEGAL 

Previously,  cold-  and  warm-adapted  groups  have  been  compared  at  a  given  tem- 
perature or  between  temperatures  by  arbitrarily  choosing  a  weight  and  determining 
the  oxygen  consumption  for  each  group.  It  is  also  profitable  to  choose  an  arbitrary 
rate  of  oxygen  consumption  and  determine  the  approximate  weight  of  animal  in 
each  group  for  which  this  rate  is  obtained  (Figs.  1  and  3).  As  a  generalization,  a 
large  cold-adapted  cockroach  consumes  about  as  much  oxygen  as  a  small  warm- 
adapted  one.  For  example,  a  0.4-gram  nymph  (26°  C.),  a  0.7-gram  nymph  (16° 
C.)  and  a  0.9-gram  nymph  (10°  C.)  when  measured  at  20°  C.  consume  equal 
amounts  of  oxygen  per  unit  weight.  Similarly,  it  is  possible  to  determine  the  tem- 
peratures at  which  cold-  and  warm-adapted  roaches  consume  the  same  amount  of 
oxygen  (Fig.  2).  On  this  basis,  cold-adapted  animals  consume  at  15°  C.  slightly 
more  oxygen  than  warm-adapted  animals  consume  at  20°  C. 

Sayle  (1928)  tested  the  effect  of  low  temperature  on  carbon  dioxide  production 
of  dragon  fly  nymphs  (Acschna  umbrosa).  She  lowered  the  temperature  from 
22°  C.  to  13°  C.  (three  days  at  17°  C.  and  three  days  at  13°  C.)  and  found  that 
carbon  dioxide  production  was  about  the  same  at  the  lower  temperature  as  the  ini- 
tial production  at  17°  C.  after  the  first  day.  The  major  portion  of  acclimation  of 
these  nymphs  was  evident  within  forty-eight  hours.  It  is  not  unreasonable  to  ex- 
pect that  the  rate  of  acclimation  in  Pcriplaneta  aincricana  is  equally  as  rapid  since 
no  further  change  was  evident  after  six  days.  In  addition,  animals  measured  at 
20°  C.  after  spending  a  number  of  hours  at  30°  C.  consume  less  oxygen  than  ani- 
mals measured  at  20°  C.  directly  from  26°  C.  Such  time  courses  as  found  in  these 
animals  compare  favorably  with  that  shown  for  other  species  (Behre,  1918; 
Planaria  dorotocephala ;  Roberts,  1952,  Pachygrapsns  crassipcs;  Segal,  1955, 
Acmaea  limatula). 

Bullock  (1955)  has  thoroughly  reviewed  the  known  cases  of  acclimation  to 
temperature  at  the  several  levels  of  organization  (molecular,  cellular,  tissue  and  or- 
gan system).  He  does  cite  several  negative  instances  in  which  animals  fail  to  show 
acclimation.  However,  the  evidence  from  widely  divergent  groups,  involving  dif- 
ferent physiological  systems,  suggests  to  us  that  compensatory  responses  to  environ- 
mental stresses  are  inherent  components  of  protoplasmic  systems.  Negative  cases 
as  cited  by  Bullock  (1955)  do  not  invalidate  this  idea.  Such  instances  suggest  to 
us  that  animals,  in  which  no  acclimation  was  found  in  the  particular  physiologic 
system  studied,  might  show  compensation  to  stress  in  another  system  or  at  a  differ- 
ent level.  Compensatory  responses  to  temperature  are  most  often  described,  but 
other  environmental  parameters  (osmotic  pressure,  drugs,  oxygen  tension;  see 
Prosser,  1955)  equally  as  important  may  evoke  such  adaptation.  If  this  phenome- 
non is  a  universal  component  of  living  systems  and  permits  animals  to  assume  de- 
grees of  environmental  independence,  it  goes  far  to  explain  their  survival  and  distri- 
bution. Within  limits  it  accomplishes  the  same  results  as  homiothermism  accom- 
plishes for  the  warm  blooded  animals. 

SUMMARY 

1.  Oxygen  consumption  has  been  studied  in  cultures  of  nymphal  and  adult 
cockroaches,  Pcriplaneta  amerlcana,  that  have  been  maintained  at  two  experimental 
temperatures  (10°  and  16°  C.)  and  the  control  temperature  (26°  C.)  for  a  period 
of  one  to  three  weeks. 


ACCLIMATION  IN  THE  COCKROACH  61 

2.  It  has  been  shown  that  the  oxygen  consumption  of  equal-weight   nymphs 
when  measured  at  20°  C.  is  higher  in  animals  that  have  been  maintained  at  the  lower 
temperatures. 

3.  Comparison  of  cold-   (10°  C.)   and  warm-adapted   (26°  C.)   nymphs  when 
measured  at  a  series  of  temperatures   (  10°  to  25°  C.)   demonstrates  that  cold-ac- 
climated animals  consume  more  oxygen  per  gram  per  hour  than  equal  weight  warm- 
adapted  ones. 

4.  Adult  cockroaches  show  acclimation  of  their  oxygen  consumption  to  tempera- 
ture.    However,  there  is  a  differential  response  with  respect  to  size ;  small  adults 
acclimate  to  a  greater  degree  than  large  ones.     Further,  all  sizes  of  nymphs  show 
a  greater  degree  of  acclimation  than  all  sizes  of  adults. 

LITERATURE  CITED 

BATEI.LI,   F.,   AND  L.   STERX,    1913.     Intensitat  des   respiratorischen   Gaswechsels   der   Insekten. 

Biochem.  Zcitschr.,  56:  50-5cS. 
BEHRE,  E.  H.,  1918.     An  experimental  study  of  acclimation  to  temperature  in  Planaria  doroto- 

ccphala.     Blol.  Bull.,  35:  277-317.  " 
BULLOCK,  T.  H.,  1955.     Compensation  for  temperature  in  the  metabolism  and  activity  of  poikilo- 

therms.     Biol.  Rcr.,  30:  311-342. 
CLOUDSLEY-THOMPSOX,  J.  L.,   1953.     Studies  in  diurnal  rhythms.     III.   Photoperiodism  in  the 

cockroach  Pcriplcincta  americana   (L.).     Ann.  Mag.  Nat.  Hist.,  6:  705-712. 
DEHXEL,  P.  A.,  1955.     Rates  of  growth  of  gastropods  as  a  function  of  latitude.     Ph\siol  Zool., 

28:  115-144. 
EDWARDS,  G.  A.,  1953.     Quoted  in  Chapter  5,  Insect  Physiology,  edited  by  K.  D.  Roeder.     J. 

Wiley  and  Sons,  New  York. 
GROEBBELS,    F.,    1925.     Untersuchungen    uber    Wachstum,    Entwicklung   und    Stoffwechsel    von 

Froschlarven  unter  verschiedenen   Bedingungen  der  Erhahrung.     Arch.   gcs.   Physiol., 

208 :  718-729. 
HELLER,    J.,    1930.     Sauerstoffverbrauch    der    Schmetterlingspuppen    in    Abhangigkeit    von    der 

Temperatur.     Zcitschr.  i'crg.  Physiol. .  11  :  448—460. 

LUDWIC,  D.  J.,  1931.     Studies  on  the  metabolism  of  the  Japanese  beetle  (Popillia  japonica  New- 
man).    I.  Weight  and  metabolism  changes.     /.  E.vp.  Zool.,  60:  309-323. 
LUHMANN,   M.,   AND   O.   DREES,    1952.     Uber   die   Temperaturabhangigkeit   der   Atmung   som- 

merschlafender  Blattkafer.     Zool.  Anz.,  148:  13-22. 
MARZUSCH,  K.,  1952.     Untersuchungen  iiber  die  Temperaturabhangigkeit  von  Lebensprozessen 

bei    Insekten    unter    besonderer    Beriicksichtigung    winterschlafender     Kartoffelkafer. 

Zeitschr.  verg.  Physiol.,  34 :  75-92. 
PARHON,  M.,  1909.     Les  echanges  nutritifs  chez  les  abeilles  pendant  les  quatre  saisons.     Ann. 

Set.  Nat.   (ser.  9),  Zool,  9:  1-58. 
PARKER,  J.  R.,   1930.     Some  effects  of  temperature  and  moisture  upon  Mclanoplus  mexicanus 

mexicanus  Saussure  and  Cauuuila  pcllucida  Scudder    (Orthoptera).     Univ.   Montana, 

Agri.  Exp.  Sta.,  Bull.  223,  1-132. 

PROSSER,  C.  L.,  1955.     Physiological  variation  in  animals.     Biol.  Rev.,  30 :  229-262. 
ROBERTS,  J.   L.,   1952.     Studies   on  acclimatization   of  respiration  to   temperature   in   the   lined 

shore  crab,  Pachygrapsus  crassipcs  Randall.     Ph.D.  dissertation,   Univ.  of  California, 

Los  Angeles. 
SAYLE,  M.  H.,  1928.     Factors  influencing  the  rate  of  metabolism  of  Aeschna  umbrosa  nymphs. 

Biol  Bull,  54 :  212-230. 
SCHOLAXDER,  P.  F.,  W.  FLAGG,  V.  WALTERS  AND  L.  IRVING,  1953.     Climatic  adaptation  in  arctic 

and  tropical  poikilotherms.     Physiol.  Zool,  26 :  67-92. 
SEGAL,  E.,  1955.     Microgeographic  variation  as  thermal  acclimation  in  an  intertidal  gastropod. 

Ph.D.  dissertation,  Univ.  of  California,  Los  Angeles. 

THOMPSON,  V.,  1937.     Effects  of  temperature  on  movements  of  embryos    (Acrididae,  Orthop- 
tera).    Physiol  Zool,  10:  21-30. 
WIGGLESWORTH,  V.  B.,  1950.     Insect  physiology.     E.  P.  Dutton  and  Co.,  Inc.,  New  York. 


NEUROSECRETORY  CELL  TYPES  AND  THEIR  SECRETORY 
ACTIVITY  IN  THE  CRAYFISH1-2 

JAMES  B.  DURAND 
Department  of  Bioloyy,  College  of  Soutli  Jersey,  Rutgers  University,  Camden  2,  Nczv  Jersey 

It  is  now  well  known  that  physiologically  active  substances  are  produced  in 
neurosecretory  cells  located  throughout  the  nervous  svstems  of  crustaceans  ( Bliss, 
1951,  1952,  1953;  Bliss,  Durand  and  Welsh,  1954;  Bliss  and  Welsh,  1952;  Carlisle, 
1953;  Enami,  1951;  Passano,  1951a,  1952,  1953).  Furthermore,  the  neurosecre- 
tory cells  are  distributed  as  distinct  groups  (Bliss,  Durand  and  Welsh,  1954  ;  Enami, 
1951),  at  least  in  the  eyestalk  and  brain.  Relatively  little  is  known  about  the 
specific  localization  of  the  sources  of  the  neurohormones  affecting  particular  physio- 
logical processes;  however,  Passano  (1951a,  1951b,  1952,  1953)  has  shown  that  the 
x-organ  in  crustaceans  produces  a  substance  that  is  capable  of  inhibiting  molt. 

Neurosecretory  cells  have  been  described  for  the  x-organ  (Bliss,  1952;  Bliss, 
Durand  and  Welsh,  1954;  Bliss  and  Welsh,  1952;  Carlisle  and  Passano,  1953; 
Enami,  1951  ;  Passano,  1953)  ;  but,  with  the  exception  of  Enami's  work  on 
Scsanna  (1951),  there  is  little  information  concerning  the  different  types  of  neuro- 
secretory cells  present  in  crustaceans.  Furthermore,  there  is  no  cytological  evi- 
dence available  to  indicate  which  of  the  different  neurosecretory  cell  types  are  in- 
volved in  the  physiology  of  molt.  It  is  apparent  that  work  along  these  lines  is 
needed,  particularly  in  view  of  the  fact  that  cytological  differences  in  cell  types  often 
go  hand  in  hand  with  differences  in  function. 

The  present  paper  will  be  concerned  with  a  histological  study  of  the  neurosecre- 
tory system  of  the  crayfish,  Orconcctcs  ririlis  (formerly  Cainbanis  ririlis)  in  rela- 
tion to  the  molting  cycle. 

MATERIALS  AND  METHODS 
1.  Animals 

The  animals  used  in  this  study  were  mature  males,  approximately  five  centi- 
meters in  carapace  length,  all  collected  from  Hobb's  Brook  Reservoir,  Lincoln, 
Mass.,  in  the  summer  of  1954.  Mature  crayfish  were  collected  on  the  dates  shown 
in  Table  I.  With  the  exception  of  May  animals,  which  had  been  kept  in  the  labora- 
tory for  three  to  four  months  and  fed  weekly  on  clam  and  fish,  eyestalks  and  brains 
were  removed  and  fixed  on  the  same  day  the  animals  were  collected. 

1  This  work  constitutes  a  portion  of  a  thesis  submitted  in  partial  fulfillment  of  the  require- 
ments for  the  Ph.D.  degree  from  Harvard  University.     The  writer  wishes  to  express  his  sin- 
cere thanks  to  Professor  John  H.  Welsh  under  whose  direction  this  work  was  carried  out. 

2  The  preparation  of   the  manuscript  was  aided   by  a   grant   from   the   Research    Council, 
Rutgers  University. 

62 


NEUROSECRETION  IN  THE  CRAYFISH  63 

2.  Dissections 

All  dissections  were  performed  in  crayfish  perfusion  fluid  (van  Harreveld, 
1936).  A  pair  of  fine  iridectmnv  scissors,  jeweler's  forceps  and  cuticle  scissors 
were  used  in  the  dissections. 

Eyestalk.  The  eyestalk  was  removed  by  cutting  the  pedunculus  lobi  optici  with 
a  pair  of  small  cuticle  scissors.  Next,  the  chitinous  exoskeleton  was  cut  the  full 
length  of  the  eyestalk  on  each  side.  The  eyestalk  was  then  pinned  ventral  side  down 
by  means  of  size  0  insect  pins  in  a  Syracuse  watch  glass,  half  filled  with  paraffin  and 
containing  crayfish  perfusion  fluid.  The  remainder  of  the  dissection  was  carried 
out  with  the  aid  of  a  binocular  dissecting  microscope. 

After  a  cut  was  made  across  the  dorsal  half  of  the  retina,  the  proximal  end  of 
the  top  half  of  the  exoskeleton  was  lifted  and  the  hypodermis  was  carefully  scraped 
from  the  exoskeleton.  Great  care  was  taken  in  this  step  to  prevent  excess  stretch- 
ing of  the  nerve  tissue. 

The  cut  end  of  the  pedunculus  lobi  optici  was  grasped  with  fine  forceps,  and  the 
eyestalk  contents  were  separated  from  the  underlying  exoskeleton.  The  whole 
content  of  the  eyestalk  was  then  placed  in  a  vial  containing  fixative.  With  practice, 
this  procedure  could  lie  accomplished  within  two  to  three  minutes.  Excellent  fixa- 
tion was  obtained  in  all  cases. 

Brain.  The  head  of  the  animal  was  removed  by  a  cut  just  posterior  to  the  brain 
and  mouth.  The  exposed  parts  were  immediately  rinsed  thoroughly  with  perfu- 
sion fluid  to  remove  any  stomach  contents,  pieces  of  hepatopancreas,  or  urine 
released  after  puncture  of  the  bladders.  Frequent  changes  of  the  perfusion  fluid 
were  made  throughout  the  dissection.  The  rostrum  of  the  animal  was  next  in- 
serted in  a  piece  of  modeling  clay  in  such  a  manner  that  the  open  end  of  the  head 
was  facing  up.  In  this  way,  the  animal's  head  served  as  a  miniature  dissecting 
vessel.  The  remainder  of  the  procedure  was  carried  out  with  the  aid  of  a  dissecting 
microscope. 

After  removal  of  the  stomach,  pieces  of  hepatopancreas,  and  green  glands,  the 
brain  was  rinsed  thoroughly  with  perfusion  fluid.  All  nerves  leading  from  the 
brain  and  the  connective  tissue  sheath  surrounding  the  brain  were  cut  away,  and 
the  brain  was  placed  in  a  vial  containing  fixative.  The  brain  was  lifted  by  means  of 
the  circumoesophageal  connectives.  This  procedure  required  about  three  to 
four  minutes. 

3.  Histologicol  procedure 

The  fixatives  employed  in  this  study  were  Helly's  fluid  (fixing  time,  eight  hours) 
and  Benin's  plus  one  per  cent  calcium  chloride  (fixing  time,  twenty-four  hours). 
Tissues  were  dehydrated  in  alcohol,  cleared  in  cedar  oil  and  embedded  in  Tissue- 
mat  (melting  point  56-58°  C.).  Sections  were  cut  at  6/t  and  stained  with  aldehyde 
fuchsin  (Gomori,  1950)  according  to  the  schedule  of  Halmi  (1952),  but  with  modifi- 
cations by  Dawson  (1953).  This  procedure  involved  a  permanganate  oxidation 
prior  to  staining  and  will  be  referred  to  in  the  text  as  PAF.  Sections  were  also 
stained  with  chrome-alum-hematoxylin-phloxin  (Gomori,  1941)  as  adapted  by 
Bargmann  (1949).  This  technique  is  referred  to  in  the  text  as  CHP. 


64 

4.  Cell  counts 


JAMES  B.  DURAND 


A  study  of  cell  types  revealed  that  secretory  material  was  present  as  small  gran- 
ules or  droplets  within  the  cells.  The  secretory  activity  of  a  group  of  cells  could  be 
judged,  therefore,  by  counts  of  cells  which  appeared  histologically  to  be  in  a  given 
stage  of  the  secretory  cycle. 


VOL 


FIGURE  1.  Drawings  of  neurosecretory  cell  types  in  the  eyestalk  and  brain  of  the  crayfish. 
Numbers  along  the  left  column  indicate  cell  types.  Letters  indicate  cells  in  successive  stages 
of  the  secretory  cycle,  ax.  axon  ;  dr,  droplet;  gr,  granule;  va,  vacuole. 


NEUROSECRETION  IN  THE  CRAYFISH  65 

Type  1  cells  of  the  x-organ,  in  a  stage  of  the  secretory  cycle  similar  to  that  shown 
in  Figure  2,  were  counted.  These  cells  are  large  enough  so  that  they  can  be  rec- 
ognized from  section  to  section  and  were  counted  only  when  the  nucleus  was  in- 
cluded in  the  section.  In  this  way  no  cell  could  be  counted  twice. 

Type  2  cells  in  the  x-organ  were  also  counted.  In  this  case,  cells  which  con- 
tained both  a  nucleus  and  a  secretory  droplet  (Fig.  1,  Cells  2b,  c)  in  the  same 
section  were  counted.  The  nuclei  of  these  cells  are  small  enough  so  that  a  section 
near  the  center  of  the  nucleus  would  be  present  only  once  per  cell.  This  method 
of  counting  resulted  in  minimum  counts  of  the  cells  in  that  particular  stage  of  the 
secretory  cycle.  Type  2  neurosecretory  cells  as  shown  in  Figure  1,  Cell  2a,  were  not 
counted.  The  marked  uniformity  of  cell  counts  during  all  months  except  May  and 
June  indicates  that  consistent  results  can  be  obtained  in  this  manner. 

This  method  of  counting  could  not  be  applied  to  the  other  neurosecretory  cell 
groups  because  the  secretory  material  in  those  groups  is  freely  distributed  through- 
out the  cytoplasm  in  the  form  of  fine  granules  (see  below). 

RESULTS 

Studies  of  serial  sections  of  eyestalks  and  brains,  stained  with  CHP  and  PAF, 
have  revealed  the  presence  of  large  groups  of  cells  (see  also  Bliss,  Durand  and 
Welsh,  1954)  that  are  histologically  different  from  the  hundreds  of  ordinary 
ganglion  cells  present  throughout  the  eyestalk  and  brain.  These  cells  are  always 
larger  than  the  ordinary  ganglion  cells.  Most  of  them  possess  large  nuclei,  have 
abundant  cytoplasm,  and  are  characterized  by  the  presence  in  the  perikaryon  and 
axon  of  droplets  of  a  material  which  stains  conspicuously  with  aldehyde  fuchsin 
and  chrome-hematoxylin.  Not  all  the  cells  have  the  same  appearance  as  to  the 
quantity  and  size  of  these  droplets.  These  characteristics  lead  to  the  conclusion 
that  the  cells  are  neurosecretory  cells  as  defined  by  E.  Scharrer  and  B.  Scharrer 
(1945)  and  described  in  a  great  variety  of  animals  by  numerous  authors  (see  es- 
pecially Scharrer  and  Scharrer,  1954;  Gabe,  1954). 

There  appear  to  be  four  neurosecretory  cell  types  (Fig.  1)  found  in  the  eye- 
stalk  and  brain  of  the  crayfish.  0.  -drills.  Size,  general  shape  of  the  cell  body, 
presence  or  absence  of  vacuoles  in  the  cytoplasm  and  the  appearance  of  the  secretory 
product  were  used  as  the  main  criteria  in  separating  the  cell  types.  Since  large 
numbers  of  the  cells  were  found  to  form  more  or  less  distinct  subdivisions  of  larger 
units  in  the  case  of  two  cell  types,  and  possessed  a  fairly  uniform  set  of  the  char- 
acteristics listed  above,  it  is  believed  that  the  cells  are  truly  of  different  types  and 
have  not  been  confused  with  various  stages  of  the  secretory  cycle  present  in  a  given 
cell  type.  The  only  cells  that  others  might  possibly  find  difficult  to  recognize  are 
those  similar  to  Type  1  (a)  and  Type  3  (d)  (Fig.  1). 

Cell  types 

Cell  Type  1.  The  distribution  of  this  cell  type  is  somewhat  limited;  it  is  most 
numerous  in  the  x-organ  and  lies  as  a  distinct  subgroup  in  the  most  distal  portion  of 
the  x-organ.  The  cell  bodies  are  large,  40-60  p.  in  length,  possess  much  cytoplasm 
and  contain  a  large  nucleus,  15//,  in  diameter.  In  the  material  used  in  this  study. 
Type  1  cell  bodies  have  extremely  irregular  outlines  which  are  very  likely  caused  by 
shrinkage  during  fixation  (Fig.  1).  The  nucleus  may  often  contain  two  and  some- 


66 


JAMES  B.  DURAND 


NEUROSECRETION  IX  THE  CRAYFISH  67 

times  three  nucleoli.  Moreover,  nucleoli  are  usually  peripheral,  lying  against  the 
nuclear  membrane.  Further,  not  all  of  the  cells  show  the  presence  of  large  amounts 
of  secretory  material  at  any  one  time.  This,  however,  is  to  be  expected,  for  ap- 
parently some  cells  are  at  the  peak  of  their  secretory  processes  while  others  are  in  a 
quiescent  state.  The  secretory  product  consists  of  a  great  number  of  aggregations 
of  small  ( 0.5-1. Oju)  granules  that  stain  with  aldehyde  fuchsin  (Figs.  1  and  2). 
It  frequently  appears  as  though  the  aggregations  are  located  on  the  surface  of  small 
clear  spaces  in  the  cytoplasm.  In  cells  that  do  not  contain  large  amounts  of  secre- 
tory material,  aggregations  may  not  be  present.  In  these  cases  secretory  material 
is  scattered  in  the  cytoplasm  as  fine  granules  about  the  size  of  those  that  make  up  the 
aggregations.  The  cytoplasm  is  generally  flaky  in  appearance  and,  in  cells  con- 
taining many  granules,  may  sometimes  be  stained  a  red-purple  by  the  PAF  tech- 
nique. However,  many  of  the  cells  do  not  show  this  cytoplasmic  staining;  this  is 
probably  because  the  cells  are  in  different  stages  of  the  secretory  cycle. 

In  some  sections,  secretory  material  may  also  be  seen  at  a  point  where  the  axon 
leaves  the  cell  body  and  along  the  axon  for  a  short  distance.  When  it  is  found 
along  the  axon,  the  secretory  material  appears  as  a  number  of  small  granules  strung 
out  along  the  axon.  Farther  from  the  cell  bodies,  though,  it  appears  to  consist  of  a 
more  finely  divided  suspension  somewThat  dispersed  in  the  axons. 

Cell  Type  2.  This  cell  type  is  a  smaller  cell  which  is  also  restricted  in  its  distri- 
bution. These  cells  are  arranged  in  the  proximal  part  of  the  x-organ  as  a  cluster 
of  grapes  as  described  by  Hanstrom  (1931 ) .  The  cell  body  measures  about  30  ^  in 
length  and  is  slightly  narrower,  20-25 /*,  than  it  is  long  (Fig.  1).  It  possesses  a 
large  nucleus,  but  none  has  been  observed  to  contain  more  than  one  nucleolus.  The 
nucleolus  here  is  also  near  the  nuclear  membrane.  The  cytoplasm  is  somewhat 
vacuolated,  although  the  vacuoles  appear  to  be  a  result  of  fixation;  they  do  not 
possess  any  definite  shape.  In  February,  these  cells  possess  small  vacuoles  with 
granules  of  secretory  material  located  peripherally.  Some  cells  in  February  have 
granules  contained  within  vacuoles.  At  other  times  of  the  year  the  material  is 
present  as  quite  large,  4^,  distinct  droplets  (Figs.  1  and  9)  as  contrasted  with  the 
granules  present  in  Type  1  cells.  The  droplets,  usually  one  or  two  per  cell,  are 
almost  always  round  and  are  usually  located  in  the  axon  hillock  or  in  the  axon. 
Sometimes  many  drops  may  be  seen  along  the  bundles  of  axons  as  they  leave  the  cell 
group  (Fig.  3). 

FIGURE  2.  Type  1  neurosecretory  cell  in  advanced  stage  of  the  secretory  cycle.  Note  the 
aggregations  of  granules.  Cells  with  this  appearance  were  counted  as  indicating  the  secretory 
activity  of  this  cell  type.  Bouin's  plus  \%  calcium  chloride;  permanganate-aldehyde-fuchsin, 
1300  X. 

FIGURE  3.  Type  2  neurosecretory  cell  containing  many  droplets  of  secretory  material  in 
its  axon  hillock.  Bouin's  plus  \%  calcium  chloride;  permanganate-aldehyde-fuchsin ;  1300  X. 

FIGURE  4.  Type  3  neurosecretory  cell  showing  peripheral  arrangement  of  vacuoles  (top 
of  photograph)-  Bouin's  plus  \%  calcium  chloride;  permanganate-aldehyde-fuchsin;  1300  X. 

FIGURE  5.  Type  3  neurosecretory  cell.  Note  centrally  located  vacuole  with  granules  of 
secretory  material  located  on  the  surface.  Bouin's  plus  \%  calcium  chloride;  permanganate- 
aldehyde-fuchsin;  1400  X. 

FIGURE  6.  Group  of  Type  4  neurosecretory  cells  in  the  eyestalk.  Note  scanty  cytoplasm 
and  scarcity  of  secretory  material.  Bouin's  plus  \%  calcium  chloride;  permanganate-aldehyde- 
fuchsin;  700  X. 

FIGURE  7.  Group  of  Type  4  neurosecretory  cells  in  the  brain.  Note  large  content  of 
secretory  material.  Bouin's  plus  \%  calcium  chloride;  permanganate-aldehyde-fuchsin;  1200  X. 


68 


JAMES  B.  DURAND 


Cell  Type  3.  This  cell  type  is  distributed  freely  throughout  the  neurosecretory 
cell  groups  in  the  eyestalk  and  brain  with  the  exception  of  the  x-organ.  These 
cells  are,  on  the  average,  slightly  larger  than  the  Type  2  cells  (Figs.  1,  4  and  5). 
They  are  generally  tear-drop  shaped,  although  not  as  distinctly  so  as  the  Type  2 


PLO 


VENTRAL 


DORSAL 


E2 


A 

A 


TYPE 
// 


VENTRAL 


DORSAL 


I 

2 
3 
4 


FIGURE  8.  Diagrammatic  representation  of  the  distribution  of  neurosecretory  cell  types 
in  the  brain  and  eyestalk  of  the  crayfish.  Compare  with  Enami's  Figure  11  (1951).  Bl 
through  B5  designate  groups  of  neurosecretory  cells  in  the  brain.  El  through  E4  designate 
groups  of  neurosecretory  cells  in  the  eyestalk.  CC,  circumoesophageal  connectives :  ME, 
medulla  externa ;  MI,  medulla  interna ;  MT,  medulla  terminalis ;  OL,  olfactory  lobes;  PLO. 
pedunculus  lohi  optici ;  SG,  sinus  gland ;  XO,  x-organ. 


NEUROSECRETION  IN  THE  CRAYFISH  69 

cells.  Two  characteristics  distinguish  these  cells  from  the  Type  2  cells.  The  first 
characteristic  is  the  nature  of  the  vacuoles.  The  vacuoles  are  rather  large,  up  to 
//A,  and  most  of  the  time  may  be  seen  around  the  periphery  of  the  cell  (Figs.  1  and 
4)  although  sometimes  they  may  be  located  more  centrally  in  the  cytoplasm  (Figs. 
1  and  5).  The  vacuoles  are  sharply  delimited  from  the  cytoplasm.  In  this  way 
they  are  markedly  different  from  those  usually  found  in  Type  2  cells.  The  second 
characteristic  is  the  appearance  of  the  secretory  product.  Thus,  in  the  Type  3 
cells,  the  secretory  product  consists  of  fine  granules  which  are  never  clumped  in  as 
large  numbers  as  they  are  in  the  Type  1  cells  (Fig.  2).  Furthermore,  secretory  ma- 
terial is  never  present  in  the  form  of  large  droplets  as  it  is  in  Type  2  cells.  Granules 
are  scattered,  apparently  at  random,  throughout  the  cytoplasm  or  they  may  be  found 
on  the  surface  of  vacuoles  or,  sometimes,  as  a  small  drop  in  the  center  of  one  of 
the  vacuoles  (Fig.  1).  Vacuolated  cells  of  this  type  are  sometimes  found  to  con- 
tain no  signs  of  secretion. 

Cell  Type  4.  Cells  of  this  type  are  located  in  all  neurosecretory  cell  groups  of 
the  eyestalk  and  brain  except  the  x-organ  and  group  E3  (Fig.  8).  The  Type  4 
cells  are  small,  about  13ju  in  diameter,  possess  a  small  nucleus,  \Q /j.  in  diameter, 
and,  as  is  obvious  from  the  measurements,  very  little  cytoplasm  (Figs.  1  and  6). 
They  are  classified  as  neurosecretory  cells  since  preliminary  studies  show  that, 
under  certain  conditions,  some  of  the  cells  undergo  changes  in  the  amount  of  se- 
cretory material  they  contain  (Fig.  7).  Furthermore,  they  are  similar  to  the 
gamma  neurosecretory  cells  described  by  Enami  (1951)  and  are  found  only  within 
the  neurosecretory  cell  groups.  Generally  they  show  little  sign  of  secretory  ac- 
tivity but  differ  from  the  ordinary  ganglion  cells  of  the  eyestalk  in  that  they  possess 
more  cytoplasm  and  cell  boundaries  which  are  easily  demonstrated  by  the  tech- 
niques used  in  these  studies.  The  boundaries  of  the  ordinary  ganglion  cells  are 
extremely  difficult  to  detect  with  these  techniques. 

The  distribution  of  neurosecretory  cell  types  is  shown  in  Figure  8.  It  should 
be  noted  that  certain  cell  groups  of  the  crayfish  differ  in  their  distribution  from  that 
reported  in  a  previous  account  (Bliss,  Durand  and  Welsh,  1954).  The  earlier  ac- 
count is  essentially  correct.  However,  groups  B2  and  B3  of  the  earlier  account 
most  likely  constitute  one  group  of  cells.  The  group  was  previously  reported  to  lie 
lateral  to  the  olfactory  lobes.  Actually  it  is  located  medial  to  the  olfactory  lobes  on 
the  lateral  side  of  the  main  mass  of  fibers  of  the  brain.  This  distribution  of  neuro- 
secretory cells  brings  the  neurosecretory  system  of  the  crayfish  into  fairly  close 
agreement  with  that  of  Sesaniw  (Enami.  1951). 

Secretory  activity 

No  published  observations  on  the  normal  molting  cycle  of  0.  t'in'lis  are  available. 
However,  the  following  information,  although  incomplete,  shows  that  there  is  a 
single  molting  time  per  year  for  crayfish  of  the  size  and  sex  used  in  this  study.  All 
animals  collected  on  June  28  were  soft ;  the  cuticle  was  parchment-like.  Consider- 
able resorption  of  calcium  had  occurred  from  all  parts  of  the  exoskeleton  and  es- 
pecially from  the  ischiopodite  of  the  cheliped.  Further,  all  of  the  animals  possessed 
well  developed  gastroliths,  about  3  mm.  in  diameter,  contained  within  the  gastro- 
lith  sac. 

Similarly,  all  animals  collected  on  July  23  were  soft;  their  exoskeletons  were 


70 


JAMES  B.  DURAND 


thin  and  parchment-like,  but  none  possessed  any  signs  of  gastroliths.  All  animals 
collected  on  August  14  had  hard  exoskeletons  and  showed  no  signs  of  an  approach- 
ing molt. 

Since  the  gastroliths  disappear  very  shortly  after  molt,  the  observations  indi- 
cate that  these  animals  had  molted  some  time  between  June  28  and  July  23.  Fur- 
thermore, the  observations  show  that  the  adult  male  animals  used  in  this  study  were 
highly  synchronized  in  their  molting  period,  for  none  appeared  to  be  approaching  a 
molt  on  any  date  after  June  28. 


0 


FIGURE  9.  Type  2  neurosecretory  cells  in  the  x -organ  of  a  crayfish  just  prior  to  molt. 
Note  the  large  number  of  secretory  droplets.  Bouin's  plus  \%  calcium  chloride;  permanganate- 
aldehyde-fuchsin ;  700  X. 

FIGURE  10.  Type  2  neurosecretory  cells  in  the  x-organ  of  a  crayfish  shortly  after  molt. 
Note  the  lack  of  secretory  material.  Bouin's  plus  \%  calcium  chloride;  permanganate-aldehyde- 
fuchsin;  700  X. 

For  the  greater  part  of  the  year,  the  number  of  cells  that  contained  secretory  ma- 
terial was  remarkably  constant.  However,  a  striking  increase  in  the  number  of 
Type  2  cells  that  contained  droplets  of  secretory  material  took  place  some  time  be- 
fore May  6.  From  Table  I  it  will  be  observed  that  more  than  twice  as  many  Type 
2  cells  contained  secretory  droplets  on  May  6  and  June  28  ( Fig.  9)  than  at  any  other 
time  of  sampling  (Fig.  10). 

Table  I  shows  that  the  number  of  Type  1  cells  that  contained  secretory  ma- 
terial did  not  change  appreciably  throughout  the  year. 

The  other  neurosecretory  cell  groups  in  the  eyestalk  were  examined  carefully, 
but  no  apparent  histological  changes  occurred  in  these  secretory  cells  during  the 


NEUROSECRETION  IN  THE  CRAYFISH 


71 


year.  The  scarcity  of  secretory  material,  relative  to  the  amounts  present  in  cell 
Types  1  and  2,  and  its  occurrence  as  small  granules  made  it  difficult  to  determine 
what  proportion  of  cells  showed  secretory  activity.  It  was  concluded,  however, 
that  no  major  histological  changes  occurred  in  the  other  neurosecretory  cell  groups 
in  the  course  of  this  study. 

DISCUSSION 
Cell  types 

A  few  comments  should  be  made  regarding  a  comparison  of  the  neurosecretory 
cell  types  of  the  crayfish  with  those  described  for  other  crustaceans  by  Enami 
(1951),  Matsumoto  (1954),  and  Carlisle  and  Passano  (1953).  Although  these 
authors  studied  brachyurans  and  used  fixatives  other  than  those  used  by  the  present 
writer,  their  findings  bear  similarities  with  those  reported  here  for  the  crayfish. 

TABLE  I 

Counts  of  Type  1  and  Type  2  neurosecretory  cells  containing  secretory  material  in  the 

brain  and  eyestalk  of  Orconectes  virilis 


Date 

Number  of  animals 

Cell  type 

Mean  count  and 
standard  error 

May  6 

2 

2 

117±1 

June  28 

4 

1 

43±1 

4 

2 

125±8 

July  23 

4 

1 

46±2 

4 

2 

53±5 

August  14 

3 

1 

51±2 

3 

2 

66±4 

August  31 

4 

1 

38±2 

4 

2 

38±4 

September  22 

3 

1 

40±1 

3 

2 

38  ±6 

Of  the  four  neurosecretory  cell  types  described  above  for  the  crayfish,  it  has 
been  shown  that  the  Type  1  and  Type  2  cells  are  practically  restricted  in  their 
distribution  to  the  x-organ ;  only  a  few  Type  1  cells  are  found  in  other  neurosecre- 
tory cell  groups.  A  study  of  Enami's  figures  reveals  that  in  Sesanna,  the  giant  beta 
neurosecretory  cell  is  the  only  type  found  in  the  x-organ  of  that  animal.  Smaller 
beta  neurosecretory  cells  are  present  as  a  small  paired  group  in  the  supraoesophageal 
ganglion  of  Sesarma.  Enami  reports  that  the  cytoplasma  of  the  beta  neurosecretory 
cells  is  fairly  homogeneous  and  of  compact  appearance,  showing  but  slight  con- 
traction upon  fixation.  It  is  apparent  from  other  figures  in  Enami's  paper  that  the 
Type  2  cells  in  the  crayfish  are  similar  to  the  giant  beta  cells  in  Sesanna. 

No  Type  2  neurosecretory  cells  were  found  in  the  brain  of  the  crayfish.  The 
crab,  Sesarma,  would  appear  to  differ  from  the  crayfish  in  that  the  crab  possesses 
a  paired  group  of  beta  neurosecretory  cells  in  the  supraoesophageal  ganglion.  In 
addition.  Enami  describes  no  cells  in  Sesarma  which  are  comparable  to  the  Type  1 
cells  of  the  crayfish. 

The  Type  3  cells  of  the  crayfish  are  comparable  to  the  alpha  cells  of  Sesarma 


72  JAMES  B.  DURAND 

in  their  distribution  and  in  some  of  their  cytological  details.  In  both  animals  they 
are  found  in  all  neurosecretory  cell  groups  except  the  x-organ.  Both  cell  types  are 
rich  in  cytoplasm  and  are  characterized  by  the  presence  of  vacuoles  which  are 
sharply  delimited  from  the  cytoplasm. 

The  Type  4  neurosecretory  cells  of  the  crayfish  are  similar  to  the  gamma  cells 
of  Sesanna.  They  correspond  in  all  features  to  the  gamma  cells.  Small  size,  little 
cytoplasm  relative  to  the  size  of  the  nucleus,  and  scarcity  of  secretory  material  are 
characteristic  of  these  cells  in  both  animals. 

Of  the  four  neurosecretory  cell  types  described  by  Matsumoto  (1954)  for 
Eriochcir  japonicus,  he  compares  only  his  C  cells,  located  in  the  ventral  ganglion, 
with  Enami's  beta  cells.  However,  Enami  has  shown  that  no  beta  neurosecretory 
cells  occur  in  the  ventral  ganglion  of  Sesanna,  Judging  from  the  figures  in  their 
papers  and  the  cell  types  observed  in  the  crayfish,  it  appears  possible  that  Matsu- 
moto's  C  cells  might  be  more  comparable  to  Enami's  alpha  cells  and  to  the  crayfish 
Type  3  cells. 

Carlisle  and  Passano  (1953)  found  three  types  of  neurosecretory  cells  in  the 
x-organs  of  most  species  of  crustaceans  they  examined.  However,  the  number  of 
cell  types  later  was  reduced  to  two  (Carlisle,  1953).  These  authors  showed  that 
in  the  Natantia,  the  x-organ  is  divided  into  two  portions,  the  pars  ganglionaris 
which  is  located  on  the  medulla  terminalis  and  the  pars  distalis  which  is  located 
elsewhere  in  the  eyestalk.  The  Brachyura  and  the  crayfish,  in  contrast  to  the 
Natantia,  possess  an  undivided  x-organ.  Also,  Carlisle  and  Passano  found  one 
neurosecretory  cell  type  to  be  located  in  the  pars  ganglionaris  x-organi  and  the  other 
in  the  pars  distalis  x-organi.  The  cells  of  the  pars  ganglionaris  x-organi  are  com- 
parable to  the  giant  beta  neurosecretory  cells  of  Sesanna,  and  Carlisle  and  Passano 
referred  to  them  as  the  x-organ  neurosecretory  cells.  It  is  evident  that,  since  the 
Type  2  neurosecretory  cells  of  the  crayfish  are  comparable  to  the  giant  beta  neuro- 
secretory cells  of  Sesanna,  they  are  also  similar  to  the  x-organ  neurosecretory  cells 
described  in  the  Natantia  by  Carlisle  and  Passano. 

There  is  a  close  parallelism  in  the  arrangement  of  neurosecretory  cell  groups  of 
the  crayfish  and  the  land  crab,  Gccarc'miis  (Bliss,  Durand  and  Welsh,  1954).  Fur- 
thermore, a  comparison  of  Figure  8  of  the  present  paper  with  Figure  1 1  in  Enami's 
paper  (1951)  has  already  revealed  that  there  is  a  remarkable  similarity  in  the 
distribution  of  neurosecretory  cell  types  in  the  crayfish  and  Sesanna.  The  paral- 
lelism in  the  distribution  of  neurosecretory  cell  types  in  the  crayfish  and  Sesanna 
is  particularly  interesting  when  the  physiological  role  of  these  cells  is  considered. 
This  is  discussed  in  the  next  section. 

Secretory  activity 

When  considering  the  increase  in  secretory  activity  that  was  observed  in  one 
type  of  neurosecretory  cell  in  May,  it  should  be  remembered  that  the  animals  used 
in  May  had  been  kept  in  the  laboratory  for  three  to  four  months.  There  is  evi- 
dence that  crustaceans  kept  in  the  laboratory  for  long  periods  of  time  are  different 
from  those  freshly  collected.  The  molt-promoting  effects  of  constant  darkness  on 
Gecarcinus  (Bliss,  1954)  are  slowed  down  or  delayed  when  freshly  collected  crabs 
are  used.  Animals  long  maintained  in  the  laboratory  respond  quickly  (Bliss, 
personal  communication).  0.  ririlis,  kept  in  the  laboratory,  have  been  observed  to 


NEUROSECRETION  IX  THE  CRAYFISH 

molt  in  fairly  large  numbers  in  May  and  the  first  part  of  June.  Although  no  ob- 
servations were  made  on  the  molting  of  0.  vinlis  in  the  field  in  May  and  in  early 
June,  it  seems  reasonable  to  assume  that  the  laboratory  stock  animals  molt  at  an 
earlier  date  than  animals  in  the  field  because  of  the  higher  temperatures  and  more 
regular  food  supply  that  probably  exist  under  laboratory  conditions.  Therefore, 
data  from  the  February  and  May  animals  used  in  this  study  may  not  be  strictly 
comparable  to  data  from  crayfish  that  were  freshly  collected. 

The  results  included  in  Table  I  raise  a  question  concerning  the  physiological 
significance  of  the  increased  content  of  stainable  material  in  the  Type  2  neurosecre- 
tory  cells  of  the  x-organ  just  prior  to  molt.  The  idea  that  the  sinus  gland  is  a  stor- 
age-release center  for  neurosecretory  products  (Bliss,  1951,  1953;  Bliss,  Durand 
and  Welsh,  1954;  Bliss  and  Welsh,  1952;  Passano,  195la,  1951b,  1952,  1953) 
implies  that  there  is  a  mechanism  whereby  the  rate  of  release  of  the  substances  can 
be  controlled.  Indeed,  the  well  known  reactions  of  certain  crustaceans  to  back- 
ground color  are  evidence  that  the  release  of  certain  neurosecretory  products,  e.g., 
chromatophorotropins,  is  precisely  regulated.  Since  a  molt-inhibiting  substance  is 
produced  in  the  x-organ  and  passed  to  the  sinus  gland  for  release  into  the  blood 
stream,  it  is  necessary  to  assume  that  at  some  time  before  the  animal  molts  there  is  a 
decreased  synthesis  of  this  substance  in  the  cells  of  the  x-organ,  a  decreased  re- 
lease from  the  sinus  gland,  or  both.  It  is  assumed  here  that  the  release  of  the  molt- 
inhibiting  substance  is  decreased  before  molt. 

It  is  known  that  the  pars  ganglionaris  x-organi  of  the  Natantia  produces  a 
molt-inhibiting  hormone  (Carlisle,  1954)  and  is  comparable  to  a  portion  of  the 
x-organ  in  the  crayfish  (this  paper).  Since  the  crayfish  x-organ  probably  produces 
a  molt-inhibiting  hormone  and  since  the  only  neurosecretory  cells  present  in  the 
pars  ganglionaris  x-organi  of  the  Natantia  are  comparable  to  the  Type  2  neuro- 
secretory cells  of  the  crayfish,  it  is  conceivable  that  this  neurosecretory  cell  type  is 
the  source  of  the  molt-inhibiting  hormone.  If  this  is  so,  then  the  accumulation  of 
stainable  material  found  in  the  Type  2  neurosecretory  cells  just  before  molt  can 
be  considered  evidence  of  more  (1)  precursor  of  the  molt-inhibitor  substance,  (2) 
carrier  substance,  or  (3)  active  material. 

It  is  evident  from  the  cell  counts  of  the  May  animals  that  an  assumed  reduction 
in  the  rate  of  release  must  occur  over  a  rather  long  period  before  molt.  In 
adult  crayfish  large  amounts  of  secretory  material  are  present  in  these  cells  early 
in  May,  and  signs  of  this  increase  are  found  in  February  in  laboratory  crayfish. 
Preliminary  studies  show  that  in  immature  crayfish  possessing  an  intermolt  period 
of  approximately  thirty-five  days,  increased  amounts  of  secretory  material  are  pres- 
ent in  Type  2  neurosecretory  cells  of  the  x-organ  at  least  five  days  before  molt. 

Fewer  Type  2  neurosecretory  cells  contain  secretory  material  after  molt.  This 
could  result,  if.  after  molt,  there  is  a  sudden  release  of  stored  material  from  the 
sinus  gland  and  a  rapid  transfer  of  material  from  the  cell  bodies  to  the  sinus  gland 
for  further  release.  Pyle  (1943)  found  pronounced  changes  after  molt  in  both  the 
amount  and  staining  qualities  of  the  sinus  gland  material.  He  fixed  eyestalks 
from  0.  virilis  a  few  hours  before  molt  and  after  the  animals  had  completed  molt. 
He  found  that  there  was  a  sharp  reduction  in  the  number  of  secretory  granules  pres- 
ent in  the  sinus  glands  after  molt.  Since  the  secretory  masses  he  refers  to  in  his 
photographs  are  identical  in  appearance  with  similar  masses  observed  by  the  present 


74 


JAMES  B.  DURAND 


author  in  neurosecretory  fiber  endings  in  the  sinus  glands,  it  is  possible  that  practi- 
cally all  of  the  material  in  a  given  axon  ending  is  released  after  molt.  In  the  cray- 
fish this  release  takes  place  in  a  period  of  not  more  than  a  few  hours  (Pyle,  1943). 
The  accumulation  of  stainable  material  in  the  Type  2  neurosecretory  cells  of  the 
x-organ  prior  to  molt  has  been  explained  on  the  basis  of  a  hypothetical  witholding 
of  molt-inhibiting  hormone  by  the  sinus  gland  and  a  continued  synthesis  of  hormone 
or  its  precursor  in  Type  2  neurosecretory  cell  bodies  of  the  x-organ.  The  secretory 


ae 


A 


B 


C 


FIGURE  11.  Hypothetical  scheme  for  the  secretory  activity  of  Type  2  neurosecretory  cells. 
A.  During  the  intermolt  period,  a  slow  release  of  secretory  material  into  the  blood  and  syn- 
thesis of  the  material  in  Type  2  neurosecretory  cell  bodies  continues.  B.  Shortly  before  molt, 
release  of  neurosecretory  material  into  the  blood  is  decreased ;  synthesis  of  the  material  in  the 
Type  2  neurosecretory  cell  bodies  continues.  Material  thus  accumulates  in  the  axon  endings 
and  in  the  cell  bodies.  C.  Immediately  after  molt,  a  sudden  release  of  secretory  material  into 
the  blood  occurs  ;  cell  body  secretory  material  is  transferred  quickly  to  the  axon  endings  for 
release,  ae,  axon  ending ;  bs,  blood  sinus. 


activity  of  the  Type  2  neurosecretory  cells  is  summarized  in  Figure  11.  This  is 
in  complete  agreement  with  the  existing  hypothesis  on  the  control  of  molt  in 
crustaceans  (Bliss,  1953;  Bliss,  Durand  and  Welsh,  1954;  Bliss  and  Welsh,  1952; 
Passano,  1953).  It  is  interesting  that  the  only  neurosecretory  cells  of  the  eyestalk 
that  show  histological  changes  correlated  with  molt  are  restricted  to  the  x-organ, 
the  only  cell  group  so  far  proved  to  be  effective  in  the  prevention  of  molt  (Passano, 
1953).  As  to  the  functions  of  the  other  neurosecretory  cell  types  in  the  crayfish, 
no  information  was  obtained  in  this  studv. 


NEUROSECRETION  IN  THE  CRAYFISH 

SUMMARY 

1 .  There  are  four  cytologically  distinct  types  of  neurosecretory  cells  in  the  eye- 
stalk  and  brain  of  Orcoucctcs  I'irilis.     Two  of  these  neurosecretory  cell  types  are 
restricted  in  their  distribution  to  the  x-organ.     The  other  two  cell  types  occur  in 
all  neurosecretory  cell  groups  in  the  eyestalk  and  brain  except  the  x-organ. 

2.  The  distribution  of  neurosecretory  cell  types  has  been  compared  with  that 
described  by  Enami  (1951)  for  Scsanna. 

3.  The  Type  2  neurosecretory  cells  are  the  only  neurosecretory  cells  that  un- 
dergo histologically  demonstrable  changes  in  secretory  activity  in  relation  to  the 
molting  cycle.     It  is  suggested,  therefore,  that  the  Type  2  neurosecretory  cells  are 
the  source  of  the  molt-inhibiting  hormone. 

4.  Arguments  are  presented  in  favor  of  the  view  that  at  some  time  before  molt 
a  decrease  occurs  in  the  rate  of  release  of  molt-inhibiting  hormone  from  the  axon 
endings  of  the  Type  2  neurosecretory  cells.     This  decrease  seems  to  be  correlated 
with  a  concurrent  accumulation  of  stainable  material  observed  in  Type  2  neuro- 
secretory cell  bodies. 

LITERATURE  CITED 

BARGMAXX,    W.,    1949.     Uber    die    neurosekretorische    Yerkniipfung    von    Hypothalamus    und 

Neurohypophyse.     Zcitschr.  f.  ZcIIf.  it.  mikr.  Anat.,  34:  610-634. 
BLISS,   DOROTHY   E.,   1951.     Metabolic  effects  of  sinus  gland  or   eyestalk  removal  in  the  land 

crab,  Gecarcinus  latcralis.     Anat.  Rcc.,  Ill  :  86. 
BLISS,    DOROTHY    E.,    1952.     Endocrine    control    of    metabolism    in    the    decapod    crustacean, 

Gecarcinus  latcralis.     Thesis  for  Ph.D.  degree,  Radcliffe  College. 
BLISS,  DOROTHY  E.,  1953.     Endocrine  control  of  metabolism  in  the  land  crab,  Gccarcinits  later- 

alis    (Freminville).     I.    Differences    in    the    respiratory    metabolism    of    sinusglandless 

and  eyestalkless  crabs.     Biol.  Bull..  104:  275-296. 
BLISS,  DOROTHY  E.,  1954.     Light  inhibition  of  regeneration  and  growth  in  the  crab,  Gecarcinus 

latcralis.     Anat.  Rcc.,  120:  742. 
BLISS,    DOROTHY    E.,   J.    B.    DURAXD   AND  J.    H.    WELSH,    1954.     Neurosecretory    systems    in 

decapod  Crustacea.     Zcitschr.  f.  Zcllf.  n.  mikr.  Anat..  39:  520-536. 
BLISS,    DOROTHY    E.,    AND   J.    H.    WELSH,    1952.     The   neurosecretory    system    of    brachyuran 

Crustacea.     Biol.  Bull.,  103:   157-169. 
CARLISLE,   D.    B.,    1953.     Studies   on   Lysinata   scticaitdata   Risso    (Crustacea   Decapoda).     VI. 

Notes   on  the   structure  of   the   neurosecretory   system   of   the   eyestalk.     Pitbbl.    Stas. 

Zool.  Napoli,  24 :  435-447. 
CARLISLE,  D.  B.,  1954.     The  x  organ-sinus  gland  complex,  somatotropin,  the  ovarian  inhibiting 

hormone,  and  sex  reversal  in  Lysinata    (Natantia,  Hippolytidae) .     Pubhl.  Staz.  Zool. 

Napoli,  Suppl.  24:  79-80. 

CARLISLE,  D.  B.,  AND  L.  M.  PASSANO,  1953.     The  x-organ  of  Crustacea.     Nature.  171 :   1070. 
DAWSON,  A.  B.,  1953.     Evidence  for  the  termination  of  neurosecretory  fibers  within  the  pars 

intermedia  of  the  hypophysis  of  the  frog,  Rana  pipicns.     Anat.  Rcc..  115:  63-69. 
ENAMI,  M.,   1951.     The  sources  and  activities  of  two  chromatophorotropic  hormones  in  crabs 

of  the  genus  Sesarma.     II.  Histology  of  incretory  elements.     Biol.  Bull..  101  :  241-258. 
GABE,  M.,  1954.     La  neuro-secretion  chez  les  invertebres.     Ann.  Bin!.,  30:  5-62. 
GOMORI,  G.,  1941.     Observations  with  differential  stains  on  human  islets  of  Langerhans.    Amer. 

J.  Path.,  17:  395-406. 
GOMORI,   G.,   1950.     Aldehyde  fuchsin :   a  new   stain  for   elastic   tissue.     Amcr.  J.   Clin.   Path., 

20:  665-666. 
HALMI,  N.  S.,   1952.     Differentiation  of  two  types  of  basophils  in  the  adenohypophysis  of  the 

rat  and  mouse.     Stain   Tech.,  27  :  61-64. 
HANSTROM,    B.,     1931.     Neue  .Untersuchungen    iiber    Sinnesorgane    und    Nervensystem    der 

Crustaceen.     I.  Morphol.  n.  Okol.  d.  Ticrc.  23  :  80-236. 


76  JAMES  B.  DURAND 

MATSUMOTO,  K.,  1954.     Neurosecretion  in  the  thoracic  ganglion  of  the  crab,  Eriocheir  japonicns. 

Biol.  Bull.,  106:  60-68. 
PASSANO,   L.   M.,    195 la.     The   X   organ-sinus   gland  neurosecretory   system   in   crabs.     Anat. 

Rec.,  Ill:  502. 
PASSANO,    L.    M.,    1951b.     The   x   organ,   a   neurosecretory    gland   controlling   molt   in   crabs. 

Anat.  Rec.,  Ill :  143. 
PASSANO,    L.    M.,    1952.     The    x    organ-sinus    gland    complex    of    brachyuran    crustaceans,    a 

neurosecretory,  molt-controlling  gland.     Thesis  for   Ph.D.   degree,  Yale  University. 
PASSANO,   L.    M.,    1953.     Neurosecretory  control   of  molting   in   crabs   by  the   x   organ-sinus 

gland  complex.     Physiologica  Comparata  ct  Occologia,  3  :   155-189. 
PYLE,  R.  W.,   1943.     The  histogenesis  and  cyclic  phenomena  of  the   sinus  gland  and  x-organ 

in  Crustacea.     Biol.  Bull.,  85 :  87-102. 

SCHARRER,  E.,  AND  BERTA  SCHARRER,  1945.       Neurosecretion.     Physio].  Rev.,  25:   171-181. 
SCHARRER,    E.,    AND    BERTA    SCHARRER,    1954.     Hormones    produced    by    neurosecretory    cells. 

(Laurentian  Hormone  Conference.)     Recent  Progress  in  Hormone  Research,  10:  183- 

240. 
VAN  HARREVELD,  A.,   1936.     A  physiological  solution  for  fresh  water  crustaceans.     Proc.  Soc. 

E.rf.  Biol.  Med.,  34:  428-432. 


V. 


THE  PRESENCE  AND  SIGNIFICANCE  OF  RESPIRATORY  ME- 
TABOLISM IN  STREAK-FORMING  CHICK  BLASTODERMS  1 

RONALD  C.  PHASER 
Department  of  Zooloi/y  and  Entomology,  I'nii'ersity  of  Tennessee.  Knoxville,  Tennessee 

The  value  of  simple  sugars,  particularly  glucose,  in  early  chick  development  is 
\vellknown  (Needham  and  Nowinski.  1937;  Spratt,  1949;  Taylor  and  Schechtman. 
1949;  Fraser,  1954a).  Needham  (1931)  and  Romanoff  and  Romanoff  (1949) 
list  glucose  as  a  free  constituent  of  egg  yolk,  and  recent  investigation  (Fraser,  un- 
published results)  has  shown  that  glucose  is  the  only  free  monosaccharide  in  egg 
white  dialysate  detectable  by  chromatographic  procedure.  In  1938  Jacobson  found 
that  there  was  a  marked  glycolysis  in  involuting  mesodermal  cells  at  gastrulation. 
Such  studies  have  illustrated  the  importance  of  carbohydrate  metabolism  in  early 
chick  embryogenesis. 

While  the  importance  of  carbohydrate  utilization  is  generally  recognized,  the 
manner  in  which  it  is  metabolized  has  been  disputed.  Novikoff,  Potter  and  LePage 
(1948)  stand  against  the  contention  of  Needham  and  Nowinski  (1937)  that  there  is 
a  non-phosphorylating  glycolytic  scheme  in  young  chick  blastoderms.  The  former 
authors  were  able  to  detect  assorted  phosphorylated  carbohydrate  components  in 
embryos  of  three  to  ten  days  incubation.  Needham  and  Nowinski  were  unable  to 
find  an  increase  in  oxygen  consumption  either  in  whole  embryos  or  homogenate, 
when  phosphorylated  sugars  were  added. 

Aside  from  the  energetics  involved  at  this  level  of  glycolysis,  terminal  oxida- 
tion with  molecular  oxygen  by  cytochrome  oxidase  has  been  followed.  Using 
manometric  means.  Potter  and  DuBois  (1942)  found  the  first  evidence  of  this 
enzyme  in  the  six-day  embryo.  Albaum  and  Worley  (1942)  were  able  to  detect 
activity,  as  measured  by  oxygen  uptake,  in  the  embryo  of  four  days.  By  soaking 
blastoderms  in  solutions  containing  dimethyl-p-phenylenediamine  and  alpha  naphthol 
(nadi  reagent),  followed  by  visual  inspection,  Moog  (1943)  has  been  able  to  show 
that  cytochrome  oxidase  is  present  even  in  head  process  stages.  Sodium  azide- 
treated  embryos  lost  much  of  the  respiratory  activity  seen  in  the  experimental 
group. 

Moog  also  found  that  this  enzyme  activity  was  expressed  in  a  morphological  pat- 
tern similar  to  that  displayed  by  reducing  enzymes  (Spratt,  1951a),  sensitivity  to 
respiratory  poisons  (Hyman,  1927;  Spratt,  1950b),  anaerobiosis  (Spratt,  1950a) 
and  starvation  (Spratt,  1951b;  Fraser,  1954a).  In  general,  these  experiments  have 
revealed  that  the  node  and  fore-brain  are  regions  of  high  metabolic  activity.  I 
(Fraser,  1954a)  have  been  able  to  demonstrate  that  the  node  is  very  susceptible 
to  degenerative  changes  on  starvation,  while  the  brain  is  relatively  refractory  to 
such  treatment. 

It  is  the  purpose  of  the  present  paper  to  determine  if  there  is  indophenol  oxidase 

1  This  investigation  was  supported  by  research  grant  NSF  G-1406  from  the  National  Science 
Foundation. 

77 


78  RONALD  C.  FRASER 

(cytochrome  oxiclase  according  to  Keilen  and  Hartree,  1938)  activity  in  earlier, 
streak-forming  stages  of  the  chick,  and  if  so,  if  there  is  some  pattern  in  its  distribu- 
tion. Further,  if  certain  cells  do  show  a  greater  respiratory  metabolism,  another 
objective  in  mind  is  to  investigate  the  possibility  that  it  has  some  significance  in  the 
differentiation  of  these  cells. 

The  results  from  the  present  work  permit  the  following  statements.  Cytochrome 
oxidase  is  detectable  in  streak-forming  chick  blastoderms,  particularly  in  the  newly 
involuted  mesoderm  cells.  There  is  no  change  in  the  activity  of  this  enzyme  in 
embryos  on  the  nadi  reagent  following  pretreatment  with  cytochrome  c  or  albumen, 
but  it  increases  appreciably  in  blastoderms  starved  for  five  hours  in  saline.  There 
is  a  striking  decrease  in  its  activity,  as  measured  by  indophenol  formation,  in  ex- 
plants  treated  mildly  with  hydroquinone,  a  reductant  presumably  for  cytochrome  c. 
The  augmented  respiratory  metabolism  seen  in  newly  formed  mesoderm  cells  is  re- 
lated in  some  manner  with  differentiative  ability  at  the  trunk  level  of  the  chick 
blastoderm,  since  fragments  of  involuted  mesoderm  expressed  ability  to  form  meso- 
derm tissue,  while  potential  mesoderm  fragments  (axial  epiblast  in  broad-  and  in- 
termediate-streak embryos)  failed  in  this  respect. 

EXPERIMENTAL  PROCEDURES  AND  RESULTS 

Newly  laid  fertile  eggs  were  obtained  from  the  poultry  farm  of  the  University 
of  Tennessee.  Most  of  the  eggs  used  were  from  White  Leghorn  chickens,  al- 
though a  few  were  from  Rhode  Island  Red  stock.  They  were  stored  in  the  refriger- 
ator at  18°  C.  upon  receipt  until  the  time  (within  five  days)  they  were  used  in  the 
experiments.  The  procedure  for  removing  blastoderms  from  the  yolk  and  for  ex- 
planting  them  in  culture  has  been  given  previously  by  Spratt  (1947).  Eggs  were 
incubated  in  a  forced  draft  incubator  at  37.8°  C.,  while  explants  were  cultured  at 
37.6°  C.  The  temperature  in  both  incubators  was  maintained  at  a  constant  value 
by  mercury  type  thermoregulators.  Manipulation  of  the  blastoderms  was  carried 
out  under  Ringer's  solution.  Specimens  for  histological  inspection  were  fixed  in 
Bouin's  fluid  and  stained  with  Delafield's  hematoxylin.  Over  six  hundred  blasto- 
derms were  used  in  the  course  of  this  investigation. 

A.  Aerobic  enzyme  pattern  in  early-streak  blastoderms 

Pre-,  intermediate-  and  definitive-streak  (DPS)  embryos  corresponding  to 
stages  1,  2-3  and  4  of  Hamburger  and  Hamilton  (1951),  respectively,  were  removed 
from  the  yolks  under  saline,  and  explanted  onto  freshly  prepared  nadi  reagent,  made 
in  the  following  manner.  Five  ml.  of  0.08  M  dimethyl-p-phenylenediamine  in 
chick  Ringer's,  5  ml.  of  0.08  M  alpha  naphthol  in  20'  r  ethyl  alcohol-Ringer's,  1  ml. 
of  bicarbonate  buffer  and  2  ml.  of  phosphate  buffer  were  added  to  27  ml.  of  Ringer's 
containing  300  ing.  agar,  after  the  saline-agar  had  been  heated  and  then  cooled  to 
approximately  50°  C.  In  every  experiment  outlined  here  and  subsequently  in 
which  the  nadi  reagent  was  used,  fresh  preparations  of  dimethyl-p-phenylenediamine 
and  alpha  naphthol  were  made  up  immediately  before  use,  because  the  former  com- 
pound is  oxidized  rather  rapidly  by  atmospheric  oxygen.  Preparation  of  the  buf- 
fers used  has  been  described  previously  (Fraser,  1954a).  At  this  time,  however, 
saturation  of  the  bicarbonate  buffer  with  CO._.  was  achieved  by  passing  the  gas  from 
a  tank  through  a  nozzle  into  the  solution.  The  final  pH  of  the  0.01  M  nadi  reagent 


RESPIRATION  IN  THE  EARLY  CHICK 


79 


TABLE  I 

Pattern  of  cytochrome  o.vidasc  activity  in  early  chick  emlvy<>s  c.\'plantcd  onto  media  containing 
dimethyl-p-phenylenediamine  and  alplia  naphthol  (nadi  rca<icnt)   and  nadi  rcaucnt 

•i\.'ith  sodium  aside 


NO. 


GENERALIZED        RESULTS 


NADI  RE  AGENT  (.  01 M) 


NADI  REAGENT    (.01  M) 
No  AZIDE  (.005  M) 


34 


38 


80  RONALD  C.  FRASER 

medium  was  7.1.  The  preparation  was  poured  immediately  into  watch  glasses  held 
in  petri  dishes  by  moisture-saturated  cotton  rings.  Gelation  of  the  medium  occurred 
within  a  few  minutes.  A  control  medium  containing  sodium  azide  at  a  final  con- 
centration of  0.005  M  was  made  up  in  a  similar  manner. 

The  embryos  were  explanted  onto  the  nacli  reagent  or  nadi  reagent-azide  media 
and  incubated  for  fifteen  minutes  at  37.6°  C.  At  precisely  this  time  they  were  re- 
moved from  the  media  by  pipette  and  examined  under  saline  against  a  white  back- 
ground through  a  dissecting  microscope. 

The  generalized  observations  are  shown  in  Table  I.  Cells  in  the  opaque  area 
peripherad  to  the  germ  wall  in  all  three  stages  tested  showed  a  dark  blue-purple 
coloration  on  the  nadi  reagent.  No  other  pattern  of  coloration  indicative  of  cellular 
respiration  could  be  found  in  pre-streak  embryos.  A  dark  color  was  apparent, 
however,  in  the  streak-forming  region  of  the  intermediate-streak  blastoderm  and 
along  the  streak  and  node  area  in  the  DPS  explants.  Other  pellucid  area  tissues 
were  very  faintly  stained.  On  the  azide-bearing  medium,  the  yolk-laden  cells  in 
the  area  opaca  retained,  in  large  part,  a  deep  coloration,  while  streak  tissues  were 
essentially  colorless.  In  fact,  it  was  extremely  difficult  to  see  the  developing  or  full 
streak  against  the  white  background  in  embryos  removed  from  the  medium  contain- 
ing the  azide. 

The  logical  interpretation  to  be  made  from  the  above  results  is  that  the  coloration 
expressed  in  the  vitelline  cells  surrounding  the  embryo  is  due  mainly  to  a  non-enzy- 
matic mechanism.  Since  interest  was  directed  toward  a  localization  of  activity  in 
the  embryo  proper,  this  issue  was  not  pressed,  although  it  may  be  conjectured  that 
the  amount  of  tissue  and  yolk  in  this  region  may  be  sufficient  to  soak  up  enough 
indophenol  from  the  surrounding  fluid  to  give  this  appearance.  Other  interpreta- 
tions may  be  advanced,  but  failure  of  the  azide  to  prevent  coloration  must  mean  that 
known  oxidative  enzymes  are  not  involved.  Furthermore,  the  marked  depression 
in  indophenol  formation  in  streak  tissues  on  the  azide  medium  must  indicate  that 
this  coloration  is  mediated  by  enzyme  action.  There  is  good  evidence  that  azide 
inhibits  the  action  of  indophenol  oxidase  (cytochrome  oxidase)  which  is  directly 
responsible  for  the  formation  of  the  bluish-purple  indophenol  (Keilin,  1936)  as 
well  as  transphosphorylation  and  ATPase  activity  (Meyerhoff,  1945). 

To  insure  that  the  darker  color  in  the  region  of  the  forming  and  full  streak  was 
not  due  simply  to  the  presence  of  more  cells  compacted  at  this  region,  fragments  of 
tissue  of  comparable  thickness  were  removed  from  streak  epiblast,  mesoderm  and 
hypoblast  for  inspection.  For  comparison,  fragments  of  non-axial  epiblast  were 
also  examined.  These  pieces  were  placed  side  by  side  on  a  microscope  slide  in  a 
small  amount  of  fluid  and  covered  with  a  cover  slip.  The  stained  tissues  prepared 
in  this  manner  were  examined  for  intracellular  indophenol  deposition. 

Figures  2  and  3  will  reveal  that  enzymatic  activity  is  greater  in  involuted  meso- 
dermal  cells  than  in  overlying  epiblast  (potential  mesoderm  in  intermediate-streak 
embryos)  cells.  Attention  is  drawn  to  the  fact  that  indophenol  is  produced  at  the 
surface  of  small  droplets  in  the  cells.  The  cytoplasm  of  the  cells  is  relatively  free 
from  coloration.  This  is  typical  of  all  cells  observed.  It  may  also  be  seen  that 
while  the  number  of  droplets  is  essentially  the  same  in  both  epiblast  and  mesoderm 
cells,  the  enzymatic  activity  is  greater  on  the  surface  of  those  in  newly  involuted 
cells.  These  globules  are  readily  stained  with  Sudan  III,  indicative  of  a  lipid  con- 


RESPIRATION   IX  THE  EARLY  CHICK  81 

tent.  These  photographs  are  of  living  cells  removed  from  a  streak-forming  blasto- 
derm. In  obtaining  the  photographs  care  was  taken  to  make  sure  that  identical 
conditions,  such  as  illumination,  exposure  time,  time  of  processing,  etc.,  were  main- 
tained. The  similar  appearance  in  the  photographs  of  a  defect  in  the  lens  of  the 
photographic  equipment  will  attest  this.  Such  inspection  revealed  that  as  far  as 
indophenol  oxidase  activity  is  concerned,  streak  hypoblast  and  all  epiblast  cells 
tested  were  the  same.  It  is  clear  that  the  darker  coloration  of  the  region  of  the  form- 
ing streak  or  in  the  node  and  full  streak  of  the  older  blastoderms  is  due  solely  to  a 
greater  respiratory  activity  in  newly  involuted  mesoderm  cells.  This  observation 
is  in  conformity  with  that  of  glycogen  utilization  by  invaginating  mesoderm  made 
by  Jacobson  (1938). 


B. 


Modification  of  cytoclirouic  o.vidasc  activity  by  prctrcatincnt 


We  are  dealing  here  with  an  enzyme  which  has  as  its  substrate  the  nadi  reagent 
under  experimental  conditions  and  presumably  cytochrome  c  in  normal  cellular 
respiration.  Therefore,  on  theoretical  grounds  at  least,  it  should  be  possible  to 
modify  the  reaction  between  the  enzyme  and  the  nadi  reagent  by  the  addition  of  the 
normal  substrate.  If  living  cells  behave  as  does  mammalian  heart  muscle  extract, 
according  to  the  observations  of  Keilen  and  Hartree  (1938),  we  should  expect  an 
increase  in  oxidation  of  the  diamine  on  addition  of  cytochrome.  At  the  same  time, 
a  depletion  of  readily  metabolizable  food  reserves  in  the  cell,  resulting  in  a  de- 
pressed enzyme  activity,  could  also  conceivably  lead  to  greater  nadi  oxidation,  and 
hence  augmented  coloration.  These  ideas  were  followed  by  the  following  experi- 
mentation. Four  dozen  eggs  were  supported  on  their  sides  in  a  tray  and  left  in 
the  refrigerator  at  18°  C.  overnight,  so  that  the  position  of  the  blastoderm  would 
be  known.  On  the  following  day  0.2  ml.  of  1.4  X  10~4  717  cytochrome  c  was  injected 
into  the  yolk  sacs  of  two  dozen  eggs,  while  a  similar  quantity  of  2%  sodium  suc- 
cinate  was  injected  into  the  other  two  dozen.  Based  on  previous  measurements  of 
frozen  eggs  a  needle  of  sufficient  length  was  chosen  so  that  the  injected  materials 
would  be  placed  about  one  quarter  inch  from  the  blastoderm.  The  needle  was  in- 
serted vertically  from  the  lower  side  of  the  egg  to  avoid  possible  injury  to  the  blasto- 
derm. Both  preparations  that  were  injected  were  sterilized  by  filtration.  The 
concentration  of  the  cytochrome  c,  prepared  according  to  Umbreit  ct  al.  (1949), 
was  established  by  use  of  the  Beckman  spectrophotometer.  The  eggs  thus  treated 
were  incubated  for  ten  hours  after  which  they  were  explanted  onto  the  Nadi  reagent 
in  the  manner  described  above. 

The  results  were  rather  disappointing  in  that  there  was  no  difference  in  enzy- 
matic activity  in  either  of  these  groups  of  embryos  as  compared  to  normal,  non-in- 
jected controls.  It  became  clear  that  the  question  as  to  whether  the  negative  results 
were  due  to  the  inactivity  of  the  materials  on  the  blastoderms  or  to  the  failure  of  the 
materials  to  reach  the  embryos  could  not  be  resolved  by  this  procedure,  so  it  was 
abandoned. 

Next,  early  streak  blastoderms  were  removed  from  the  yolk  in  the  usual  manner 
after  ten  hours  of  incubation,  and  incubated  in  various  liquid  preparations.  These 
included:  (1)  saline.  (2)  albumen,  (3)  albumen-cytochrome  c,  (4)  albumen-cyto- 
chrome  r-hydroquinone  and  (5)  albumen-hydroquinone.  The  final  concentration 
of  cytochrome  c  was  1.4  X  10~5M,  that  of  hydroquinone  10~3M.  The  albumen  con- 


82 


RONALD  C.  FRASER 


centration  was  the  same  as  previously  employed  in  agar  gels.  Pretreatment  time 
for  those  in  the  first  three  media  indicated  was  five  hours.  It  has  been  shown 
(Spratt,  1951b;  Fraser,  1954a)  that  permanent  damage  occurs  in  embryos  ex- 
planted  on  non-nutrient  media  for  intervals  longer  than  this.  Blastoderms  were 
pretreated  in  the  media  containing  hydroquinone  for  one  hour,  since  beyond  this 
time  cell  dispersal  began.  Twenty-four  embryos  were  placed  in  each  medium,  in- 
cubated for  the  period  indicated  above,  removed,  washed  thoroughly  in  saline  and 
immediately  explanted  onto  a  nadi  reagent-bearing  agar  medium.  On  this  they 
were  again  incubated  for  fifteen  minutes  after  which  they  \vere  removed,  placed  in 
Ringer's  and  inspected.  Embryos  of  the  same  age  were  removed  directly  from  eggs 
and  treated  on  the  nadi  reagent  for  the  same  length  of  time  for  comparison. 

TABLE  II 

Relative  expression  of  cytochrome   o.ridasc  activity   in  streak-forming   chick   blastoderms  pre- 
treated -n'itli   materials  indicated 


GENERALIZED     RESULTS 


PRETREATMENT 
MEDIUM 


SALINE 


ALBUMEN 

ALBUMEN-CYTOCHROME   C 
CONTROL 


ALB- CYT.C-HYDROQUINONE 
ALBUMEN  -  HYDROQUINONE 


NUMBER 


24 


60 


48 


The  results  are  shown  in  generalized  form  in  Table  II.     To  avoid  confusion, 
the  three  groups  of  media  producing  different  results  will  be  treated  separately. 

( 1)  Albumen,  albumen-cytochrome  c,  control  (no  previous  treatment)  :  the  sixty 
blastoderms  in  this  group  were  all  similar  to  those  described  previously  with  respect 
to   indophenol   oxidase   pattern   and   intensity   of   color.     It   appears   obvious   that 
neither  the  albumen  nor  the  cytochrome  c  had  any  affect  on  the  enzyme  activity 
when  presented  to  the  embryos  in  this  manner. 

(2)  Saline  (non-nutrient)  :  Blastoderms  incubated  in  this  fluid  stained  most  in- 
tensely by  nadi  reagent.     All  embryonic  tissues  were  slightly  darker  than  those  on 
media  listed  above,  but  streak  tissue  was  considerably  more  colored.     A  compari- 
son of  cellular  details  in  fragments  of  tissues  from  streak  epiblast,  mesoderm  and 
hypoblast  between  these  and  albumen-treated  embryos  revealed  that  coloration  was 
darker  in  all  three  germ  layers  in  prestarved  embryos,  with  newly  involuted  meso- 
derm again  showing  the  greatest  indophenol  deposition  (Fig.  4). 


RESPIRATION  IN  THE  EARLY  CHICK 

(3)  Albumen-cytochrome  c-hydroqninone,  albnmen-hydroquinone:  After  pre- 
treatment  in  these  media,  blastoderms  showed  a  striking  decrease  in  indophenol 
coloration  when  incubated  on  the  nadi  reagent.  All  tissues  seemed  to  be  stained 
somewhat  less,  but  again  the  streak-forming  tissues  seemed  most  influenced  by 
pretreatment.  Although  not  nearly  as  faintly  colored  as  those  on  an  azide  medium, 
these  tissues  nevertheless  were  considerably  lighter  in  appearance  than  in  control 
blastoderms. 

In  all  cells  observed,  the  blue  color  was  localized  on  the  surface  of  intracellular 
globules,  even  in  starved  embryos.  It  is  interesting  to  note  that  cells  showing  the 
greatest  enzyme  activity  are  those  of  starving  blastoderm  mesoderm,  and  that  this 
activity  is  on  the  surface  of  lipid  material.  The  significance  of  this  and  of  other 
observations  made  at  this  time  will  be  discussed  more  fully  later.  It  will  suffice 
to  point  out  here  that  the  less  intense  color  on  the  droplets  in  cells  of  hydroquinone- 
treated  animals  represents  a  decrease  in  dimethyl-p-phenylenediamine-alpha  naph- 
thol  oxidation.  This  is  what  one  would  expect  if  one  assumed  that  the  hydroquinone 
acts  specifically  as  a  reductant  (as  has  been  shown  by  Krahl  and  co-workers,  1941, 
in  sea  urchin  eggs)  for  intracellular  cytochrome  c  and  not  indophenol,  and  provided 
that  the  cells  were  not  killed  by  such  treatment. 

Considering  the  first  assumption,  it  was  determined  that  hydroquinone,  in  the 
concentration  used  in  the  experiment,  and  even  in  much  greater  concentration, 
could  neither  prevent  the  formation  of  indophenol  from  fresh  nadi  reagent,  nor 
could  it  reduce  indophenol  to  the  leuco  form  in  vitro.  Secondly,  other  blastoderms 
treated  with  hydroquinone  as  indicated  were  subsequently  washed  thoroughly  and 
explanted  onto  an  albumen-agar  medium.  These  were  then  incubated  for  twenty- 
four  hours.  Although  development  did  not  proceed  as  in  normal  explants,  there 
was  some  slight  morphogenesis,  and  tissues  did  not  have  an  opaque  appearance 
characteristic  of  death  of  the  cells. 

C.  Non-autonomy  of  the  increase  in  cytochrome  o.vidase  activity 

The  question  arose  as  to  whether  the  increase  in  enzymatic  activity  seen  in  in- 
voluted mesoderm  was  a  function  of  time  or  of  location  of  tissue.  Preceding  state- 
ments have  indicated  that  after  a  pretreatment  interval  of  five  hours,  there  was  less 
indophenol  localized  in  epiblast  cells  than  in  mesodermal  cells.  But  coincident  with 
change  in  time  there  has  been  some  involution  during  pretreatment. 

Small  fragments  of  tissue  taken  from  streak  epiblast  and  streak  mesoderm  were 
removed  from  streak-forming  blastoderms  and  cultured  under  albumen  (prepared  as 
previously  outlined)  for  intervals  from  five  to  ten  hours.  These  were  then  washed 
in  saline  and  explanted  onto  the  nadi  reagent  medium  for  fifteen  minutes  and  in- 
spected. Similar  fragments  removed  from  blastoderms  of  the  same  age,  but  re- 
moved directly  from  eggs,  were  stained  as  controls. 

The  cultured  streak  epiblast  tissue  had  the  same  blue  indophenol  coloration  as 
the  controls.  Streak  mesoderm  cells  from  both  groups  also  looked  identical,  al- 
though darker  in  appearance  than  epiblast  cells. 

By  preventing  involution  in  this  manner  in  epiblast  tissue  (prospective  meso- 
derm), cultured  for  a  sufficient  period  of  time  for  this  basic  morphogenetic  phenome- 
non to  have  occurred,  it  was  thus  possible  to  show  that  increase  in  indophenol  oxi- 
dase  activity  is  not  autonomous  in  this  tissue.  It  seems  clear  that  the  gain  in 


84 


RONALD  C.  FRASER 


respiratory  activity  is  either  due  to  movement  of  cells  through  the  streak  in  gastrula- 
tion  or  to  the  influence  of  surrounding  cells  in  a  new  mesodermal  location.  The 
following  experiments  are  directed  toward  this  question. 

D.  Significance  of  increased  respiratory  activity  of  cells  in  histogenesis 

If  an  increase  in  enzyme  activity  in  cells  is  due  simply  to  placement  of  the  cells, 
it  should  conceivably  occur  in  streak  epiblast  cells  implanted  in  a  mesodermal  loca- 
tion in  early  chick  embryos.  Eighteen  fragments  of  such  tissue  were  implanted 
through  small  tears  in  the  hypoblast  into  positions  indicated  in  Figure  1.  Frag- 
ments were  removed  from  stage  2  (Hamburger  and  Hamilton,  1951)  embryo  streak 
epiblast,  while  hosts  were  of  stages  2  and  4.  Blastoderms  with  implanted  tissue 
were  then  incubated  for  six  hours  on  a  regular  albumen-agar  medium,  after  which 


DONOR 


HOSTS 


FIGURE  1.  Illustration  of  sites  for  implanting  fragments  of  streak  epiblast  into  mesodermal 
locations  of  intermediate-streak  and  DPS  blastoderms. 

they  were  removed  and  explanted  onto  the  nadi  medium  and  reincubated  for 
fifteen  minutes.  The  implants,  when  found,  were  then  removed  under  saline  and 
mounted  on  microscope  slides  along  with  nadi-stained  fragments  of  epiblast  removed 
directly  from  streak-forming  embryos  for  comparison.  About  one-third  of  the  im- 
plants were  extruded  during  incubation  on  albumen  and  were  hence  lost. 

Fragments  placed  at  mesodermal  positions  2  and  4  in  Figure  1  looked  identical 
to  control  pieces.  There  had  been  no  increase  in  enzyme  activity  in  cells  placed 
in  these  positions.  From  this  and  the  foregoing  observation,  we  might  conclude 
that  the  increase  in  cytochrome  oxidase  activity  in  newly  involuted  cells  is  neither 
a  direct  function  of  time  nor  location,  but  is  tied  in  with  the  morphogenic  move- 
ment of  the  cells  through  the  streak.  This  at  least  seems  true  for  cells  at  the  level 
of  the  streak  in  the  early  chick  blastoderm. 

It  was  rather  surprising  to  see  that  implants  into  mesoderm  in  the  prospective 
head  region  ( positions  1  and  3  in  Figure  1 )  were  considerably  darker  than  the  con- 
trols. On  the  cell  level,  there  was  perceptibly  more  indophenol  on  the  globules  in 
cells  of  the  implants  than  of  the  control  fragments.  At  this  region  of  the  embryo,  it 
appears  that  the  development  of  intracellular  catalytic  activity  is  related  to  location 
of  the  cells. 


RESPIRATION  IN  THE  EARLY  CHICK 


85 


These  experiments  were  then  followed  by  others  to  test  the  significance  of  this 
increase  in  observed  enzymatic  activity  with  respect  to  differentiative  ability  of  the 
tissues  involved.  Fragments  of  streak  epiblast  were  excised  from  streak-forming 
embryos  and  marked  very  lightly  with  diluted  India  ink  under  saline.  They  were 
then  placed  in  saline  and  larger  particles  of  carbon  were  removed  with  a  steel 
needle.  After  a  final  washing,  the  pieces  of  tissue  were  implanted  into  mesodermal 
sites  indicated  in  Figure  1,  in  both  streak-forming  and  DPS  hosts.  Implantation 
occurred  through  small  tears  made  in  the  hypoblast  at  the  desired  regions.  In  a 
similar  manner,  small  pieces  of  newly  involuted  mesoderm  from  the  same  embryos 
were  implanted  in  the  same  regions  with  the  exception  of  the  head  region  in  early 
streak  embryos.  The  host  blastoderms  were  then  cultured  on  albumen-agar  for 
twenty-four  hours  at  36.7°  C,  fixed  with  Bouin's  fluid  and  prepared  for  paraffin 
impregnation.  The  serial  sections  were  made  12  microns  in  thickness.  Eighteen 
implants  were  made  into  each  site  indicated. 

TABLE  III 

Summary  of  results  from  implanting  fragments  of  streak  epiblast  of  streak-forming  (SF) 
chick  embryos  into  mesodermal  sites  of  host  embryos. 


Type  of  fragment 

Site  of  implantation 

Fragments 
recovered* 

Results 

Epiblast 

Head  SF 

12 

Head  mesoderm  and  pharynx 

Epiblast 

Head  DPS 

9 

Head  mesodern  and  pharynx 

PIpiblast 

Flank  SF 

15 

Isolated  ball  degenerative  cells 

Epiblast 

Flank  DPS 

•  13 

Isolated  ball  degenerative  cells 

Streak  meso. 

Head  DPS 

12 

Head  mesoderm  and  pharynx 

Streak  meso. 

Flank  SF 

10 

Ball  of  living  cells 

Flank  mesoderm 

Streak  meso. 

Flank  DPS 

10 

Ball  of  living  cells 

Flank  mesoderm 

*  Number  of  blastoderms  showing  some  evidence  that  tissue  had  been  implanted. 

The  results  are  given  in  Table  III.  It  will  become  immediately  evident  that 
there  is  a  good  correlation  between  increased  cytochrome  oxidase  activity  and  dif- 
ferentiative ability.  Fragments  of  epiblast  placed  in  prospective  head  mesoderm 
not  only  show  an  increase  in  metabolic  (oxidative)  activity  but  also  display  an  ex- 
panded capacity  for  differentiation.  Implants  in  flank  regions  failed  to  show7  any 
increase  in  enzymatic  activity  coincident  with  a  failure  to  produce  mesodermal 
structures.  It  is  also  apparent  that  involuted  mesoderm  has  the  capacity  to  form 
both  mesoderm  and  endoderm  in  the  head  region  as  well  as  flank  mesenchyme. 
There  are  limitations  to  this  ability  at  the  trunk  level,  however.  Figures  5-10  are 
photographs  illustrating  the  results  obtained.  Engulfed  particles  located  in  head 
mesoderm  and  pharynx  are  taken  as  evidence  that  these  cells  had  differentiated  from 
implanted  tissue. 

DISCUSSION 

The  work  of  Moog  (1943)  has  established  that  cytochrome  oxidase  is  present 
in  chick  embryos  in  stages  as  early  as  those  possessing  a  head  process.  In  view 
of  the  fundamental  importance  of  this  enzyme  in  many  diverse  organisms,  there 


86 


RONALD  C.  ERASER 


FIGURE  2.  Living  nacli  reagent-treated  cells  from  streak  epiblast  of  an  intermediate-streak 
blastoderm.  Note  that  indophenol  is  deposited  on  the  surface  of  intracellular  droplets.  <  960. 

FIGURE  3.  Photomicrograph  of  living  cells  stained  on  the  nadi  reagent.  These  cells  are 
from  newly  involuted  mesoderm  of  an  intermediate-streak  embryo.  X  960. 

FIGURE  4.  Stained  streak  mesoderm  cells  from  a  pre-starved  intermediate-streak  blastoderm. 
X960. 

FIGURE  5.  Carbon-marked  cells  in  pharyngeal  endoderm  of  a  stage  2  explant  after  twenty- 
four  hours  of  subcultivation.  The  brain  at  this  level  has  not  rolled  completely  into  a  tube. 
X  120. 

FIGURE  6.  Carbon  engulfed  by  mesoderm  and  pharynx  of  a  DPS  explant  after  one  day  of 
subcultivation.  X120. 


RESPIRATION  IN  THE  EARLY  CHICK 


87 


8 


10 


(g 


FIGURE  7.  Remnant  of  an  epiblast  implant  grafted  into  flank  mesoderm  of  a  DPS  host 
followed  by  twenty-four  hours  of  incubation.  X 120. 

FIGURE  8.  Photomicrograph  of  an  isolated  ball  of  degenerative  epiblast  cells  in  flank 
mesoderm  of  a  stage  2  blastoderm  host  after  one  day  of  subcultivation.  <  120. 

FIGURE  9.  Isolated  mass  of  viable  cells  in  mesoderm  initially  implanted  in  the  flank  region 
of  a  DPS  host.  Photograph  taken  twenty-four  hours  postoperatively.  Donor  cells  were  from 
newly  involuted  mesoderm  of  a  stage  2  blastoderm.  X  120. 

FIGURE  10.  Photomicrograph  of  carbon-marked  flank  mesoderm  cells  in  a  stage  2  host 
following  one  day  of  subcultivation.  Donor  tissue  was  newly  involuted  mesoderm  from  a  stage 
2  embryo.  X  120. 


RONALD  C.  FRASER 

is  no  reason  to  believe  that  it  may  not  be  present  in  even  earlier  chick  blastoderms. 
It  has  been  detected  by  Krahl  and  co-workers  (1941)  in  pre-  and  post-fertilized 
Arbacia  eggs.  Regardless  of  the  type  of  food  being  utilized,  as  long  as  aerobic  oxi- 
dation occurs  it  seems  likely  that  this  terminal  enzyme  will  be  involved.  It  is  com- 
mon knowledge  that  the  chick  embryo  uses  oxygen  continuously  following  laying 
of  the  egg.  The  results  of  the  present  investigation  have  shown  that  cytochrome 
oxidase  is  indeed  present  in  very  early  embryos,  detectable  by  the  nadi  reagent 
under  the  conditions  utilized  even  in  streak-forming  stages.  Before  this  time  it  is 
probable  that  this  enzyme  is  present  but  is  not  specifically  outstandingly  active  at 
any  localized  region. 

The  specific  significance  of  the  observed  rise  in  activity  of  cytochrome  oxidase  in 
involuting  cells  can  only  be  conjectured.  Certainly  it  is  associated  in  some  manner 
with  the  differentiative  process,  since  it  is  accompanied  by  an  expansion  of  possible 
fates  in  cells  in  which  it  occurs.  It  may  well  be  that  the  increased  energy  liberated 
in  these  cells  at  this  time  may  be  directed  toward  anabolic  processes.  That  dif- 
ferentiation is  accompanied  by  aerobic  oxidation  has  very  recently  been  pointed 
out  by  Warburg  (1956).  The  correlation  between  oxidative  activity  and  histo- 
genesis  appears  of  prime  significance. 

Previously  (Waddington,  1932;  Eraser,  l()54b)  it  has  been  shown  that  an  in- 
terchange of  cells  between  head  endoderm  ( pharynx )  and  head  mesenchyme  is 
much  in  evidence  in  early  chick  embryos.  It  may  well  be  that  tissues  in  this  region 
are  more  influenced  in  their  differentiation  by  cells  about  them  than  they  are  at 
lower  (trunk)  levels.  No  evidence  has  been  found  in  the  present  investigation  to 
support  the  observations  of  Waddington  and  Taylor  (1937)  that  epiblast  tissue  im- 
planted at  lower  regions  of  the  chick  blastoderms  would  form  mesodermal  structures. 

Mention  should  be  made  of  the  results  obtained  in  experiments  dealing  with 
nadi  oxidation  in  blastoderms  pretreated  with  various  materials.  If  we  assume 
that  there  is  competition  between  the  nadi  reagent  and  cytochrome  c  for  the  enzyme 
cytochrome  oxidase,  then  certain  interpretations  of  the  results  can  fruitfully  be  made. 
This  assumption  is  in  sharp  contrast  to  the  ideas  of  Keilen  and  Hartree  (1938),  but 
see  below.  It  is  well  known  that  both  of  these  materials  are  oxidized  by  this  en- 
zyme in  the  presence  of  molecular  oxygen  to  indophenol  and  oxidized  cytochrome 
r,  respectively.  If  we  accept  the  assumption  of  a  competition  of  substrates,  the 
increase  in  nadi  oxidation  in  starved  embryos  could  mean  that  normal  oxidation 
through  cytochrome  c  is  curtailed,  presumably  due  to  exhaustion  of  utilizable  car- 
bohydrate reserves  (free  hexoses).  Dimethyl-p-phylenediamine-alpha  naphthol 
would  therefore  be  oxidized  more  readily,  leading  to  the  more  pronounced  coloration 
observed. 

Indophenol  intensity  was  the  same  in  embryos  pretreated  with  albumen-cvto- 
chrome  c  as  in  controls.  At  the  same  time,  embryos  incubated  in  albumen-cyto- 
chrome  c-hydroquinone  and  albumen-hydroquinone  were  perceptibly  less  colored. 
If  cytochrome  r  could  enter  the  cells,  we  should  expect  a  decrease  in  nadi  oxidation. 
The  fact  that  hydroquinone  in  the  absence  of  cytochrome  c  produced  the  same  re- 
sult as  with  it  suggests  that  the  cytochrome  is  not  gaining  entrance  to  the  cells. 
Krahl  ct  a!.  (  1941 )  and  Keilen  and  Hartree  (1938)  using  preparations  of  Arbacia 
eggs  and  mammalian  heart  muscle,  respectively,  have  shown  that  there  is  an  in- 
crease in  cytochrome  oxidase  activity  proportional  to  the  amount  of  cytochrome 
c  added.  The  size  of  the  cytochrome  (molecular  weight  of  13,000  according  to 


RESPIRATION  IN  THE  EARLY  CHICK  89 

Potter,  1950)  should  not  be  a  serious  detriment  to  cell  entry,  since  larger  com- 
pounds are  suspected  of  entering  cells.  Nevertheless,  the  results  would  indicate 
that  it  did  not  enter  the  cells.  It  is  obvious,  however,  that  the  hydroquinone  had. 
In  view  of  the  fact  that  this  material  will  not  reduce  indophenol,  but  has  been  shown 
to  reduce  cytochrome  c  (Krahl  ct  <//.,  1941)  one  is  led  to  the  conclusion  that  the 
hydroquinone  selectively  and  persistently  reduces  the  normally  present  intracellular 
cytochrome  c,  and  hence  the  affinity  for  the  oxidase  with  the  nadi  reagent  is 
reduced. 

There  is.  however,  an  alternative  explanation  which  is  more  in  keeping  with 
the  conclusions  of  Keilen  and  Hartree  (1938).  These  workers  found  that  the  cata- 
lytic action  of  cytochrome  oxidase  on  p-phenylenediamine  was  greatly  enhanced  by 
the  addition  of  cytochrome  c,  and  that  this  aromatic  amine  was  oxidized  much  more 
readily  by  the  enzyme  than  were  other  compounds,  including  hydroquinone.  They 
state  further  that  the  rate  of  catalytic  hydroquinone  oxidation  may  be  increased  30- 
to  40-fold  on  the  addition  of  cytochrome  c  (10~r>  to  10"1  M)  to  the  preparation. 
Thus,  rather  than  there  being  a  competition  between  cytochrome  c  and  hydroquinone 
for  the  enzyme,  in  heart  muscle  preparations  at  least,  there  is  a  dependency  on  the 
presence  of  the  cytochrome  for  the  catalytic  oxidation  of  hydroquinone.  Assuming 
this  and  again  considering  only  the  hydroquinone  as  entering  the  cell,  it  must  be 
that  intracellularly  the  hydroquinone  has  more  affinity  for  the  enzyme  than  has  the 
nadi  reagent.  This  is,  of  course,  somewhat  at  variance  with  the  English  workers' 
observations.  It  may  be  that  the  difference  in  results  lies  in  the  materials  and  meth- 
ods used.  Intracellularly,  structure  may  provide  results  differing  from  those  ob- 
tained in  I'itro.  At  any  rate,  this  interpretation  is  also  in  keeping  with  observations 
made. 

Finally,  consideration  should  be  given  to  the  perplexing  problem  of  starvation 
of  the  chick  embryos  whose  cells  are  amply  supplied  with  high  energy  food  material. 
Reference  has  already  been  made  to  the  fact  that  chick  blastoderms  soon  die  when 
explanted  on  non-nutrient  media.  Spratt  (1951b)  has  shown  that  recovery  is  pos- 
sible in  embryos  starved  for  six  hours  on  a  saline-agar  medium,  when  they  are 
returned  to  an  albumen  substrate.  I  have  found  (Fraser,  1954a)  that  certain  de- 
generative features  are  obvious  in  explants  starved  for  ten  hours.  In  checking  re- 
cently, I  have  found  that  in  embryos  starved  for  ten  hours,  there  are  still  many  in- 
tracellular Sudan  III-stainable  globules.  It  thus  becomes  evident  that  lipids  are 
not  utilized,  at  least  to  any  appreciable  degree,  by  early  chick  blastoderms.  This 
conclusion  had  been  drawn  previously  by  Xeedham  (1931).  This  author,  using 
R.O.  determinations  as  a  basis,  stated  that  during  chick  embryogeny  carbohydrates 
are  utilized  for  the  first  seven  days  of  incubation  (R.Q.  =:  1 ).  proteins  are  used  next 
and  finally  lipids  are  used  only  near  the  time  of  hatching. 

SUMMARY 

1 .  Cytochrome  oxidase  has  been  detected  in  chick  blastoderms  as  early  as  the  in- 
termediate-streak stage,  by  use  of  the  explanting  procedure  on  an  agar  medium  con- 
taining dimethyl-p-phenylenediamine-alpha  naphthol  (nadi  reagent).  Intracellular 
indophenol  deposition  was  localized  on  the  surface  of  lipid  droplets,  particularly  in 
newly  involuted  mesodermal  cells.  Enzymatic  activity  was  negligible  in  embryos 
explanted  on  a  similar  medium  containing  sodium  azide. 


90  RONALD  C.  ERASER 

2.  Nadi  oxidation  was  augmented,  notably  in  streak  mesoderm  of  early  explants 
after  such  blastoderms  had  been  starved  in  saline  for  a  period  of  five  hours.     Em- 
bryos pretreated  in  albumen-saline,  or  albumen-saline-cytochrome  c  for  a  similar 
interval  showed  no  increase  or  decrease  in  intracellular  enzymatic  activity  as  com- 
pared to  controls,  when  they  were  subsequently  explanted  onto  the  nadi-bearing 
medium.     However,   diamine   oxidation   in  blastoderms   treated   in   solutions  con- 
taining albumen-cytochrome  r-hydroquinone  and  albumen-hydroquinone  was  per- 
ceptibly decreased. 

3.  The  development  of  the  ability  to  oxidize  the  nadi  reagent  was  not  autonomous 
in  fragments  of  streak  epiblast   (prospective  mesoderm),  but  required  normal  in- 
volution at  gastrulation.     This  was  shown  by  pieces  of  this  tissue  implanted  into 
trunk-level  mesoderm.     When  implanted  in  a  future  head  mesoderm  location,  how- 
ever, such  fragments  did  reveal  an  increase  in  enzymatic  activity.     When  incubated 
in  albumen-saline  for  intervals  of  time  up  to  ten  hours,  small  pieces  of  epiblast  did 
not  show  an  increase  in  nadi  oxidation. 

4.  These  results  were  correlated  with  the  ability  of  the  tissue  fragments  to  form 
mesodermal  and  endodermal  structures.     Implants  of  epiblast  placed  in  prospective 
head  mesoderm  of  streak-forming  and  definitive  primitive  streak  hosts  were  in- 
corporated  into   head   mesenchyme   and   pharyngeal   tissue.     Similar   tissue    when 
placed  with  other  mesoderm  at  trunk  levels  failed  to  differentiate  into  mesenchyme. 
Newly  involuted  mesoderm  from  streak-forming  blastoderms  had  the  same  fate  as 
did  epiblast  fragments,  when  implanted  in  a  future  head  mesoderm  location.     At 
the  trunk  level  this  tissue  became  integrated  into  mesoderm  cells  about  it  or  formed 
semi-isolated  balls  of  living  tissue. 

5.  The  significance  of  the  observations,  with  respect  to  nutritional  requirements 
of  early  chick  blastoderms  and  the  relationship  between  oxygen  utilization  and  dif- 
ferentiation, is  discussed  briefly. 

LITERATURE  CITED 

ALBAUM,  H.  G.,  AND  L.  G.  WORLEY,  1942.  The  development  of  cytochrome  oxidase  in  the 
chick  embryo.  /.  Biol.  Chan..  144:  697-700. 

ERASER,  R.  C.,  1954a.  The  utilization  of  some  carbohydrates  by  in  vitro  cultured  chick  blasto- 
derms in  wound  healing.  Biol.  Bit!!.,  106:  39—47. 

ERASER,  R.  C.,  1954b.  Studies  on  the  hypoblast  of  the  young  chick  embryo.  /.  R.vp.  ZooL.  126: 
349-400. 

HAMBURGER,  V.,  AND  H.  L.  HAMILTON,  1951.  A  series  of  normal  stages  in  the  development 
of  the  chick  embryo.  /.  MorphoL,  88:  49-92. 

HYMAN,  L.  H.,  1927.  The  metabolic  gradients  of  vertebrate  embryos.  III.  The  chick. 
Biol  Bull.  52:  1-39. 

JACOBSON,  W.,  1938.  The  early  development  of  the  avian  embryo.  II.  Mesoderm  formation 
and  the  distribution  of  presumptive  embryonic  material.  /.  MorphoL,  62:  445-501. 

KEILEN,  D.,  1936.  The  action  of  sodium  azide  on  cellular  respiration  and  on  some  catalytic  oxi- 
dation reactions.  Proc.  Roy.  Soc.  London,  Scr.  B ,  121  :  165-173. 

KEILEN,  D.,  AND  E.  F.  HARTREE,  1938.  Cytochrome  oxidase.  Proc.  Roy.  Soc.  London,  Scr.  B, 
125:  171-186. 

KRAHL,  M.  E.,  A.  K.  KELTCH,  C.  E.  NEUBECK  AND  G.  H.  A.  CLOWES,  1941.  Studies  on  cell 
metabolism  and  cell  division.  V.  Cytochrome  oxidase  activity  in  the  eggs  of  Arbacia 
punct»htta.  J.  Gen.  Physio/..  24:  597-617. 

MEYERHOFF,  O.,  1945.  The  origin  of  the  reaction  of  Harden-Young  in  cell-free  alcoholic  fermen- 
tations. /.  Biol  Chan..  157:  105-119. 

MOOG,  F.,  1943.  Cytochrome  oxidase  in  early  chick  embryos.  /.  Cell.  Coinp.  Plivsiol.,  22:  223- 
231. 


RESPIRATION  IN  THE  EARLY  CHICK  91 

NEEDHAM,  J.,   1931.     Chemical  embryology.     Cambridge  Univ.   Press,   London. 

NEEDHAM,  J.,  AND  W.  NOWINSKI,  1937.  Intermediary  carbohydrate  metabolism  in  embryonic 
life.  I.  General  aspects  of  anaerobic  glucolysis.  Biochem.  J.,  31:  1165-1184. 

NOVIKOFF,  A.,  V.  POTTER  AND  G.  LE  PAGE,  1948.  Phospborylating  glycolysis  in  the  early 
chick  embryo.  /.  Biol.  Chcin.,  173:  239-252. 

POTTER,  V.  R.,  1950.  Respiratory  enzymes.  Edit,  by  H.  A.  Lardy.  Chapt.  7,  pp.  151-157. 
Burgess  Pub.  Co.,  Minneapolis. 

POTTER,  V.  R.,  AND  K.  P.  Du  Bois,  1942.  The  quantitative  determination  of  cytochrome 
c.  J.  Biol.  Chan..  142:  417-426. 

ROMANOFF,  A.,  AND  A.  ROMANOFF,  1949.     The  avian  egg.     J.  Wiley,  New  York. 

SPRATT,  N.  T.,  JR.,  1947.  A  simple  method  for  explanting  and  cultivating  early  chick  in  vitro. 
Science,  106 :  452. 

SPRATT,  N.  T.,  JR.,  1949.  Nutritional  requirements  of  the  early  chick  embryo.  I.  The  utiliza- 
tion of  carbohydrate  substrates.  /.  Ex  p.  Zoo/.,  110  :  273-298. 

SPRATT,  N.  T.,  JR.,  1950a.  Nutritional  requirements  of  the  early  chick  embryo.  II.  Differential 
nutrient  requirements  for  morphogenesis  and  differentiation  of  the  heart  and  brain. 
/.  Exp.  Zoo!..  114:  375-402. 

SPRATT,  N.  T.,  JR.,  1950b.  Nutritional  requirements  of  the  early  chick  embryo.  III.  The  meta- 
bolic basis  of  morphogenesis  and  differentiation  as  revealed  by  the  use  of  inhibitors. 
Biol.  Bull.,  99  :  120-135. 

SPRATT,  N.  T.,  JR.,  195  la.  Demonstration  of  spatial  and  temporal  patterns  of  reducing  enzyme 
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methylene  blue.  Anat.  Rcc..  109:  384. 

SPRATT,  N.  T.,  JR.,  1951b.  Effects  of  starvation  on  development  and  on  reducing  enzyme  activi- 
ties of  early  chick  blastoderms.  Anat.  Rcc.,  Ill  :  553. 

TAYLOR,  K.  M.,  AND  A.  M.  SCHECHTMAX.  1949.  In  vitro  development  of  the  early  chick  em- 
bryo in  the  absence  of  small  organic  molecules.  /.  /I.v/>.  Zoo/.,  Ill  :  227-253. 

UMBREIT,  W.  \Y.,  R.  H.  BURRIS  AND  J.  F.  STAUFFER,  1949.  Manometric  techniques  and  tissue 
metabolism.  Burgess  Pub.  Co.,  Minneapolis. 

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WADDINGTON,  C.  H.,  AXD  J.  TAYLOR,  1937.  Conversion  of  presumptive  ectoderm  to  mesoderm 
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WARBURG,  O.,  1956.     On  the  origin  of  cancer  cells.     Science,  123:  309-314. 


THE  LOCATION  OF  CONTACT  CHEMORECEPTORS  SENSITIVE 
TO  SUCROSE  SOLUTIONS  IN  ADULT  TRICHOPTERA  1 

HUBERT  FRINGS  AND  MABLE  FRINGS 

Department  of  Zoology  and  Entomology,  The  Pennsylvania  State  University,  University  Park, 
Pennsylvania,  and  Alt.  Desert  Island  Biological  Laboratory,  Salisbury  Cove,  Maine 

Descriptions  of  the  mouth-parts  and  feeding  of  adult  Trichoptera  in  recent 
American  text  books  and  other  general  works  on  entomology  are  not  consistent. 
Folsom  and  Wardle  (1934,  pp.  20,  39),  Frost  (1942.  p.  89),  Comstock,  (1950,  p. 
555),  Metcalf,  Flint  and  Metcalf  ( 1951,  p.  229)  and  Ross  ( 1948.  p.  367)  described 
the  mouth-parts  as  "vestigial,"  "rudimentary,"  "suhatrophied,"  or  "greatly  re- 
duced." These  views  carried  as  a  corollary  the  belief  that  the  adults  take  little  or 
no  nourishment,  and  Brues  (1946,  p.  44)  listed  caddis-flies  with  the  "aphagia" :  in- 
sects "which  do  not  feed  at  all  after  maturity."  Borror  and  DeLong  (1955,  p.  437), 
however,  described  the  mouth-parts  as  "chewing  type,  with  the  palpi  well  developed 
but  with  the  mandibles  much  reduced,"  and  stated  that  "the  adults  feed  principally 
on  liquid  foods."  Swain  (1948,  p.  79)  also  stated  that  adult  caddis-flies  take  liquid 
food,  but  termed  the  mouth-parts  a  "short,  uncoiled  proboscis." 

A  review  of  earlier  accounts  reveals  a  similar  lack  of  agreement.  Reaumur  ( 1737, 
pp.  175-176)  wrote  that  the  mouth-parts  of  Trichoptera  are  for  sucking  and  lapping, 
like  those  of  Diptera.  Kirby  and  Spence  (1826,  p.  464,  PI.  VII,  Fig.  1),  on  the 
other  hand,  regarded  the  mouth-parts  as  modified  mandibulate.  Burmeister  (  1832, 
pp.  68 ;  377-378)  stated  that  the  mouth-parts  are  intermediate  between  the  mandibu- 
late and  haustellate  types,  comparing  them  with  those  of  bees.  Lucas  ( 1893)  made 
a  detailed  study  of  the  mouth-parts  of  Anabolia  }urcata(  --  laci'is).  He  found  the 
mandibles  to  be  atrophied,  the  labrum  and  maxillae  reduced,  and  the  labium  devel- 
oped into  a  sucking  organ,  the  haustellum.  Ulmer  (1904)  and  Cummings  (1913, 
1914)  reported  a  well  developed  haustellum  to  be  present  in  every  family  of  Tri- 
choptera. These  facts  are  reported  in  the  special  works  on  Trichoptera  by  Betten 
(1934,  pp.  19-22).  Ross  (  1<>44.  p.  4)  and  Mosely  and  Kimmins  (1953,  pp.  10-11), 
and  in  the  text  books  of  Packard  ( 189S.  pp.  74-75  ).  Weber  ( 1933,  pp.  66-68 ;  1954. 
pp.  297-298  ),  and  Imms  (1948,  pp.  19,  411-412). 

Reaumur  (1737,  pp.  175-176)  stated  that  adult  Trichoptera  take  liquid  foods, 
and  Burmeister  (1832,  pp.  377-378)  reaffirmed  this,  reporting  that  he  found  them 
feeding  on  nectar  of  flowers.  Lucas  (1893)  reported  finding  tiny  particles  like 
pollen  in  the  folds  of  the  haustellum.  and  he  therefore  believed  that  they  feed  on  nec- 
tar. There  were  other  workers  who  made  observations,  often  quite  casual,  that 
confirmed  or  contradicted  these  ideas.  These  are  reviewed  in  the  papers  of  Siltala 
(1907)  and  Dohler  (1914).  Siltala  (1907)  gave  adult  Pliryc/anea  striata  and 
Limnephilus  rJwuibicus  only  water  for  three  or  four  days,  then  placed  a  flowering 

1  Paper  No.  2048  in  the  Journal  Series  of  the  Pennsylvania  Agricultural  Experiment  Sta- 
tion;  supported  in  part  by  Research  Grant  No.  E-802  from  the  National  Microbiological  In- 
stitute of  the  National  Institutes  of  Health,  Public  Health  Service. 

92 


CHEMORECEPTORS  IN  TRICHOPTERA  93 

branch  of  Spirca  near  them.  They  flew  to  the  flowers  and  fed  on  the  nectar,  thus 
confirming  the  reports  of  feeding  by  adults. 

Dohler  (1914)  made  many  observations  that  leave  little  doubt  that  adult  Tri- 
choptera  feed.  He  fed  them  on  sucrose  solution  to  which  litmus  was  added  and 
followed  the  changes  in  acidity  in  the  gut  as  an  indication  of  digestion.  He  fed  fer- 
ric lactate  in  water  and  demonstrated  the  absorption  of  iron  by  the  gut-wall.  When 
he  gave  only  water  to  23  individuals  of  Limnephilus  flarifornis,  they  survived  for 
19-40  days.  When  he  gave  sugar-water  to  19  others,  they  lived  45-105  days.  He 
observed  feeding  closely  by  seizing  the  wings  of  the  insects  and  holding  them  as  he 
brought  drops  of  sugar-water  to  their  mouth-parts.  He  found,  in  L.  flavicornis, 
such  greedy  acceptance  of  the  food  that  some  individuals  ruptured  the  intestine 
through  over-feeding.  All  these  laboratory  observations  led  Dohler  to  conclude  that 
adult  Trichoptera  feed  in  nature,  and  he  supported  this  by  reference  to  Siltala's  ob- 
servations and  those  of  earlier  workers  who  reported  finding  caddis-flies  on  flowers 
or  at  sweet  baits  used  to  lure  moths. 

Lucas,  Siltala  and  Dohler,  thus,  seem  to  have  shown  convincingly  that  the  mouth- 
parts  of  some  larger  adult  Trichoptera  are  functional.  The  labrum  is  reduced,  the 
mandibles  rudimentary,  the  maxillae  modified,  the  maxillary  and  labial  palpi  well 
developed,  and  the  hypopharynx  or  labium  or  both  developed  into  an  extensible 
haustellum.  These  species  of  Trichoptera,  at  least,  probably  feed  in  nature  on  nec- 
tar and  other  sweet  substances. 

The  present  paper  reports  experiments  on  four  species  of  Trichoptera  from  two 
families,  and  observations  on  two  other  species  from  two  more  families.  The  pur- 
pose of  the  experiments  was  to  discover  the  location  of  the  contact  chemoreceptors 
mediating  feeding  responses  when  stimulated  with  sucrose  solution.  The  results 
further  support  the  belief  that  the  mouth-parts  of  adult  Trichoptera  are  functional 
and  that  the  insects  feed  in  the  adult  stage. 

MATERIALS  AND  METHODS 

The  following  species  of  Trichoptera  were  studied  experimentally.  Identifica- 
tions were  made  by  the  authors  with  the  aid  of  works  of  Betten  (1934),  Milne 
(1934-36)  and  Ross  (1944).  Dr.  H.  H.  Ross  kindly  checked  the  identifications, 
and  we  wish  to  thank  him  for  this. 

Family  :  Phryganeidae 

Banksiola  sinithi — 2  males,  4  females 

Ptilostoinis  ocellifcra — 1  male,  6  females 

Phryganea  sayi — 4  males,  8  females 
Family :  Limnephilidae 

Platycentropus  radiatus — 8  males,  9  females 

All  the  experimental  subjects  were  captured  when  they  came  to  lights  at  night. 
They  were  lightly  anaesthetized  with  ether  and  mounted  alive  by  fastening  the  dorsal 
side  of  the  thorax  and  the  wings  to  a  wax  block  on  the  end  of  a  glass  rod  (Fig.  1). 
These  are  relatively  large  (B.  sniilhi  about  15  mm.  long;  the  others  20-25  mm. 
long),  and  when  they  were  thus  mounted  could  easily  be  observed.  Longevity  was 


94  HUBERT  FRINGS  AND  MABLE  FRINGS 

good  if  the  animals  were  fed  and  watered  daily ;  even  with  legs  and  other  parts  re- 
moved they  lived  for  up  to  26  days. 

The  contact  chemoreceptors  were  located  by  the  methods  described  in  detail  in 
Frings  and  Frings  (1949).  The  animals  were  tested  daily  after  night  fall,  for 
they  responded  more  actively  at  night,  even  with  the  necessary  artificial  illumination, 
than  in  day  light,  as  Dohler  (1914)  also  noted.  Before  each  daily  series  of  tests, 
the  animals  were  given  water  to  satiety.  It  was  essential  that  water-satiety  be  main- 
tained in  the  subjects,  because  the  tests  involved  discrimination  between  water  and 
water  with  sucrose  added.  When  the  animals  had  taken  all  the  water  they  would 
take,  water  was  brought  to  the  locus  being  tested  on  an  artist's  brush  or  glass  mi- 


FIGURE  1.     A  caddis-fly    (Platycaitrapiis  nnliatns)    mounted  alive  on  a  paraffin  block  on  the 

end  of  a  glass  rod  (3  X). 

croneedle  and  the  reaction  noted  under  a  binocular  dissecting  microscope.  This  was 
followed  by  a  similar  trial  with  1  AI  sucrose  solution,  and  the  reaction  again  noted. 
These  presentations  were  repeated  a  sufficient  number  of  times  to  be  sure  that  the 
insect  responded  similarly  or  differently  to  the  two  stimuli.  Such  a  series  of  trials 
constituted  one  test.  There  were  variable  numbers  of  tests  carried  out  at  each 
testing  period.  At  the  end  of  each  daily  testing  period,  the  insects  were  fed  to 
satiety  on  the  sucrose  solution.  They  imbibed  heavily,  but  did  not  damage  them- 
selves, as  Dohler  reported  for  the  animals  he  tested. 

When  preliminary  experiments  had  revealed  possible  loci  of  contact  chemore- 
ceptors, the  structures  were  removed  and  the  animals  retested  similarly.  Opera- 
tions were  performed  under  light  anaesthesia  with  paired  controls  anaesthetized 


CHEMORECEPTORS  IN  TRICHOPTERA  95 

and  sham  operated.     For  microscopic  examination  of  possible  end-organs  on  the 
experimentally  determined  loci,  the  structures  were  removed  in  70%  ethyl  alcohol, 
transferred  to  95%  and  thence  to  Diaphane  on  micro  slides. 
The  following  species  were  observed  unmounted  : 

Family :  Leptoceridae 

Oecetis  cincrascens 
Family :  Hydroptilidae 

Orthotrichia  aincricana 

Attempts  to  mount  and  test  about  30  individuals  of  the  first-named  were  made,  but 
these  did  not  survive  more  than  one  day.  Both  of  these  species  were  very  common 
and,  as  described  by  Dohler,  highly  attracted  to  sugar-water.  They  came  onto  the 
laboratory  table  and  could  be  given  water  and  sucrose  solutions  while  they  scurried 
about.  By  carefully  controlling  the  placement  of  droplets  near  them,  it  was  pos- 
sible to  test  them  and  to  observe  feeding.  O.  cincrascens  is  large  enough  (about 
12  mm.  long)  to  be  observed  with  the  naked  eye.  0.  aincricana,  like  all  Hydroptili- 
dae, is  quite  small  (about  3  mm.  long).  It  was  necessary,  therefore,  to  observe  it 
with  a  dissecting  microscope.  Luckily  the  insects  came  right  onto  the  brushes  and 
needles  used  in  testing  the  larger  forms,  and  they  were  thus  easily  observed. 

RESULTS 

With  B.  smithi  only  gross  localization  tests  were  made,  using  brushes  with  water 
and  1  M  sucrose  solution  applied  to  various  organs  of  the  intact,  mounted  animals. 
The  palpal  tips  proved  to  be  quite  sensitive :  touching  them  with  sugar-water 
brought  about  extension  of  the  haustellum.  The  tarsi,  likewise,  had  contact  chemo- 
receptors :  touching  them  with  a  brush  bearing  sugar-wrater  induced  eager  reaching 
with  the  palpi  toward  the  brush.  With  \vater  alone  on  a  brush,  in  each  case,  with- 
drawal or  neutral  reactions  were  elicited.  Touching  the  antennae  with  water  or 
sugar-water  resulted  in  withdrawal  of  the  antennae,  indicating  that  these  lack  con- 
tact chemoreceptors  sensitive  to  sucrose. 

With  Pt.  occllifcra,  P.  sayi  and  P.  radiatns  more  detailed  experiments  were  car- 
ried out :  with  Pt.  ocellijera  about  100  tests  were  made  over  a  period  of  10—26  days ; 
with  P.  sayi,  about  200  tests  over  16  days;  with  P.  radiatns  about  300  sets  of  tests 
over  16  days.  These  three  species  reacted  almost  exactly  alike,  and  the  results  are 
thus  given  together.  Figure  2  is  a  photograph  of  the  head  and  mouth-parts  of 
P.  radiatns  to  show  the  well  developed  haustellum  and  palpi. 

The  first  series  of  experiments  was  designed  to  give  the  general  locations  of  the 
contact  chemoreceptors.  Using  brushes  in  paired  trials  with  water  and  1  M  su- 
crose solution,  no  evidence  of  discrimination  was  found  when  the  antennae  were 
tested.  If  the  insects  were  "thirsty,"  however,  the  antennae  were  quite  sensitive 
to  water  vapor.  If  a  brush  bearing  water  was  brought  near  to  but  not  in  contact 
with  the  antennae,  the  insects  almost  immediately  began  to  reach  excitedly  with  the 
palpi.  Once  sated  with  water,  however,  this  ceased,  and  the  only  reaction  to  con- 


96  HUBERT  FRINGS  AND  MABLE  FRINGS 

tact  with  a  brush  moistened  with  water  or  sugar-water  was  withdrawal  of  the  an- 
tennae. The  conclusion  that  the  antennae  lack  contact  chemoreceptors,  however, 
must  be  stated  cautiously,  for  recent  experiments  wTith  some  Lepidoptera  (Frings 
and  Frings,  1956)  show  that  reactions  mediated  by  the  antennae  may  depend  upon 
presence  or  absence  of  contact  chemoreceptors  on  other  parts  of  the  body. 

Contact  of  the  tarsi  of  water-sated  individuals  with  sugar-water  elicited  reach- 
ing with  the  maxillary  and  labial  palpi  and  partial  extension  of  the  haustellum. 
Ordinarily  the  palpi  were  folded  against  the  head,  and  this  reaction  was  quite  clear- 
cut.  Touching  only  the  ventral  surface  of  the  tarsus  of  one  fore  leg  with  the  brush 
mediated  the  same  response.  It  was  impossible,  however,  to  touch  the  other  tarsi  in 


FIGURE  2.     Head  and  mouth-parts  of  an  adult  caddis-fly   (P.  radiatus)   showing  the  well  devel- 
oped maxillary  and  labial  palpi  and  the  medial  haustellum  (25  X). 

an  intact  animal  without  having  the  fore  tarsi  also  brought  to  the  brush.  The  palpi 
likewise  proved  to  be  receptive :  touching  them  with  sugar-water  elicited  spreading 
of  the  haustellum.  If  1  M  NaCl  solution  was  used  instead  of  sucrose  on  the  tarsi, 
there  was  no  reaching  with  the  palpi,  and  if  it  was  used  on  the  palpi,  they  were  with- 
drawn sharply. 

Further  experiments  on  intact  animals  were  made  with  glass  micro-needles  bear- 
ing water  and  sugar-water.  The  results  with  the  antennae  and  tarsi  were  the  same 
as  when  brushes  were  used.  Touching  only  the  tips  of  the  maxillary  palpi  with 
sugar-water  elicited  reaching  with  both  sets  of  palpi  and  partial  spreading  of  the 
haustellum,  much  like  the  reaction  obtained  by  touching  the  tarsi.  Bringing  sucrose 
solution  to  any  part  of  a  maxillary  palpus  other  than  the  tip  of  the  terminal  segment 
brought  about  withdrawal,  just  as  with  wrater.  Touching  the  maxillary  palpal  tips 


CHEMORECEPTORS  IN  TRICHOPTERA  97 

with  NaCl  solution  elicited  a  sharp  retraction  of  the  palpi.  Touching  the  tips  of  the 
lahial  palpi  with  sugar-water  on  a  needle  elicited  spreading  of  the  haustellum  in 
preparation  for  feeding.  Other  parts  of  the  lahial  palpi  seemed  not  to  be  sensitive, 
as  with  the  maxillary  palpi.  With  XaCl  solution,  these  palpi  were  also  drawn  away. 
The  feeding  reaction,  therefore,  seems  to  occur  in  two  stages :  1 )  exploration  with 
the  palpi  when  an  acceptable  solution  touches  the  tarsi  or  maxillary  palpal  tips,  and 
2)  extension  and  spreading  of  the  haustellum  when  the  solution  touches  the  tips  of 
the  labial  palpi. 

Following  these  tests,  operations  were  performed  to  enable  us  to  test  parts  that 
could  not  be  touched  without  interference  by  kno\vn  receptors.  The  forelegs  were 
removed  first.  Using  the  brushes,  the  middle  and  hind  tarsi  together  were  found 
to  be  sensitive  to  sucrose.  With  care  the  middle  tarsi  together  or  singly  could  be 
touched,  and  this  elicited  the  usual  response.  With  the  fore  and  hind  legs  removed, 
the  middle  tarsi  were  easily  tested  and  found  to  bear  contact  chemoreceptors  sensi- 
tive to  sucrose.  With  the  fore  and  middle  legs  removed,  the  hind  tarsi  together 
or  singly  were  also  found  to  be  sensitive.  All  the  tarsi,  thus,  bear  the  receptors. 
Using  microneedles,  the  receptors  were  found  on  the  ventral  surfaces  of  the  tarsi 
and  not  on  the  other  parts  of  the  legs,  but  the  exact  segments  of  the  tarsi  bearing 
them  were  not  determined. 

With  the  last  segments  of  the  maxillary  and  labial  palpi  removed,  the  reactions 
to  contact  of  the  palpi  with  sucrose  solutions  were  abolished,  thus  confirming  previ- 
ous observations  that  the  receptors  were  confined  to  these  segments.  To  test  the 
sensitivity  of  the  haustellum,  the  fore  and  middle  legs  and  the  palpi  were  removed, 
and  the  animals  offered  water  and  sugar-water  on  brushes.  A  little  difficulty  was 
encountered  at  first,  because  the  haustellum  became  covered  with  clotted  hemolymph 
from  the  cut  ends  of  the  palpi.  After  this  was  washed  off,  however,  the  animals 
were  able  to  feed.  They  obviously  could  distinguish  sucrose  solution  from  water, 
spreading  the  haustellum  and  drinking  the  former  when  sated  with  water.  If  NaCl 
was  used  instead  of  sucrose,  they  refused  to  drink.  If  they  were  drinking  sugar- 
water  and  NaCl  solution  was  suddenly  substituted,  they  reacted  by  immediate  with- 
drawal of  the  haustellum  and  often  by  violent  retraction  of  the  head.  Thus  the 
receptors  could  distinguish  acceptable  from  unacceptable  materials  in  solution.  The 
receptors  were  not  located  exactly  on  the  haustellum,  but  they  would  seem  to  be 
near  the  distal  margins,  for  application  to  the  tip  of  the  haustellum  of  a  droplet  of 
sugar- water  on  a  needle  brought  about  almost  immediate  extension. 

The  parts  of  the  body  bearing  contact  chemoreceptors  sensitive  at  least  to  sucrose 
and  NaCl,  therefore,  are  the  ventral  sides  of  all  the  tarsi,  the  tips  of  the  terminal  seg- 
ments of  the  maxillary  and  labial  palpi  and  the  haustellum.  Generally  the  reaction 
to  appropriate  stimulation  of  the  tarsal  or  maxillary  palpal  receptors  is  reaching 
with  and  vibration  of  the  palpi  and  partial  extension  of  the  haustellum.  The  reaction 
to  appropriate  stimulation  of  the  labial  palpal  tips  or  the  haustellum  is  extension  and 
spreading  of  the  haustellum  and  feeding.  No  differences  were  noted  between  the 
reactions  of  males  and  females. 

The  tarsi,  palpi  and  haustellum  of  these  three  species  were  mounted  on  slides 
and  examined  in  an  attempt  to  find  the  possible  end-organs  involved.  On  the  ven- 
tral surfaces  of  the  tarsi  there  are  many  short,  thin  walled,  trichoid  sensilla  among 
the  longer  hairs  and  spines.  These  are  quite  similar  to  the  probable  receptors  in 


98  HUBERT  FRINGS  AND  MABLE  FRINGS 

Lepidoptera  and  Diptera  (Eltringham,  1933;  Frings  and  Frings,  1949;  Grabowski 
and  Dethier,  1954;  Hayes  and  Liu,  1947;  Lewis,  1954a,  1954b ;  Tinbergen  1939). 
On  the  palpi  there  are  many  trichoid  sensilla  en  all  the  segments,  but  there  is  no 
way  at  present  to  select  any  as  possible  receptors.  On  the  posterior  face  of  the 
haustellum,  there  are  basiconic  and  trichoid  sensilla  in  small  numbers.  Either  of 
these  might  be  involved,  because  no  other  obvious  sensilla  are  present,  but  the  data 
do  not  warrant  any  definite  selection.  It  is  probable,  therefore,  that  the  receptors 
on  the  tarsi  and  palpi  are  trichoid  sensilla,  while  those  on  the  haustellum  are  either 
trichoid  or  basiconic. 

With  0.  cincrasccns  only  a  few  tests  were  made  on  mounted  animals  before  their 
untimely  deaths.  It  was  obvious  that  they  had  tarsal  and  palpal  receptors  like  the 
others  tested,  but  these  were  not  further  localized.  With  this  species  and  with 
0.  americana  many  observations  were  made  on  unmounted  individuals  that  visited 
the  laboratory  table  where  the  others  were  being  tested  or  came  onto  the  brushes  and 
needles  used  in  the  experiments.  They  scurried  about  in  characteristic,  excited 
manner,  turning  to  and  fro,  antennae  vibrating.  If  a  droplet  of  water  was  in  their 
way,  they  usually  stopped  as  soon  as  they  touched  it  and  drank.  When  sated  with 
water,  they  no  longer  stopped  at  these  droplets.  If  a  droplet  of  sucrose  solution  was 
placed  in  their  way,  however,  they  stopped  as  soon  as  the  tarsi  touched  it  and  turned 
round  and  round  reaching  with  the  palpi.  As  soon  as  the  palpi  touched  the  droplet 
of  sugar-water  the  haustellum  was  extended  and  the  insect  fed. 

DISCUSSION 

These  observations  and  experiments  on  adults  of  six  species  of  Trichoptera  rep- 
resenting four  families  sho\v  that,  in  these  at  least,  the  mouth-parts  are  functional, 
modified  for  sucking,  and  that  the  adults  feed.  These  results  are  fully  concordant 
with  the  reports  of  Lucas  (1893),  Siltala  (1907)  and  Dohler  (1914).  Ulmer 
(1904)  and  Cummings  (1913,  1914)  reported  that  only  a  few  species  from  one  or 
two  families  lack  a  well  developed  haustellum.  While  all  the  observations  on  living 
animals  have  been  made  on  representatives  from  only  four  families  and  mostly  from 
two  families,  the  conclusions  may  have  validity  for  Trichoptera  generally.  At  least 
no  one  has  shown  experimentally  that  any  species  does  not  feed  in  the  adult  state. 

The  presence  of  tarsal  contact  chemoreceptors  also  indicates  affinity  with  the 
haustellate  insects.  Those  studied  to  date  (Hemiptera,  Lepidoptera,  Diptera,  Hy- 
menoptera),  in  contrast  with  the  mandibulate  forms,  have  contact  chemoreceptors 
on  the  tarsi  (Frings  and  Frings,  1949).  The  near  certainty  that  the  tarsal  and 
palpal  end-organs  of  Trichoptera  are  trichoid  sensilla  further  allies  them  with  the 
typical  haustellate  forms.  Haustellate  species,  in  general,  have  trichoid  sensilla  for 
trophic  contact  chemoreception  and  mandibulate  forms  basiconic  sensilla  (Frings 
and  Frings,  1949).  In  the  presence  of  contact  chemoreceptors  on  the  palpi  and  the 
absence  on  the  antennae,  the  Trichoptera  are  more  like  the  Diptera  than  the  Lepidop- 
tera (Frings  and  Frings,  1949,  1956).  The  general  form  of  the  mouth-parts  in 
Trichoptera  also  is  more  like  that  of  Diptera  than  Lepidoptera.  How  much  weight 
can  be  attached  to  evidence  such  as  this  in  determining  relationships  among  larger 
groups  of  insects  we  do  not  know.  Certainly,  however,  further  comparative  studies 
of  contact  chemoreception  in  Trichoptera,  as  well  as  other  haustellate  groups, 
would  be  desirable. 


CHEMORECEPTORS  IN  TRICHOPTERA  99 

SUMMARY 

The  loci  of  contact  chemoreceptors  sensitive  to  sucrose  and  NaCl  in  solution 
and  mediating  feeding  responses  were  determined  experimentally  in  adult  Trichop- 
tera  of  four  species  from  the  families,  Phryganeidae  and  Limnephilidae.  The  re- 
ceptors are  on  the  ventral  surfaces  of  all  the  tarsi,  the  tips  of  the  maxillary  and  labial 
palpi  and  the  haustellum.  The  animals  feed  on  liquids,  and  these  receptors  allow 
them  to  distinguish  acceptable  from  non-acceptable  materials  in  solution.  Less 
precise  observations  on  two  other  species  from  two  other  families  showed  a  similar 
situation  in  these.  The  end-organs  are  probably  trichoid  sensilla.  This  fact,  along 
with  the  presence  of  tarsal  contact  chemoreceptors,  places  adult  Trichoptera  of  these 
species,  at  least,  among  typical  haustellate  insects,  most  nearly  resembling  many 
Diptera  in  locations  of  the  receptors  and  feeding  reactions. 

LITERATURE  CITED 

BETTEN,  C,  1934.     The  caddis  flies  or  Trichoptera  of  New  York  State.     N.  Y.  State  Mits.  Bull., 

No.  292. 
BORROR,  D.  J.,  AND  D.  M.  DELoxo,  1955.     An  introduction  to  the  study  of  insects.     Rinehart  & 

Co.,  New  York. 

BRUES,  C.  T.,   1946.     Insect  dietary.     Harvard  University  Press.     Cambridge. 
BURMEISTER,  H.,  1832.     Handbuch  der  Entomologie.     Erster  Band.     Allgemeines  Entomologie. 

G.  Reimer.     Berlin. 
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THE  FIREFLY  PSEUDOFLASH  IN  RELATION  TO  PHOTOGENIC 

CONTROL  * 

J.  WOODLAND  HASTINGS  AND  JOHN  BUCK 

Department  of  Biological  Sciences,  Nortlnvcstcrn  University.  Evanston,  III.,  and  Laboratory  of 
Physical  Biology,  National  Institutes  of  Health,  Bethesda  14,  Md. 

INTRODUCTION 

The  normal  flashes  of  many  fireflies  are  short  sharp  bursts  of  light  lasting  about 
a  tenth  of  a  second,  with  essentially  total  darkness  between.  The  problem  of  how 
this  light  emission  is  so  precisely  controlled  has  long  appealed  to  investigators  in- 
terested in  biological  trigger  mechanisms.  Two  basic  facts  have  become  firmly  es- 
tablished :  that  nervous  activity  can  initiate  luminescence  and  that  oxygen  is  es- 
sential for  light  production  both  in  the  intact  firefly  and  in  cell-free  extracts  (for 
review  see  Buck,  1948;  Harvey,  1952;  McElroy  and  Hastings,  1955;  Buck,  1955). 

There  are  two  principal  theories  concerning  the  control  of  normal  flashing.  One 
postulates  direct  nervous  stimulation  of  the  photogenic  cell,  and  the  other  proposes 
nervous  control  of  the  oxygen  supply  to  the  cell.  There  has  been  little  empirical 
evidence  bearing  on  the  idea  that  the  nerve  impulse  acts  on  the  photogenic  cell  di- 
rectly. The  theory  of  oxygen  limitation,  on  the  other  hand,  has  been  widely  sup- 
ported on  both  physiological  and  anatomical  grounds.  Actually,  as  pointed  out 
previously  (Buck,  1948),  all  the  experimental  findings  that  have  been  ascribed  to 
direct  oxygen  control  can  be  equally  well  interpreted  as  effects  on  nervous  control. 
The  anatomical  evidence  is  more  persuasive,  though  circumstantial.  It  consists  of 
the  facts  (a)  that  tracheal  end  cells  under  the  light  microscope  appear  to  have  a 
structure  which  can  be  interpreted  as  valvular  (Dahlgren,  1917),  (b)  that  end  cells 
are  present  in  the  photogenic  organs  of  flashing  types  of  fireflies  and  absent  in  types 
that  produce  only  sustained  glows,  and  (c)  that  the  end  cells  are  strategically  situ- 
ated on  the  tracheae  at  the  points  where  the  tracheoles  enter  the  photogenic  tissue. 

The  principal  experimental  support  for  the  idea  that  the  end  cell  functions  as  a 
valve  comes  from  the  work  of  Snell  (1932)  and  Alexander  (1943).  They  found 
that  fireflies  exposed  to  low  oxygen  concentrations  developed  a  dull  "anoxic  glow" 
(=  our  "hypoxic  glow"),  and  when  suddenly  re-exposed  to  air  produced  a  brilliant 
"pseudoflash"  lasting  a  second  or  more.  They  interpreted  these  events  as  follows : 
The  low  oxygen  narcotizes  the  normally  closed  end  cell  valves  and  causes  them  to 
open,  allowing  the  ambient  gas  to  enter  the  previously  anaerobic  photogenic  cyto- 
plasm. Since  the  oxygen  concentration  is  low,  only  a  dull  glow  develops.  When 
air  is  subsequently  admitted  the  higher  oxygen  concentration  permits  a  more  bril- 
liant luminescence,  wrhich,  however,  then  quickly  dies  out  as  the  valves  recover  and 
close  in  the  higher  oxygen  concentration.  From  these  hypoxic  glow-pseudoflash 
responses  it  was  argued  that  normal  flashing  could  also  be  controlled  by  end  cell 
limitation  of  oxygen.  It  should  be  noted,  however,  that  all  the  experiments  in- 

1  This  work  was  supported  in  part  by  a  grant  from  the  National  Science  Foundation. 

101 


102  J.  WOODLAND  HASTINGS  AND  JOHN  BUCK 

volved  markedly  unnatural  conditions  and  hence  are  not  necessarily  relevant  to  nor- 
mal flash  control. 

Recent  experiments  of  McElroy  and  associates  on  cell-free  extracts  of  firefly 
photogenic  tissue  appear  to  bear  directly  on  the  question  of  oxygen  limitation  versus 
nerve  stimulation.  When  all  the  components  required  for  light  emission  (luciferin, 
luciferase,  Mg++,  adenosine  triphosphate  and  oxygen)  are  mixed  together  a  flash  of 
light  occurs,  its  intensity  reaching  a  peak  in  less  than  0.1  second  and  then  declining 
to  a  low  sustained  level  within  the  next  10-20  seconds.  Evidence  too  detailed  to 
present  here  indicates  that  this  in  vitro  flash  involves  several  reactions  (McElroy 
and  Hastings,  1955;  McElroy,  personal  communication).  First,  it  is  believed, 
luciferin,  luciferase  and  ATP  react  to  form  a  luciferin-adenylic  acid-enzyme  "active 
intermediate."  The  intermediate  is  then  oxidized  rapidly  and  irreversibly,  with 
emission  of  light.  This  oxidation  corresponds  to  the  initial  rise  of  luminescence  in 
the  flash.  However,  the  oxyluciferin  formed  during  luminescence  undergoes  a 
slower,  reversible  phosphorylation  by  ATP  to  form  oxyluciferin-adenylic  acid,  which 
strongly  inhibits  the  enzyme  in  the  active  intermediate.  This  inhibition  accounts 
for  the  decline  in  light  intensity  after  the  initial  peak — a  decline  which  takes  place  in 
the  presence  of  excess  oxygen.  The  eventual  low  level  plateau  of  luminescence  thus 
reflects  the  low  concentration  of  uninhibited  enzyme  available  once  a  steady  state 
among  the  various  reactions  is  established. 

The  concentration  of  the  active  intermediate — which  may  be  considered  to  be 
the  substrate  of  the  light-producing  reaction — can  be  changed  in  two  significantly 
different  ways :  ( 1 )  If  oxygen  concentration  is  greatly  decreased  the  rate  of  the 
oxidative  reaction  is  decreased,  resulting  in  decreased  luminescence  and  accumu- 
lation of  active  intermediate.  When  air  is  readmitted  the  accumulated  intermediate 
is  rapidly  oxidized,  resulting  in  a  flash  of  light.  This  shows  that  it  is  possible,  by 
changing  oxygen  concentration,  both  to  limit  luminescence  and  to  cause  a  flash. 
The  ''oxygen  flash"  of  the  extract  has  a  remarkable  quantitative  resemblance  to 
the  pseudoflash  of  the  intact  firefly,  which,  it  will  be  remembered,  is  induced  by  a 
similar  sequence  of  changes  in  oxygen  concentration.  This  suggests  that  both 
types  of  flash  are  due  to  oxidation  of  accumulated  active  intermediate,  and  that 
neither  of  them  needs  be  oxygen-limited  during  its  decay  phase.  (2)  The  concen- 
tration of  the  active  intermediate  may  also  be  increased  by  addition  of  pyrophos- 
phate,  which,  by  opposing  the  formation  of  *he  oxyluciferin-adenylic  acid  inhibitor, 
frees  active  enzyme.  Since  this  reversal  of  enzyme  inhibition  is  rapid,  lasts  only 
until  the  added  pyrophosphate  is  used  up,  and  does  not  involve  any  change  in  oxygen 
concentration,  it  provides  a  possible  model  for  the  mechanism  which  induces  the 
normal  flash. 

The  in  vitro  system  therefore  suggests  that  the  flashing  of  the  firefly  need  not 
normally  be  controlled  by  oxygen  concentration  even  though  light  production  may, 
under  some  artificial  conditions,  become  oxygen-limited.  In  view  of  this  possibility, 
and  of  the  ambiguity  of  previous  oxygen-limitation  experiments  on  intact  fireflies, 
it  is  important  to  re-examine  the  evidence  purporting  to  demonstrate  end-cell  con- 
trol of  luminescence. 

MATERIALS  AND  METHODS 

The  forms  investigated  were  adults  of  the  lampyrid  fireflies  Photnris  sp.  and 
Photinus  pyralis  from  the  Baltimore-Washington  area,  adults  of  the  elaterid  firefly 


CONTROL  OF  FIREFLY  PSEUDOFLASH  103 

Pyrophorus  atlanticiis  from  Florida,  and  larvae  of  Photuris.  In  the  males  of  the 
first  two  species,  as  in  many  lampyrid  fireflies,  the  photogenic  tissue  occupies  the 
ventral  surfaces  of  abdominal  segments  6  and  7.  In  Pyrophorus  we  investigated 
the  small  circular  organs  at  the  posterior  dorsal  corners  of  the  prothorax.  In  the 
Photuris  larva  the  photogenic  organs  are  a  pair  of  small  lateral  plaques  on  the 
ventral  side  of  abdominal  segment  8.  Similar  organs  exist  in  the  larva  and  pupa 
of  Photinus  pyralis,  and  sometimes  persist  into  the  adult  where  they  function  inde- 
pendently of  the  main  organs. 

Different  gas  mixtures  were  prepared  by  passing  various  gases  through  cali- 
brated flow  meters  into  a  mixing  chamber  and  thence  to  the  exposure  chamber, 
which  was  a  5  cm.  length  of  glass  tubing  of  6  mm.  bore.  For  visual  observation 
up  to  three  specimens  were  accommodated  in  the  chamber,  separated  by  wire  screen 
partitions.  A  flow  rate  of  300-400  mL  per  minute  was  used  and  a  reversing  stop- 
cock between  mixing  and  exposure  chambers  permitted  quick  shifting  from  one  gas 
to  another,  the  unused  gas  being  vented.  Light  intensity  wras  measured  with  a 
photomultiplier  tube  apparatus  (Hastings,  McElroy  and  Coulombre,  1953)  and  re- 
corded with  an  oscilloscope  camera  or  a  graphic  meter,  the  firefly  being  held  in 
position  against  the  wall  of  the  exposure  chamber  with  a  loose  cotton  plug. 

RESULTS 

1.  Responses  of  intact  lampyrid  fireflies  to  varied  oxyyen  concentration 

When  adults  of  Photinus  and  Photuris  were  exposed  to  various  low  oxygen 
concentrations  a  dim  hypoxic  glow  usually  developed,  and  when  such  glowing  fire- 
flies were  suddenly  re-exposed  to  air  a  pseudoflash  occurred.  Figure  1  illustrates  a 
response  of  this  sort,  the  hypoxic  glow  being  represented  by  BC  and  the  pseudo- 
flash  by  CDE.  Figure  2  shows  the  pseudoflash  portion  in  more  detail.  An  "oxy- 
gen flash,"  which  occurs  when  air  is  readmitted  to  an  anaerobic  cell-free  extract  of 
photogenic  tissue,  is  reproduced  in  Figure  3  for  comparison.  About  50  records  of 
the  hypoxic  glow  and  pseudoflash  have  been  made  and  analyzed,  supplemented  by 
many  hundred  visual  observations.  These  in  general  confirm  the  findings  of  Snell 
and  of  Alexander,  but  certain  differences  were  noted.  For  example,  if  a  firefly  was 
left  in  the  low  oxygen  mixture  after  the  hypoxic  glow  had  reached  its  plateau  level, 
instead  of  then  exposing  it  to  air,  the  intensity  of  the  glow  usually  decreased  over 
the  course  of  several  minutes  (Fig.  1,  dotted  line,  FG).  It  was  also  observed  that 
some  individuals  failed  to  give  a  pseudoflash,  or  both  hypoxic  glow  and  pseudoflash, 
and  that  there  was  considerable  variation  in  the  length  of  the  period  between  hypoxic 
exposure  and  beginning  of  the  hypoxic  glow  (Fig.  1,  AB),  and  in  the  intensities  of 
both  glow  and  pseudoflash.  Also,  as  in  nature,  normal  individuals  sometimes 
showed  an  initial  constant  dim  glow  in  air  which  made  it  difficult  to  recognize  the 
start  of  the  hypoxic  glow. 

In  spite  of  these  variations  in  response,  a  number  of  quantitative  relations  \vere 
apparent.  First,  in  fireflies  which  had  been  exposed  to  a  particular  low  oxygen 
gas  mixture,  the  peak  intensity  of  the  pseudoflash  in  air  was  approximately  pro- 
portional to  the  intensity  of  the  hypoxic  glow  in  the  low  oxygen  mixture  just  prior 
to  admission  of  air.  Before  the  beginning  of  the  hypoxic  glow  no  pseudoflash  could 
be  elicited ;  during  the  dimmer  periods  of  the  hypoxic  glow,  either  before  or  after 
the  maximum,  pseudoflashes  of  low  intensity  occurred ;  and  during  the  brighter  pe- 


104 


J.  WOODLAND  HASTINGS  AND  JOHN  BUCK 


riods  of  the  hypoxic  glow  more  brilliant  pseudoflashes  occurred.  In  35  experiments 
in  which  the  hypoxic  glow  was  induced  with  0.25%  oxygen,  the  ratio  of  pseudoflash 
intensity  to  hypoxic  glow  intensity  varied  only  from  30  to  120,  and  the  range  for 
repeated  measurements  on  single  individuals  was  even  smaller.  Thus  both  pseudo- 
flash  and  hypoxic  glow  intensities  pass  through  a  maximum  with  time. 

A  second  finding  was  that  the  ratio  of  pseudoflash  intensity  to  hypoxic  glow 
intensity  varied  with  the  oxygen  concentration  used  to  induce  the  hypoxic  glow. 
For  example,  in  one  typical  individual  the  pseudoflash  intensity  in  air  was  1.5  times 


10 


8 


Z 

LJ 

?    4 

h- 

O     2 


D 


FLASH 
600 


01234 
TIME-  MINUTES 

FIGURE  1.  Hypoxic  glow-pseudoflash  response  of  intact  male  of  Photinus  pyralis,  dia- 
grammed from  graphic  meter  record.  Ordinate,  light  intensity  in  arbitrary  units.  At  A 
(zero  time)  Y±%  oxygen  was  introduced.  At  B  the  hypoxic  glow  began,  AB  representing  the 
latent  period.  At  C  air  was  flushed  through  the  chamber  and  a  pseudoflash  of  600  units  rela- 
tive intensity  occurred.  Had  the  firefly  been  left  in  V±%  oxygen  at  C,  the  hypoxic  glow  would 
have  continued  (FG),  slowly  diminishing  in  intensity. 

as  great  as  the  hypoxic  glow  elicited  with  2A%  oxygen;  -  when  1%  oxygen  was 
used  the  pseudoflash  was  10  times  as  bright  as  the  glow;  when  0.25%  oxygen  was 
used  the  pseudoflash  was  60  times  as  bright ;  and  when  0.05%  oxygen  was  used  the 
pseudoflash  was  5000  times  as  bright.  The  increase  in  this  ratio  with  progressively 
lower  oxygen  concentrations  was  evidently  due  both  to  diminution  in  hypoxic  glow 
intensity  and  to  an  increase  in  the  absolute  pseudoflash  intensity. 

2  No  systematic  attempt  was  made  to  find  the  upper  oxygen  concentration  limit  for  pseudo- 
flash  occurrence,  but  it  is  certainly  higher  than  the  limit  set  by  Snell  (about  %%). 


CONTROL  OF  FIREFLY  PSEUDOFLASH 


105 


A  third  characteristic  of  the  hypoxic  glow-pseudoflash  response  is  that  the 
pseudoflash  was  remarkably  constant  in  duration  and  in  form  (rates  of  accretion  and 
decay  of  intensity),  regardless  of  variations  in  both  degree  and  duration  of  hypoxia 
prior  to  readmission  of  air.  This  was  true  both  in  repeated  measurements  with 
one  individual  and  in  records  from  different  individuals.  Intensity  variations  of 
well  over  a  thousand-fold  occurred  without  difference  in  duration.  If  the  pseudo- 
flash  were  being  controlled  by  some  sort  of  oxygen-sensitive  effector,  such  as  Snell 


FIGURES  2,  3,  8,  9,  10,  11.  Various  luminous  responses  photographed  from  the  oscilloscope 
screen.  Ordinate,  light  intensity ;  abscissa,  time,  with  sweep  going  from  left  to  right.  Further 
descriptions  in  text.  Time  scale  in  seconds.  FIG.  2.  Pseudoflash  of  intact  adult  male  of 
Photinus  pyralis.  FIG.  3.  Oxygen  flash  of  cell-free  extract  of  P.  pyralis.  FIG.  8.  Spontaneous 
flashing  of  intact  female  of  Plwtnris.  FIG.  9.  Spontaneous  glow  of  intact  Photuris  larva. 
FIG.  10.  Pseudoflash  of  Photuris  larva.  Temporary  depression  in  trace  following  flash  repre- 
sents period  during  which  photocell  power  supply  was  switched  off.  FIG.  11.  Spontaneous 
flash  of  intact  male  of  P.  pyralis. 

supposed  the  end  cell  to  be,  it  would  be  remarkable  that  this  degree  of  constancy  of 
response  could  be  achieved,  particularly  in  view  of  the  individual  variability  in  in- 
tensity and  latency  of  hypoxic  glow,  and  in  intensity  of  pseudoflash. 

2.  Oxygen  responses  in  relation  to  structure 

It  was  shown  previously  (Buck,  1948)  that  there  is  no  correlation  between  the 
state  of  spiracular  valves  and  the  times  of  occurrence  or  characteristics  of  the  normal 


106 


J.  WOODLAND  HASTINGS  AND  JOHN  BUCK 


flash  of  Photinus  pyralis,  and  that  when  intact  fireflies  are  tested  with  progressively 
falling  oxygen  concentration  the  spiracles  open  well  before  the  hypoxic  glow  begins 
and  close  after  it  ceases.  Evidence  that  the  spiracles  have  no  immediate  influence 
on  the  hypoxic  glow-pseudoflash  response  was  obtained  in  the  present  study  by 
testing  adults  of  Photuris  and  Photinus  in  which  the  spiracles  of  the  luminous 
segments  had  been  made  inoperative  by  cautery  with  an  electrically  heated  needle 
or  by  insertion  of  a  short  length  of  human  baby  hair.  Although  these  specimens 
often  showed  a  continuous  glow  in  air,  presumably  caused  by  the  mechanical  dis- 


100  r 


CO 
£ 


50 


h- 
x 

o 

J 


8 


5  10          15 

%    OXYGEN 


20 


FIGURE  4.  Relation  between  oxygen  concentration  and  glow  intensity  ( in  per  cent  of  in- 
tensity in  air)  of  smeared  photogenic  tissue  of  males  of  P.  pyralis.  Data  from  several 
experiments. 

turbance,  they  gave  pseudoflashes  similar  to  those  in  individuals  with  normal  spiracles. 
Likewise,  it  was  observed  that  spiracular  opening  is  regularly  induced  by  exposure 
to  5%  oxygen,  whereas  the  hypoxic  glow  usually  requires  that  the  ambient  oxygen 
concentration  be  reduced  to  the  order  of  1%  to  2%.  Absence  of  spiracular  or  in- 
deed any  sort  of  valvular  influence  is  also  seen  in  dead  fireflies  which,  if  prevented 
from  drying  out,  may  exhibit  a  constant  dim  air  glow  for  a  day  or  more  after  all 
visible  signs  of  life  have  disappeared.  Such  dead  specimens  have  permanently  open 
spiracles  yet  give  a  pseudoflash  response. 

A  series  of  experiments  was  performed  in  which  increasing  degrees  of  interfer- 


CONTROL  OF  FIREFLY  PSEUDOFLASH 


107 


ence  with  possible  central  nervous  or  tracheal  control  of  luminescence  were  achieved 
by  (a)  decapitation,  (b)  cutting  off  the  abdomen  at  the  junction  of  the  fifth  and 
sixth  segments,  (c)  excising  the  photogenic  organ  alone,  and  (d)  smearing  the 
photogenic  tissue  on  glass.  None  of  these  preparations  produced  normal  flashes,  or 
indeed  any  continued  spontaneous  luminescence,  except  for  the  smeared  organs, 
which  exhibited  a  continuous  dim  glow  in  air,  decreasing  in  intensity  very  gradually 


10 


>  8 


LJ 

H 

-  4 


I 

/ 


1        1        1        1 

i        i 

i 

i 

2               4 

6 

8 

TIME -MINUTES 


FIGURE  5.  Changes  in  light  intensity  of  smeared  photogenic  tissue  of  male  of  P.  pyralis  in 
varied  oxygen  concentrations.  Tissue  in  air  for  first  three  minutes.  At  A,  8.3%  oxygen  was 
introduced.  At  B,  air  was  readmitted,  inducing  a  pseudoflash-like  excess  luminescence. 

(30  minutes  or  more  to  extinction).  The  first  three  types  of  preparations  re- 
sponded to  changes  in  ambient  oxygen  concentration  just  like  intact  fireflies,  i.e.,  in 
low  oxygen  concentrations  they  developed  hypoxic  glows  and  when  air  was  read- 
mitted they  produced  typical  pseudoflashes.  It  is  thus  clear  that  nerve  impulses 
originating  in  the  central  nervous  system  play  no  role  in  the  photogenic  response 
to  hypoxia.  It  is  also  apparent  that  none  of  the  tracheae  external  to  the  light  organ 
is  involved. 

The  intensity  of  the  glow  of  the  smeared  organ  is  proportional  to  ambient  oxy- 


108 


J.  WOODLAND  HASTINGS  AND  JOHN  BUCK 


gen  concentration  below  21%  (Fig.  4)  and  also  increases  greatly  in  pure  oxygen. 
Figure  5  shows  the  time  course  of  the  luminescence  when  a  smeared  organ  was 
exposed  (at  A)  to  8.3%  oxygen  and  then  re-exposed  (at  B)  to  air.  The  changes 
in  luminescence  are  qualitatively  very  similar  to  those  which  occur  when  the  same 
procedure  is  carried  out  with  cell-free  extracts  (Hastings,  McElroy  and  Coulombre, 
1953).  The  smeared  preparation  differs  from  both  the  extracts  and  the  intact  or- 
gan in  that  its  pseudoflash  has  a  longer  duration  and  is  not  so  bright  relative  to  the 


^ 

I 


200 


h; 

CO 

LJ 
h- 


150 


J 1 


I      I      I     I      I      I     I      I     I 


30  60  90 

TIME-SECONDS 


120 


FIGURE  6.  Relation  between  duration  of  hypoxia  and  intensity  of  pseudoflash  in  air  of 
smeared  photogenic  tissue  of  P.  pyralis.  Each  point  represents  pseudoflash  intensity,  in  per 
cent  of  glow  intensity  in  air,  in  an  experiment  similar  to  that  diagrammed  in  Figure  5.  The 
hypoxic  mixture  used  contained  \%  oxygen. 

intensity  of  the  hypoxic  glow.  These  differences  in  glow  and  flash  are  to  be  ex- 
pected if  oxygen  has  become  limiting  in  the  luminescent  reaction,  and  presumably  it 
is  the  disruption  of  the  tracheal  supply  within  the  photogenic  tissue  itself  which  is 
responsible  for  this  oxygen-limitation.  Similar  "slow"  flash  responses  have  been 
demonstrated  in  extracts  under  conditions  of  oxygen  limitation  (McElroy  and 
Hastings,  unpublished).  On  the  basis  of  the  biochemical  reactions  already  dis- 
cussed, the  intensity  of  the  pseudoflash  of  the  smeared  organ  is  presumably  a 
measure  of  the  amount  of  active  intermediate  which  has  accumulated.  The  de- 


CONTROL  OF  FIREFLY  PSEUDOFLASH 


109 


pendence  of  this  accumulation  upon  both  time  of  hypoxia  and  oxygen  concentration 
in  the  hypoxic  gas  mixture  is  illustrated  in  Figures  6  and  7. 

In  normal  males  of  Plwthnts  and  Plwtiiris  all  the  photogenic  tissue  in  both  lu- 
minous segments  ordinarily  participates  in  each  flash,  and  apparently  simultaneously. 


200 


UJ 

I- 


I 

CO 


150 


10 


15 


20 


OXYGEN-°7o 


FIGURE  7.  Relation  between  intensity  of  pseudoflash  in  air  of  smeared  photogenic  tissue 
of  P.  pyralis,  and  degree  of  hypoxia.  Tissue  exposed  to  each  oxygen  concentration  for  two 
minutes,  then  flushed  with  air.  Intensity  plotted  on  ordinate  in  per  cent  of  glow  intensity  in  air. 

In  some  instances,  however,  it  was  observed  that  both  spontaneous  flashes  and  vari- 
ous types  of  induced  luminescence  involved  only  one  of  the  segments,  or  only  parts 
of  one  or  both.  Furthermore,  the  type  of  luminescence  displayed  sometimes  differed 
in  different  regions  of  a  single  organ  or  even  changed  in  the  course  of  an  experiment. 


110  J.  WOODLAND  HASTINGS  AND  JOHN  BUCK 

The  flash  of  the  female  of  Photnris  is  usually  too  sharp  and  brilliant  for  reliable 
visual  observation  of  heterogeneity,  but  may  possibly  also  involve  asynchronous 
luminescence  (Fig.  8). 

In  instances  in  which  only  a  portion  of  a  photogenic  organ  gave  the  hypoxic 
glow-pseudoflash  response,  the  portion  which  failed  to  respond  often  developed  a 
dull  glow  in  air  after  the  pseudoflash  in  the  other  portion  had  ceased.  Since  the 
photocell  integrates  all  the  light  emitted,  one  needs  to  be  aware  of  the  possibility 
that  intensity  X  time  recordings  of  luminescence  (e.g..  Figs.  1,  2)  may  be  the  re- 
sultant of  two  quite  different  sorts  of  things,  namely,  change  in  light  intensity  per 
unit  organ  area,  and  change  in  area  active.  We  cannot  exclude  the  possibility  that 
an  occasional  heterogeneous  response  of  this  type  was  recorded  in  our  work,  but 
do  not  believe  that  any  of  our  present  interpretations  is  in  error  because  of  such 
an  accident. 

3.  Oxygen  effects  on  Induced  glows 

Bright  steady  glows  can  be  induced  in  both  intact  fireflies  and  isolated  abdomens 
by  air  passed  through  cotton  soaked  in  ethyl  ether  or  over  potassium  cyanide  crystals 
(i.e.,  without  change  in  ambient  oxygen  concentration).  The  dosage  must  be 
chosen  to  avoid  either  premature  recovery  of  the  animal  or  rapid  destruction  of  the 
photochemical  system  (Buck,  1948).  When  fireflies  which  were  glowing  from  ex- 
posure to  ether  or  cyanide  vapor  were  exposed  to  low  oxygen  concentrations,  lumi- 
nescence abruptly  declined  to  a  low  level,  then  rose  somewhat  as  an  hypoxic  glow. 
When  air  was  readmitted  a  typical  pseudoflash  occurred.  This  illustrates  the  oc- 
currence of  a  flash  under  conditions  in  which  tracheal  end  cells  would  be  expected 
to  be  inactivated. 

4.  Oxygen  responses  of  Pyroplwrus,  Plwturls  larva  and  persistent  pupal  organ  of 
Plwt in  its  pyralis 

The  photogenic  organs  of  the  large  elaterid  firefly  Pyropliorus  and  of  the  larvae 
and  pupae  of  lampyrid  fireflies  offer  an  interesting  contrast  to  the  organs  of  adult 
lampyrid  fireflies  in  two  respects.  First,  they  never  normally  flash,  but  emit  light 
in  long-sustained  glows  at  irregular  intervals.  Second,  they  lack  the  tracheal  end 
cells  which  are  characteristic  of  the  flashing-type  adult  lampyrid  organ.  The  pupal 
organs  frequently  persist  into  the  adult,  thus  combining  both  organ  types  in  the 
same  individual. 

Observation  of  oxygen  effects  in  Pyroplwrus  is  complicated  by  the  fact  that  the 
glow  normally  fluctuates  cyclically  in  intensity,  at  frequencies  varying  from  about 
one  peak  per  second  to  one  per  five  seconds  or  slower,  as  observed  also  in  a  Cuban 
species  by  Harvey  (1931).  In  addition,  the  intensity  of  the  glow  increases  mark- 
edly when  the  creature  is  disturbed.  Thus  hypoxia  sometimes  proves  sufficiently 
irritating  that  the  light  emitted  is  at  first  actually  brighter  than  in  air,  and  there  is 
no  initial  decline  due  to  oxygen  limitation  as  in  adult  lampyrids  glowing  in  air.  The 
luminescence  is  only  oxygen-limited  at  ambient  concentrations  of  1%  or  lower. 
PyropJiorus  in  low  oxygen  concentrations  responds  to  a  sudden  increase  in  oxygen 
by  emitting  a  pseudoflash  which  is  qualitatively  similar  to  the  typical  adult  lampyrid 
pseudoflash,  but  often  appears  after  a  quite  long  latent  period  (up  to  20  seconds)  and 
lasts  much  longer. 


CONTROL  OF  FIREFLY  PSEUDOFLASH  111 

The  minute  photogenic  organs  of  the  larva  of  Photnrls  and  the  persistent  pupal 
organs  of  Photinns  pyralis  emit  a  fluctuating  luminescence  strikingly  similar  to  that 
of  Pyrophorus  (Fig.  9).  Their  responses  to  oxygen  have  not  been  followed  in  de- 
tail except  to  confirm  Buck's  observation  (1946,  1948)  that  low  ambient  oxygen 
induces  an  hypoxic  glow  and  subsequently  raised  oxygen  elicits  a  pseudoflash.  A 
record  reproduced  in  Figure  10  shows  that  the  larval  pseudoflash  closely  resembles 
that  of  the  adult.  The  persistent  pupal  organ  in  the  adult  usually  (but  not  always) 
gives  an  hypoxic  glow-pseudoflash  response  in  parallel  with  that  of  the  adult  organ. 

DISCUSSION 

Evidence  presented  above  has  shown  that  neither  spiracle,  main  trachea  nor 
central  nervous  system  is  necessary  for  either  the  appearance  or  disappearance  of 
luminescence  in  the  usual  type  of  hypoxic  glow-pseudoflash  response.  The  possi- 
bility that  tracheal  end  cell  valves  might  be  involved  in  the  response  is  likewise  all  but 
eliminated  by  the  following  considerations:  (a)  Pseudoflashes  occur  in  Pyrophorus, 
the  Plwturis  larva  and  the  persistent  pupal  organ  of  Photinns  pyralis  (all  of  which 
lack  end  cells),  and  in  lampyrid  fireflies  treated  with  cyanide  and  ether  (where  end 
cell  valves  should  be  inactivated)  ;  (b)  as  already  pointed  out,  the  constancy  of  pseu- 
doflash duration  makes  it  difficult  to  believe  that  an  end  cell  mechanism  is  functioning 
in  the  control ;  (c)  recent  electron  microscopy  by  Beams  and  Anderson  (1955)  casts 
grave  doubt  on  there  being  any  valvular  structure  in  the  end  cell.  In  fact  the  induc- 
tion of  pseudoflashes  in  dead  fireflies  makes  it  unlikely  that  this  response  depends 
upon  active  participation  of  any  part  of  either  tracheal  or  nervous  systems. 

Even  with  end  cell  control  excluded  there  remains  the  question  of  whether  the 
hypoxic  glow-pseudoflash  response  might  nevertheless  be  controlled  by  oxygen  limi- 
tation. We  have  seen  that  in  the  glow  of  smeared  tissue  and,  within  a  narrow  con- 
centration range,  in  the  hypoxic  glow  itself,  oxygen  does  appear  to  be  a  limiting  re- 
actant.  However,  the  hypoxic  glow  is  actually  induced  not  by  increase  in  oxygen 
concentration  but  by  decrease,  and  the  pseudoflash  dies  away  (i.e.,  is  controlled) 
under  conditions  in  which  ambient  oxygen  concentration,  if  changing  at  all.  must  be 
rising.  When  we  add  to  these  paradoxes  the  fact  that  glowing  can  be  induced  by 
pure  oxygen  (Alexander)  and  by  a  wide  variety  of  physical  and  chemical  agencies, 
and  that  even  the  hypoxic  glow  can  change  spontaneously  in  intensity  without  any 
change  in  ambient  gas  concentration,  it  becomes  very  difficult  to  visualize  oxygen  as 
playing  any  consistent  role  in  either  initiating  or  stopping  these  induced  types  of 
luminescence. 

For  reasons  discussed  by  Buck  (1955)  the  striking  kinetic  similarity  between 
pseudoflash  and  oxygen  flash  (Figs.  2,  3)  does  not  necessarily  indicate  the  same 
causation.  However,  the  detailed  parallels  between  the  two  responses  leave  little 
doubt  that  the  pseudoflash  of  the  intact  organ  involves  the  photochemical  system 
identified  in  the  cell-free  extract.  Thus  active  intermediate  can  be  presumed  to  ac- 
cumulate in  the  photogenic  tissue  during  hypoxia,  and,  upon  readmission  of  air,  to 
be  concurrently  oxidized  and  inhibited  with  production  of  a  pseudoflash  (see  In- 
troduction). Similarly,  assuming  that  liberation  of  active  intermediate  would  con- 
tinue in  the  organ  of  a  dead  firefly  until  autolysis  supervened,  the  ability  of  some 
dead  individuals  to  glow  and  to  give  pseudoflashes  could  be  explained.  Further- 
more, the  in  vitro  system  is  free  from  all  the  morphological  objections  to  end  cell 


112  J.  WOODLAND  HASTINGS  AND  JOHN  BUCK 

involvement  discussed  above  and  it  is  consistent  with  the  constant  duration  of  the 
pseudoflash,  which  is  particularly  difficult  to  account  for  on  the  basis  of  valvular 
control.  Such  constancy,  in  other  words,  is  precisely  what  would  be  expected  if 
the  luminescence  decays  primarily  because  of  an  enzyme-inhibiting  reaction  rather 
than  because  of  oxygen  limitation. 

The  conclusion  that  no  end  cell  valve  functions  in  the  hypoxic  glow-pseudoflash 
response  does  not,  of  course,  exclude  the  possibility  that  normal  flashing  is  con- 
trolled by  such  a  mechanism.  Since,  however,  the  induction  of  the  pseudoflash  has 
been  the  principal  experimental  support  for  belief  in  end  cell  control,  the  existence  of 
a  more  reasonable  alternate  explanation  of  the  pseudoflash  leaves  little  ground  for 
favoring  end  cell  involvement  in  the  normal  flash.  Furthermore,  oxygen  control 
by  whatever  method  appears  intrinsically  less  tenable  than  enzymatic  control  on  at 
least  two  counts :  First,  if  the  flash  were  oxygen-limited  the  photogenic  tissue  would 
have  to  be  hypoxic  throughout  the  long  interflash  periods.  This  would  be  unlikely 
on  physiological  grounds,  even  if  respiration  had  a  lower  oxygen  requirement  than 
luminescence  (actually,  in  all  forms  thus  far  studied  luminescence  persists  at  oxygen 
concentrations  far  lower  than  will  support  any  significant  respiration).  If,  however, 
the  normal  flash  were  controlled  by  temporary  reversal  of  enzyme  inhibition,  rather 
than  by  oxygen  limitation,  the  tissues  could  remain  fully  aerated  at  all  times.  Sec- 
ond, the  normal  flash  of  many  lampyrid  fireflies  is  so  short  as  to  cast  doubt  on  the 
possibility  of  control  by  diffusive  gas  transfer.  Even  in  the  relatively  slow  flash  of 
Photinns  p\ralis  (Fig.  11)  the  average  rise  time  is  0.075  second,  and  the  response 
of  individual  photogenic  units  is  almost  certainly  much  faster  (Buck,  1948,  p.  446). 
In  the  female  of  Photnris  (Fig.  8)  the  rise  time  is  not  more  than  0.03  second  and 
the  decay  of  luminescence  is  almost  equally  rapid.  This  shows  that  an  efficient 
mechanism  for  oxygen  removal  would  need  to  be  present  as  well  as  one  for  suddenly 
supplying  oxygen.  A  priori,  therefore,  it  would  be  expected  that  an  intracellular 
mechanism  involving  enzyme  inhibition  and  activation  would  be  better  suited  to 
the  required  response  velocities  than  one  involving  passage  of  oxygen  between 
tracheae  and  cell. 

The  fact  that  intact  fireflies  are  able  to  extinguish  their  light  completely  between 
flashes,  whereas  low-level  luminescence  continues  in  the  in  vitro  system,  need  not 
be  unduly  disturbing  since  the  intact  cell  presumably  has  more  efficient  methods  of 
shifting  the  chemical  equilibria  concerned  and  of  sequestering  reactants.  It  has  been 
suggested  (Buck,  1948,  1955)  that  the  photogenic  tissue  is  ordinarily  kept  dark  by 
some  sort  of  aerobic  metabolic  process  and  that  light  is  produced  only  when  this 
process  is  interfered  with.  Such  a  mechanism  would  account  for  both  "abnormal" 
and  normal  luminescence,  since  it  should  be  inhibited  by  very  diverse  agencies  such 
as  hypoxia,  various  poisons  (e.g.,  pure  oxygen,  cyanide)  and  anesthetics  ( ether )- 
allowing  light  to  be  produced — while  at  the  same  time  forming  a  likely  type  of  sys- 
tem to  be  integrated  with  the  normal  biological  trigger,  the  nerve  impulse.  A  ten- 
tative biochemical  pathway  has  recently  been  suggested  (McElroy  and  Hastings, 
1955)  by  which  a  nerve  impulse  might  lead  to  a  rapid  temporary  increase  in  active 
intermediate  concentration — i.e.,  to  a  flash.  Whether  or  not  the  precise  mechanism 
suggested  proves  to  be  correct,  this  general  type  of  linkage  of  stimulatory  and  re- 
sponse systems  deserves  special  attention  because  it  provides  an  endogenous  mecha- 
nism capable  of  the  observed  rapidity  and  precision  of  photogenic  control. 


CONTROL  OF  FIREFLY  PSEUDOFLASH  113 

SUMMARY 

As  reported  by  Snell  and  Alexander,  lampyrid  fireflies  exposed  to  oxygen  con- 
centrations of  the  order  of  2%  or  lower  develop  a  sustained  "hypoxic  glow,"  and 
when  subsequently  re-exposed  to  air  emit  a  much  brighter  and  shorter  "pseudoflash." 
We  find  that  these  responses  can  be  independent  of  the  spiracles,  and  are  given  by 
decapitated  fireflies,  isolated  abdomens  and  excised  photogenic  organs,  showing 
their  independence  of  central  nervous  system  and  tracheae.  The  hypoxic  glow- 
pseudoflash  response  is  also  given  by  the  elaterid  firefly  Pyrophorus  and  by  the  larval 
and  pupal  photogenic  organs  of  lampyrid  fireflies.  Since  all  these  organs  lack 
tracheal  end  cells,  these  cells  cannot,  as  Snell  and  Alexander  believed,  control  this 
type  of  light  production.  This,  together  with  other  evidence,  makes  it  clear  that 
luminescence  is  rarely  oxygen-limited.  Rather,  all  our  observations  are  consistent 
with  enzyme  activation  and  inhibition  in  a  system  of  photochemical  reactions  of  the 
sort  proposed  by  McElroy  and  Hastings  (1955). 

LITERATURE  CITED 

ALEXANDER,  ROBERT  S.,  1943.     Factors  controlling  firefly  luminescence.     J .  Cell.  Coinf*.  Plivsiol.. 

22:  51-71. 
BEAMS,  H.  W.,  AND  EVERETT  AXDERSON,   1955.     Light  and  electron  microscope  studies  on  the 

light  organ  of  the  firefly  (Pliotiuns  pyralis).     Biol.  Bull.,  109:  375-393. 
BUCK,   JOHN    B.,    1946.     The   spiracular   factor   in   the   control   of   luminescence   in   the   firefly. 

Anat.  Rcc.,  96:  51. 
BUCK,  JOHN  B.,  1948.     The  anatomy  and  physiology  of  the  light  organ  in  fireflies.     Ann.  N.  Y. 

Ac  ad.  Sci..  49  :  397-482. 
BUCK,  JOHN,   1955.     Some  reflections  on  the  control  of  bioluminescence.     Pp.  323-332  in  The 

Luminescence  of  Biological   Systems    (Ed.   F.   H.  Johnson),  Amer.  Assoc.  Adv.   Sci., 

Washington. 

DAHLGREN,  ULRIC,  1917.     The  production  of  light  by  animals.     /.  Franklin  Inst..  183  :  323-348. 
HARVEY,  E.  X.,  1931.     Photocell  analysis  of  the  light  of  the  Cuban  elaterid  beetle,  Pyrofhorus. 

J.  Gen.  Physio!.,  15:  139-145." 

HARVEY,  E.   N.,  1952.     Bioluminescence.     Academic   Press,  New  York. 
HASTINGS,  J.  WOODLAND,  WILLIAM  D.  MCELROY  AND  JANE  COULOMBRE.   1953.     The  effect  of 

oxygen    upon    the    immobilization    reaction    in    firefly    luminescence.     /.    Cell.    Comp. 

Physiol..  42  :  137-150. 
McELROY,  W.  D.,  AND  J.  W.  HASTINGS,  1955.     Biochemistry  of  firefly  luminescence.     Pp.  161- 

198  in  The  Luminescence  of  Biological  Systems    (Ed.  F.  H.  Johnson),  Amer.  Assoc. 

Adv.  Sci.,  Washington. 
MCELROY,  YV.   I)..  J.  W.   HASTINGS,  JANE  COULOMBRE  AND  VALERIE  SONNENFELD,   1953.     The 

mechanism  of  action  of  pyrophosphate  in  firefly   luminescence.     Arch.  Biochcm.   Bio- 

phys.,  46:  399-416. 
SNELL,   PETER  A.,   1932.     The  control  of  luminescence  in   the  male   lampyrid   firefly,   Photnris 

pennsylranica.  with  special  reference  to  the  effect  of  oxygen  tension  on   flashing.     /. 

Cell.  Camp.  Physiol.,  1  :  37-51. 


HOW  SEA  STARS  OPEN  BIVALVES1 

MARCEL  E.  LAVOIE  - 
Syracuse  University,  Syracuse,  N.  V. 

The  damage  inflicted  upon  the  oyster  and  clam  industries  by  sea  star  predation 
(Galtsoff  and  Loosanoff,  1939;  Barnes,  1946)  has  stimulated  much  interest  in  the 
method  employed  by  asteroids  to  open  the  shells  of  bivalve  molluscs.  The  many 
solutions  proffered  in  the  past  were  reduced  to  two  probable  alternatives  within  the 
last  sixty  years:  (1)  the  "toxin"  theory  which  proposes  that  sea  stars  secrete  a 
substance  which  produces  relaxation  of  the  adductor  muscles  of  their  victims ;  and 
(2)  the  "mechanical"  theory  which  credits  the  sea  stars  with  the  ability  to  pull 
the  valves  of  the  molluscan  shell  apart  by  means  of  their  tube  feet. 

The  first  hypothesis  was  proposed  originally  by  Eudes-Deslongchamps  (1826). 
Most  of  its  advocates  (including  Hesz,  1878;  Figuier,  1891;  Pieron,  1913;  Cahn, 
1950;  Korringa,  1953  and  Aldrich,  1954)  postulated  that  the  chemical  agent  was 
secreted  by  the  digestive  organs  of  the  sea  stars.  Van  der  Heyde  (1922)  and 
Sawano  and  Mitsugi  (1932)  supported  this  view  with  experiments  which  demon- 
strated that  extracts  of  asteroid  stomach  and/or  pyloric  caeca  produce  tetanus  and, 
often,  permanent  cessation  of  cardiac  beat  when  poured  over  the  hearts  of  living 
molluscs. 

The  mechanical  theory,  advanced  originally  by  Fischer  (1864)  and  Bell  (1892), 
was  established  firmly  by  Schiemenz  (1895)  who  demonstrated  experimentally  that 
the  valves  of  the  clam  Venus  vcrrncosa  could  be  separated  by  a  pull  of  900  grams, 
while  a  clam  held  by  the  tube  feet  of  an  Asterias  could  be  released  only  if  a  pull 
of  more  than  1000  grams  was  applied  to  it.  He  concluded  that  the  sea  star  could 
exert  a  pull  greater  than  that  which  could  be  sustained  by  Venus,  but  he  failed  to 
note  that  he  had  measured  only  the  adhesive  capacity  of  the  echinoderm's  tube  feet. 
He  did  not  show  that  the  sea  star  possessed  the  ability  to  produce  sufficient  muscular 
force  to  open  bivalves.  However,  it  is  believed  that  the  data  presented  below 
demonstrate  the  existence  of  such  forces  and  render  the  toxin  theory  less  tenable. 

MATERIALS  AND  METHODS 

The  two  groups  of  experimental  procedures  employed  were  designed  to  de- 
termine (1)  the  effects  of  sea  star  extracts  upon  a  representative  bivalve,  and  (2) 
whether  sea  stars  actually  pull  upon  the  valves  of  their  prey. 

1.  Procedures  for  determining  effects  of  extracts 

The  stomach  and/or  pyloric  caeca  of  Asterias  forbesi  (obtained  from  the  Marine 
Biological  Laboratory  at  Woods  Hole)  were  excised  and  ground  with  a  Pyrex  glass 

1  This  investigation  is  a  portion  of  a  dissertation  submitted  in  partial  fulfillment  of  the 
requirements  for  the  degree  of  Doctor  of  Philosophy  in  the  Department  of  Zoology  at  Syracuse 
University  in  September,  1955. 

~  Present  address :  Department  of  Zoology,  University  of  New  Hampshire,  Durham,  New 
Hampshire. 

114 


HOW  SEA  STARS  OPEN  BIVALVES 


115 


homogenize!  in  the  cold.  Enough  sea  water  or  distilled  water  was  added  to  make 
up  10(/r  solutions  relative  to  the  wet  weight  of  the  organs  used.  (Other  concen- 
trations were  tested  and,  generally,  yielded  similar  results.)  Extraction  was  al- 
lowed to  proceed  for  varying  times  (5  minutes  to  48  hours)  and  the  tissue  debris 
was  removed  by  filtration  or  centrifugation.  Other  extraction  methods  were  em- 
ployed to  test  the  possibilities  that  the  alleged  toxin  might  be  only  poorly  soluble  in 
water,  that  it  might  occur  in  bound  form,  or  that  it  might  require  activation.  Thus, 
some  extractions  were  made  with  fat  solvents,  some  extracts  were  dialyzed,  others 
were  frozen  and  thawed  before  use,  and  some  were  mixtures  of  homogenates  from 
different  organs. 


rff 


I 


FIGURE  1.  Constant  stress  apparatus.  Each  800-gram  weight  was  suspended  by  a  cord 
passing  over  a  ball-bearinged  pulley  to  a  double  hook  inserted  into  notches  filed  in  the  beak  of 
the  mussel  shell.  Another  hook,  also  made  from  two  bent  pins,  was  soldered  to  the  bottom  of 
the  pan  and  passed  through  the  same  notches.  Gapes  were  measured  by  means  of  a  calibrated 
metal  triangle  which  could  be  slipped  in  between  the  valves  near  the  hooks. 

All  extracts  were  tested  on  the  common  sea  mussel,  M\tilus  cdulis.  In  most 
cases  0.5  ml.  of  the  clear  extract  was  injected  by  means  of  hypodermic  syringe  into> 
the  mantle  cavity  or  0.15  ml.  was  injected  directly  into  the  posterior  adductor  muscle 
by  way  of  a  notch  filed  in  the  shell's  dorsal  edge.  Each  mussel  had  been  pre-tested 
to  insure  that  its  physiological  condition  was  approximately  comparable  to  that  of 
the  other  experimental  animals.  The  pre-test  was  accomplished  by  exerting  a  pull 
of  800  grams  on  the  valves  for  five  minutes ;  only  mussels  which  gaped  less  than  one 
mm.  were  used  for  injection  tests.  After  being  injected,  each  mussel  was  subjected 
to  a  steady  pull  of  800  grams  on  its  valves  (Fig.  1)  for  45  minutes  during  which 
measurements  of  the  gape  were  made  at  regular  intervals. 


116 


MARCEL  E.  LAVOIE 


In  some  cases,  the  extract  was  merely  added  to  the  sea  water  into  which  the  mus- 
sel was  placed  after  having  been  kept  out  of  water  for  12  hours,  and  the  gape  was 
determined  after  5  and  10  minutes.  In  other  experiments,  the  mussel  heart  was 
exposed  and  perfused  with  the  extracts  while  kymograph  records  were  made  of  the 
effects  on  the  beat.  Controls  for  all  types  of  tests  were  treated  with  solvent  only 
(sea  water  or  distilled  water)  or  with  extracts  of  other  sea  star  organs  or  extracts  of 
the  digestive  organs  of  other  invertebrates. 


FIGURE  2.  Apparatus  for  measuring  sea  star  pulling  force.  The  device  and  the  mussel 
are  represented  at  approximately  actual  size.  a.  calibrated  capillary  tube;  b.  water  column; 
c.  cut  posterior  adductor  muscle ;  d.  steel  coil  spring  ;  c.  bolt ;  /.  metal  plate  soldered  to  the 
spring;  g.  plugged  end  of  water-filled  rubber  tube:  h.  cut  umbo.  In  some  experiments  this 
manometric  unit  was  replaced  by  a  plastic  cylinder  which  fitted  between  the  two  bolts. 

FIGURE  3.  Increasing  load  stress  apparatus.  </.  calibrated  water  jar;  b.  control  valve;  c. 
pulley ;  d.  waxed  cardboard  container ;  c.  mussel.  The  approximate  total  load  applied  to  the 
shell  was  computed  by  adding  the  container  weight  to  the  weight  of  the  water  poured  into  it 
from  the  calibrated  jar. 


2.  Procedures  for  determining  sea  star  [>itllinf/  ulnlity 

The  adductor  muscle  of  medium-sized  Myti/its  was  severed  with  a  thin  razor 
blade  and  the  valves  were  then  made  to  shut  firmly  by  means  of  an  "artificial 
muscle."  This  consisted  of  a  tightly  coiled  steel  spring  about  l/2  inch  long  with  a 
metal  plate  soldered  at  each  end.  The  spring  was  held  in  place  (Fig.  2)  by  short 
bolts  inserted  through  holes  bored  in  the  valves.  The  metal  plates  were  bent  so  as 
to  compress  the  sealed  end  of  a  water-filled  rubber  tube  which  passed  out  of  the  shell 
through  a  hole  effected  by  breaking  off  one  tip  of  the  umbo.  The  distal  end  of  the 
rubber  tube  was  slipped  over  the  end  of  a  graduated  capillary  tube.  Any  outward 


HOW  SKA  STARS  OPEN  BIVALVES 


117 


pull  on  the  valves  was  reflected  in  the  stretching  of  the  spring  and,  consequently, 
in  an  increased  volume  of  the  ruhber  tubing  and  a  lowering  of  the  water  level  in  the 
manometer  tube.  The  variations  in  water  level,  produced  by  a  sea  star  humped 
over  a  mussel  containing  this  apparatus,  could  be  duplicated  by  inserting  the  mussel, 
afterward,  in  the  stress  apparatus  illustrated  in  Figure  3.  In  some  instances,  the 
severed  adductor  muscle  was  replaced  by  a  threaded  plastic  cylinder  so  that  the 
valves  could  be  bolted  together  firmly  or  allowed  to  separate  only  slightly. 

TABU    I 

Gaping  of  Mytilns  undc>-  stress.      These  raw  data  arc  f row  two  representative  groups  of  experiments 

involving  the  application  of  stress  to  the  shells  after  injections  into  the  adductor  muscles.     The 

apparatus  permitted  the  testing  of  10  mussels  simultaneously;  generally,  five  were  treated 

with  extract  and  five  with  control  solutions.     Shells  ranged  in  size  from  43  X  22  mm. 

to  55  X  30  mm. 


Gape  in  millimeters  after 

Injected  with 

5  min. 

10  min. 

15  min. 

20  min. 

25  min. 

45  min. 

Distilled  water 

0 

0 

0 

0 

0 

0 

0 

0 

0.7 

2.0 

0.2 

2.0 

0 

0 

4.8 

4.0 

5.8 

7.8 

0.5 

0 

9.6 

0.2 

2.8 

2.2 

0.9 

2.8 

1.7 

1.8 

2.8 

1.8 

2.2 

2.5 

2.7 

2.7 

2.5 

2.5 

2.8 

2.9 

3.8 

3.0 

3.8 

3.2 

3.5 

3.2 

3.2 

3.8 

3.5 

4.0 

3.8 

4.8 

4.9 

5.0 

5.0 

5.5 

3.8 

4.0 

3.8 

4.8 

5.7 

6.2 

Pyloric  caeca  in 

0 

0 

0 

0 

0 

2.2 

distilled  water 

0 

0 

0 

0 

0 

4.8 

0 

0 

0 

0.8 

3.7 

4.8 

1.8 

1.8 

1.9 

3.0 

3.0 

2.8 

1.7 

3.0 

3.2 

3.0 

3.0 

2.0 

1.8 

1.8 

1.8 

1.8 

1.9 

5.8 

2.1 

2.1 

2.1 

2.2 

2.5 

3.0 

3.8 

3.8 

3.8 

5.0 

5.3 

5.4 

4.0 

4.0 

3.8 

4.0 

4.8 

4.8 

4.8 

5.7 

5.8 

5.8 

5.8 

4.8 

RESULTS 

1.  Effects  of  sea  star  extracts 

As  reported  by  previous  investigators,  extracts  of  the  digestive  organs  of  sea 
stars  generally  produce  tetanus  in  molluscan  hearts.  But  so  do  other  substances 
including  sea  water.  Furthermore,  any  suggestion  that  an  asteroid  secretion  may 
affect  the  adductor  muscle  indirectly  by  stopping  the  heart  seems  untenable  in  view 
of  the  observation,  made  in  some  of  these  experiments,  that  Mytilns  whose  hearts 
are  excised  may  continue  to  maintain  their  valves  tightly  shut  for  two  or  three  days. 

M \tilits  placed  in  sea  water  contaminated  with  sea  star  extracts  usually  "taste" 
the  medium  and  then  close  their  valves  firmly.  The  degree  of  gaping,  among  the 


118 


MARCEL  E.  LAVOIE 


sixty  mussels  tested  in  this  manner,  was  less  for  specimens  exposed  to  diluted  ex- 
tracts than  for  those  placed  in  sea  water  alone.  This  seems  to  indicate  that  no 
muscle-relaxing  toxin  was  present  in  the  extracts. 

Gape  measurements  made  on  mussels  injected  with  extracts  or  control  solu- 
tions revealed  that  the  rate  of  shell  opening  varied  through  a  very  narrow  range 
for  all  tests.  The  average  value  of  the  rate  of  gaping  for  mussels  which  were  not 
injected  was  almost  identical  to  that  of  mussels  whose  mantle  cavity  or  adductor  had 
been  injected  with  sea  water  or  distilled  water  or  with  one  of  the  various  types  of 
extracts  (see  representative  data  in  Table  I).  Over  1000  mussels  were  tested  in 


FIGURE  4.  Astcrias  feeding  upon  decoy  mussels.  The  rubber  tube  leading  to  the  mano- 
metric  recorder  is  covered  with  a  glass  sleeve  near  the  mussel  in  order  to  prevent  compression 
of  the  tube  by  the  sea  star's  antimeres.  The  asteroid  on  the  left  is  in  the  process  of  inserting  its 
stomach  into  a  shell  whose  valves  are  tightly  bolted  together  by  means  of  plastic  cylinder. 

this  manner  and  the  data  can  only  lead  to  the  conclusion  that  the  extracts  did  not 
contain  any  substance  which  could  be  considered  effective  in  inducing  relaxation 
of  the  bivalve  adductor  muscles. 

2.  Observation  of  sea  star  pulling  ability 

Sea  stars,  kept  in  20-gallon  tanks  of  circulating  sea  water,  were  presented  live 
mussels  whose  adductors  had  been  replaced  by  springs  or  cylinders  as  described 
previously.  The  soft  parts  of  most  of  these  mussels  were  reached  by  the  asteroid 
stomachs  and  were  partly  or  wholly  digested.  Unquestionably,  no  secretion  of  the 
sea  stars  could  have  had  any  weakening  effect  upon  the  ''artificial  muscles"  holding 
the  pelecypod  valves  closed.  The  following  cases,  selected  from  several  dozen  ob- 
servations, illustrate  the  significance  of  the  results  obtained : 


HOW  SEA  STARS  OPEN  BIVALVES  119 

1 )  A  sea  star  was  observed  while  it  approached  a  mussel  containing  the  spring 
device  and  while  it  humped  over  its  victim  in  the  typical  predatory  position  (Fig.  4). 
During  the  five  minutes  it  required  to  settle  in  an  advantageous  position    (and, 
probably,  to  extrude  its  stomach)  there  was  no  change  in  the  water  level  of  the 
recording  apparatus.     During  the  next  three  minutes,  however,  the  level  dropped 
rapidly ;  at  the  end  of  this  time  the  sea  star  was  removed  from  the  aquarium  and  its 
arms  were  peeled  back  forcibly  in  order  to  expose  the  mussel.     The  valves  were 
found  closed  tightly  upon  the  sea  star's  stomach,  most  of  which  was  inserted  into 
the  shell.     In  this  case,  the  drop  in  the  recording  tube  was  duplicated  later  with  a 
load  of  1200  grams  on  the  shell's  valves;  but  spring-containing  mussels  requiring 
2600  to  3000  grams  pull  to  open  0.1  mm.  were  also  successfully  preyed  upon  by 
the  sea  stars. 

TABLE  II 
Summary  of  pulling  forces  exerted  by  a  sea  star  upon  a  spring-containing  mussel 

Time  (in  minutes)  Pulling  force  (in 

from  beginning  of  grams)  applied  by 

observation  the  sea  star 

0  440 

5  740 

10  620 

14  710 

15  560 
20  620 
60  470 
90  0 

115  650 

135  0 

150  680 

155  800 

158  0 

159-165  Sea  star  moved  oft"  mussel 

2)  Another  Astcrias  was  observed  for  almost  three  hours  after  it  was  found 
humped  over  a  prepared  mussel.     During  that  time,  the  water  level  of  the  recording 
tube  varied  through  three  irregular  cycles  of  rises  and  falls.     When  these  variations 
were  duplicated  later  by  placing  the  mussel  in  the  stress  apparatus,  it  was  seen  that 
they  represented  the  pulling  forces  shown  in  Table  II.     When  the  mussel  was 
opened  it  was  found  to  be  partly  digested.     This,  and  many  similar  observations, 
seems  to  indicate  that  the  sea  star's  pull  is  not  applied  steadily. 

3)  A  mussel  whose  valves  were  bolted  together  very  firmly  so  that  no  space 
could  be  discerned  between  them  under  9  X  magnification,  was  loosened  forcibly 
from  the  grasp  of  a  sea  star  that  had  humped  over  it  for  several  hours.     The 
asteroid's  stomach  was  mostly  inside  the  shell  and  it  did  not  slip  out  again  during 
the  next  hour  while  the  sea  star  dragged  the  shell  along  the  bottom  of  the  aquarium. 
Later,  when  the  shell  was  exposed  to  increasing  loads  in  the  stress  apparatus,  the 
valves  were  bent  enough  by  a  load  of  3100  grams  to  produce  an  opening  between 
them  of  0.1  mm. 

4)  Several  mussels  whose  valves  were  tied  together  so  as  to  open  only  0.1  mm. 
were  invaded  by  sea  stars  whose  stomachs  were  seen  to  slip  out  of  the  shells  when  the 
echinoderms'  arms  were  pulled  away  from  the  shells. 


120  MARCEL  E.  LAVOIE 

DISCUSSION 

The  negative  results  of  the  experiments  involving  sea  star  extracts  are  not 
proof  that  asteroids  do  not  secrete  a  toxin  during  predation,  but  they  do  indicate 
that  no  such  substance  can  be  separated  from  the  sea  star  organs  by  the  extraction 
methods  used.  Furthermore,  a  muscle-relaxing  secretion  would  seem  superfluous, 
at  least  in  the  predation  of  Asterias  forbcsi  upon  Mytilns  ediilis,  since  it  was  shown 
above  that  this  asteroid  is  capable  of  producing  a  pulling  force  which  is  transmitted 
to  the  valves  of  mussels  by  the  anchoring  action  of  the  tube  feet. 

It  may  be  questioned  whether  some  species  of  pelecypods  which  are  attacked  by 
sea  stars  might  not  require  stronger  pulls  to  open  than  those  that  can  be  mustered 
by  Asterias.  Reese  ( 1942)  showed  that  3750  grams  could  be  withstood  for  several 
days  by  some  Venus  and  Ostrea;  Tamura  (1929)  reported  that  the  Japanese  oyster 
may  sustain  15,000  grams  pull  for  as  long  as  five  minutes;  Galtsoff  (1952)  referred 
to  the  ability  of  oysters  to  withstand  6000  grams  for  several  hours;  Plateau  (1884) 
computed  Ostrea's  "absolute  resistance"  (equal  to  the  force  required  to  open  its  shell 
one  mm.)  at  5026  grams,  while  Marceau  (1909)  reported  that  Mytilns  could  with- 
stand a  pull  of  11.3  kg./sq.  cm.  of  its  adductor  muscle  tissue. 

These  impressive  figures  seem  to  preclude  any  possibility  that  sea  stars  pull  open 
the  shells  of  Ostrea  and  Venus.  But,  on  closer  examination,  Plateau's  "absolute 
resistance"  appears  outstandingly  significant — if  a  force  of  5026  grams  can  produce 
an  opening  of  one  mm.  in  Ostrea,  might  not  a  lesser  pull  be  sufficient  to  open  the 
shell  0.1  mm.,  the  smallest  measured  gape  through  which  sea  stars'  stomachs  have 
been  observed  to  penetrate?  Many  of  the  objections  to  the  mechanical  theory  in 
the  past  have  been  based  on  the  supposition  that  much  larger  gapes  would  be  re- 
quired (Reese  proposed  7  mm.  as  a  minimum),  and  the  fact  that  such  wide  openings 
could  be  effected  only  by  tremendously  strong  forces  which  a  sea  star  could  not 
be  expected  to  exert.  The  experimental  results  described  above  have  shown  that 
Asterias  is  capable  of  producing  pulling  forces  equivalent  to  3100  grams.  It  seems 
likely  that  even  greater  forces  could  be  demonstrated  with  adequate  apparatus. 
Therefore,  there  is  little  reason  to  suppose  that  the  usual  bivalve  prey  of  sea  stars 
cannot  be  opened  by  the  attached  tube  feet,  at  least  enough  for  the  insinuation  of  the 
stomach.  According  to  this  view,  only  very  large  and  highly  resistant  molluscs 
would  be  immune  to  sea  star  predation.  In  fact,  the  larger,  more  resistant 
Mvtilns  cdulis  are  seldom  attacked  successfully  by  sea  stars.  However,  Feder 
(1955)  reports  that  the  larger  M\'tilus  ealifornianus  are  eaten  by  asteroids,  but 
that  entry  into  the  shell  is  gained  by  way  of  the  mussel's  byssus  "door"  which  is 
relatively  wide  in  that  species.  By  contrast,  only  one  among  the  hundreds  of  east 
coast  Asterias  observed  during  this  research  was  seen  to  have  employed  this  ap- 
proach. Feder  also  measured  forces  and  shell  openings  which  closely  approximate 
the  figures  reported  herein. 

It  must  be  emphasized  that  the  observations  made  during  this  investigation  do 
not  support  the  popularly  accepted  notion  that  the  process  of  predation  is  a  "tug-of- 
war"  in  which  the  sea  star  becomes  the  victor  by  virtue  of  its  persistence  and  greater 
endurance.  The  penetration  is  effected,  as  shown  above,  quite  rapidly  and  as  the 
result  of  a  sudden  overwhelming  force,  which  is  relaxed  and  re-applied  at  intervals 
until  digestion  of  the  soft  parts  of  the  bivalve  has  proceeded  to  the  point  where  the 
adductor  muscle  is  rendered  ineffective. 


HOW  SEA  STARS  OPEN  BIVALVES  121 

The  exact  mechanism  responsible  for  the  pulling  force  has  not  been  established. 
However,  it  is  thought  to  reside  in  the  musculature  of  the  tube  feet  described  in  de- 
tail by  Smith  (1937,  1947).  Once  humped  over  the  bivalve,  the  asteroid's  body 
moves  very  little  or  not  at  all,  but  the  tube  feet  are  very  active,  protracting  and  re- 
tracting in  such  a  way  that  they  give  the  impression  of  operating  in  relays.  Each 
tube  foot's  muscular  tissue  is  ample  to  overcome  the  29  grams  of  adhesiveness  of  the 
base  (Paine,  1926).  If  this  value  is  used  as  a  criterion,  then,  it  would  appear  that 
a  sea  star  would  need  to  employ  less  than  one-fourth  of  all  its  tube  feet  simultane- 
ously to  produce  pulls  of  over  5000  grams. 

SUMMARY 

1.  An  investigation  was  made  into  the  possibility  that  sea  stars  secrete  a  sub- 
stance which  is  toxic  or  anesthetic  for  bivalves.     Extracts  prepared  from  the  organs 
of  feeding  and  non-feeding  Astcrias  forbcsi  were  introduced  into  the  adductor  muscle 
and  the  mantle  cavity,  or  perfused  over  the  beating  heart,  of  Mytilits  edulis.     The 
effects  of  such  solutions  were,  generally,  identical  to  those  produced  by  sea  water 
or  distilled  water. 

2.  Sea  stars  were  induced  to  feed  upon  specially  prepared  mussels,  so  that  the 
forces  which  their  tube  feet  exerted  on  the  shells  could  be  measured  manometrically. 
The  adductors  of  the  mussels  used  in  such  experiments  had  been  severed  and  re- 
placed by  steel  springs  or  plastic  cylinders  which  could  not  be  affected  by  any  al- 
leged toxin.     It  was  found  that  the  tube  feet  did  pull  the  valves  apart  and  forces 
of  over  3000  grams  were  recorded.     It  was  observed  also  that  a  very  minute  open- 
ing between  the  valves   (0.1  mm.)  was  sufficient  to  permit  the  insinuation  of  the 
asteroid  stomach. 

3.  The  common  interpretation  of  the  mechanical  theory,  which  asserts  that  the 
sea  star  "fatigues"  the  mollusc,  appears  inaccurate  in  view  of  the  findings  of  this 
research.     There  is  evidence  that  the  opening  of  the  valves  is  a  rapid  process  in- 
volving overwhelming,  discontinuous  forces,  so  that  the  predator  may  be  considered 
to  relax  its  pull  upon  the  valves  at  intervals  and  to  allow  its  stomach  to  be  com- 
pressed between  the  valves  until  it  pulls  them  apart  again. 

LITERATURE  CITED 

ALDRICH,  F.  A.,  1954.     On  the  functional  morphology  of  the  alimentary  canal  of  the  sea  star 

Astcrias  forbesi  Desor.     Ph.D.  thesis,  Rutgers  University. 
ALLEN,    E.    J.,    1896.     How    do   starfishes   open   oysters?     /.    Mar.    Biol.    Assoc.,   4:    266-285. 

(English  translation  of  a  paper  by  Schiemenz,  1895.) 
BARNES,   E.   W.,   1946.     Starfish  menace   in   Southern   Mass,   in    1931.     Bull.   Bingham   Ocean. 

Coll.,  9 :  38-43. 
BELL,  F.  J.,  1892.     Catalogue  of  the  British  echinoderms  in  the   British   Museum.     Longmans 

and  Co.,  London. 

CAHN,  A.  R.,  1950.     Oyster  culture  in  Japan.     Fisheries  Leaflet.  383  :   1-80.     Washington. 
EUDES-DESLONGCHAMPS.    H.,    1826.     Notes    sur    1'Asterie    commune.     Ann.    Sci.    Nat.    Paris 

(Zoologie),  9:  219-221. 
FEDER,  H.  M.,  1955.     On  the  methods  used  by  the  starfish  Pisastcr  ochraceus  in  opening  three 

types  of  bivalve  molluscs.     Ecology.  36 :  764-767. 
FIGUIER,  L.,  1891.     The  ocean  world.     Cassell  and  Co.,  London. 
FISCHER,   P.,   1864.     Faune  conchiologique   marine   du   Department   de   la   Gironde.     Act.   Soc. 

Linn.  Bordeaux,  25  :  257-344. 


122  MARCEL  E.  LAVOIE 

GALTSOFF,  P.  S.,  1952.     How  strong  is  the  oyster?     Addresses  delivered  to  the  National  Shell 

Fisheries  Association,  pp.  51-53. 
GALTSOFF,   P.   S.,  AND  V.   L.   LOOSANOFF,   1939.     Natural  history  and  control  of  the  starfish. 

U.  S.  Bur.  Fish,  Bull.,  31 :  75-132. 

HESZ,  W.,  1878.     Die  werbellosen  Tiere  des  Meeres.     Hanover. 
VAN  DER  HEYDE,   H.   C.,   1922.     On  the  physiology  of  digestion,   respiration,  and  excretion  in 

echinoderms.     C.  de  Boer  Jr.,  Amsterdam. 
KORRINGA,  P.,  1953.     Oysters.     Sci.  Amcr.,  189:  86-91. 

MARCEAU,  F.,  1909.     Contraction  of  molluscan  muscle.     Arch.  Zool.  Exp.  Gen.,  2:  295-469. 
PAINE,  V.  L.,  1926.     Adhesion  of  the  tube  feet  in  starfishes.     /.  Exp.  Zool.,  45:  361-366. 
PIERON,  H.,  1913.     Sur  la  maniere  dont  les  poulpes  viennent  a  bout  de  leur  proie,  des  lamelli- 

branches  en  particulier.     Arch,  dc  Zool.  E.rp.  Gen.,  53  :  1-13. 
PLATEAU,  F.,  1884.     Force  absolue  des  muscles  des  invertebres.     Arch,  dc  Zool.  E.\-p.,  12:  145- 

170. 

REESE,  A.  M.,  1942.     The  old  starfish-clam  question.     Science,  96:  513. 
SAWANO,  E.,  AND  K.  MITSUGI,  1932.     Toxic  action  of  the  stomach  extracts  of  the  starfishes  on 

the  heart  of  the  oyster.     Sci.  Rep.  Tohoku  Imp.  Unit'.,  1 :  79-88. 
SCHIEMENZ,   P.,   1895.     Wie  off  en  die   Seestern   Austern?     Mittheilungen  des   Deutschen   See- 

fishcherievereins  Bd.   12  No.  6:    102-118.     (Translated  into  English  by  Allen,   1896.) 
SMITH,  J.  E.,  1937.     The  structure  and  function  of  the  tube  feet  in  certain  echinoderms.    /.  Mar. 

Biol,  Assoc.,  22  :  345-357. 
SMITH,  J.  E.,  1947.     The  activities  of  the  tube  feet  of  Astcrias  rubcns  L. ;   I.   The  mechanics 

of  movement  and  posture.      Quart.  J.  Micr.  Sci.,  88  :  1-14. 
TAMURA,  T.,  1929.     The  power  of  the  adductor  muscle  of  the  oyster,  Ostrca  circumpicta.     Sci. 

Rep.  Tohoku  Imp.  Univ.,  4:  259-279. 


STUDIES  ON  MARINE  BRYOZOA.     VIII.  EXOCHELLA 
LONGIROSTRIS  JULLIEN  1888 

MARY  ROGICK 
College  of  AYn-  Rochdlc.  New  Rochdlc.  N.  V. 

The  writer  wishes  to  express  deep  gratitude  to  the  National  Science  Foundation 
for  research  grants  aiding  this  and  other  studies  and  to  the  Smithsonian  Institution, 
U.  S.  N.  M.,  for  the  loan  of  bryozoan  specimens  collected  during  the  U.  S.  Navy's 
1947-48  Antarctic  Expedition  by  Comdr.  David  C.  Nutt. 

The  purpose  of  this  study  is  to  report  E.rochclla  longirostris  Jullien  1888  (order 
Cheilostomata,  Family  Exochellidae)  from  the  Antarctic,  to  raise  some  questions 
about  its  synonymy  and  to  add  further  morphological  and  ecological  data  to  the 
limited  information  existing  on  this  species. 

E.rochclla  longirostris  Jullien  1888 

(Figures  1  A-J) 
Synonymy  and  distribution  data: 

1888.     E.vocliclla  longirostris.     Jullien  pp.  55-56,  PI.  3,  Figs.  1-4;  PI.  9.  Fig.  2. 

From  He  Hoste,  baie  Orange,  Canal  du  Beagle,  He  Gable,  Tierra  del 

Fuego.     19  meters. 

1904.     E.  longirostris.     Calvet  p.  29.     Magellan  Straits,  Punta  Arenas. 
1908.     E.  longirostris.     Canu  p.  300,  PI.  VI,  Fig.  13.     From  Post-Pampeen  de 

Punta  Borja,  Puerto  Militar,  Bahia  Blanca  (Argentina). 
1937.     E.  longirostris.     Marcus  pp.  82-83;  PI.   17,  Fig.  43.     Bay  of  Santos, 

Brazil ;  20  meters. 
1941.     E.    longirostris.     Marcus    p.    22;    Fig.    16.     Sta.    Catharina,    Parana; 

Guaratuba. 
1949.     E.  longirostris.     Marcus  p.  1.     South  of  Victoria,  Espirito  Santo,  Lat. 

20°33'S.,  Long.  40°14'W. ;  35  meters. 
1952.     E.  longirostris.     Mawatari  p.  265.     Wakayama  Prefecture,  Shirahama 

and  Tonda,  Japan. 

Some  difficulty  was  encountered  in  the  identification  of  this  species  because 
lullien's  original  description  was  inadequate.  Externally,  the  USNM  specimens 
resemble  those  pictured  by  Jullien  (1888)  and  Canu  (1908)  but  these  authors  did 
not  figure  the  internal  aspect  of  the  primary  orifice,  a  very  important  diagnostic 
character.  Levinsen  (1909,  p.  321;  PI.  17,  Figs.  6a— c)  beautifully  and  completely 
described  an  E. \~ochella  longirostris  from  Challenger  Sta.  315,  Falkland  Islands. 
However,  it  is  not  at  all  certain  that  Levinsen  and  Jullien  were  describing  the  same 
species.  Levinsen  pictured  a  distinct  lyrula  on  the  proximal  border  of  the  primary 

123 


FlCURE    1, 


.MARINE  BRYOZOA,  VIII.  125 

orifice,  while  Jullien  stated  that  the  orifice  is  rounded  and  that  its  peristome  is  pro- 
longed forward  and  backward.  The  USNM  specimens  do  not  show  such  a  promi- 
nent structure  so  immediately  within  the  primary  orifice  but  do  show  the  peristome 
thickened  medially  to  simulate  a  lyrula  a  little  in  front  of  the  primary  orifice  border. 
Waters's  notations  on  this  species  (1889  E.  longirostris  and  1906  Sniittia  longi- 
rostris}  are  not  precise  enough  for  one  to  be  able  to  determine  if  he  actually  had 
Jullien's  species,  so  are  not  included  in  the  present  synonymy.  Marcus'  Bay  of 
Santos  specimens  are  considerably  smaller  in  all  parts  (zooecia,  apertures,  ovicells, 
avicularia)  than  the  USNM  specimens,  judging  from  the  scale  accompanying 
Marcus'  PI.  17,  Fig.  43.  His  avicularia  seem  much  thinner  than  those  of  the 
present  specimens.  Finally,  the  peristomial  processes  appear  to  be  thinner  and 
sharper  than  those  of  the  USNM  material.  In  spite  of  these  differences,  it  is  be- 
lieved that  these  belong  to  the  same  species  and  that  the  differences  are  due  to  eco- 
logical and  geographical  factors,  the  USNM  Antarctic  specimens  showing  the  stur- 
diest and  largest  specimens  of  this  variable  form. 

Diagnosis:  Zoarium  encrusting,  heavily  calcified.  Zooecial  boundaries  distinct. 
Convex  frontal  an  areolate  pleurocyst,  somewhat  ribbed  in  old  zoids.  Ovicells  non- 
porous,  covered  over  by  the  frontal  of  the  next  distal  zoid.  Avicularia  adventitious, 
pointed,  medium-sized,  frontal,  not  peripheral  nor  over  an  areolar  pore;  one,  two 
or  none  may  occur  on  a  zoid.  Mandible  long,  triangular,  sharply  pointed.  Peri- 
stome incomplete  distally  in  ovicelled  zoids.  Raised  peristome  develops  a  mucro, 
sometimes  medially  thickened  to  simulate  a  lyrula.  Peristomial  sinus  on  each  side 
of  the  mucro.  Peristomial  side  walls  raised,  sometimes  pressing  inward.  Primary 
orifice  has  a  hemispherical  vestibular  arch.  Immediate  lyrula  and  cardelles  absent. 

All  figures  are  drawn  with  the  aid  of  a  camera  lucida. 

FIGURE  1.     Exochella  longirostris  Jullien  1888,  from  the  Antarctic. 

A.  A  zooecium  at  growing  edge  of  colony.     Thin  young  peristome  still  incomplete  in  back, 
with  two  sinuses  and  a  lip-like  mucro  proximally.     Mandible  opened.     One  dietella  (broad  dis- 
tal pore)  shown  at  top.     Drawn  to  the  0.2  mm.  scale  above. 

B.  Operculum,  drawn  to  the  Figure  G  scale. 

C.  Mandible,  drawn  to  the  0.1  mm.  scale  at  left. 

D.  Avicularium  with  membranous  part  burned  off.     Drawn  to  the  Figure  G  scale. 

E.  An  old  ovicelled  zooecium.     The  very  thick  peristome  is  worn  away  in  front  and  at 
right.     The  projection  simulating  a  lyrula  is  not  a  true  lyrula  but  a  thickened  "core"  of  the 
mucro,  a  thickening  of  the  proximal  peristomial  wall  and  characteristic  of  the  most  heavily 
calcified  zooecia.     Drawn  to  the  Figure  A  scale. 

F.  Another  young  incomplete  peristome.     The  mucro  is  more  pointed  and  the  side  walls 
press  in  more  acutely  than  in  Figure  A.     Drawn  to  the  0.1  mm.  scale  above. 

G.  An  avicularium  with  mandible  in  place.     Membranous  "back"  area  in  black.     Drawn  to 
the  0.2  mm.  scale  at  right. 

H.  The  primary  orifice  in  black,  vestibular  arch  above,  and  compensation  sac  area  below, 
as  seen  from  inside  the  zooecium.  Drawn  to  the  Figure  F  scale. 

I.  Four  zooecia  seen  from  the  attached  basal  side.  Each  has  four  dietellae  (large  heavily 
stippled  distal  pores).  Five  small  areolar  pores  are  on  the  lower  corner  of  the  left  zoid  whose 
basal  wall  has  broken  away  there.  The  zooecium  at  right  has  the  remains  of  the  operculum 
and  tentacular  sheath  suspended  from  the  orifice.  Compensation  sac  area  also  plainly  visible  in 
all.  Drawn  to  the  0.5  mm.  scale  below. 

J.  Nine  old  zooecia.  The  upper  three  and  the  lowest  one  are  non-ovicelled.  The  remain- 
ing five  have  non-porous  ovicells  more  or  less  undistinguishable  from  the  frontal  of  their  dis- 
tal zooecia.  They  can  be  recognized  by  the  incomplete  distal  wall  of  the  peristome.  One,  two 
or  no  avicularia  may  be  present  per  zooecium.  They  are  not  areolar  but  are  more  central  in 
location.  Drawn  to  the  Figure  I  scale. 


126  MARY  ROGICK 

Operculum  forms  three  fourths  of  a  circle,  with  proximal  edge  bevelled.     Compen- 
sation sac  about  the  size  of  the  primary  orifice.     Three  to  five  dietellae. 

Measurements.  The  first  figures  are  the  minimum,  the  next  the  maximum  and 
the  last,  in  parentheses,  the  average  of  10  readings  for  each  structure  (except  for 
the  avicularia  whose  averages  are  based  on  30  readings).  Length  and  width  are 
abbreviated  to  L  and  W.  Readings  are  in  millimeters. 

0.734-0.979  (0.888)  Zooecia  L 

0.605-0.922  (0.736)  Zooecia  W 

0.158-0.259  (0.204)  Avicularia  L 

0.072-0.130  (0.102)  Avicularia  W 

0.115-0.147  (0.131)  Primary  orifice  L 

0.144-0.166  (0.155)  Primary  orifice  W 

0.144-0.173  (0.153)  Secondary  orifice  L,  including  sinus 

0.101-0.144  (0.124)  Secondary  orifice  L,  exclusive  of  sinus 

0.144-0.173  (0.153)  Secondary  orifice  W 

0.302-0.360  (0.334)  Ovicell  L 

0.360-0.418  (0.382)  Ovicell  \Y 

0.137-0.158  (0.147)  Operculum  L 

0.130-0.173  (0.154)  Operculum  W 

0.128-0.151  (0.137)  Mandible  L 

0.058-0.073  (0.068)  Mandible  W 

0.115-0.158  (0.130)  Compensation  sac  area  L 

0.122-0.158  (0.143)  Compensation  sac  area  W 

Zoariiun.  The  ivory-colored,  heavily  calcified  zoarium  is  sturdy  and  sometimes 
extensive.  A  25  X  36-mm.  pebble  had  one  surface  completely  encrusted  by  one 
colony.  Colonies  are  unilaminate,  forming  a  thick  crust,  usually  numbering  many 
zoids.  Polypide  remains  present  in  some. 

Zooecia.  The  hexagonal  zooecia  are  distinct  and  sizable.  Some  are  ovicelled, 
some  not;  some  have  avicularia,  others  do  not.  From  the  basal  aspect  (Fig.  1,1). 
the  three  distal  walls  are  convex,  the  three  proximal  walls  concave.  The  thick 
frontal  is  a  granular  to  beaded  pleurocyst.  Ridges  arise  between  its  closely  spaced 
elliptical  areolar  pores  and  continue  part  way  up  the  frontal  (Fig.  1,  F,  J).  The 
compensation  sac  area  is  small  and  immediately  below  the  orifice  (Fig.  1,  H,  T). 
The  basal,  attached  zooecial  surface  has  3  to  5,  usually  4,  large  oval  dietellae 
(Fig.  1,  I). 

Avicularia.  One  or  two  frontal  avicularia  occur  on  many  of  the  zoids.  Their 
orientation  is  variable  on  the  solid  part  of  the  frontal.  They  are  not  oral  nor  areolar 
though  some  occur  fairly  close  to  the  zooecial  edge.  Others  are  more  central  (Fig. 
1,  J).  The  small  avicularial  chamber  tips  the  beak  upward  along  a  modest  slope. 
The  avicularia  are  always  of  the  same  type  and  of  fairly  uniform  size.  Their  back 
area  is  hemispherical,  the  beak  triangular  and  longer.  The  mandible  is  a  narrow 
triangle,  with  the  two  long  sides  concave  (Fig.  1,  C,  G),  and  edges  reinforced.  The 
USNM  avicularia,  though  larger  in  actual  measurements  than  those  of  Marcus' 
species,  are  smaller  in  proportion  to  the  rest  of  the  /.ooecial  front  than  are  Marcus' 
specimens. 

Orifice.     The  orifice  is  not  terminal  but  a  slight  distance  short  of  that.     Its 


MARINE  BRYOZOA,  VIII.  127 

distal  wall  is  not  formed  by  the  next  distal  zoid.  The  deeply  set  primary  orifice  is 
slightly  more  than  hemispherical,  with  a  handsome  vestibular  arch  and  a  nearly 
straight  proximal  border  (Fig.  1,  H).  The  chitin-rimmed  operculum  has  the  same 
shape  (Fig.  1,  B).  Lyrula  and  cardelles  are  absent  in  the  primary  orifice  but  the 
peristome  immediately  in  front  of  the  operculum  simulates  a  lyrula.  This  appears 
to  be  at  variance  with  Levinsen's  figures  which  show  a  lyrula  apparently  right  on  the 
border  of  the  primary  aperture.  Whether  or  not  this  is  the  condition  of  Jullien's 
original  material  is  unknown.  The  secondary  orifice  shape  is  variable,  depending 
on  the  degree  of  calcification,  being  sometimes  trifoliate,  sometimes  horseshoe- 
shaped  (Fig.  1,  E,  F,  J).  The  distal  peristome  wall  is  entire  in  mature  non-ovi- 
celled  zoids  but  interrupted  by  the  ovicell  in  fertile  ones.  The  peristome  thickens 
considerably  with  age.  Proximally  the  peristome  develops  a  tab-like  mucro  (Figs. 
A,  F)  bordered  on  each  side  by  a  sinus.  The  mucro  may  thicken  medially  to  such 
an  extent  inward  that  it  could  be  easily  mistaken  for  a  lyrula  (Fig.  1,  E).  Later- 
ally, the  peristomial  wall  may  or  may  not  pinch  in  (Fig.  1,  F). 

Oricclls.  Young  ovicells  are  salient,  old  ones  heavily  calcified  and  immersed. 
They  are  not  porous  but  some  are  bordered  laterally  by  a  few  areolar  pores  which 
do  not  penetrate  the  ovicell  wall  proper.  The  ovicell  surface  is  granular  to  beaded, 
occasionally  irregularly  ridged  (Fig.  1,  E).  No  avicularia  occur  on  the  ovicells  nor 
does  the  peristome  encroach  upon  them  but  the  frontal  of  the  next  distal  zoid  covers 
the  ovicell  front  completely. 

Distribution  and  ecology.  This  species'  most  northerly  record  (and  the  only 
one  for  the  northern  hemisphere)  is  that  of  Mawatari  (1952)  from  Japan.  All 
other  previous  records  are  from  the  southern  hemisphere,  ranging  from  20°33'S. 
Lat.  (Marcus,  1949,  south  of  Victoria,  Brazil)  to  about  55°40'S.  Lat.  (Jullien, 
1888,  Tierra  del  Fuego,  He  Hoste). 

The  USNM  specimens  appeared  on  a  rock  from  Sta.  184  and  on  pebbles  Nos. 
2,  3,  4,  12,  13  and  16  from  an  unidentified  Antarctic  locality  (Comdr.  D.  C.  Nutt, 
U.  S.  Navy's  1947-48  Antarctic  Expedition).  Station  184  was  at  Marguerite  Bay, 
Antarctica,  location  approximately  6S°30'W.  Long,  and  68°30'S.  Lat.,  bottom 
dredge  haul,  depth  85-100  fathoms,  water  temperature  30.2°  F.,  Feb.  19,  1948. 
This  represents  the  most  southerly  and  deepest  record  for  the  species  and  the  first 
time  it  was  collected  well  within  the  Antarctic  Circle.  Some  of  the  USNM  col- 
onies have  grown  over  Foraminifera,  incorporating  their  shells  within  the  zooecial 
base.  Sponge  spicules  are  matted  over  one  colony,  calcareous  worm  tubes  and  oc- 
casional bryozoan  zoids  (of  other  species)  are  present  on  other  colonies.  How- 
ever, most  of  the  colony  surface  is  free  of  extraneous  growths.  The  Antarctic 
specimens  appear  to  be  much  larger,  thicker-walled  and  more  sturdy  than  those 
from  warmer  localities.  The  present  study  specimens  are  on  deposit  at  the  U.  S. 
Nat.  Museum,  Smithsonian  Institution,  Cat.  Nos.  11325,  11326,  11327  and  11328. 

Affinities.  E.vochcUa  lonf/irostris  Jullien  1888  and  a  fossil  species  E.  grandis 
Canu  and  Bassler  (1935,  p.  32,  PI.  9,  Fig.  3)  from  the  Tertiary  Balcombian  Beds 
of  Muddy  Creek.  Victoria,  Australia,  appear  to  be  closely  related.  The  USNM 
specimens  are  similar  in  size  and  measurements  to  E.  (/nindis  but  lack  the  prominent 
mural  thread  and  the  very  conspicuous  beading  of  the  pleurocyst.  In  E.  grandis 
the  avicularia  replace  the  areolar  port's  but  in  the  USXM  /:.  longirostris  they  gen- 
erally do  not  and  are  less  peripheral. 


128  MARY  ROGICK 

SUMMARY 

1.  The  geographic  range  of  Exochclla  longirostris  is  extended  to  the  Antarctic. 

2.  The  Antarctic  specimens  are  sturdier,  larger  and  thicker-walled  than  those 
of  the  same  species  from  warmer  waters  and  have  avicularia  which  are  a  bit  smaller 
proportionately,  although  larger  in  actual  measurements. 

3.  Numerous  measurements  of  various  structures  and  zooecia  are  included,  to 
show  the  range  of  variation  for  this  species. 

LITERATURE  CITED 

CALVET,  L.,  1904.     Bryozoen.     Hamburg.  Magalhaens.  Sammelreise,  45  pp. 

CANU,  F.,  1908.  Iconographie  des  Bryozoaires  fossiles  de  1'Argentine.  An.  Mus.  Nac.  Buenos 
Aires,  vol.  17,  (ser.  3,  vol.  10)  :  245-341. 

CANU,  F.,  AND  R.  S.  BASSLER,  1935.  New  species  of  tertiary  cheilostome  bryozoa  from  Vic- 
toria, Australia.  Sniithson.  Misc.  Coll..  93  (3)  :  1-54.  Publ.  3302. 

JULLIEN,  J.,  1888.     Bryozoaires.     Miss,  du  Cap  Horn,  6(1,):  1-92. 

LEVINSEN,  G.  M.  R.,  1909.  Morphol.  and  systematic  studies  on  cheilostom.  bryozoa.  Copen- 
hagen. 431  pp. 

MARCUS,  E.,  1937.  Bryoz.  marin.  brasileiros,  I.  Univ.  Sao  Paulo,  Bol.  Fac.  Philos.,  Sci.  c 
Lctr.  1,  Zoo/.,  1 :  5-224. 

MARCUS,  E.,  1941.  Bryoz.  marin.  do  litoral  paranaense.  Arq.  do  Mus.  Paranaense,  I  (1): 
7-36. 

MARCUS,  E.,  1949.  Some  bryozoa  from  the  Brazilian  coast.  Comm.  Zool.  Mus.  Nat.  Hist. 
Montevideo,  3  (53)  :  1-33. 

MAW  ATARI,  S.,  1952.     Bryozoa  of  Kii  Peninsula.     Publ.  Seto  Mar.  Biol.  Lab.,  II   (2)  :  261-288. 

WATERS,  A.  W.,  1889.  Bryozoa  from  New  South  Wales.  Ann.  Mag.  Nat.  Hist.,  (4)  4:  1-24, 
PI.  1-3. 

WATERS,  A.  W.,  1906.  Bryozoa  from  Chatham  .  .  .  Islands.  Ann.  May.  Nat.  Hist.,  (7)  17  : 
12-23,  PI.  1. 


MICROGEOGRAPHIC  VARIATION  AS  THERMAL  ACCLIMATION 

IN  AN  INTERTIDAL  MOLLUSC 

EARL  SEGAL  L  - 
Department  of  Biology,  Kansas  State  Teachers  College,  Emporia,  Kansas 


• 


A  growing  body  of  literature  suggests  that  many  poikilotherms  are  able  to  regu- 
late, to  a  remarkable  degree,  their  physiological  activity  rates.  Regardless  of  the 
latitude  over  which  certain  species  are  distributed  or  the  seasonal  temperature 
change  to  which  they  are  subjected,  their  physiological  rates  converge  towards  a 
mean  value.  To  accomplish  this  relative  constancy,  northern  populations  and 
winter  forms  often  have  higher  rates  of  activity,  metabolism  and  development  than 
southern  populations  and  summer  forms  when  measured  at  the  same  temperature. 
Animals  thermally  conditioned  in  the  laboratory  have  also  shown  this  compensatory 
phenomenon. 

Ample  documentation  is  provided  in  the  comprehensive  review  of  Bullock 
(1955),  who  also  presents  data  showing  temperature  adaptation  at  the  tissue,  cellular 
and  enzyme  level,  and  in  that  of  Prosser  (1955),  who  summarizes  evidence  of  com- 
pensatory adjustment  to  oxygen  tension,  osmotic  pressure  and  drugs,  as  well  as  to 
temperature.  Roberts  (1952)  and  Dehnel  (1955),  who  themselves  have  con- 
tributed studies  of  this  problem,  give  additional  references. 

In  1953,  Segal,  Rao  and  James  extended  the  known  cases  of  intraspecific  physio- 
logical differentiation  with  respect  to  temperature  to  include  microgeographically 
separated  individuals  of  the  species.  The  heart  rate  in  the  limpet  Acmaea  limatula 
and  water  propulsion  in  the  mussel  Mytilus  californianus  were  faster  in  samples 
from  low  intertidal  levels  than  from  high  levels  at  any  given  temperature. 

In  the  present  study,  an  attempt  has  been  made  to  corroborate  and  extend  the 
initial  findings  on  A.  limatula  and  to  ascertain  whether  the  significant  parameter  of 
the  difference  in  microhabitats  is  temperature.  Besides  heart  rate,  differences  in 
gonad  size  and  spawning  readiness  have  been  investigated.  A  major  portion  of  this 
study  constitutes  an  attempt  to  test  the  hypothesis  that  we  are  dealing  with  individual 
adaptations  to  habitat  temperature. 

MATERIALS  AND  METHODS 
Habitat 

The  aspidobranch  gastropod  Acmaea  limatula  Carpenter  is  a  eurytopic  intertidal 
species  which  at  Palos  Verdes,  California  (Lat.  33°  43'  N.,  Long.  118°  16'  W.)  has 
a  vertical  distribution  of  approximately  1%  meters  from  a  mid-tidal  to  a  low-tidal 
level.  Highest  and  lowest  individuals  may  be  separated  by  as  much  as  20  meters 

1  The  work  was  performed  in  the  Department  of  Zoology,  University  of  California,  Los 
Angeles,  Calif. 

- 1  wish  to  thank  Dr.  T.  H.  Bullock  for  his  encouragement  and  guidance  throughout  the 
course  of  this  investigation. 

129 


130  EARL  SEGAL 

of  sloping,  rocky  beach ;  the  nature  of  the  beach  prohibits  interchange  between 
higher  and  lower  levels. 

At  each  of  four  collecting  sites  (referred  to  in  the  text  as  sites  1,  2,  3,  and  4) 
the  low-level  specimens  were  taken  from  below  zero  datum  (mean  lower  low  water: 
U.  S.  Coast  and  Geodetic  Survey  Tide  Tables,  Pacific  Coast)  where  they  are  at  the 
temperature  of  the  surf  but  for  a  few  hours  each  month.  The  higher  level  speci- 
mens were  taken  1  to  ll/2  meters  above  zero  datum  where  they  are  subject  to  ex- 
posure about  50%  of  the  time. 

Collection  and  care  of  animals 

Animals  were  removed  from  the  substrate  with  a  thin  spatula  and  transported 
wet  to  the  laboratory  in  enamel  or  plastic  trays.  In  the  laboratory  the  animals  were 
covered  with  fresh  sea  water,  aerated,  and  refrigerated  at  temperatures  approxi- 
mating the  average  ocean  temperature  for  that  season  (see  Fig.  7).  Each  day  the 
water  was  replaced  with  fresh  sea  water  at  the  same  temperature. 

During  the  winter  and  spring  months  an  attempt  was  made  to  approximate  the 
natural  exposure  time  for  high-level  individuals.  The  water  was  poured  off  and 
the  animals  allowed  to  warm  to  room  temperature  and  stand  for  5-6  daylight  hours. 

Heart  exposure 

No  later  than  24  hours  after  collection,  the  heart  was  exposed  by  cutting  a  hole  in 
the  shell  to  the  left  and  slightly  posterior  to  the  shell  apex  with  a  fine  toothed 
trephine.  This  tool  was  designed  to  take  different  sized  cutting  heads :  3  mm.  in 
diameter  for  cutting  small  shells,  41/v>  mm.  in  diameter  for  cutting  larger  shells.  A 
retractable  pin  in  the  center  of  the  trephine  prevented  the  cutting  edge  from  wander- 
ing. The  surface  of  the  mantle  exposed  by  the  hole  was  flushed  clean  of  shell  par- 
ticles with  a  fine  stream  of  sea  water. 

Each  animal  was  numbered  with  colored  lacquer,  then  placed  in  a  10-inch  finger 
bowl  (15  to  20  individuals  per  bowl)  which  contained  l-l1/^  inches  of  sea  water. 
The  animals  were  returned  to  the  refrigerator  for  one-two  days  to  allow  the  gut  to 
empty  and  permit  recovery  of  the  animals  from  any  possible  operative  shock. 

Recording  procedure 

The  day  heart  beats  were  counted,  two  finger  bowls  were  placed  on  a  wire  mesh 
platform  two  inches  below  the  surface  in  a  15-gallon  aquarium.  Water  circulated 
through  the  aquarium  at  a  constant  temperature  ±  0.2°  C. 

Initially,  the  water  bath  was  at  the  refrigerator  temperature  and  was  then  gradu- 
ally lowered  to  4°,  7°,  or  9°  C.  Two  to  four  hours  were  allowed  for  the  animals  to 
reach  the  lower  temperatures.  Temperatures  were  raised  by  increments  of  5°  C. 
to  a  maximum  of  29°  C.  Animals  were  allowed  two  hours  to  reach  each  tempera- 
ture. 

Using  a  stop-watch,  the  number  of  seconds  required  for  10  heart  beats  was 
counted  by  eye.  At  lower  temperatures  a  reading  was  taken  of  each  animal  in 
the  group  and  this  was  repeated  a  second  and  third  time.  At  higher  temperatures, 
because  of  the  greater  possibility  of  error  due  to  the  increased  heart  rate,  the  read- 


MICROGEOGRAPHIC  VARIATION 


131 


ings  were  taken  5  times.     Plotted  points  (see  Figs.  1,  3,  and  4),  are  the  average  of 
these  data  converted  to  beats  per  minute. 

The  basic  measurement  used  in  this  study  is  stable  and  reliable.  A  few  minutes 
after  the  operation,  which  does  not  break  the  mantle,  the  heart  rate  settles  to  a 
value  which  is  consistent  over  many  hours  and  even  days.  Those  animals  showing 
excessive  locomotor  activity  or  irregularity  of  heart  activity  were  discarded. 


50  - 


30 


40 


530 

UJ 

X 


£8° 


low-  level 


high-  level 


sample    of 
Dec.   17,  1953 


low-  level 


high-  level 


sample     of 
Oct.  7,  1953 


0.3  0.4  0.5  0.6         0.7        0.8      0.9     1.0     I.I     1.2 

WET      WT.      OF       SOFT        PARTS       IN      GRAMS 

FIGURE  1.  Relation  between  heart  rate  and  wet  weight  of  soft  parts  at  14°  C.  for  vertically 
separated  A.  liinatnla  from  Palos  Verdes.  Points  are  averages  of  3  to  5  readings  of  the  number 
of  seconds  per  10  beats  for  one  individual.  The  December  collection  shows  a  near  maximum 
excursion  of  the  difference  in  heart  rate  between  samples.  The  October  collection  shows  the 
minimum  difference.  Co-ordinates  are  logarithmic ;  equal  percentage  deviation  is,  therefore, 
shown  by  equal  spatial  spread  from  the  hand-drawn  regression  lines  anywhere  on  the  graph. 
All  curves  are  eye-fitted. 

RESULTS 
Effect  of  Intcrtidal  Height  on  Heart  Rate 

When  the  animals'  heart  rates  are  measured  at  temperatures  from  4°-29°  C..  it  is 
found  that  the  lower  in  the  range  of  distribution  an  animal  lives  the  faster  is  its  heart 
rate.  A  comparison  is  made  of  the  heart  rates  of  "highs"  and  "lows"  and  the  near 
maximum  and  the  minimum  difference  in  rate  between  samples  are  illustrated  in 
Figure  1 .  At  lower  temperatures  the  absolute  difference  in  heart  frequency  between 
"highs"  and  "lows"  is  less  than  it  is  at  higher  temperatures. 

When  heart  rate  is  plotted  against  temperature,  it  can  be  seen  that  the  curve 
for  a  low-level  animal  is  above  that  for  an  equal  weight  individual  from  high-level 


132 


EARL  SEGAL 


(Fig.  2).  Since  heart  frequency  will  be  shown  to  vary  with  habitat  and  season, 
rates  at  successive  collections  could  not  be  combined.  The  pair  of  rate/temperature 
curves  in  Figure  2  have  been  selected  from  the  many  pairs  calculated  because  they 
show  the  response  both  over  the  greatest  range  of  temperatures  and  the  largest 
number  of  temperature  points. 

When  both  groups  are  tested  at  any  temperature,  within  the  physiological  ranges 
of  temperatures  of  the  species,  heart  rates  of  low-level  animals  are  as  much  as  30  to 
40%  faster  than  those  of  equal  weight  animals  from  high-level.  Heart  rates  of 
equal  value  for  "highs"  and  "lows"  are  obtained  when  the  high-level  individuals  are 


140 
120 

100 
90 

80 

z 

S    70 
<"  60 


UJ 

CD  50 


40 


4 
cc 


30 


K 

< 

id 
X 


20  - 


low-  level 


high-  level 


from      somples      of 
Dec.    17,  1953 


24 


2  9 


TEMPERATURE 


FIGURE  2.  Heart  rate  as  a  function  of  temperature  for  equal  weight  (0.6  gm.)  A.  limatula 
from  December  collection  of  high-  and  low-level  samples.  Points  represent  intersection  of 
perpendicular,  erected  at  0.6  gm.,  with  weight  regression  curves  at  each  temperature. 

measured  at  temperatures  l°-5%°  C.  above  that  of  low-level  individuals.  In  Feb- 
ruary, for  example  (not  graphed),  a  low-level  individual  of  average  weight  (0.6 
gm.)  has  a  heart  rate  of  53  beats  per  minute  at  14°  C.  An  average  high-level  indi- 
vidual of  equal  weight  shows  the  same  rate  when  measured  at  19.5°  C. 

In  addition  to  the  difference  in  position  of  the  rate-temperature  curves,  there 
appears  to  be  a  reliable  difference  in  the  slopes  as  measured  by  the  Q10.  Between 
9°  and  19°  C.,  perhaps  even  above  19°  C.,  the  O10  of  the  heart  rate  is  consistently 
lower  for  low-level  animals  during  the  winter  and  spring  months  (Table  I). 

It  would  be  of  value  to  be  able  to  state  conclusively  whether  high-level  indi- 
viduals of  the  species  are  living  at  a  warmer  temperature  than  low-level  individuals, 
at  least  within  the  local  coastal  area.  An  attempt  will  be  made  to  establish  this 
point. 


MICROGEOGRAPHIC  VARIATION 


133 


High-level  individuals  are  submerged  approximately  50f/r  of  the  time.  During 
the  hours  of  exposure  these  individuals  are  subjected  to  air  temperatures  which 
fluctuate  about  the  prevailing  water  temperatures.  A  series  of  readings  (taken 
with  a  thermistor  probe,  one  mm.  in  diameter,  inserted  under  the  foot  of  limpets  in 
place  in  the  field)  on  a  sunny  day  in  late  October  show  that  body  temperatures 

TABLE  I 

Qio  values  of  the  heart  rate  for  0.6  gm.  high-  and  low-level  A.  limatula  over  the  temperature  ranges 
indicated.      Values  calculated  for  equal  weight  animals  on  the  weight  regression  curves  at  each 

collection  period 


Qio 

Date 

High-level 

Low-level 

7/27/53 

9-14 

3.40 

3.14 

14   19 

2.56 

2.53 

19-24 

1.99 

1.88 

10/7/53 

9-14 

3.22 

3.10 

14-19 

2.38 

2.89 

19-24 

1.73 

1.77 

12/17/53 

9-14 

2.62 

2.30 

14-19 

2.34 

1.98 

19-24 

2.02 

1.97 

1/4/54 

9-14 

2.70 

2.49 

14-19 

2.18 

2.16 

19-24 

1.80 

1.62 

2/1/54 

9-14 

2.57 

2.34 

14-19 

2.54 

1.97 

19-24 

1.77 

1.80 

2/15/54 

9-14 

2.67 

2.54 

14-19 

2.38 

2.05 

19-24 

1.83 

1.95 

4/6/54 

9-14 

2.74 

2.37 

14-19 

2.02 

1.78 

19-24 

1.59 

1.73 

5/8/54 

9-14 

2.51 

2.37 

14-19 

2.11 

2.02 

19-24 

1.76 

1.65 

reached  a  high  of  30°  C.  in  the  sun  and  21.5°  C.  in  the  shade.  Body  temperatures 
may  have  reached  higher  values  since  the  animals  were  exposed  for  an  additional 
two  hours.  Over  a  20-year  period  (U.  S.  Weather  Bureau,  personal  communica- 
tion) 40  to  50%  of  the  days,  from  October  through  April,  have  been  sunny  at  the 
local  beaches.  From  November  through  April  the  surface  water  temperature 
averages  slightly  over  14.5°  C. 


134  EARL  SEGAL 

Reference  to  Figure  8  shows  that  the  annual  range  of  the  inshore  surface  water 
temperatures  averages  approximately  7°  C.  Air  temperatures,  taken  a  few  feet 
above  ground,  show  daily,  and  therefore  monthly,  fluctuations  exceeding  the  yearly 
temperature  range  of  the  inshore  waters.  Of  course,  air  temperatures  a  few  feet 
above  ground  give  only  a  rough  directional  estimate  of  microhabitat  temperatures 
one  cm.  above  ground.  Over  a  period  of  \%  years,  dry  bulb  recordings  have  shown 
the  microhabitat  temperatures  to  be  consistently  higher  than  prevailing  air  tem- 
peratures. 

Minimum  air  temperatures  falling  below  ocean  temperatures  are  encountered 
primarily  from  late  spring  to  early  autumn  when  low  tides  generally  expose  high- 
level  animals  during  late  evening  and  early  morning  hours.  During  the  part  of  this 
period  when  the  ocean  temperature  is  above  17°  C.,  the  difference  in  heart  rate  be- 
tween high-  and  low-level  animals  is  at  a  minimum.  The  critical  exposure  occurs 
during  winter  and  early  spring  in  the  late  morning  and  afternoon  hours.  During 
this  period  the  difference  in  heart  rate  is  maximal. 

It  is  of  considerable  interest,  in  this  regard,  that  high-level  animals  show  lower 
Q]0's  of  the  heart  rate  between  7°  and  9°  C.  than  low-level  animals  (low-level  = 
3.55,  3.66;  high-level  --  2.00,  2.15 — two  experimental  recordings).  It  suggests  that 
low-level  animals  are  approaching  cold  depression  at  a  higher  temperature  than  are 
the  high-level  animals.  The  physiological  temperature  range  is  therefore  believed 
to  be  wider  for  the  relatively  warm  adapted  high-level  animals. 

Influence  of  Certain   1  \iriahles  on  Hear/  Rate 

Body  size.  Among  the  numerous  factors  bearing  influence  upon  physiological 
rate  functions,  size  has  been  found  to  contribute  to  the  variation  in  heart  rate  in  the 
species  under  investigation.  Size  has  been  measured  by  the  wet  weight  of  soft 
parts.  Within  the  weight  range  of  0.3  to  1.2  gm.,  larger  animals  show  consistently 
slower  rates  at  all  temperatures  from  4°  to  29°  C. 

The  regression  of  rate  with  weight  is  without  apparent  systematic  variation  over 
the  year  and  is  not  significantly  different  between  "highs"  and  "lows"  at  14°  C. 
(P  ==  .35  for  the  difference  between  mean  regression  coefficients  of  1 1  high-level  and 
12  low-level  samples).  Ten  of  the  23  samples  show  no  reliable  difference  in  re- 
gression from  9°-24°  C.  The  remaining  13  samples  have  larger  negative  b  values 
on  either  side  of  14°  and  19°  C.  Hence,  no  single  expression  is  available  to  describe 
fully  the  effect  of  weight  on  heart  rate  in  this  species. 

Within  the  weight  range  of  0.4  to  1.0  gm..  the  regression  is  usually  linear  when 
plotted  on  logarithmic  coordinates  and  varies  from  --  0.043  to  --  0.172.  However, 
it  is  non-linear  on  either  side  of  this  weight  range.  It  is  as  if  we  were  plotting  only 
a  segment  of  a  large  parabola  (see  Figs.  1  and  3). 

Both  the  factors  of  size  and  individual  variation  contribute  to  the  scatter  about 
each  regression  line.  Size,  however,  is  the  major  factor  producing  the  scatter. 
Since  weight  and  rate  are  inversely  related,  rate  differences  are  meaningful  only 
with  essentially  equal  weight  animals. 

Sc.v.  Sexes  in  A.  liinatitla,  as  in  all  species  of  the  genus,  are  separate,  but  three 
rather  than  two  sexual  states  are  present.  To  the  conventional  male  and  female  is 
added  the  condition  of  indeterminacy.  The  latter  is  simply  the  post-spawning  phase 
of  the  male  or  female  in  which  gametes  are  absent  (see  section  on  gonad  size). 


MICROGEOGRAPHIC  VARIATION  135 

It  has  not  been  possible  to  find  a  differential  effect  upon  the  heart  rate  that  can 
be  attributed  to  any  of  the  sexual  states  excluding  the  pre-spawning  animals  heavy 
with  gonads  (see  below). 

Gonad  size.  An  analysis  of  possible  reproductive  patterns  will  be  presented  in  a 
following  section.  It  is  obvious  from  the  data  that  the  size  and  condition  of  the 
gonads  vary  over  the  year  and  between  vertically  separated  individuals.  Size  of 
the  gonad  as  such  has  a  negligible  effect  on  the  heart  rate ;  the  condition  of  the 
gonads  is,  however,  of  importance. 

On  occasion  when  as  many  as  50%  of  a  sample  wrere  possessed  of  insignificant 
gonads  or  were  of  indeterminate  sex,  and  the  remainder  showed  gonads  weighing 
up  to  20%  of  the  body  weight,  the  heart  frequency  of  all  animals  fell  within  the 
scatter  of  either  group.  On  the  other  hand,  pre-spawning  buildup  of  gonadal  tis- 
sue, regardless  of  the  size  attained,  rendered  the  heart  beat  erratic  and  not  reliably 
measureable  under  our  conditions.  This  was  found  in  July  for  both  high-  and  low- 
level  individuals.  In  the  latter  part  of  the  same  month,  when  comparing  heart  rates 
of  samples  having  approximately  equal  gonads  by  weight,  high-level  individuals 
showed  slower  rates  consistent  with  the  difference  between  groups  throughout  the 
year. 

Diurnal  rJiythins.  Two  groups  of  5  animals  each  were  maintained  at  14°  C. 
and  the  heart  beat  counted  at  one-hour  intervals  over  a  20-hour  period.  The  ani- 
mals were  under  constant  illumination.  Under  these  conditions,  the  presence  of  a 
day-night  rhythm  could  not  be  demonstrated  among  either  the  high-  or  low-level 
individuals. 

Effect  of  Transplantation  on  Heart  Rate 

Twenty-nine  days.  During  March,  1953,  42  high-level  and  42  low-level  lim- 
pets were  reciprocally  transposed  at  site  1 .  Fifty  control  specimens  from  each  level 
were  handled  in  like  manner  but  returned  to  their  natural  positions  (see  section  on 
behavioral  response).  The  numbers  of  recoveries  are  presented  below. 

Individuals  transposed  from  low-level  to  high  52% 

Individuals  transposed  from  high-level  to  low  57% 

Control  individuals  from  high-level  84% 

Control  individuals  from  low-level  60% 

Twenty-nine  days  after  reciprocal  transplantation,  the  heart  rate,  when  meas- 
ured from  4°-29°  C.,  appeared  to  have  undergone  a  complete  reversal.  Figure  3 
shows  the  heart  rate  response  of  the  transplants  and  controls  at  three  selected  tem- 
peratures. Heart  rates  of  high-level  individuals  introduced  into  the  low-level  tide 
pool  show  a  remarkable  degree  of  overlap  with  those  of  the  tide  pool  controls.  An 
exception  may  be  noted  in  the  case  of  three  individuals  above  1.00  gm.  in  weight. 
However,  since  the  rates  for  these  animals  fall  within  the  variation  of  all  transplants 
about  the  regression  line,  no  significance  has  been  attached  to  them. 

Tide  pool  individuals  transposed  to  high-level  show  heart  rates  close  to  but  con- 
sistently faster  than  those  of  the  high-level  controls.  Since  animals  transposed  to 
the  high-level  position  moved  from  their  sites  of  placement  to  more  protected  posi- 
tions (see  below),  the  migrants  have  acclimated  to  a  temperature  somewhat  lower 
than  the  "living"  temperature  of  high-level  controls. 

Fourteen  days.     During  January,  1954,  a  second  reciprocal  transplantation  was 


136 


EARL  SEGAL 


performed  at  site  4.  Recovery  was  exceptionally  poor.  Of  50  high-level  indi- 
viduals transposed  to  low-level  and  50  low-level  controls,  all  but  2  and  5.  respec- 
tively, were  lost.  Of  the  same  number  of  low-level  individuals  transposed  to  high- 
level,  20%  were  recovered  as  compared  with  50%  of  the  high-level  controls.  Heart 
rates  of  low-level  controls  at  the  end  of  the  experimental  period  and  high-level  trans- 
plants to  low-level  were  therefore  not  available  for  comparison  in  significant 
numbers. 


X 
en 


Ld 
OD 


90 

80 
70 

60 
50 

40 
30- 


UJ 

I 


20 


10 


24  "C 


TRANSPLANTS  -  HIGH     TO      LOW 


HIGH       CONTROLS 


0.4  0.5        0.6       0.7     0.8    0.9  1.0          1.2 

WET      WT.      OF      SOFT      PARTS      IM      GRAMS 

FIGURE  3.     Relation  between  heart  rate  and  wet  weight  of  soft  parts  of  reciprocal  transplants 
and  controls  29  days  after  transplantation.     Eacli  point  represents  one  individual. 

Comparing  animals  of  equal  weight,  it  is  evident  that  the  transplants  to  high- 
level  have  a  lower  heart  frequency  than  initial  low-level  controls  and  a  higher  fre- 
quency than  high-level  controls  at  any  temperature  from  9°-24°  C.  (Fig.  4).  The 
degree  of  acclimation  of  the  transplants  cannot  be  stated  in  equivalent  °C..  since 
the  thermal  history  in  the  field  cannot  be  given  in  simple  terms.  We  only  know 
that  partial  acclimation  has  occurred. 

If  we  calculate  the  change  in  heart  frequency  of  the  transplants  as  a  percentage 
of  the  difference  in  the  frequencies  of  the  low-  and  high-level  controls,  at  the  begin- 


MICROGEOGRAPHIC  VARIATION 


137 


100 
90 
80 

70 
'•  60 


10 

250 

m 


40 


LJ 


<t 
CC 


30 


UJ 

I 


20 


LOW   CONTROLS 


TRANSPLANTS-    LOW      TO      HIGH 


„      HIGH 


--9°C 


0-3      0.4     0.5   0.6   0.7  0.8  0.9  1.0 
WET   WT   OF   SOFT   PARTS  IN  GRAMS 

FIGURE  4.  Relation  between  heart  rate  and  wet  weight  of  soft  parts  for  second  experimental 
transplantation.  Duration  of  experiment  14  days.  Each  point  represents  one  individual. 
Low-level  controls  measured  at  start  of  experimental  period;  high-level  controls  measured  at 
end. 


TABLE  II 

Calculation  of  per  cent  acclimation  after  transplantation  from  low-  to  high-level.     Heart  rate  values  arc 

from  equal  weight  (0.6  gm.)  high-  and  low-level  controls  at  start  of  experiment  and  high  controls  and 

transplants  to  high-level  after  14  days.     Rate  values  are  taken  from  Figure  4.     Per  cent 

acclimation  of  transplants  calculated  from  the  difference  in  rate  of  low-  and  high-level 

controls  at  beginning  and  end  of  experiment.     Performed  in  January  1954. 


Temp.  °  C. 

Mean  heart  rates  of  average  0.6  gm.  animals  in  beats/min. 

%  acclimation 

High  controls 
12/17/54 

Low  controls 
12/17/54 

Transpl.  to  high 

1/4/54 

High  controls 
1/4/54 

9 

21.0 

33.0 

25.0 

22.5 

76.0 

14 

34.0 

49.0 

41.0 

37.0 

66.5 

19 

52.0 

69.0 

59.0 

54.0 

66.5 

24 

73.0 

97.0 

81.0 

72.5 

65.5 

29 

92.0 

126.0 

106.0 

95.0 

64.5 

138 


EARL  SEGAL 


ning  and  end  of  the  experimental  period,  we  can  roughly  compare  the  response  after 
14  days  with  that  after  29  days  (Table  II).  At  the  5  temperatures  shown,  an  aver- 
age of  68%  of  the  difference  in  heart  frequency  between  the  groups  has  been  achieved 
by  the  transplants  in  14  days.  However,  since  the  transplants  are  acclimating  to 
a  temperature  lower  than  that  of  the  high-level  habitat  (see  section  on  behavioral 
response),  68%  may  be  too  low  a  figure. 


100 
90 

80 
70 

60 


Z 

i 


c/l 

"5    50 


40 


bJ 

I 


20 


24°  C 


19°  C 


14°  C 


low- level  >^ 

controls 


transplonts    to     high-level 


9°C 


I 


I 


high  -  level 

controls — »* 


024  8  14 

TIME     IH     DAYS      AFTER       TRANSPLANTATION 


FIGURE  5.  Acclimation  of  heart  rate  in  low-level  transplants  to  high-level  2,  4,  8,  and  14 
days  after  transplantation.  Points  represent  equal  weight  (0.6  gm.)  animals  taken  from  the 
weight  regression  curve  of  each  sample.  Low-level  controls  measured  at  beginning  of  experi- 
ment, high-level  controls  at  end  and  low-level  transplants  to  high-level  at  days  indicated. 


It  is  of  interest  to  notice  that  there  is  a  proportionately  greater  change  in  heart 
frequency  of  the  transplants  at  9°  C.  than  at  any  of  the  higher  temperatures  up  to 
29°  C.  This  differential  response  may  have  as  a  basis  the  difference  in  slope  of  the 
rate/temperature  curves  of  high-  and  low-level  controls.  The  Q10's  of  the  heart 
rate  show  the  low-level  or  cold  acclimated  group  to  be  less  sensitive  to  temperature 
change  at  lower  temperatures,  i.e.,  the  curve  is  flatter  between  9°  and  14°  C.  An 
average  0.6-gm.  animal  from  low-level  has  a  O10  of  2.33,  while  that  of  equal  weight 


MICROGEOGRAPHIC  VARIATION 


139 


TABLE  III 

Calculation  of  per  cent  acclimation  14  days  after  transplantation  from  low-  to  high-level  and  calculation 

of  Qio  at  several  temperatures,  2,  4,  8,  and  14  days  after  transplantation.     Heart  rates  obtained 

as  in  Table  II.     Per  cent  acclimation  calculated  as  in  Table  II.     Performed  February  1954. 


Heart  rates  of  average  0.6  gm.  animals  in  beats/min. 

.        .. 

Temp. 
°  C. 

High  cont. 

Low  cont. 

Trans,  to  high.     Days  after  trans. 

High  cont. 

after 
14  days 
% 

2/1/54 

2/1/54 

2/15/54 

2 

4 

8 

14 

9 

20.0 

34.0 

29.5 

27.5 

25.0 

24.0 

20.5 

74.0 

14 

32.0 

52.0 

48.0 

45.0 

42.0 

39.0 

33.0 

68.5 

19 

51.0 

73.0 

69.0 

65.0 

60.0 

57.5 

51.0 

70.5 

24 

67.0 

98.0 

95.0 

90.0 

82.0 

78.0 

69.0 

67.5 

Qio  values  from  these  figures 


9-14 

2.56 

2.34 

2.65 

2.68 

2.80 

2.64 

2.67 

14-19 

2.54 

1.97 

2.07 

2.08 

2.04 

2.17 

2.38 

19-24 

1.77 

1.80 

1.90 

1.92 

1.87 

1.86 

1.83 

high-level  and  transplant  to  high-level  is  2.70  and  2.69,  respectively,  after  14  days. 
The  difference  in  Q1(,  decreases  with  increasing  temperature.  The  change  in  slope 
of  the  rate/temperature  curve  requires  a  proportionately  greater  change  in  heart 
frequency  at  lower  temperatures. 


60 


50 


40 


UJ 

K 

ct 


low-  level     samples 


high-level      samples 


o. 


5/8/54 


2/13/53 


16 


21 


TIME       IN      LABORATORY  -    DAYS 

FIGURE  6.  Heart  rate  during  laboratory  acclimation.  High-  and  low-level  samples  kept 
cool  (14°  C.)  and  without  food.  Samples  taken  on  days  indicated;  heart  rate  recorded  at  ac- 
climation temperature  (14°  C.).  Points  represent  equal  weight  (0.5  gm.)  "average"  animals 
taken  from  weight  regression  curves. 


140 


EARL  SEGAL 


110 
100 

90 
80 

70 

?     60 


50 


BD 

.• 


UJ 

•- 


cc 


30 


or 

UJ 

o. 


20 

;'i 

>  19 

i  '8 
:    17 

15 

14 


X'Ai  ,., 


low- level      A.    limotulo 


i       i       i       i       i 


J_ 


J 1 1 1 I I I 


_L 


FEB    MAR    APR    MAY   JUN     JUL    AUG    SEP    OCT    MOV    DEC    JAN     FEB    MAR   APR    MAY    JUN 


mean    monthly    inshore     surface 
sea     water     temperatures     for 
the    years     1953-54 


9-14    14-19    19-24 
TEMP    °C 


J I I L 


J I I L 


JUL   AUG   SEP  OCT  NOV  DEC   JAN   FEB  MAR  APR  MAY  JUN 
TIME      IN     MONTHS 

FIGURE  7. 


MICROGEOGRAPHIC  VARIATION  141 

Fourteen-days:  time  course.  In  early  February,  1954,  100  low-level  limpets 
were  moved  to  high-level  at  site  4.  A  total  of  60%  were  recovered.  Of  50  low- 
and  50  high-level  controls,  50 %  and  60%,  respectively,  were  recovered.  Trans- 
plants were  collected  2,  4,  8,  and  14  days  after  transplantation. 

Within  two  days  a  decrease  in  the  heart  rate  of  transposed  individuals  is  notice- 
able. The  further  decrease  in  heart  rate  is  non-linear  plotted  semilogarithmically, 
declining  most  rapidly  in  the  first  8  days  (Fig.  5). 

Roughly  70%  of  the  difference  in  heart  rate  was  achieved  by  14  days  (Table 
III).  This  figure  is  arrived  at  by  using  the  high-level  control  values  (at  14  days) 
as  a  criterion  of  complete  acclimation.  However,  for  the  reason  given  above  (see 
second  experimental  transplantation),  70%  is  too  low  a  figure. 

The  response  of  the  transplants  to  short  term  temperature  steps  is  remarkably 
similar  in  both  this  and  the  previous  experiment.  Not  only  is  there  a  proportion- 
ately greater  decrease  in  heart  frequency  at  9°  than  at  24°  C.  after  14  days,  but  the 
differential  response  is  apparent  after  two  days.  Table  III  shows  the  calculated 
Qio's  of  the  heart  rate  for  equal  weight  controls  and  transplants.  The  change  in 
slope  at  the  lower  end  of  the  rate/temperature  curve  is  conspicuous  \vithin  two  days. 

Behavioral  Response  to  Transplantation 

On  all  three  occasions,  when  low-level  limpets  were  transposed  to  the  level  of 
high-water  individuals,  it  was  observed  that  the  transplants  moved  from  the  site  of 
placement. 

In  the  first  group  of  low-  to  high-level  transplants,  recovered  on  April  11,  1953, 
29  days  after  transplantation,  all  surviving  animals  were  found  buried  beneath  a 
shell  and  gravel  deposit  at  depths  to  6  inches.  High-level  controls  were  recovered 
from  the  basalt  outcroppings  where  they  were  naturally  located,  whereas  the  trans- 
plants, originally  placed  in  close  proximity  to  the  controls,  had  migrated  vertically 
downward  from  1-6  inches  out  of  the  direct  sunlight  and  into  the  damp  deposit.  A 
return  to  their  old  level  is  considered  to  be  impossible  on  the  boulder  strewn  beach. 

The  identical  behavioral  response  was  elicited  from  transplants  in  subsequent 
experiments.  All  surviving  animals  were  found  to  have  moved  either  vertically 
downward  or  horizontally  under  overhanging  rocks,  in  both  cases  into  cooler  and 
damper  regions.  During  the  closely  watched  experiment,  migratory  movements 
were  found  to  be  complete  two  days  after  placement.  High-level  individuals,  again, 
remained  where  they  had  been  placed,  in  some  cases  in  the  identical  spot. 

The  behavioral  response  to  transplantation  has  bearing  upon  the  heart  frequency- 
relations  and  is  discussed  in  connection  with  those  measurements. 

FIGURE  7.  Heart  rate  as  a  function  of  season  for  low-level  A.  liwatitla.  Upper  four  curves 
— horizontal  lines  connect  points  for  equal  weight  (0.6  gin.)  animals  taken  from  weight  regres- 
sion curves.  Vertical  lines  denote  total  variation  around  regression  lines  within  the  weight 
range  of  0.4-0.8  gm.  The  weight  selected  for  comparison  is  from  the  middle  of  the  usual  range 
of  weight  at  any  collection  period  and  from  the  comparable  linear  segment  of  the  regression 
curves.  Fifth  curve — mean  monthly  inshore  surface  water  temperatures  in  °C.  from  Redondo 
Beach,  California  (area  adjacent  to  northernmost  collection  site)  for  the  years  1953-1954. 
Points  are  monthly  means  calculated  from  the  values  of  four  daily  recordings  made  between 
9  :00  A.M.  and  6  :00  P.M.  Lower  two  curves — Q10  values  calculated  from  the  data  above  (upper 
4  curves)  for  the  temperature  intervals  shown.  Inset — QIO  plotted  against  temperature  to  com- 
pare summer  and  winter  averages. 


142  EARL  SEGAL 

Laboratory  Studies 

Additional,  though  indirect,  laboratory  evidence  substantiates  the  field  studies 
on  acclimation.  High-  and  low-level  A.  liniatula  were  maintained  at  14°  C.  in  the 
laboratory  without  food  for  periods  up  to  22  days.  Five  to  10  individuals  wrere 
withdrawn,  one  day  after  collection  and  at  intervals  thereafter,  and  the  heart  beat 
counted  at  14°  C.  The  heart  rate  of  an  average  weight  low-level  animal,  selected 
from  the  weight  regression  curves,  decreased  25 T^  of  the  initial  rate  after  22  days. 
A  similarly  obtained  equal  weight  animal  from  high-level  showed  no  appreciable 
decrease  in  rate  in  the  same  number  of  days  (Fig.  6). 

At  the  time  of  collection,  low-level  animals  were  living  in  the  field  at  approxi- 
mately the  temperature  of  the  experiment.  The  decrease  in  heart  rate  is  presumed 
to  be  due  to  starvation  uncomplicated  by  a  tendency  to  acclimate.  High-level  ani- 
mals, on  the  other  hand,  were  living  in  the  laboratory  at  a  temperature  lower  than 
that  in  the  field.  At  14°  C.  two  opposing  forces  are  at  work  on  these  animals: 
starvation  tending  to  decrease  the  heart  rate  and  acclimation  to  a  lower  temperature 
tending  to  increase  it. 

Effect  of  Season  on  Heart  Rate 

Low-level  population.  Low-level  A.  liniatula,  collected  during  the  winter  and 
spring,  have  faster  heart  rates  at  all  temperatures  from  4°  to  29°  C.  than  animals 
of  equal  weight  collected  in  summer  (Fig.  7).  The  seasonal  trend  in  heart  fre- 
quency is  in  good  agreement  with  the  change  in  the  mean  monthly  surface  water 
temperature.  In  general,  there  is  an  inverse  relationship  such  that  an  increase  in 
temperature  is  followed  by  a  decrease  in  heart  rate  which  increases  again  with 
waning  temperatures.  The  difference  in  winter  and  spring  recordings  of  1953  and 
1954  has  a  basis  in  the  habitat  differences  of  the  samples  taken  during  those  periods. 
Animals  were  collected  from  different  sites  during  the  comparable  periods  of  each 
year.  Low-level  animals  collected  in  1953  were  apparently  slightly  more  subject 
to  warming  than  were  those  animals  collected  in  1954. 

The  mean  absolute  change  in  rate  (from  winter  to  summer)  at  24°  C.  is  greater 
than  at  lower  temperatures  but  the  proportionate  change  at  9°  C.  exceeds  that  at 
higher  temperatures.  At  9°  C.  there  is  a  maximum  46%  change  in  rate  as  com- 
pared with  29^o  at  24°  C. 

Temperature  sensitivity  of  the  heart  rate  (as  measured  by  the  O10)  similarly 
changes  with  season  (see  Fig.  7  and  Table  I).  Winter  and  spring  animals  show 
lower  OU)'s  and  thus  decreased  sensitivity  to  temperature  change  between  9°  and  24° 
C.  With  increasing  temperature  the  difference  in  Q1(,  between  winter  and  summer 
animals  decreases.  There  is  no  appreciable  difference  if  we  compare  Q1(1's  at  tem- 
peratures at  which  winter  and  summer  animals  show  equal  rates  (at  14°  C.  or  above 
for  winter  animals). 

High-lercl  population.  When  the  seasonal  change  in  heart  rate  of  high-  level 
forms  is  examined,  the  picture  is  less  clear  than  that  obtained  for  low-level  forms 
(Fig.  8).  There  is  a  suggestion  of  the  inverse  relationship  with  seasonal  tempera- 
ture change,  but  the  range  in  heart  frequency  is  smaller  and  a  response  to  short 
term  temperature  fluctuations,  of  the  order  of  several  days  to  a  week,  is  evident 
(see  February,  1954.  Fig.  8).  Although  high-level  animals  were  always  chosen 
from  the  upper  extreme  of  the  intertidal  range  of  the  species,  the  possibility  of  habi- 
tat differences  between  samples  cannot  be  ruled  out. 


MICROGEOGRAPHIC  VARIATION 


143 


z 

2 


90 
80 
70 

60 
50 


o 

2    40 

LU 

i— 
< 


30 


cr 

< 


20 


26 

24 
22 

o  20 
o 

I 
UJ 

5   16 

H 
< 
°C  14 

bJ 
Q_ 

3 '2 

t— 

10 
8 


9  °C 


high-  level        A     limatula 


I          I          I          I          I          I          I          I          I          I          I          I          I          1          I          I          I 

FEB    MAR    APR    MAY    JUN     JUL     AUG     SEP    OCT    NOV    DEC    JAN     FEB    MAR   APR    MAY 


low  tides    generally    expose     animals        low    tides     generally     expose 


during      late     evening      and     early 


morning      and       afternoon 
hours 


monthly     mean     maximum 
air     temperature 

possible  average 
effective  acclimation 
temperature  (estimated  I 


monthly    mean 
nshore      surface     water 
temperature 


~\monthly      mean 
minimum     air     temperature 


TIME       IN        MONTHS 

FIGURE  8.  Heart  rate  as  a  function  of  season  for  high-level  A.  limatula.  Parameters  of 
upper  4  curves  are  the  same  as  in  Figure  7.  Lower  series  of  curves  show  (1)  mean  maximum 
and  minimum  monthly  air  temperatures  (— O— )  taken  4  times  daily  at  Santa  Montica,  Cali- 
fornia;  (2)  monthly  mean  inshore  surface  water  temperatures  (  9  )  as  in  Figure  7  but  on 
reduced  scale;  and  (3)  possible  average  effective  acclimation  temperature  (estimated)  based 
on  the  heart  rate  response  ( ) . 


144 


EARL  SEGAL 


High-level  animals  begin  pre-spawning  buildup  of  gonadal  tissue  in  July  and  ap- 
pear to  maintain  ripe  gonads  through  October  (see  section  on  gonad  size).  The 
rather  rapid  heart  rates  in  late  July  and  possibly  also  in  early  October  may  be  due 
to  an  increased  metabolic  activity  associated  with  gametogenesis. 

Table  I  shows  the  QIO  values  calculated  from  the  curves  in  Figure  8.  Samples 
taken  during  "characteristic"  winter  months  (January,  April)  show  decreased  tem- 
perature sensitivity  (lower  Q10's)  than  samples  taken  in  "characteristic"  summer 
months  (August).  However  there  is  a  marked  lack  of  consistency  in  the  tempera- 
ture sensitivity  of  the  heart  rates  of  high-level  animals.  Samples  taken  during 

TABLE  IV 
Gonad  size  as  a  function  of  season  in  high-  and  low-level  A.  limatula 


Average  gonad  size  in  %  wet  weight  of  soft  parts 

Date  of  collection 

High  level 

Gonads  turgid 

No.  of 
specimens 

Low  level 

Gonads  turgid 

No.  of 
specimens 

2/13/53 

8.6 

20 

29.3 

26 

3/13/53 

7.6 

14 

16.3 

12 

4/11/53 

0.0 

22 

16.1 

21 

7/12/53 

— 

X 

18 

— 

X 

18 

7/27/53 

21.8 

X 

14 

20.2* 

7 

9/25/53 

28.lt 

X 

6 

** 

12 

10/  7/53 

29.4 

12 

17.1 

15 

12/17/53 

6.6 

13 

21.7 

19 

I/  4/54 

7.6 

20 

31.1 

12 

2/  1/54 

0.0 

16 

20.4 

15 

2/15/54 

0.0 

12 

15.9 

10 

4/  6/54 

0.0 

14 

17.0 

18 

5/  8/54 

0.0 

8 

15.6 

11 

6/12/54 

15.6 

14 

16.1 

12 

*  Six  additional  animals  spawned  in  field — weights  not  taken. 
*  All  animals  spawned  in  field — weights  not  taken, 
f  Five  additional   animals   spawned   in   laboratory — weights   not   taken. 


months  of  unseasonal  temperature  fluctuations  (February,  1954)  do  not  show  the 
Q10  associated  with  adaptation  of  the  heart  rate  to  a  warmer  temperature,  although 
the  rates  themselves  have  adapted. 

Gonad  Sise 

In  relation  to  inter  tidal  height.  Gonad  size,  as  per  cent  of  the  wet  weight  of 
soft  parts,  has  not  been  found  to  vary  systematically  with  the  size  of  the  organism 
over  the  range  of  0.3-1.2  gm.  Therefore,  the  mean  gonad  weight,  as  per  cent  of  the 
body  weight,  of  the  animals  of  each  collection  period  was  calculated.  These  values 
are  presented  in  Table  IV. 

Excluding  the  period  roughly  from  early  June  to  mid-October,  low-level  A.  lima- 
tula appeared  to  maintain  a  larger  gonad  than  high-level  forms  of  the  species.  Mean 
gonad  weight  of  low-level  individuals  did  not  fall  below  15%  of  the  body  weight  dur- 


MICROGEOGRAPHIC  VARIATION 


145 


ing  the  1^-year  period  of  observation.  High-level  individuals,  from  approximately 
November  until  June,  possessed  either  small  or  negligible  amounts  of  gonadal  tis- 
sue with  as  many  as  60%  showing  indeterminate  sex.  Indeterminate  sex  among 
low-level  individuals  was  observed  during  the  month  of  September  after  spawning 
occurred  in  the  field. 

In  relation  to  season.  Natural  spawning  among  high-  and  low-level  individuals 
does  not  appear  to  occur  at  similar  times.  Turgidity  of  the  gonad  (regardless  of 
size)  and  deformation  of  the  female  gametes  have  been  used  to  indicate  the  presence 

TABLE  V 

Effect  of  transplantation  on  size  of  gonad  in  A.  limatula 


Date  of 
collection 

Average  gonad  size  in  %  wet  weight  of  soft  parts 

Controls 

Transplants 

High  level 

No. 

Low  level 

No. 

Low  to 
high 

No. 

High  to 
low 

No. 

Experiment  I — 29  days 


3/13/53 
4/11/53 

7.6 
0.0 

14 
22 

16.3 
16.1 

12 
21 

0.0 

18 

15.9 

20 

Experiment  II — 14  days 


12/17/53 
I/  4/54 

6.6 
7.6 

13 
20 

21.7 
31.1 

19 
13 

21.9 

10 

Experiment  III — at  intervals  to  14  days 


2/  1/54 

0.0 

16 

20.4 

15 

2/  3/54 

19.9 

9 

2/  5/54 

21.4 

9 

2/  9/54 

21.2* 

4 

2/15/54 

0.0 

12 

15.9 

10 

7.3** 

4 

*  Six  other  animals  spawned  in  laboratory. 

**  Six  other  animals  without  weighable  gonads  at  time  of  collection. 

of  pre-spawning  ripeness — in  agreement  with  Fritchman  (1953).  In  July,  samples 
from  both  high-  and  low-level  revealed  this  condition  (see  Table  IV).  Later  in 
the  same  month,  partial  or  complete  spawning  of  50%  of  the  low-level  sample  had 
occurred.  Spawning  of  the  entire  low-level  population  is  assumed  to  have  taken 
place  before  late  September. 

High-level  individuals,  on  the  other  hand,  showed  a  persistent  turgidity  of  the 
gonads  throughout  late  September  in  the  Ik-Id,  although  under  maintained  tempera- 
ture (17°)  in  the  laboratory,  50r/,  of  the  September  sample  .spawned.  In  early 
October,  weight  of  the  gonads  of  high-level  forms  had  not  significantly  changed, 
but  turgidity  was  no  longer  apparent.  By  December,  50'/>  of  the  high-level  sample 


146  EARL  SEGAL 

was  devoid  of  weighable  gonadal  tissue ;  by  February,  \00%  of  the  animals  showed 
this  condition  and  remained  so  until  the  following  June.  After  September,  spawn- 
ing among  the  low-level  population  was  apparently  at  an  end ;  the  gonads  then  re- 
turned to  approximately  the  average  weight  for  non-spawning  months.  However, 
a  second  buildup  of  the  gonads  occurred  in  midwinter  (January  and  February  of 
two  consecutive  years)  which,  though  of  substantial  weight,  did  not  show  the  char- 
acteristic pre-spawning  turgidity. 

Effect  of  transplantation.  Gonads  of  transposed  animals  were  examined  sub- 
sequent to  recording  of  the  heart  rate.  Data  are  available  from  three  experimental 
transplantations  and  have  been  summarized  in  Table  V. 

1.  After  29  days,  in  the  spring,  a  complete  reversal  of  the  gonad  size  was  ob- 
tained.    High-level  transplants  to  low-level  developed  gonads  whose  average  size 
was  not  significantly  different  from  that  of  the  low-level  controls. 

2.  During  January,  1954,  the  second  gonadal  buildup  occurred  among  low-level 
individuals.     While  the  average  gonadal  weight  of  low-level  controls  increased  by 
aproximately  50%  in  14  days,  that  of  the  transplants  from  low-  to  high-level  did 
not  increase ;  rather  they  averaged  the  same  size  as  before  transplantation. 

3.  During  February,  1954,  animals  were  collected  2,  4,  8,  and  14  days  after 
transplantation  from  low-  to  high-level.     No  change  was  observed  in  the  size  of  the 
gonads  up  to  S  days  after  transplantation  although  60%  of  the  individuals  from  the 
eighth  day  of  collection  spawned  in  the  laboratory  ( 19°  C. ) .     At  the  end  of  14  days, 
60%  of  the  transplanted  individuals  possessed  negligible  gonads  and  the  weights 
of  the  remaining  40%  averaged  less  than  one-half  that  of  the  low-level  controls  on 
the  same  date. 

DISCUSSION 

Microgeographic  variation.  The  preceding  data  show  that  the  microgeographic 
intertidal  distribution  of  a  gastropod,  Acinaca  liniatitla,  is  reflected  in  certain  physio- 
logical and  morphological  differences  (see  Segal,  Rao  and  James.  1953,  pre- 
liminary report).  The  differences  found  in  relation  to  shell  and  body  size  have  been 
reported  previously  (Segal,  1956).  High  intertidal  A.  liniatnla  are  found  about  one 
meter  above  zero  datum;  low  intertidal  A.  liniatnla  are  found  at  zero  datum  and  be- 
low. High-level  forms  show  a  slower  heart  rate  than  low-level  forms  when  both 
are  measured  at  any  given  temperature  from  7°  to  29°  C.  Comparisons  show  that 
high  intertidal  animals  are  exposed  about  50%  of  the  time  and  are  subjected  to  air 
temperatures  which  rise  above  and  fall  below  that  of  the  ocean.  Low  intertidal  ani- 
mals are  submerged  over  90%>  of  the  time  and  live  essentially  at  the  temperature  of 
the  ocean. 

We  do  not  have  the  complete  curve  of  temperature  against  time  for  the  high-level 
animals ;  therefore,  we  do  not  know  how  the  temperatures  they  are  acclimated  to  are 
related  to  the  temperature  fluctuations  they  are  subjected  to.  We  only  know  that 
these  animals  respond  as  if  they  are  living'  at  a  higher  temperature  than  that  of  the 
ocean.  Tn  this  regard,  Kirberger  (1953)  maintained  an  annelid.  Lnnibn'cnlns 
raricgalns,  for  12  hours  alternately  at  In "  and  23°  ('.  for  S  to  14  days.  She  com- 
pared the  O,  consumption  of  these  animals  with  that  of  two  groups  kept  solely  at  16° 
and  at  23°  C.  for  the  same  period  of  time.  Those  kept  at  the  alternating  tempera- 


MICROGEOGRAPHIC  VARIATION  147 

tures  averaged  the  fluctuations  and  responded  as  if  they  were  adapted  to  19°  C. 
Animals  maintained  at  the  constant  temperatures  showed  the  typical  compensa- 
tory response,  i.e.,  those  animals  from  16°  C.  consumed  more  O2  than  those  from 
23°  C.  when  measured  at  the  same  temperatures. 

Numerous  studies  have  shown  similar  physiological  differences  to  exist  between 
macrogeographically  distributed  populations  of  a  species.  The  question  has  been 
raised  as  to  whether  these  animals  are,  in  fact,  members  of  the  same  species.  Such 
latitudinal  studies  where  the  physiological  differences  are  clearly  correlated  with 
habitat  temperatures  (Mayer,  1014;  Sparck,  1936;  Fox  and  Wingfield,  1937;  Fox, 
1939;  Roberts,  1952;  Dehnel,  1955)  are  sufficiently  similar  to  intertidal  micro- 
geographic  studies  to  warrant  the  suggestion  that  the  same  compensatory  phenome- 
non is  involved.  High  intertidal  individuals,  similar  to  warm  seas  populations,  be- 
have as  though  they  are  warm-adapted  relative  to  low  intertidal  individuals  and 
cold  seas  populations.  Rao  (1953),  and  Segal,  Rao  and  James  (1953).  in  the  only 
studies  where  microgeographic  and  macrogeographic  physiological  differences  have 
been  compared  in  the  same  species  (Mytilus  calif  ornianus) ,  show  that  2l/>  feet  of 
vertical  separation  is  equivalent  to  about  350  miles  in  latitudinal  separation.  The 
rate  of  water  propulsion  in  low-level  northern  mussels  differs  as  much  from  that  in 
low-level  southern  mussels  as  the  rate  in  low-level  southern  mussels  differs  from  that 
in  high-level  southern  mussels  2%  feet  higher  in  the  intertidal  zone.  The  data  sug- 
gest that  we  are  dealing  with  the  phenomenon  of  individual  adjustment  to  habitat 
temperatures ;  in  short  we  may  hypothesize  that  this  is  a  phenotypic  adaptation. 
In  the  present  study  we  have  made  a  more  direct  test  of  this  hypothesis. 

If  a  physiological  rate  character  is,  in  time,  readily  reversible  under  changed 
temperature  conditions,  we  may  say  that  this  rate  attribute  is  acquired  during  the 
ontogeny  of  the  individual.  These  changed  temperature  conditions  may  be  arti- 
ficially imposed  by  laboratory  acclimation  or  by  transplantation  of  the  organism  in 
the  field  ;  they  may  be  naturally  imposed  by  the  changing  season. 

When  low-level  A.  limatida  were  transposed  to  high-level,  slowing  of  the  heart 
rate  was  evident  in  two  days  and  full  adaptation  was  accomplished  within  14  to  29 
days.  The  reverse,  adaptation  to  cold,  was  also  complete  within  14  to  29  days 
(Figs.  3,  4,  and  5).  Thus,  the  difference  in  heart  rate  of  individuals  at  different 
intertidal  levels  was  shown  to  be  reversible  under  habitat  conditions. 

There  are  few  published  reports  on  transplanting  individuals  of  a  species  from 
one  habitat  to  another,  using  some  physiological  rate  character  as  a  measure  of  ad- 
justment. Sumner  and  Lanham  (1942)  and  Loosanoff  and  Nomejko  (1951)  re- 
port instances  of  transplantation  with  no  apparent  acclimation.  These  results  may 
be  due  either  to  the  inability  of  a  homogeneous  species  to  acclimate  as  in  the  first  ref- 
erence cited,  or  to  the  existence  of  true  physiological  races  as  in  the  second  reference. 
Physiological  races  have  been  demonstrated  previously  both  among  field  and  labora- 
tory populations  (Brown,  1929;  Goldschmidt,  1932,  1934;  Hovanitz,  1947;  Stauber, 
1950).  The  transplantation  method  seems  to  be  effective  for  revealing  the  nature 
of  intraspecific  physiological  and  morphological  differentiation  (Moore,  1934; 
Segal,  1956). 

Acclimation  of  the  heart  rate  has  also  been  shown  to  occur  with  the  seasonal 
change  in  temperature.  Low-level  animals  have  about  the  same  rate  in  winter 
and  in  summer  at  their  respective  field  temperatures  (Fig.  7).  High-level  animals 


148  EARL  SEGAL 

show  responses  to  unseasonal  air  temperature  fluctuations  which  tend  to  mask  the 
seasonal  acclimation  (Fig.  8).  In  substantiation  of  the  field  studies,  acclimation 
to  cold  has  also  been  demonstrated  in  the  laboratory  (Fig.  6). 

Temperature  sensitivity.  The  sensitivity  of  the  heart  rate  to  temperature  change, 
measured  by  Q10,  has  also  been  shown  to  vary  with  intertidal  height.  Between  9° 
and  19°  C.,  but  not  as  clearly  above  19°  C.,  low-level,  cold  adapted  organisms  show 
lower  O10's  than  equal  weight  high-level  organisms  (the  rate  of  change  over  tem- 
perature intervals  is  vised  rather  than  at  temperature  points  because  it  is  believed 
that  rates  of  1°  C.  increments  are  necessary  for  a  reliable  estimation  of  the  change 
in  rate  at  a  given  temperature).  Belehradek  (1935),  using  examples  taken  from 
data  of  various  investigators,  points  out  that  temperature  coefficients  commonly  in- 
crease with  the  adaptation  of  the  protoplasm  to  higher  temperatures.  This  thesis  is 
further  strengthened  through  additional  evidence  of  Rao  (1953)  and  recalculations 
by  Rao  and  Bullock  (1954)  of  earlier  equivocal  data. 

Of  interest  are  the  temperature  relations  of  high  and  low  intertidal  groups. 
Although  low-level  animals  respond  as  though  they  are  cold  acclimated  relative  to 
high-level  animals,  environmental  temperatures  below  13°  C.  are  probably  rarely 
encountered  in  this  area.  High-level  animals  do  meet  with  such  temperatures  dur- 
ing the  winter  and  spring  months  when  the  higher  of  the  two  low  tides  and  the 
lower  of  the  two  high  tides  of  each  day  are  of  insufficient  magnitude  to  cover  the 
animals.  It  is  worthy  of  note  that  on  the  two  occasions  when  heart  rates  were 
measured  at  7°  C.  (at  4°  C.  both  groups  show  cold  depression  and  cessation  of  beat 
in  a  fair  percentage  of  each  sample),  low-level  animals  show  higher  O1()'s  between  7° 
and  9°  C.  It  indicates  that  the  low-level,  cold  acclimated  group  is  paradoxically 
approaching  cold  depression  at  a  higher  temperature  than  the  relatively  warm  ac- 
climated, high-level  group.  It  further  suggests  that  the  physiological  temperature 
range  (that  range  of  temperatures  over  which  there  is  no  observable  indication  of 
depression)  extends  farther  into  the  cold  in  the  warm  acclimated  group.  Above 
29°  C.  heart  beats  could  no  longer  be  counted  with  accuracy,  but  the  very  fact  that 
high-level  animals  have  been  found  with  higher  body  temperatures  in  the  field,  while 
the  surface  ocean  temperature  rarely  if  ever  has  exceeded  24°  C.  in  this  locale,  per- 
mits the  interpretation  that  the  physiological  range  similarly  extends  farther  into  the 
warm.  Dehnel  (1955)  reaches  the  same  conclusion  for  optimal  temperature 
range  of  larval  growth  within  the  species  in  populations  from  Southern  California 
and  Alaskan  waters. 

Additional  confirmation  exists  for  the  thesis  that  cold-adapted  organisms  show 
lower  Q10's  and  thus  greater  independence  to  temperature  change.  Winter  animals 
as  compared  with  summer  animals  have  lower  Ou,'s,  at  least  from  9°  to  24°  C.,  and 
animals  transplanted  from  low-level  (cold)  to  high-level  (warm)  show  an  in- 
creased temperature  dependence  (higher  O10's)  writhin  two  days. 

Scholander  et  al.  (1953),  in  a  metabolic  study  of  arctic  and  tropic  poikilotherms, 
suggests  that  a  low  Q10  would  only  be  advantageous  to  offset  the  effects  of  changes 
in  temperature  due  to  diurnal,  seasonal,  or  migratory  factors.  The  authors  state 
that  no  such  adaptation  was  found  among  the  species  which  would  profit  from  a 
low  O10 :  temperate  water  forms,  fresh  water  forms,  and  terrestrial  forms.  Rao  and 
Bullock  (1954)  agree  that  at  present  no  general  case  can  be  made  for  lower  O10's 
in  forms  exposed  to  changing  temperature  but  argue  that  cold  adapted,  e.g.,  arctic 


MICROGEOGRAPHIC  VARIATION  149 

species  as  compared  with  tropic  species,  do  show  lower  Q,0's  even  though  they  may 
not  be  normally  exposed  to  changes  in  temperature. 

The  present  study  is  unique  in  that  comparisons  are  made  between  animals 
which  are  living  under  fluctuating  temperature  conditions  (high-level,  exposed)  and 
animals  which  are  living  under  relatively  constant  temperature  conditions  (low- 
level,  submerged).  In  this  locale  the  high-level  forms  are  exposed  to  considerably 
higher  temperatures  than  that  of  the  ocean  and  these  animals  act  as  though  warm 
adapted.  As  shown,  the  warm  adapted  animals  have  the  higher  Q10  in  spite  of  the 
fluctuating  temperature  of  the  habitat.  Similarly,  the  summer  forms  from  both 
high-  and  low-level  have  higher  O10's. 

These  differences  could  not  have  been  expected  simply  from  measurements  of 
the  Qln  of  the  species  at  any  one  time.  If  individuals  of  a  species  residing  in  differ- 
ent microhabitats  and  from  one  season  to  another  show  variations  in  temperature 
sensitivity,  a  great  burden  is  placed  upon  comparisons  between  species.  Other 
than  for  species  living  in  arctic  and  tropical  seas,  with  their  almost  constant  tempera- 
tures, it  is  doubtful  whether  Q10  values  (or  any  temperature  coefficient  describing 
sensitivity  to  changes  in  temperature)  of  a  physiological  rate  activity  are  meaningful 
except  in  very  limited  comparisons.  The  thermal  history  of  one  segment  of  a  spe- 
cies is  not  the  thermal  history  of  that  species.  The  range  of  O10  values  (at  a  given 
temperature)  permissible  within  the  genetic  makeup  of  a  species  would  describe  the 
temperature  sensitivity  of  that  species. 

S/nm'H/m/  and  yonad  sise.  If  the  data  on  spawning  represent  normal  behavior, 
then  low-level  A.  limatula  spawn  before  high-level  A.  limatula.  Now  the  question 
remains  as  to  whether  high-level  A.  limatula  actually  spawn.  All  low-level  animals 
show  partial  or  complete  spawning  by  late  August.  High-level  animals,  on  the 
other  hand,  if  they  do  spawn,  do  so  sometime  between  October  and  December.  We 
are  not  sure  that  high-level  animals  spawn  because  gonadal  turgor  and  deformation 
of  the  female  gametes,  which  Fritchman  (1953)  considers  as  indicative  of  pre- 
spawning  ripeness,  were  not  present  in  October  although  the  gonads  of  high-level 
animals  were  of  large  size.  Therefore,  we  must  assume  either  that  ( 1 )  high-level 
animals  do  not  spawn  in  the  field,  or  (2)  pre-spawning  ripeness  is  not  a  necessary 
condition.  Again,  if  spawning  occurs,  it  is  out  of  phase  with  that  of  the  low-level 
population.  Yet,  Fritchman  (1953),  working  with  high-  and  low-level  members 
of  two  species  of  the  same  genus  in  central  California  (A.  fenestnita  cribraria  and 
A.  tcstudinalis  scutum},  did  not  find  a  difference  in  spawning  time. 

With  the  warming  of  the  ocean  in  May  and  June  (Fig.  8),  gametogenesis  is 
stimulated  in  both  high-  and  low-level  populations.  By  July,  all  animals  showed 
the  gonadal  turgor  and  deformation  of  the  female  gametes  associated  with  the  pre- 
spawning  ripe  condition.  Spawning  occurs  in  the  low-level  population ;  this  popu- 
lation is  submerged  and  therefore  subjected  to  the  more  constant  temperatures  of 
the  ocean.  High-level  animals,  which  are  only  submerged  50^  of  the  time,  do  not 
spawn  during  this  period  of  warmest  average  sea  water  temperatures  (July,  August, 
September:  21°-19°  C).  But,  by  October,  the  characteristics  associated  with  pre- 
spawning  ripeness  have  disappeared  although  the  gonads  are  still  large.  From 
October  to  December,  when  the  average  inshore  surface  water  temperatures  have 
fallen  to  17°  C.  and  below,  the  high-level  animals  lose  their  gonads.  The  trigger 
mechanism  necessary  to  initiate  spawning  may  well  be  a  required  time  interval  spent 


150  EARL  SEGAL 

at  a  given  temperature  rather  than  the  reaching  or  exceeding  of  that  temperature  for 
a  short  interval  of  time.  Fifty  per  cent  of  the  high-level  animals  (September  collec- 
tion; mean  ocean  temperature  19°  C.)  spawned  in  the  laboratory  after  three  days  at 
17°  C. 

The  loss  of  the  gonad  sometime  between  October  and  December  coincides  with 
the  seasonal  tidal  change ;  from  October  through  April  high-level  animals  are  ex- 
posed during  the  late  morning  and  afternoon  hours  (Fig.  8).  It  is  during  these 
hours  that  these  animals  are  subjected  to  direct  solar  radiation  and  to  heat  conduc- 
tion from  the  exposed  rock  substratum.  The  presence  of  a  large  gonad,  or  for  that 
matter  any  gonadal  material,  would  decrease  the  area  under  the  shell  available  for 
water  and  thereby  decrease  the  animals'  ability  to  avoid  desiccation  (Segal,  1956). 
Low-level  animals  transplanted  to  high-level  lost  the  gonadal  material  within  two 
weeks  (Table  V).  The  evidence  suggests  that  we  are  dealing  with  a  non-breeding 
population  living  at  the  extreme  of  the  intertidal  distribution  of  the  species. 

SUMMARY 

1.  Highest  and  lowest  members  of  a  eury topic  intertidal  species,  A.  limatula, 
have  been  compared  in  the  following :  heart  rate,  gonad  size,  and  spawning  behavior. 

2.  Within  the  weight  range  of  0.4  to  1.0  gm..  the  heart  rate  varies  inversely  with 
increasing  weight.     The  regression  coefficients  fall  between  •-  0.042  and    -  0.172; 
thus  no  single  expression  is  available  to  describe  fully  the  effect  of  weight  on  heart 
rate  in  this  species. 

3.  Sex  and  size  of  gonad  ( as  divorced  from  turgidity  )  have  not  been  found  to 
contribute  to  the  variation  in  heart  rate  between  samples. 

4.  Comparing  equal  weight  animals,  it  is  found  that  low  intertidal  individuals 
have  faster  heart  rates  than  high  intertidal  individuals  at  any  temperature  from  4°  to 
29°  C. 

5.  From  data  on  field  temperatures  it  is  suggested  that  the  significant  parameter 
of  the  intertidal  difference  is  temperature.     High-level  animals  are  subjected  to  con- 
siderable periods  of  warmer  as  well  as  to  some  periods  of  cooler  temperatures  than 
are  low-level  animals. 

6.  An  attempt  was  made  to  characterize  the  difference  in  heart  rate  by  :  trans- 
planting the  animals  in  the  field,  following  the  seasonal  changes,  and  maintaining 
samples  of  both  populations  in  the  laboratory  at  a  cool  temperature  (14°  C.)  and 
without  food. 

7.  When  low-level  animals  are  transplanted  to  high-level  their  heart  rates  slow 
so  that  within  29  days  it  is  equal  to  that  of  the  high-level  animals  when  measured 
at  any  given  temperature.     The  half-acclimation  time  was  about  6  days.     In  the 
field,  acclimation  to  cold  was  also  shown  to  be  complete  within  29  days. 

8.  Comparisons  of  the  heart  rate  during  winter  and  summer  showed  that  both 
high  and  low  intertidal  animals  have  faster  rates  in  winter  at  any  given  temperature 
from  9°  to  29°  C.     Acclimation  to  cold  was  also  shown  in  the  laboratory. 

9.  The  above  results  lead  to  the  interpretation  that  the  microgeographic  differ- 
ence in  heart  rate  is  a  phenotypic  expression  of  a  compensatory  phenomenon  op- 
erating to  maintain  approximately  equal  heart  activity  in  spite  of  the  habitat  tem- 
perature differences.     Latitudinal  differences  in  physiological  rate  activities  which 


MICROGEOGRAPHIC  VARIATION  151 

are  clearly  correlated  with  habitat  temperature  are  sufficiently  similar  to  the  inter- 
tidal  differences  reported  here  to  warrant  the  suggestion  that  the  same  phenomenon 
is  involved. 

10.  Low-level  and  winter  animals  show  a  heart  rate  that  is  less  dependent  on 
temperature  changes  in  the  range  from  9°  to  19°  C.     This  same  response  is  not  ob- 
served consistently  above  19°  C.     While  both  high-  and  low-level  animals  appear 
to  be  approaching  cold  depression  below  9°  C.,  the  low-level  animals  are  cold  de- 
pressed at  a  higher  temperature  than  the  relatively  warm-adapted  high-level  animals ; 
the  low-level  animals  possess  a  higher  OUI  in  this  range.     It  is  suggested  that  the 
physiological  temperature  range  of  the  warm-acclimated  group  extends  both  higher 
and  lower  than  that  of  the  cold-acclimated  group.     In  the  field,  the  Q10  of  the  heart 
rate  changes  within  two  days  after  the  animals  are  transplanted. 

11.  The  size  of  the  gonad  also  varies  with  intertidal  height.     Low-level  animals 
maintain  a   larger  gonad   during  winter   and   spring  than   do   high-level   animals. 
Transplantation  also  reveals  this  difference  to  be  reversible. 

12.  Analysis  of  spawning  behavior  (using  turgidity  as  the  criterion  of  pre-spa\vn- 
ing  readiness )  presents  the  possibility  that  either  ( 1 )  the  two  groups  spawn  a  num- 
ber of  months  out  of  phase  with  each  other,  or   (2)  high-level  individuals  do  not 
contribute  to  the  breeding  population. 

LITERATURE  CITED 

BELEHRADEK,   J.,    1935.     Temperature  and   living  matter.     Protoplasma-Monogr,     Berl.   8. 

BROWN,  L.  A.,  1929.  The  natural  history  of  cladocerans  in  relation  to  temperature.  I.  Distri- 
bution and  the  temperature  limits  for  vital  activity.  Amcr.  Nat.,  63:  248-264. 

BULLOCK,  T.  H.,  1955.  Compensation  for  temperature  in  the  metabolism  and  activity  of  poikilo- 
therms.  Biol.  Rev..  30  :  311-342. 

DEHXEL,  P.  A.,  1955.  Rates  of  growth  of  gastropods  as  a  function  of  latitude.  Physiol. 
Zool..  28:  115-144. 

Fox,  H.  M.,  1939.  The  activity  and  metabolism  of  poikilothermal  animals  in  different  latitudes. 
V.  Pror.  Zool.  Soc.  Loud..  109:  141-156. 

Fox,  H.  M.,  AND  C.  A.  WINGFIELD.  1937.  The  activity  and  metabolism  of  poikilothermal  ani- 
mals in  different  latitudes.  II.  Prof.  Zool.  Soc.  Loud..  107  :  275-282. 

FRITCHMAN,  H.  K.,  1953.  A  study  of  reproductive  cycles  in  the  California  Acmaeidae.  Ph.D. 
thesis,  Univ.  of  Calif.,  Berkeley. 

GOLDSCHMIDT.  R.,  1932.  Untersuchungen  zur  Genetik  der  geographischen  Variation.  V. 
Arch.  f.  En  hi'.,  126:  674-768. 

GOLDSCHMIDT,  R.,  1934.     Lymantria.     Bibliographia  Gcnctica,  11  :   1-186. 

HOVANITZ,  W.,  1947.  Occurrence  of  parallel  series  of  associated  physiological  and  morpho- 
logical characters  in  diverse  groups  of  mosquitos  and  other  insects.  Contrib.  Lab. 
Vert.  Biol.,  Univ.  Mich.,  No.  32:  1-24. 

KIRBERGER,  C.,  1953.  Untersuchungen  iiber  die  Temperaturabhangigkeit  von  Lebensprozessen 
bei  verschiedenen  Wirbellosen.  Zcitschr.  vcrgl.  Physiol.,  35:  175-198. 

LOOSANOFF,  V.  L.,  AND  C.  A.  NoMEjKO,  1951.  Existence  of  physiologically  different  races  of 
oysters,  Crassostrca  virginica.  Biol.  Bull.,  101  :  151-156. 

MAYER,  A.  G.,  1914.  The  effect  of  temperature  upon  tropical  marine  animals.  Pap.  Tortugas 
Lab.,  6 :  3-24. 

MOORE,  H.  B.,  1934.  The  relation  of  shell  growth  to  environment  in  Patella  vnlgata.  On 
"ledging"  in  shells  at  Port  Erin.  Proc.  Malacol.  Soc.  Land.,  21  :  217-222. 

PROSSER,  C.  L.,  1955.     Physiological  variation  in  animals.     Biol.  Rev.,  30:  229-262. 

RAO,  K.  P.,  1953.  Rate  of  water  propulsion  in  Mytilus  californianus  as  a  function  of  latitude. 
Biol,  Bull.,  104:  171-181. 


152  EARL  SEGAL 

RAO,  K.  P.,  AND  T.  H.  BULLOCK,  1954.     Q10  as  a  function  of  size  and  habitat  temperature  in 

poikilotherms.     Amer.  Nat.,  88:  33-44. 
ROBERTS,  J.  L.,  1952.     Studies  on  acclimatization  of  respiration  to  temperature  in  the  lined  shore 

crab,  Pachygrapsus  crassipcs  Randall.     Ph.D.  thesis,  Univ.  of  Calif.,  Los  Angeles. 
SCHOLANDER,  P.  F.,  W.  FLAGG,  V.  WALTERS  AND  L.  IRVING,  1953.     Climatic  adaptation  in  arctic 

and  tropical  poikilotherms.     Physiol.  Zool.,  26 :  67-93. 
SEGAL,   E.,   1956.     Adaptive   differences   in   water-holding   capacity   in   an   intertidal   gastropod. 

Ecology,  37  :  174-178. 
SEGAL,  E.,  K.  P.  RAO  AND  T.  W.  JAMES,   1953.     Rate  of  activity  as  a  function  of  intertidal 

height  within  populations  of  some  littoral  molluscs.     Nature,  172:   1108-1109. 
SPARCK,    R.,    1936.     On    the    relation    between    metabolism    and    temperature    in    some    marine 

lamellibranchs  and  its  ecological  and  zoogeographical  importance.     A",  dansk.  i>idcnsk. 

Sclsk.,  Biol.  Medd.,  13  :  1-27. 
SUMNER,  F.  B.,  AND  U.  N.  LANHAM,  1942.     Studies  of  the  respiratory  metabolism  of  warm  and 

cold  spring  fishes.     Biol.  Bull,  82:  313-327. 
STAUBER,  L.  A.,  1950.     The  problem  of  physiological  species  with  special  reference  to  oysters 

and  oyster  drills.     Ecology,  31  :  109-118. 


MEMBRANE  POTENTIAL  AND  RESISTANCE  OF  THE  STARFISH 
EGG  BEFORE  AND  AFTER  FERTILIZATION  * 

ALBERT  TYLER,  2  ALBERTO  MONROY,»  C.  Y.  KAO,-*  AND 
HARRY  GRUNDFEST  " 

Marine  Biological  Laboratory,  Woods  Hole,  Mass. 

Unequal  distribution  of  ions  between  the  interior  and  exterior  is  characteristic  of 
living  cells.  In  many  thus  far  studied  the  concentration  of  potassium  is  much  higher 
in  the  interior  while  the  concentrations  of  sodium  and  chloride  are  lower.  This 
ionic  asymmetry  is  associated  with  a  potential  difference  across  the  plasma  mem- 
brane which  is  approximately  related  to  the  relative  concentration  of  potassium 
(Hober,  1945;  Hodgkin,  1951)  according  to  the  Nernst  equation: 

or  at  20°  C.,         E  (in  mv.)  =  -58  log 


(A+)o 

Membrane  potentials  have  been  recorded  from  many  cells  (Hodgkin,  1951  ; 
Grundfest,  1955),  including  some  whose  internal  potassium  concentration  is  known, 
by  means  of  a  fine,  saline-filled,  microcapillary  (Gelfan,  1927,  1931  ;  Ling  and 
Gerard.  1949)  inserted  through  the  cell  surface.  The  magnitude  of  this  potential 
in  different  cells  ranges  from  50  to  100  mv.,  inside  negative.  This  indicates  an  in- 
ternal excess  of  potassium  approximately  9  to  50  times  the  external  concentration, 
and  is  in  approximate  accord  with  the  observed  values  in  specific  cases  in  which 
potassium  concentration  has  been  determined. 

Several  investigators  (Gelfan,  1931;  Rothschild,  1938;  Kamada  and  Kinosita, 
1940)  had,  many  years  ago,  reported  that  they  could  find  no  potential  difference 
across  the  membrane  of  echinoderm  eggs.6  Interest  in  this  problem  has  sharpened 
recently  because  of  two  new  factors.  In  the  first  place  a  number  of  workers  (Scheer,. 

1  This  work  was  reported  at  the  General  Scientific  Session  of  the  Marine  Biological  Labora- 
tory in  1955  (Grundfest,  Kao,  Monroy  and  Tyler,  1955;  and  Tyler,  Monroy,  Kao  and  Grundfest, 
1955).  We  wish  to  thank  the  Director  and  Staff  of  the  MBL  for  the  facilities  placed  at  our 
disposal. 

-  Kerckhoff  Laboratories  of  Biology,  California  Institute  of  Technology,  Pasadena.  Work 
of  this  investigator  supported  in  part  by  research  grant  C-2302  from  the  National  Cancer  Insti- 
tute, National  Institutes  of  Health,  Bethesda,  Md. 

;;  Istituto  di  Anatomia  Comparata,  University  of  Palermo,  Italy.  Fullbright  Fellow,  Sum- 
mer, 1955. 

4  Department  of  Physiology  and  Pharmacology,  State  University  of  New  York,  College  of 
Medicine  at  New  York. 

5  Department   of   Neurology,    College   of   Physicians   and    Surgeons,    Columbia   University, 
New  York.     Supported  in  part  by  grant  (No.  1880-Penrose)  from  the  American  Philosophical 
Society  ;  and  grants  from  Muscular  Dystrophy  Association  of  New  York,  and  Process  and  In- 
struments, Brooklyn,   N.   Y.     Laboratory  facilities  were  provided  by  a  grant  from  the   MBL 
under  its  ONR  contract  (Monr  —  09703). 

6  In  a  brief  report  Taylor  and  Whitaker   (1926)   mention  experiments  on  eggs  of  the  sea 
urchin  Clypeaster  rosaceus  showing  a   potential   difference,   inside  negative,  of  about    1   mv., 
which  would  be  very  low  in  comparison  with  other  kinds  of  cells  that  have  been  investigated. 

153 


154  A.  TYLER,  A.  MONROY,  C.  Y.  KAO  AND  H.  GRUNDFEST 

Monroy,  Santangelo  and  Riccobono,  1954;  Furshpan,  1955;  Kao,  1955)  have  in- 
dependently made  similar  observations  in  several  varieties  of  marine  eggs,  using 
modern  recording  equipment  and  stable  KCl-filled  (Kamada  and  Kinosita,  1940; 
Nastuk  and  Hodgkin,  1950)  microcapillary  electrodes.  In  the  second  place,  con- 
vincing data  have  become  available  (Rothschild  and  Barnes,  1953)  showing  that  at 
least  for  the  eggs  of  the  sea  urchin  Paracentrotus  Ih'idns,  the  potassium  content  in 
the  aqueous  phase  of  the  egg  is  21  times  higher  than  in  sea  water,  while  the  internal 
sodium  and  chloride  concentrations  are,  respectively,  about  %  and  %  of  those  in 
sea  water.  It  would  therefore  seem  likely  that  in  the  sea  urchin  egg  a  membrane 
potential  of  about  80  mv..  inside  negative,  should  be  observed. 

The  persistent  failure  to  find  a  membrane  potential  prompted  a  re-examination  of 
this  problem  with  certain  technical  refinements  which  provide  definitive  verification 
of  the  entry  of  a  microelectrode  into  the  cell,  as  well  as  measurements  of  the  resist- 
ance and  capacity  of  the  membrane.  Parallel  experiments  with  microinjection 
(Tyler  and  Monroy,  1955 )  helped  to  elucidate  and  overcome  difficulties  encountered 
in  attempts  to  pierce  the  cell  membrane  of  echinoderm  eggs.  A  potential  difference 
was  thereupon  found  to  exist  across  the  membrane  of  Astcrias  eggs.  Its  magnitude 
was  found  to  be  somewhat  lower  than  would  be  expected  on  the  basis  of  the  high 
internal  K+,  which  was  also  determined  in  these  experiments.  As  in  other  kinds  of 
cells  that  have  been  investigated,  the  membrane  potential  difference  changes  reversi- 
bly  on  changing  the  external  K+  concentration. 

Although,  as  will  be  shown  below,  it  is  unlikely  that  penetration  by  the  micro- 
electrode  had  been  attained  in  earlier  work,  several  observers  (Rothschild,  1938; 
Scheer  ct  al.,  1954;  Furshpan,  1955)  have  reported  that  eggs  could  be  fertilized 
while  apparently  impaled.  Fertilization  was  also  successful  in  the  present  experi- 
ments with  the  electrode  truly  inside  the  egg.  The  effects  of  fertilization  on  the  po- 
tential and  on  the  electrical  constants  of  the  membrane  were  therefore  also  studied. 

METHODS 

Tyler  and  Monroy  (1955)  carried  out  experiments  attempting  microinjection 
of  fluids  into  eggs  of  Arbacia,  Echinarachnius  and  Astcrias.  A  smaller  number  of 
experiments  were  performed  in  the  present  series  in  an  effort  to  penetrate  eggs  of 
Arbacia  punctnlata  with  microelectrodes.  Confirming  the  experience  of  Chambers, 
(Pandit  and  Chambers,  1932,  and  personal  communication)  in  both  cases  it  was 
found  that  piercing  the  surface  is  difficult.  Microelectrodes  or  micropipettes  which 
appear  to  have  penetrated,  in  actuality  only  carry  the  membrane  before  them  even 
to  the  extent  of  creating  a  tunnel  (as  Dan.  1943.  has  also  observed)  as  the  micro- 
capillary  travels  through  the  diameter  of  the  egg.  This  was  clearly  revealed  in  the 
microinjection  experiments  of  Tyler  and  Monroy  (1955),  in  which  it  was  observed 
that  the  membrane  could  form  a  tight  sleeve  around  the  inserted  pipette,  the  latter 
then  appearing  to  be  within  the  cytoplasm  of  the  egg.  However,  injected  fluid 
(KCl-NaCl  solutions)  would  simply  expand  this  sleeve  and  flow  out  into  the  sur- 
rounding medium  rather  than  into  the  egg.  Eggs  of  Astcrias  forbcsii  behaved 
similarly,  as  illustrated  in  Figure  1 ,  but  in  view  of  their  larger  size  ( average  diam- 
eter, 146  /A)  these  were  chosen  in  preference  to  eggs  of  Arbacia  for  further  investi- 
gation. Penetration  of  these  eggs  was  accomplished  by  the  technique  of  jarring  the 
preparation  by  a  light  tap  on  the  table.  This  sudden  vibration  was  especially  ef- 


ELECTRICAL  PROPERTIES  OF  STARFISH  EGGS 


155 


fective  after  the  indented  plasma  membrane  had  formed  a  tight  sleeve  around  the 
electrode  (as  in  Figure  le)  and  was  allowed  to  remain  in  this  condition  for  a  short 
while.  Figure  2  illustrates  eggs  with  one  or  two  electrodes  that  have  penetrated 
into  the  cytoplasm. 


FIGURE  1.  Photomicrographs  showing  apparent  entry  of  a  micropipette  into  an  egg 
of  Astcrias  forbcsii  held  by  a  "sucking"  pipette.  Magnification,  104  X.  The  micropipette, 
filled  with  isotonic  XaCl-KCl  solution  containing  chlorphenol  red,  is  pushed  through  the  vitel- 
line  membrane  and  indents  the  underlying  surface  (plasma  membrane)  forming  a  large  conical 
depression  (d).  After  about  two  minutes  the  depression  closes  over  the  pipette  (c)  and  the 
latter  appears  to  be  within  the  cytoplasm.  However,  injection  of  fluid  (/  to  h)  shows  that  the 
walls  of  the  depression  had  formed  a  tight  sleeve  around  the  shaft  of  the  pipette.  The  fluid  ex- 
pands this  sleeve  and  stretches  the  vitelline  membrane,  flowing  out  through  the  latter.  Upon 
removal  of  the  pipette  (i  to  k)  the  remaining  fluid  is  expelled  as  the  egg  rounds-up  within  some 
two  minutes.  The  same  egg  with  a  fertilization  membrane  elevated  at  two  minutes  after  fertili- 
zation is  shown  in  /. 


156 


A.  TYLER,  A.  MONROY,  C.  Y.  KAO  AND  H.  GRUNDFEST 


The  experiments  were  carried  out  primarily  on  unfertilized  eggs.  A  few  meas- 
urements were  made  on  eggs  with  germinal  vesicles  or  fertilized  prior  to  impalement 
by  microelectrodes.  The  eggs  were  obtained  from  spontaneously  shedding  animals. 
They  were  kept  at  temperatures  of  18  to  20°  C.  until  used,  one  to  five  hours  after 
shedding.  The  experiments  were  carried  out  mostly  at  about  25°  C.  and  all  the  eggs 
used  appeared  to  be  normal.  All  those  so  tested,  as  well  as  parallel  samples,  were 
fertilizable.  Some  batches  of  eggs  were  stored  at  about  10°  C.  but  these  underwent 
spontaneous  activation  on  transfer  to  the  room  temperature  and  were,  therefore,  not 
used  in  the  experiments. 


FIGURE  2.  Photomicrographs  of  eggs  of  Astcrias  forhcsii  held  on  "sucker"  and  impaled 
on  microelectrodes.  Magnification:  a  and  />,  116X;  c,  200  X.  a,  an  egg  with  intact  germinal 
vesicle;  />,  another  after  dissolution  of  the  germinal  vesicle;  c.  an  egg  fertilized  after  insertion 
of  two  microelectrodes.  The  tips  of  the  microelectrodes  are  not  visible. 

All  the  impaled  eggs,  in  the  experiments  involving  fertilization,  had  undergone 
dissolution  of  the  germinal  vesicle,  and  were  in  various  stages  of  the  maturation  di- 
visions. Sperm  were  diluted  in  a  solution  of  10~3  molar  Versene  '  in  sea  wrater, 
since  the  latter  improves  the  fertilizing  power  of  dilute  sperm  suspensions  (Tyler, 
1953).  The  sperm  were  introduced  by  means  of  a  capillary  pipette  at  a  distance 
several  millimeters  from  the  egg.  The  time  at  which  sperm  were  seen  to  approach 
the  egg,  as  well  as  the  time  of  formation  of  the  fertilization  membrane  were  noted 
for  correlation  with  the  measurements  of  the  membrane  potential. 

Experimental  arrangement 

Sea  water  containing  eggs  was  placed  (Fig.  3A)  on  a  transilluminated  Incite 
plate  mounted  on  a  mechanical  stage  under  a  binocular  microscope.  The  sea  water 
was  in  continuity  with  one  end  of  a  sea  water-filled  tunnel,  into  the  other  end  of 
which  was  inserted  an  Ag-AgCl  reference  electrode.  One  or  two  microelectrodes 
(tip  diameters  less  than  0.5  //),  each  individually  carried  in  a  micromanipulator,  ap- 

7  Versene  is  the  trade  name  (Bersworth  Chemical  Co.)  of  ethylene  diamine  tetraacetic  acid. 


ELECTRICAL  PROPERTIES  OF  STARFISH  EGGS 


157 


preached  diagonally  from  above  at  a  small  angle.  Opposite  was  another  manipu- 
lator which  held  a  glass  suction  pipette.  This  device  (Tyler,  19551)),  modified  from 
the  elastimeter  of  Mitchison  and  Swann  (1954),  was  very  useful  for  holding  the  egg 
fixed  gently  but  firmly  at  one  pole  while  the  microelectrodes  were  pressed  against 
the  other  (Figs.  1  and  2).  The  polished  tip  of  the  "sucker,"  somewhat  smaller  than 
the  diameter  of  the  egg,  dipped  into  the  sea  water  containing  the  eggs.  The  other 
end  was  flexibly  coupled  to  a  vertical  glass  tube  which  could  be  raised  or  lowered  by 
a  rack  and  pinion  movement.  The  system  was  filled  with  sea  water.  By  maneu- 


rock  a 
pinion 


rubber 
tubing 


gloss 
upright 


B 

Current  El. (E,)  Voltoge  EI.(Et) 


manipulator 
clamp 


Potential 


Current 


FIGURE  3.  Diagrammatic  illustration  of  the  experimental  arrangement.  A:  The  mechani- 
cal and  optical  set-up.  The  egg  (O)  is  shown  (enlarged)  lying  in  a  drop  of  sea  water  (SW) 
on  a  lucite  plate  (P)  which  is  illuminated  from  below  (L)  bnd  observed  through  a  micro- 
scope (M).  The  sucker  (S)  has  a  rack  and  pinion  for  raising  or  lowering  the  vertical  tube. 
The  electrodes  (E)  are  inserted  into  the  egg  at  the  pole  opposite  that  held  in  the  sucker.  B: 
The  electrical  arrangement.  The  two  microelectrodes  (El.i  and  EU)  are  shown  in  the  egg  (O). 
El.i  is  the  current  electrode  fed  through  a  pulse  generator.  E1.2  is  connected  to  one  grid  of  the 
potential  recording  amplifier.  The  other  grid  connects  with  the  fluid  (SW)  as  well  as  with  a 
resistor  (R)  through  the  reference  electrode  (RE).  Across  R  is  the  amplifier  measuring  the 
current  in  the  pulse.  When  a  single  microelectrode  (E1.2)  was  used,  RE  was  grounded. 

vering  the  mechanical  stage,  any  desired  egg  in  the  drop  could  be  brought  to  the 
vicinity  of  the  tip  of  the  sucker.  Lowering  the  upright  created  sufficient  negative 
pressure  to  take  up  and  hold  the  egg  firmly  against  the  tip.  Manipulation  was  car- 
ried out  under  80  or  160  x  magnification. 

Electrical  measurements 

Determination  of  the  membrane  potential  and  the  resistance  and  capacity  of  the 
membrane  constituted  the  electrical  measurements.  For  the  former  a  single  micro- 
electrode,  drawn  prefilled  with  3  M  KG  (Kao,  1954),  was  sufficient.  This  was  con- 
nected to  a  high  impedance  negative  capacity  input  amplifier  8  and  a  cathode  ray 

8  Designed  by  Mr.  E.  Amatniek,  electronic  engineer  at  the  Dept.  of  Neurology,  Columbia 
University. 


158  A.  TYLER,  A.  MONROY,  C.  Y.  KAO  AND  H.  GRUNDFEST 

oscillograph.  The  external  medium  was  grounded  through  the  Ag-AgCl  reference 
electrode,  or  in  some  cases  the  "sucker"  was  itself  made  the  reference  system.  The 
standard  sensitivity  employed  was  20  mv./cm.  deflection  on  the  face  of  the  oscillo- 
graph tube  so  that  changes  in  potential  as  low  as  two  to  three  mv.  could  be  detected. 
Visual  observation  was  supplemented  by  photography  of  the  trace. 

Determination  of  the  electrical  constants  of  the  membrane  required  passage  of  a 
square  pulse  current  through  the  membrane  and  the  measurement  of  the  potential 
difference  created  by  this  current  across  the  resistance  and  capacity  of  the  mem- 
brane (Fig.  3B).  For  this  purpose  two  microelectrodes  were  inserted  into  the 
eggs  (Fig.  2c).  One  of  these  was  connected  to  a  pulse  generator  delivering  12  or 

WVWVVVVVVVVVVVVVWVVV 

•M 

D 


B 


FIGURE  4.  Current  and  voltage  pulses  as  recorded  before  and  after  entry  of  two  micro- 
electrodes  into  the  egg.  Left:  A  large  current  pulse  (A,  upper  trace)  caused  only  transient 
capacitative  artifacts  in  the  voltage  trace  (below)  as  long  as  the  electrodes  were  outside  the  egg. 
B,  C:  The  current  pulse  was  reduced.  B,  before  and  C,  after  penetration  of  the  electrodes.  A 
third  trace,  which  represents  the  zero  level  at  the  time  of  entry,  is  seen  at  its  correct  position  in  C. 
The  membrane  potential,  about  —  20  mv.,  has  brought  the  voltage  trace  down.  The  square 
pulse  of  current  is  reflected  in  a  membrane  IR  drop  with  retarded  onset  and  decay.  Right: 
Another  egg.  D :  The  current  pulse  caused  only  the  capacitative  artifact  on  the  voltage  trace. 
E:  When  the  two  electrodes  were  pressed  against  the  egg,  the  voltage  trace  also  recorded  a 
deflection  with  rapid  onset  and  decay.  However,  the  steady  potential  was  zero.  F :  A  few  sec- 
onds later,  the  electrodes  had  penetrated  the  egg,  causing  the  characteristically  slowed  onset  and 
decay  of  the  voltage  trace.  A  membrane  potential  of  about  —  30  mv.  is  also  seen.  Time  scale 
in  msec.,  upper  right. 

30  msec,  pulses  of  controllable  amplitude,  synchronized  in  rate  and  time  with  the 
sweep  of  the  oscillograph.  The  external  reference  electrode  was  connected  to  a  re- 
sistance, the  other  end  of  which  was  grounded,  as  was  the  return  of  the  stimulator. 
An  amplifier  across  the  resistor  recorded  the  IR  drop  in  the  latter  and  the  current, 
7,  through  the  membrane  was  calculated  from  this  measurement.  The  sensitivity  of 
the  current  trace  on  the  oscillograph  was  1  mv./cm.  and  with  a  1  megohm  resistor 
for  the  IR  drop  this  amplitude  of  deflection  corresponded  to  0.001  juA. 


ELECTRICAL  PROPERTIES  OF  STARFISH  EGGS 


159 


The  second  microelectrode  led,  as  before,  to  another  amplifier,  but  in  this  case 
recording  the  membrane  potential  differentially,  the  second  grid  being  connected  to 
the  indifferent  electrode  and  the  high  end  of  the  resistor.  The  potential  change  of 
this  amplifier  during  the  square  pulse  thus  represented  the  IR  drop  across  the  mem- 
brane in  series  with  the  resistance  of  the  fluid.  Since  the  resistance  of  the  latter 
was  small  compared  with  that  of  the  membrane,  it  was  neglected.  From  the  knowl- 
edge of  7  obtained  in  the  current  record,  R  of  the  membrane  could  be  computed. 
This  was  transformed  to  the  specific  membrane  resistance  RM  (ohm-cm.2)  by  multi- 
plying by  the  surface  area  of  the  egg  (average  diameter  =-  146 /x;  surface  ==  6.7  : 


oo-o 


-10- 


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c 
o> 


'-30 


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c 
o 

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.o 

£ 


-40 


-50 


-60 


O 

i 
i 
i 


I 
i 
I 
Electrode 

Entry 


Electrode 
E  it 


-CX 


O--O--O--C/ 


Sperm   Reached 
Added    Egg 


Membrane 
Visible 


O---O--O-O 


u 
If 
ii 

6 


I  2 

Time    After  Insemination  —  min. 


55-S2O 


FIGURE  5.  Membrane  potential  of  Asterias  egg  and  its  changes  on  fertilization.  Entry  of 
the  electrode  into  the  egg  caused  sudden  appearance  of  —  60  mv.  membrane  potential,  which  de- 
creased to  —  30  mv.  rapidly.  The  time  scale  has  its  origin  at  the  time  sperm  were  added  to  the 
sea  water.  Within  30"  sperm  were  seen  to  have  contacted  the  egg  and  at  this  time  the  previ- 
ously steady  potential  decreased  by  5  mv.  Subsequently  the  potential  again  increased  and  re- 
mained steady  at  --40  mv.  until  the  electrode  was  removed.  Absence  of  drift  in  the  system  is 
indicated  by  the  return  of  the  voltage  trace  to  the  initial  value. 

10~4  cm.2).  The  time  constant  (r)  when  the  rise  and  fall  of  the  voltage  trace  (Fig. 
4C,  F)  had  reached  67c/c  of  the  final  value  was  measured  from  the  records.  The 
membrane  capacity  (Cv)  was  determined  in  ju.F/cm.2  from  the  relation  r  --  Ru  Cu. 
Certain  precautions  have  to  be  taken  in  experiments  of  this  type.  The  current 
electrode  must  be  non-polarizable  in  the  range  of  currents  used  for  the  measure- 
ments. This  was  checked  at  the  start  and  end  of  each  experiment.  Secondly,  ap- 
plied current  must  be  rather  low.  With  a  microelectrode  tip  of  0.5  /A  diameter,  1  juA 
flow  represents  a  current  density  at  the  tip  of  about  500  A/cm.-  which  might  lead  to 


160 


A.  TYLER,  A.  MONROY,  C.  Y.  KAO  AND  H.  GRUNDFEST 


heating  and  perhaps  breaking  of  the  electrode.  Furthermore,  a  flow  of  1  juA 
through  the  whole  surface  area  of  the  egg  membrane  is  equivalent  to  a  current  of  1.5 
mA/cm.2  which  is  a  high  density,  at  least  for  the  membranes  of  excitable  tissues. 

The  use  of  the  two  microelectrodes  and  a  current  pulse  served  the  additional  im- 
portant purpose  of  providing  unequivocal  evidence  of  the  penetration  of  both  elec- 
trodes into  the  egg  (Fig.  4).  When  these  electrodes  were  in  the  fluid  the  record 
of  the  voltage  trace  differed  radically  from  the  trace  of  the  current  pulse  (  Fig.  4A,  B, 
D).  The  former  showed  only  rapid  short-lived  deflections  of  opposite  sign  at  the 
beginning  and  end  of  the  applied  pulse.  These  are  attributable  to  capacitative 


-     O— O 


-10 


1-20 


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"o 

1-30 

o. 

o 

o 

f  -40 
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-50- 


-60- 


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ode  ' 

£         \                                         Electrode 

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ry 

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0 

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Added                     ^                        j^ 

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0                             ^Q 

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Membrane           it. 
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Time   After   Insemination  -  min.                         55-523 

FIGURE  6.  Depolarizing  effect  of  externally  applied  KC1  and  its  reversibility.  The  se- 
quence up  to  one  minute  on  the  time  scale  is  similar  to  that  of  Figure  5.  When  the  membrane 
potential  of  the  fertilized  egg  had  reached  its  maximum  value,  the  sea  water  was  largely  re- 
placed with  isotonic  KC1.  This  caused  rapid  and  almost  complete  depolarization,  which  was  re- 
versed on  washing  out  the  KC1  with  sea  water.  A  drift  in  the  amplifier  of  4  mv.  negative  had 
changed  the  base  line  slightly. 


coupling  between  the  electrodes.  During  the  major  portion  of  the  applied  pulse, 
the  voltage  trace  remained  essentially  at  zero  potential,  reflecting  the  low  resistance 
of  the  sea  water  and  the  consequently  low  IR  drop  across  it.  On  occasion,  when 
both  electrodes  were  simultaneously  pressed  firmly  against  the  egg,  the  voltage 
trace  showed  a  large  deflection  which  would  be  expected  if  the  current  path  now 
included  a  high  resistance  formed  by  the  surface  of  the  egg  membrane.  However, 
this  voltage  record  was  characterized  (Fig.  4E)  by  its  faithful  reproduction  of  the 
form  of  the  current  pulse,  indicating  that  the  recording  electrode  was  not  across 


ELECTRICAL  PROPERTIES  OF  STARFISH  EGGS 


161 


the  capacity  of  the  membrane.  "When  penetration  occurred  the  voltage  pulse 
changed  in  form  characteristically,  rising  and  falling  more  slowly  than  the  current 
trace  (Fig.  4C,  F).  This  behavior,  in  view  of  our  original  uncertainty  as  to  the 
existence  of  a  potential  difference  across  the  egg  membrane,  proved  valuable  initially 
in  definitely  establishing  the  entry  of  the  microelectrodes  into  the  cytoplasm. 

Ionic  content  of  Astcrias  eggs 

Measurements  of  the  internal  ionic  milieu  of  Astcrias  eggs  were  not  found  in 
the  literature.  A  sample  of  over  two  million  eggs  was  therefore  subjected  to  analy- 
sis by  flame  photometry.  The  procedure  is  detailed  below  in  conjunction  with  the 
data  obtained. 


RESULTS 


Membrane  potential 


In  nearly  all  the  eggs  studied  no  potential  accompanied  apparent  penetration  by 
the  electrode,  but  one  or  more  taps  on  the  table  always  caused  the  sudden  appearance 
of  a  potential  difference  with  the  internal  electrode  negative,  except  in  a  number  of 
eggs  which  cytolyzed  as  the  electrode  appeared  to  enter.  The  failure  to  obtain  the 

TABLE  I 

Steady  potential  difference  observed  across  the  membrane  of  unfertilized  eggs 


P.D.  in  mv.,  inside 

negative 

10 

15 

20 

25 

30 

40 

45 

50 

Number  of  eggs 

(Total  24) 

3 

2 

5 

1 

8 

3 

1 

1 

In  one  additional  egg,  fertilized  prior  to  penetration  of  the  electrode,  the  membrane  p.d.  was 
initially  --40  mv.,  and  increased  to  —48  mv. 

In  one  other  egg  only  a  p.d.  of  +10  mv.  was  obtained. 

potential  initially  was  probably  due  to  extensibility  of  the  egg  membrane.  Jarring 
probably  caused  penetration  of  the  membrane  during  the  resultant  vibration.  The 
potential,  upon  penetration,  reached  values  up  to  60  mv.,  apparently  instantaneously, 
then  rapidly  declined  to  a  lower  steady  value  (Figs.  5,  6).  The  steady  value  of  the 
membrane  potential  ranged  in  different  experiments  from  a  low  of  10  mv.  to  a  high 
of  50  mv.  (Table  I),  with  the  majority  of  eggs  showing  potentials  of  20  to  30  mv. 

The  larger  initial  value  probably  reflects  more  nearly  the  true  potential  difference 
momentarily  disclosed  as  the  fine  tip  of  the  microelectrode  broke  through  the  egg 
membrane.  The  subsequent  release  of  tension  of  the  latter,  as  it  rounds  up  and 
moves  farther  onto  the  electrode,  could  create  an  imperfect  seal  of  the  membrane 
around  the  shaftlet  and  lead  to  partial  short  circuiting  of  the  full  potential.  In 
most  experiments  the  potential  remained  steady  at  the  lower  value  as  long  as  the 
electrode  was  left  in  the  egg.  Withdrawal  was  accompanied  by  an  abrupt  return 
of  the  oscillograph  trace  to  the  zero  potential  level.  There  seemed  to  be  no  con- 
sistent difference  in  the  value  of  the  steady  potential  if  two  electrodes  were  inserted 
simultaneously  or  sequentially.  The  number  of  experiments  of  this  type  was  too 


162  A.  TYLER,  A.  MONROY,  C.  Y.  KAO  AND  H.  GRUNDFEST 

small  and  the  scatter  of  potentials  too  great  to  employ  this  method  for  calculating  the 
possible  magnitudes  of  leaks  around  the  electrode. 

Two  eggs  included  in  Table  I,  through  which  relatively  high  currents  (0.5  to 
l.OjuA)  were  later  passed,  cytolyzed  in  the  course  of  the  experiments.  Five  cyto- 
lyzed  spontaneously,  and  two  that  had  an  intact  germinal  vesicle  cytolyzed  on  sub- 
sequent penetration  of  this  structure.  In  all  cases  cytolysis  resulted  in  disappear- 
ance of  the  membrane  potential.  In  three  cases  the  potential  disappeared  with  no 
observable  cytolytic  effects.  Movement  of  the  tip  then  again  disclosed  the  mem- 
brane potential.  These  can  be  interpreted  as  eggs  in  which  the  cytoplasm  had  be- 
come sealed  off  from  the  electrode  (Chambers,  1922)  by  a  precipitation  membrane 
(Heilbrunn,  1927,  1952;  cf.  Costello,  1932).  In  a  few  other  experiments,  all  done 
with  the  same  microelectrode,  penetration  was  indicated  at  first  by  a  small  positive 
potential  (about  10  mv.)  which  in  all  but  one  case  reversed  to  negativity.  The 
positive  potential  may  have  been  due  to  increase  of  the  electrode  resistance  by  plug- 
ging of  the  tip  as  this  pressed  into  the  egg  membrane.  With  the  grid  current 
of  the  amplifier  about  lO'^A  and  positive,  a  shift  of  +  10  mv.  would  be  caused  by 
insertion  of  a  resistance  of  10°  ohms.  Another  possibility  is  that  the  electrode  had 
penetrated  the  egg,  but  had  been  sealed  off  from  the  cytoplasm  while  some  leakage 
remained  around  the  shaft.  The  internal  negativity  would  then  register  as  posi- 
tivity  on  pickup  by  the  external  electrode. 

Membrane  potential  upon  fertilisation 

Eight  eggs  were  fertilized  while  impaled.  Insemination  was  done  usually  at 
least  5  minutes  after  impalement  in  order  to  ascertain  that  the  membrane  potential 
was  steady  and  that  the  egg  was  not  undergoing  cytolytic  changes.  When  sper- 
matozoa were  seen  to  have  reached  the  impaled  egg  (about  15  to  30  seconds  after 
insemination)  the  membrane  potential  suddenly  decreased  from  its  previously 
steady  value.  This  change  amounted  to  5  to  10  mv.,  and  was  temporary  (Figs. 
5,6).  The  membrane  potential  then  began  to  increase  gradually,  eventually  attain- 
ing a  magnitude  greater  than  the  former  steady  value,  and  in  some  cases  as  large 
as  that  momentarily  seen  during  entry  of  the  electrode.  This  increased  internal 
negativity  persisted  during  the  subsequent  period  of  observation  which,  for  the 
present  series  of  experiments,  was  not  longer  than  5  minutes.  The  new  steady 
value  of  potential,  5  to  20  mv.  higher  than  before  fertilization,  was  attained  in  1  to 
2.5  minutes,  which  was  also  the  time  at  which  the  fertilization  membrane  had  be- 
come distinctly  elevated. 

Ionic  content  of  Asterias  eggs 

Estimate  of  the  magnitude  of  the  membrane  potential  to  lie  expected  requires 
knowledge  of  the  ionic  concentration  in  the  egg.  Determinations  were  therefore 
made  of  the  K  and  Na  content  of  Asterias  eggs.  The  procedure  was  as  follows: 

A  100-ml.  suspension  of  unfertilized  eggs  was  prepared  in  sea  water.  From 
this,  a  one-mi,  sample  was  removed  by  means  of  a  wide-mouth  (2.5  mm.)  pipette. 
It  was  diluted  20-fold  and  a  one-nil,  portion  used  for  counting  the  number  of  eggs. 
During  these  procedures  precautions  were  taken  to  keep  the  suspension  of  eggs 
distributed  as  uniformly  as  possible.  The  final  one-mi,  diluted  sample  contained 


ELECTRICAL  PROPERTIES  OF  STARFISH  EGGS 


163 


1096  eggs.  Therefore  the  99  ml.  of  the  original  suspension  contained  2.17  X  10G 
eggs.  The  eggs  of  the  latter  suspension  were  allowed  to  settle,  and  most  of  the 
fluid  was  drawn  off.  The  remainder  was  then  centrifuged  for  15  minutes  at 
1500  X  g,  in  graduated  centrifuge  tubes.  The  packed  eggs  measured  6.1  ml.,  and 
above  them  was  an  additional  gelatinous,  opalescent  layer  of  2.1  ml.  representing  the 
material  of  the  gelatinous  coat  of  the  egg.  Supernatant  fluid  was  withdrawn  to 
leave  a  total  volume  of  10  ml.  of  packed  eggs,  gelatinous  layer  and  sea  water.  The 
eggs  lost  during  the  procedure  were  determined  from  counts  of  aliquots  of  the 

TABLE  II 
Measurements  of  diameters  of  eggs  of  Asterias  forbesii 


Number  of  eggs 

1 

3 

1 

1 

2 

2 

1 

3 

Total:         14 

Average  of  the  two  di- 

ameters (n) 

140 

142.5 

143.8 

145 

146.3 

147.5 

148.8 

150 

Average:  145.  9 

supernatants.  Their  number  was  2128  or  less  than  0.1^  of  the  total  in  the  packed 
eggs.  To  the  10  ml.  volume  of  packed  eggs  and  supernatant,  and  separately  to  an 
equal  volume  of  supernatant  fluid,  were  added  10  ml.  of  sulphuric  acid.  The  two 
preparations  were  allowed  to  stand  overnight,  transferred  with  distilled  water 
washings  to  digestion  flasks  and  boiled  for  about  4  hours,  one  ml.  of  30%  H2O2  be- 
ing added  to  help  clarify  the  material.  Both  preparations  were  then  transferred  to 
100-ml.  volumetric  flasks  and  made  up  to  that  volume  in  distilled  water.  The  origi- 
nal samples  had  thereby  been  diluted  10-fold.  These  were  analyzed  for  K+  and 
Na+  by  flame  photometry.9 

TABLE  III 
Determinations  of  K  and  Na  content  of  eggs  of  Asterias  forbesii 


(1)  mM  in  10  ml.  of  suspension 
containing  2.168  X  107  eggs 

(2)  mM  in  10  ml.  of  supernatant 

(3)  mM  in  6.47  ml.  of  supernatant 

(4)  mM  in  3.53  ml.  of  eggs  [(l)-(3)] 

(5)  mM/ml.  eggs 


Potassium 

0.656 
0.205 
0.133 
0.523 
0.148 


Sodium 

2.40  to      2.61 

4.00  to      4.32 

2.59  to      2.79 

-0.19  to  -0.18 


To  calculate  the  content  of  these  ions  in  the  eggs  it  was  necessary  to  determine 
the  egg  volume,  exclusive  of  interstitial  fluid.  The  diameters  of  14  eggs,  in  which 
the  difference  between  diameters  at  right  angles  was  less  than  4%,  were  measured, 
with  the  results  shown  in  Table  II.  The  flame  photometric  determinations  and 
calculated  values  of  K  and  Na  content  are  given  in  Table  III. 

On  the  basis  of  an  average  diameter  of  146 /A  the  volume  of  each  egg  is  1.63  X 
10~G  cm.3  and  that  of  the  total  number  in  the  suspension  is  3.53  ml.  The  latter 
value  is  58%  of  the  volume  (6.1  ml.)  of  the  packed  eggs  after  the  low  speed  centri- 
fugation,  and  is  in  reasonable  agreement  with  the  value  to  be  expected  from  the 
packing  of  spheres,  plus  a  small  allowance  for  adherent  jelly  coat.  The  10-ml. 

;i  We  are  indebted  to  Dr.  James  Green  of  Rutgers  University  and  to  Dr.  George  Scott  of 
Oberlin  College  for  the  analyses. 


164 


A.  TYLER,  A.  MONROY,  C.  Y.  KAO  AND  H.  GRUNDFEST 


specimen  containing  eggs  therefore  was  composed  of  35.3%  eggs  and  64.7%  inter- 
stitial fluid  (including  gelatinous  coat  material).  From  the  measurements  of  the 
parallel  sample  of  supernatant  fluid  the  6.47  ml.  of  the  supernatant  in  the  egg  sample 
contained  0.133  mM  K+.  The  3.53  ml.  of  eggs  therefore  contained  0.523  mM  or 
0.148  mM/ml.  of  eggs.  This  figure  is  about  15  times  the  K+  concentration  of  sea 
water  (0.01  mM/ml.).  On  the  basis  that  the  eggs  contain  approximately  75% 
water  by  weight  and  80%  by  volume,  the  K+  concentration  becomes  0.185  mM/ml. 

TABLE  IV 

Electrical  constants  of  Asterias  eggs 


Expt. 
No. 

Conditions 

Steady 
membrane 
potential 
(mv.) 

Maximum  applied 
current  GuA) 

Tm 

(ohms) 

R\r 

T 

(msec.) 

CM 
(/iF/cm.») 

Outward 

Inward 

A.   Maximum  currents  not  exceeding  .01 


25 

unfertilized 

* 

.006 

.006 

4.0X106 

2680 

1.28 

0.48 

fertilized 

— 

.006 

.006 

3.6 

2410 

1.25 

0.52 

26 

unfertilized 

-30 

.006 

.006 

4.97 

3330 

1.92 

0.58 

27 

unfertilized 

-15 

.005 

.01 

3.7 

2430 

1.04 

0.43 

28 

unfertilized 

-15 

.005 

.005 

5.8 

3880 

2.75 

0.71 

fertilized 

-50 

.005 

.005 

5.8 

3880 

1.6 

0.41 

B.  Higher  maximum  currents 


7 

unfertilized 

-10 

0.10 

0.06 

5.05X105 

368 

8a 

unfertilized 

-10 

0.10 

— 

11. 

737 

b 

unfertilized 

-10 

0.15 

— 

8.6 

574 

11 

unfertilized 

-20 

0.25 

— 

2.6 

172 

6 

unfertilized 

-20 

0.40 

— 

2.0 

133 

13 

unfertilized 

-30 

0.4 

0.4 

1.07 

71.6 

la 

unfertilized 

-30 

1.0 

1.0 

2.6 

174 

b 

unfertilized 

1.0 

1.0 

2.9 

194 

14 

unfertilized 

* 

1.5 

1.5 

2.5X104 

16.8 

*  Potential  not  measured  because  of  amplifier  drift. 

of  the  water  in  the  eggs.  This  corresponds  fairly  closely  with  the  value  of  0.210 
mM/mg.  of  water  found  in  eggs  of  the  sea  urchin  Paracentrotus  livid  us  (Rothschild 
and  Barnes,  1953). 

The  supernatant  contained  about  the  same  amount  of  sodium  as  does  sea  water, 
but  the  potassium  concentration  (0.02  mM/ml.)  \vas  twice  that  of  sea  water  (Table 
II).  For  the  calculation  given  above  it  was  assumed  that  the  extra  K+  derived  from 
gelatinous  material  of  the  eggs,  some  of  which  remained  in  the  supernatant.  On 
the  other  hand,  if  this  potassium  had  leaked  out  of  the  eggs  during  preparation  for 
analysis,  the  initial  concentration  of  the  ion  in  the  eggs  would  have  been  0.168 
mM/ml.  of  eggs  and  0.210  mM/ml.  of  the  water  in  the  eggs. 

Similar  calculations  for  the  Na  content  of  the  starfish  eggs,  using  either  the 
values  of  2.40  and  4.00  mM/10  ml.  of  egg  suspension  and  supernatant,  respectively, 


ELECTRICAL  PROPERTIES  OF  STARFISH  EGGS 


165 


or  2.61  and  4.32  gave  a  slightly  negative  value  (—  0.2  niM  per  3.53  ml.  eggs)  for 
this  ion.  In  view  of  the  high  content  of  Na  in  sea  water  and  therefore  in  the  inter- 
stitial fluid  of  the  egg  suspension,  the  value  for  Na  is  much  more  sensitive  to  errors 
in  determination  of  egg  volume  than  is  that  of  K.  For  example,  if  the  actual  egg- 
volume  were  13%  greater  than  determined,  the  calculated  Na  content  would  he  zero 
while  that  of  K  would  be  0.17  mM  ml.  of  the  water  in  the  eggs.  We  may  therefore 
conclude  that  the  Na  concentration  in  Asterias  eggs  is  less  than  one-twentieth, 
while  the  K  concentration  is  between  17  and  21  times,  the  values  found  in  sea 
water. 


Voltoge  -  mv. 


Rm    —  3100  ohm-cm 


30-- 


20" 


10  - 


O      o 


Cothodol 


I 


.005 


Anodol 


.005  -01 

Current  —  >jA 


--10 


-•20 


--30 


Combined  Data 
55-5 


FIGURE  7.  Membrane  current-membrane  voltage  relation  in  four  Asterias  eggs.  Two  of 
the  six  experiments  represent  measurements  done  after  fertilization.  The  slope  of  the  straight 
line  drawn  through  the  combined  data  is  the  average  resistance  (rm)  and  yields  RM  =  3100  ohm- 
cm2.  Anodal  signifies  outward  current  and  cathodal,  inward. 

Change  in  membrane  potential  on  increasing  external  K+ 

Four  experiments  served  to  test  and  demonstrate  the  sensitivity  to  K+  of  the 
potential  difference  across  the  egg  membrane,  but  the  relation  between  the  external 
K+  and  the  potential  was  not  studied  quantitatively.  Within  a  few  seconds  after 
isosmotic  KC1  was  added  to  the  sea  water  surrounding  an  impaled  egg  the  mem- 
brane potential  decreased  (Fig.  6)  and  reached  almost  complete  depolarization. 
The  K+-rich  solution  was  then  replaced  with  sea  water  and  the  initial  value  of  the 
membrane  potential  was  again  restored.  Fertilized  and  unfertilized  eggs  responded 


166 


A.  TYLER,  A.  MONROY,  C.  Y.  KAO  AND  H.  GRUNDFEST 


in  the  same  manner.  The  technical  arrangement  did  not  permit  rapid  washing  out 
of  the  excess  KC1  and  this  probably  accounts  for  the  slower  return  of  the  initial 
membrane  potential. 

Membrane  resistance  and  capacity 

These  electrical  constants  of  the  membrane  were  determined  in  10  eggs  with  the 
square  pulse  technique  described  earlier.  However,  in  six  the  maximum  current 
densities  were  rather  high  (0.5  to  1.5/xA)  and  the  results  were  probably  affected 


Voltage -mv 


Rm    ~  3830  ohm-cm2 


30-- 


20-- 


10-- 


Cathodal 


_L 


.005 


Anodol 


.005 


Current  - 


--10 


--20 


--30 


O    Unfertilized 
•    Fertilized 


55-528 


FIGURE  8.     Membrane   resistance  before   and   after   fertilization.     The   current-voltage    relation 

of  the  same  egg  before  and  after  fertilization. 

by  this  condition.  In  six  experiments  with  four  eggs  ( two  eggs  were  studied  after 
fertilization  aawell  as  before)  the  maximum  current  of  the  square  pulse  was  limited 
to  0.01  //A  and  these  results  are  shown  in  Table  IVA  and  Figures  7  and  8.  The 
average  membrane  resistance  was  3100  ohm-cm.-  In  the  two  eggs  measured  after 
the  fertilization  membrane  was  elevated  there  was  no  significant  change  in  resistance 
(Fig.  8;  Table  IVA).  In  the  six  experiments  employing  high  current  densities 
(0.5  to  1.5 /iA)  the  values  of  R^  were  markedly  smaller  (Table  IVB).  No  ex- 
planation will  be  attempted  at  present  for  this  effect.  Over  the  range  of  current 
densities  used  in  all  the  experiments  the  relation  between  membrane  voltage  and 
current  was  linear.  Assuming  a  specific  resistance  of  100  ohm-cm,  (approximately 


ELECTRICAL  PROPERTIES  OF  STARFISH  EGGS  167 

three  times  that  of  sea  water;  Cole  and  Cole,  1936a,  1936b)  for  the  cytoplasm,  the 
droplet  constituting  the  interior  of  the  egg  would  have  a  resistance  of  about  8  X  103 
ohms,  surrounded  by  a  membrane  with  a  resistance  several  orders  higher  than  this 
in  magnitude. 

Determination  of  the  time  constant  (r)  and  of  the  membrane  capacity  (d/) 
was  only  approximate,  because  the  oscillographic  records  of  the  membrane  voltage 
change  were  made  on  too  slow  a  time  base.  The  average  capacity  (0.52  /xF /cm.2) 
is  somewhat  smaller  than,  but  of  the  same  order  of  magnitude  as,  the  1.1  juF/cm.- 
obtained  by  Cole  and  Cole  (1936a)  with  eggs  of  Asterias  forbesii,  and  0.7  to 
2.7  pF  cm.-  listed  by  Cole  and  Curtis  (1950)  for  unfertilized  eggs  of  other  marine 
animals. 

DISCUSSION 

The  original  object  of  this  investigation  was  to  seek  an  explanation  for  the  re- 
ported absence  of  a  membrane  potential  in  some  echinoderm  eggs.  On  the  basis  of 
Rothschild  and  Barnes'  (1953)  finding  that  sea  urchin  eggs  contain  21-fold  higher 
concentration  of  K+  than  does  sea  water  it  was  to  be  expected  that  a  membrane  po- 
tential of  about  80  mv.  ought  to  be  present.  A  membrane  potential  has  nowr  been 
found  in  Asterias  eggs.  Its  magnitude,  at  least  60  mv.  under  what  we  believe  to  be 
the  optimum  condition,  is  smaller  by  about  15  to  20  mv.  than  might  be  expected  on 
the  basis  of  the  values  (17:  1  to  21  :  1)  that  we  obtained  for  the  ratio  of  K+  in 
Asterias  eggs  to  that  in  sea  water.  A  similar  discrepancy  is  usually  found  in 
nerve  and  muscle  fibers  (Hodgkin,  1951 :  Grundfest,  1955). 

The  reversible  depolarization  of  the  egg  membrane  in  response  to  increasing  the 
external  K+  agrees  with  the  behavior  of  other  cells  (Hodgkin,  1951),  but  the  quan- 
titative relation  between  potential  and  K+  concentration  was  not  tested  in  the  present 
experiments.  Another  point  of  similarity  relates  to  the  lowr  internal  Na+  concentra- 
tion. Asterias  eggs  contain  too  little  Na+  for  accurate  measurement  under  the 
experimental  conditions  employed.  Rothschild  and  Barnes  (1953)  found  a  con- 
centration of  52  inM/kg.  water  in  eggs  of  Paracentrotus  lividns  as  compared  with 
485  for  sea  water,  and  the  Na+  concentration  of  various  excitable  cells  is  also  con- 
siderably lower  than  in  the  fluid  surrounding  them  (  Hodgkins,  1951 ).  It  is  thereby 
likely  that  an  active  transport  mechanism  exists  in  echinoderm  eggs  as  it  apparently 
does  in  other  types  of  cells  (rf.  Brown  and  Danielli.  1954). 

Rothschild  (1938),  and  Kamada  and  Kinosita  (1940)  had  considered,  but  re- 
jected, the  possibility  that  failure  to  obtain  a  membrane  potential  might  be  due  to 
failure  of  electrode  to  penetrate  the  egg.  Their  decision  was  based  on  the  apparent 
entrance  of  injection  fluid  into  the  egg.  Furshpan  (1955)  believed  that  because  in 
many  experiments  he  had  pushed  the  electrode  clear  through  the  egg,  it  must  have 
been  in  the  cytoplasm  at  some  stage  in  the  process  and  therefore  considered  his  re- 
sults to  demonstrate  absence  of  a  membrane  potential.  However,  as  has  been  noted 
by  Dan  (1943)  and  by  Tyler  and  Monroy  (1955),  the  micropipette  can  readily 
tunnel  through  the  egg,  without  entering  the  cytoplasm  ;  the  distended  plasma  mem- 
brane on  one  side  simply  joins  that  on  the  other  and  both  are  then  perforated  with- 
out injury  to  the  egg  which  can  later  close  the  tunnel.  It  is  pertinent  to  quote  in  this 
connection  the  remarks  of  Chambers  (1922,  p.  189)  : 


168  A.  TYLER,  A.  MONROY,  C.  Y.  KAO  AND  H.  GRUNDFEST 

Pushing  a  pipette,  especially  a  comparatively  large  one,  into  an  egg  cell  frequently 
causes  the  surface  of  the  cell  to  become  invaginated  and  thus  forms  a  deep  pocket.  The 
tip  of  the  pipette,  even  if  it  should  finally  break  through  the  surface,  is  apt  to  become 
separated  from  the  protoplasm  of  the  interior  by  the  formation  of  a  new  surface  film 
continuous  with  the  original  surface  of  the  cell. 

Chambers'  conclusion  was  confirmed  by  Tyler  and  Monroy  (1955,  and  illustrated 
in  Fig.  1)  and  in  the  present  experiments.  It  is  now  also  well  known,  particularly 
from  the  work  of  Heilbrunn  (1927,  1952;  cf.  Costello,  1932),  that  when  the  cyto- 
plasm of  eggs  of  marine  animals,  or  of  other  cells,  is  brought  in  contact  with  Ca- 
containing  solution  a  surface  precipitation  reaction  occurs.  The  formation  of  such 
a  precipitation  membrane  around  the  tip  of  the  pipette  might  have  been  responsible 
for  the  lack  of  a  membrane  potential  in  the  experiments  of  Rothschild  (1938)  in 
which  injection  tests  indicated  penetration.  The  absence  of  a  potential  in  the  experi- 
ments of  Gelfan  (1931),  Kamada  and  Kinosita  (1940)  and  Scheer  et  al.  (1954) 
can  also  be  attributed  to  failure  of  penetration  or  possible  formation  of  a  precipita- 
tion membrane. 

A  small  positive  potential  was  observed  by  Gelfan  (1931)  when  the  microelec- 
trode  presumably  penetrated  the  germinal  vesicle  of  the  Astcrias  eggs.  In  our  ex- 
periments, confirming  Chambers  (1921),  puncture  of  the  germinal  vesicle  invariably 
led  to  cytolysis  of  the  egg.  The  sudden  disappearance  of  the  membrane  potential 
when  the  germinal  vesicle  was  impaled  and  cytolysis  resulted,  indeed  served  as 
additional  verification  of  penetration  in  the  experiments  reported  in  the  present 
paper.  As  noted,  and  discussed  earlier,  small  positive  potentials  were  occasionally 
observed  in  these  experiments. 

Although  the  absence  of  a  membrane  potential  in  echinoderm  eggs  reported  by 
earlier  observers  is  explained  in  the  light  of  the  present  experiments,  there  remains 
the  finding  (Kao,  1955)  that  eggs  of  the  killer  minnow,  Fundulus,  do  not  exhibit  a 
membrane  potential.  The  precautions  using  two  microelectrodes  and  an  applied 
pulse,  were  also  employed  in  those  experiments  to  ascertain  penetration  of  the  egg 
membrane.  However,  the  ionic  composition  of  Fundulus  eggs  is  unknown  and  the 
explanation  for  this  different  finding  must  remain  in  abeyance. 

For  the  most  part  measurements  of  the  membrane  potential  have  been  carried 
out  on  cells  from  tissue  aggregates.  Some  data  are,  however,  available  for  unicel- 
lular organisms.  These  are  of  interest  not  only  because  they  provide  a  rather 
closer  analog  to  eggs  than  do  tissue  components,  but  also  because  they,  too,  reflect 
the  effects  of  improvements  in  technique.  Telkes  (1931)  reported  that  amoebae 
have  a  membrane  potential  of  10  to  30  mv.,  inside  negative.  Buchthal  and 
Peterfi  (1937)  found  only  small  variable  potentials  of  either  sign  (up  to  3  mv.). 
Later,  however,  Wolfson  (1943)  succeeded  in  recording  a  membrane  potential  of  up 
to  90  mv.  in  Chaos  cJiaos.  Dr.  S.  Crain,  of  the  Department  of  Neurology,  Columbia 
University  (personal  communication)  has  obtained  similar  values  for  the  membrane 
potential  in  Chaos  chaos  and  Paramaeciuin.  As  in  eggs,  it  is  difficult  to  penetrate 
the  cell  membrane  and  Wolfson  used  the  device  of  applying  negative  pressure  on  the 
electrode,  sucking  the  amoeba  onto  the  shaftlet  and  eventually  rupturing  its  cell 
membrane.  Tauc  (1953)  found  a  membrane  potential  of  80  to  100  mv.  in  the 
plasmodium  of  a  myxomycete.  As  in  the  Asterias  egg,  the  high  initial  value  de- 
creased subsequent  to  penetration. 


ELECTRICAL  PROPERTIES  OF  STARFISH  EGGS  169 

Effects  of  fertilization  on  the  membrane  potential 

A  second  objective  of  these  experiments  was  to  examine  whether  or  not  changes 
in  membrane  potential  accompany  the  events  of  fertilization.  Peterfi  and  Rothschild 
(1935),  using  two  small  external  electrodes  placed  on  opposite  sides  of  the  surface 
of  the  frog  egg,  reported  (p.  875)  that  "there  are  strong  indications  that  the  attach- 
ment of  the  spermatozoon  to  the  egg  results  in  an  action  potential  being  propagated 
over  the  egg  surface,  the  action  potential  being  characterized  by  having  no  recovery 
phase."  Scheer  et  al.  (1954),  although  they  could  not  obtain  a  steady  membrane 
potential  with  an  electrode  apparently  inserted  into  eggs  of  Paraccntrotus  lividus  and 
Arbacia  lixnla,  reported  transient  potential  differences  upon  fertilization.  These 
changes  consisted  of  a  series  of  rapid  pulses  of  irregular  size,  ranging  from  about 
2  to  5  mv.  They  began  at  the  time  that  the  first  visible  reaction  (cortical  change) 
to  the  sperm  was  observed,  persisted  during  the  period  of  egg  contraction  and  gradu- 
ally disappeared.  In  the  case  of  Paraccntrotus  eggs  the  pulses  \vere  much  less  fre- 
quent (often  only  one  or  two)  than  in  Arbacia  (as  many  as  fifty).  Scheer  et  al. 
(1954)  point  out  that  these  changes  are  not  strictly  comparable  to  the  action  po- 
tentials of  nerve  and  muscle.  Furshpan  (1955)  saw  no  potential  changes  upon 
fertilization  of  eggs  of  the  sea  urchins  Strongylocentrotus  purpuratus  and  Lytechinus 
pictus,  but  as  in  the  case  of  the  other  observers,  neither  was  a  steady  membrane  po- 
tential obtained  with  these  eggs.  Similarly  Kamada  and  Kinosita  (1940),  tising 
an  "internal"  electrode  found  no  change  upon  fertilization,  nor  a  "resting"  potential 
in  eggs  of  the  sea  urchin  Strongyloccntrotus  pnlchcrrinnis.  An  attempt  to  examine 
possible  changes  in  membrane  potential  upon  fertilization  was  also  made  recently  by 
Allen,  Lundberg  and  Runnstrom  (1955)  with  external  electrodes  in  contact  through 
sea  water  in  agar  with  the  ends  of  a  capillary  tube  in  which  a  sea  urchin  egg  was 
elongated  and  fertilized.  They  found  no  shift  in  potential  but  concluded  that  the 
technique  proved  inadequate  for  this  problem. 

The  present  series  of  experiments  disclose  that  in  eggs  of  Asterias  jorbesii,  at 
least,  there  is  a  change  in  the  membrane  potential  beginning  with  a  sudden  de- 
crease when  spermatozoa  are  seen  to  have  made  contact  with  the  egg.  The  initial 
decrease  of  membrane  potential  is  converted  to  an  increase  which  reaches  its  maxi- 
mum and  steady  value  when  the  fertilization  membrane  is  raised.  These  changes  in 
membrane  potential  appear  to  develop  smoothly  without  the  occurrence  of  the  pulses 
recorded  by  Scheer  ct  al.  (1954)  \vith  a  capacitatively  coupled  amplifier. 

The  time  course  of  the  initial  decrease  in  potential  was  not  determined  ac- 
curately. The  optical  system  did  not  permit  identification  of  the  moment  of  sperm 
entry  into  the  egg,  and  unfortunately  photographic  recording  of  the  oscillograph 
traces  was  not  done  at  sufficiently  frequent  intervals  to  define  accurately  the  initial 
portion  of  the  membrane  potential  change.  However,  visual  observation  at  1  per 
second  sweeps,  which  was  routinely  done,  indicated  that  the  decrease  took  less  than 
10  seconds  to  reach  its  maximum  value. 

More  detailed  experiments  will  be  required  to  establish  whether  or  not  the  ob- 
served electrical  changes  in  Asterias  eggs  are  associated  in  time  with  the  optical 
changes  which  accompany  fertilization.  The  color  change  which  passes  over  the 
surface  of  the  sea  urchin  egg  within  a  few  seconds  after  the  attachment  of  the  sperm 
(Runnstrom,  1928)  is  a  propagated  response  with  a  total  conduction  time  of  about 
20  seconds  at  18°  C.  (Rothschild  and  Swann.  1949).  Birefringence  of  the  surface 


170  A.  TYLER,  A.  MONROY,  C.  Y.  KAO  AND  H.  GRUNDFEST 

disappears  in  about  the  same  time  (Monroy  and  Montalenti,  1947).  These  events 
therefore  have  about  the  same  time  course  as  the  decrease  and  subsequent  beginning 
of  the  return  of  the  membrane  potential  to  its  former  steady  amplitude. 

Studies  on  the  rate  at  which  block  to  polyspermy  is  established  in  sea  urchin 
eggs  (Rothschild  and  Swann,  1950,  1951,  1952;  Rothschild.  1953,  1954)  have  led 
to  the  conclusion  that  there  is  a  fast  reaction  which  passes  over  the  surface  of  the 
egg  in  about  two  seconds  and  that  reduces  the  chances  of  refertilization  by  a  factor  of 
20.  This  is  followed  by  a  slower  change  which  in  about  60  seconds  reduces  to  zero 
the  probability  of  a  successful  sperm-egg  collision.  The  wave  of  explosion  of  the 
"Harvey-Moser  granules"  (Harvey,  1911,  p.  523;  Moser,  1939)  takes  about  15  sec- 
onds to  traverse  the  surface  of  an  echinoid  egg  from  the  point  of  attachment  of  the 
sperm  (Endo,  1952).  The  time  course  of  these  events  is  about  the  same  in  the 
starfish  egg  judging  from  the  fact  that  the  fertilization  membrane  becomes  visibly 
elevated  at  one  to  two  minutes.  As  noted  earlier,  the  new  maximum  of  membrane 
potential  is  attained  at  about  the  time  that  the  fertilization  membrane  is  clearly 
visible. 

A  distinction  must,  however,  be  made  in  correlating  the  electrically  and  opti- 
cally observed  effects.  The  latter  are  initiated  at  the  site  of  entry  of  the  sperm  and 
are  slowly  propagated  from  there.  The  former  are  recorded  with  an  electrode 
inside  a  sphere  of  small  diameter  and  this  condition  operates  against  the  likelihood 
of  observing  discretely  localized  membrane  potential  change.  If  the  electrical 
changes  observed  during  fertilization  have  their  basis  in  localized  changes  of  the 
membrane,  these  are  probably  electronically  averaged  in  the  actual  recording  and 
would  not  reveal  clearly  a  spreading  electrical  change  which  might  accompany  the 
propagated  evolution  of  the  optically  observed  phenomena. 

The  nature  of  the  membrane  events  which  lead  to  the  electrical  changes  observed 
after  fertilization  can  only  be  speculated  upon.  Potential  change  in  excitable  tis- 
sues is  associated  with  change  in  membrane  permeability  and/or  altered  ionic  flux 
(Bernstein,  1912;  Hodgkin  and  Katz,  I'M1);  Fatt  and  Katz,  1951;  Hodgkin  and 
Huxley,  1952;  Eccles,  Fatt  and  Koketsu,  lf>54).  In  the  more  complex  bioelectric 
generators  of  frog  skin  or  the  gastric  mucosa,  changes  in  the  transport  respectively 
of  Na+  (Ussing,  1954;  Kirschner,  1955  )  and  CT  (Hogben,  1(>55)  are  involved.  At 
least  in  the  former  case,  metabolic  activity  and  hormonal  factors  play  an  important 
role.  Whether  or  not  the  electrical  changes  which  occur  upon  fertilization  are  as- 
sociated with  known  metabolic  changes  (cf.  Brachet,  1947;  Rvmnstrom.  1949;  Ty- 
ler, 1955a)  is  not  at  present  clear.  Changes  in  ionic  permeability  such  as  might  be 
indicated  by  the  electrical  data  probably  do  occur.  However,  the  time  course  of 
the  K+  accumulation  is  not  known.  Monroy-Oddo  and  Esposito  (1951)  have  re- 
ported that  sea  urchin  eggs  gain  K+  upon  fertilization,  and  such  accumulation  might 
account  for  the  late  increase  in  the  membrane  potential.  Malm  and  Wachmeister 
(1950),  on  the  other  hand,  report  a  slight  decrease  in  potassium  content,  and  a  con- 
siderable increase  in  sodium,  of  sea  urchin  eggs  upon  fertilization.  They  attribute 
this  to  the  fertilization  membrane  being  permeable  to  the  ions  in  the  surrounding- 
sea  water,  so  that  analyses  after  fertilization  would  show  an  increase  in  ions  present 
in  high  concentration  in  sea  water  and  an  apparent  decrease  in  ions  like  potassium 
previously  accumulated  by  the  egg.  Shapiro  and  Davson  (1941  )  had  also  reported 
no  significant  change  in  potassium  content  in  sea  urchin  eggs  upon  fertilization. 
They  noted,  too,  that  both  unfertilized  and  fertilized  eggs  lost  potassium  slowly 


ELECTRICAL  PROPERTIES  OF  STARFISH  EGGS  171 

(1.5  to  8f/  in  two  hours)  on  standing  in  sea  water,  and  that  eggs  in  K+-enriched 
(5  X  )  artificial  sea  water  accumulated  K+.  In  experiments  with  radioactive  cations 
Brooks  (1939)  noted  an  increase  in  the  accumulation  of  radiosodium  upon  fertiliza- 
tion in  eggs  of  Urechis  canpo.  E.  L.  Chambers  et  ol.  (1948)  reported  that  the  rate 
of  exchange  of  K4-  increased  7  to  13  times  upon  fertilization  in  eggs  of  Strongylo- 
centrotits  pnrpiiratus  and  Arbacia  punctnlata.  They  considered  (see  also  E.  L. 
Chambers,  1949)  that  only  20  per  cent  of  the  K  is  readily  exchangeable  in  the 
unfertilized  egg  and  85  to  100  per  cent  in  the  fertilized  egg.  The  rate  of  exchange 
of  "freely  diffusible"  K  is  therefore  concluded  to  be  two  to  three  times  more  rapid 
in  the  fertilized  egg.  While  there  are  some  evident  differences  in  the  results  of  the 
various  experiments  cited,  the  indications  are  that  there  are  likely  to  be  changes  in 
permeability  to  certain  ions,  occurring  upon  fertilization,  that  may  correlate  with 
the  changes  in  electrical  potential. 

On  the  other  hand,  the  electrical  changes  might  rather  be  due  to  alteration  in 
the  mechanical  properties  of  the  egg  membrane.  The  large  potential  initially  seen 
on  penetrating  the  unfertilized  egg  was  about  equal  in  magnitude  to  the  largest 
steady  potential  attained  after  fertilization.  The  decline  from  the  initial  value  has 
been  interpreted  as  being  caused  by  imperfect  sealing  of  the  plasma  membrane 
around  the  shaftlet  of  the  microelectrode.  It  is  therefore  possible  that  the  initial 
decrease  upon  fertilization  indicates  a  further  loosening  of  the  seal  and  that  the 
subsequent  rise  of  membrane  potential  only  reflects  formation  of  a  better  seal.  The 
various  physical  changes  initiated  in  the  fertilization  reaction  and  discussed  above 
might  well  be  implicated  in  an  alteration  of  the  membrane  seal  around  the  electrode. 

TJie  electrical  constants  of  the  eg;/  membrane 

The  measurements  of  membrane  resistance  and  capacity  were  in  the  present  ex- 
periments subsidiary  to  the  use  of  the  square  pulse  technique  for  ascertaining  pene- 
tration of  the  egg  surface  by  the  microelectrodes.  Therefore  the  results  are  chiefly 
indicative  of  the  orders  of  magnitude  of  these  values,  subject  to  a  more  extensive 
study.  However,  the  measurements  can  throw  some  light  on  the  more  detailed  in- 
terpretation of  the  membrane  potential  and  will  be  discussed  largely  in  this  context. 


The  unfertilized  egg 


Most  cells  thus  far  adequately  studied  have  membrane  resistances  in  the  range 
of  1000  ohm-cm.2  However,  the  values  range  from  as  low  as  0.1  ohm-cm.-  (rostral 
membrane  of  the  electoplaque  of  the  eel ;  Keynes  and  Martins-Ferreira,  1953  )  to  as 
high  as  20,000  ohm-cm.2  (activated  Fmidnlus  egg;  Kao,  1955).  The  membrane 
resistance  of  Astcrias  eggs  (average  :  3.100  ohm-cm.2)  indicates  that  ionic  transport 
is  rather  low  across  this  membrane.  In  a  tabulation  of  the  membrane  resistance  of 
various  cells.  Cole  and  Curtis  (1950)  give  an  estimate  that  the  membranes  of  both 
sea  urchin  and  starfish  eggs  have  infinite  resistance.  Earlier,  however.  Cole  and 
Cole  (1936a)  pointed  out  that  the  method  of  measurement  was  not  appropriate  for 
determinations  of  membrane  resistance.  A  2%  change  in  the  estimate  of  relative 
cell  volumes  to  that  of  external  fluid  would  have  led  to  a  calculated  value  of  mem- 
brane resistance  as  low  as  25  ohm-cm.2  Rothschild  (1938)  stated  that  while  his 
attempts  to  measure  membrane  resistance  of  Echinus  eggs  were  unsatisfactory,  the 


172  A.  TYLER,  A.  MONROY.  C.  Y.  KAO  AND  H.  GRUNDFEST 

value  was  probably  no  higher  and  perhaps  lower  than  104  ohm-cm.2  Furshpan 
(1955)  estimated  that  the  resistance  which  the  sea  urchin  egg  interposed  in  the 
microelectrode  circuit  of  his  experiments  was  about  1  megohm.  From  the  surface 
area  of  these  eggs  (diameter,  75  ju,;  area  1.8  X  10~4  cm.2)  he  calculated  the  membrane 
resistance  as  180  ohm-cm.2  However,  since  no  membrane  potential  was  obtained  in 
those  experiments,  it  is  unlikely  that  the  electrode  had  penetrated  the  egg  membrane 
and  this  might  account  for  the  low  estimate.  Cole  and  Curtis  (1938)  calculated 
membrane  resistances  from  0.2  to  10  ohm-cm.2  for  unfertilized  and  fertilized  Arbacia 
eggs  from  measurements  of  single  eggs  in  a  small  capillary  but  were  forced  to  dis- 
card them  because  the  assumption  of  no  parallel  leakage  also  gave  unreasonable 
values  for  the  membrane  capacity.  Allen,  Lundberg  and  Runnstrom  (1955)  re- 
port a  resistance  of  8.7  X  105  ohms  for  an  egg  of  Psammechinus  in  a  narrow  (57  /*,) 
capillary  but  consider  that  this  may  be  in  error  by  virtue  of  a  leakage  pathway  pro- 
vided between  the  surface  of  the  egg  and  the  walls  of  the  capillary.  They  state  (p. 
178)  that  in  later  experiments  Lundberg  (1955,  unpub.)  has  obtained  a  value  of 
1350  ohm-cm.2 

Passage  of  currents  of  0.5  to  1.5  /^A  through  the  Astcnas  egg,  corresponding  to 
current  densities  of  0.75  to  2.25  mA/cm.2.  caused  marked  decrease  of  the  membrane 
resistance  in  6  eggs.  These  results  need  further  study,  and  if  the  phenomenon  is 
established  might  yield  valuable  clues  to  membrane  properties,  since  the  change  in 
resistance  was,  at  least  in  some  eggs,  not  accompanied  by  cytolysis.  The  relatively 
short  pulses  used  in  the  experiments  did  not  appear  to  cause  any  activation  changes 
such  as  were  reported  in  the  experiments  of  Allen,  Lundberg  and  Runnstrom 
(1955). 

It  is  interesting  to  note  that  even  with  the  highest  currents  employed,  the  rela- 
tion between  membrane  current  and  voltage  was  strictly  linear  both  for  inward  and 
outward  currents.  This  is  not  the  case  in  excitable  tissues.  Outwardly  directed 
currents  of  relatively  low  magnitude  initiate  the  changes  of  membrane  potential 
inherent  in  the  local  response  and  spike  and  these  are  associated  with  a  marked  drop 
of  membrane  resistance,  whereas  inwardly  directed  currents  tend  to  increase  the  re- 
sistance (Cole  and  Curtis,  1941).  Thus,  eggs  of  Astcrias,  although  they  un- 
doubtedly undergo  excitation  by  sperm  in  the  form  of  the  reactions  of  fertilization, 
evidently  do  not  respond  to  electric  stimulation  in  the  manner  characteristic  of  elec- 
trogenic  excitable  tissues. 

It  is  of  further  interest  that  the  maximum  quantity  of  electricity  in  the  square 
pulses  used  for  the  present  experiments  was  about  5  X  W~5  coulomb/cm.2  The 
squid  giant  axon  and  other  cells,  however,  are  excited  by  10"8  to  10~9  coulomb/cm.2 
of  membrane  (Cole,  1949;  Grundfest,  1952;  Hodgkim  Huxley  and  Katz,  1952). 

Effect  of  fertilisation 

If  the  initial  decrease  and  subsequent  increase  of  the  membrane  potential  in  the 
fertilized  egg  are  consequences  of  altered  ionic  flux,  the  latter  change  should  be  re- 
flected as  a  change  of  the  membrane  resistance.  The  most  accurate  measurements 
of  this  with  respect  to  both  magnitude  and  time  course  would  be  provided  by  study 
of  the  high  frequency  impedance.  Such  measurements  were  not  done  in  the  pres- 
ent experiments  and  the  values  derived  from  the  square  pulse  technique  apply,  not 
to  the  initial  period  of  fertilization,  but  to  the  stage  when  the  fertilization  membrane 


ELECTRICAL  PROPERTIES  OF  STARFISH  EGGS  173 

had  been  lifted  and  the  membrane  potential  had  reached  its  steady  higher  value. 
At  this  time  the  membrane  resistance  of  the  two  eggs  studied  before  and  after  fer- 
tilization (Fig.  8,  Table  4A)  was  identical  with  the  initial  value.  This  may  be  taken 
to  indicate  that  the  fertilization  membrane  is  a  rather  porous  structure  which  offers 
relatively  little  impedance  to  ion  movements.  Cole  (1928),  Cole  and  Spencer 
(1938)  and  Cole  and  Guttman  (1942)  reported  that  upon  fertilization  of  sea  urchin 
or  frog  eggs  there  was  no  change  in  cortical  resistance,  and  our  data  therefore  also 
support  this  finding  as  regards  at  least  a  stage  a  few  minutes  after  fertilization. 

However,  this  constancy  poses  a  dilemma  which  we  have  not  been  able  to  re- 
solve. It  was  suggested  earlier  that  the  low  value  of  steady  membrane  potential  in 
the  unfertilized  egg,  and  its  initial  decrease  and  subsequent  increase  upon  fertiliza- 
tion, might  have  been  caused  by  alterations  in  the  seal  of  the  egg  membrane  against 
the  wall  of  the  microelectrode.  This  assumption  would  necessitate  upward  revision 
of  the  value  of  the  membrane  resistance  of  the  unfertilized  egg,  perhaps  up  to  double 
the  calculated  average  of  3100  ohm-cm.2,  since  the  leaks  decreasing  the  membrane 
potential  to  about  half  would  be  in  parallel  with  the  resistance  of  the  membrane. 
However,  the  membrane  resistance  of  the  eggs  seemed  to  be  relatively  independent 
of  their  steady  membrane  potentials  (Table  IVA),  and  therefore  no  attempt  has  been 
made  in  the  present  study  to  correct  the  calculated  values  of  R^.  Nevertheless,  the 
possibility  remains  that  the  membrane  resistance  of  the  unfertilized  egg  is  higher 
than  3000  ohm-cm.2  and  that  the  apparent  absence  of  change  after  fertilization  is 
fortuitous.  The  improved  sealing  of  the  membrane  to  the  electrode  might  have  in- 
creased the  leakage  resistance  greatly  and  thereby  have  improved  the  accuracy  of  the 
measurement  of  R3I.  In  that  case,  the  actual  membrane  resistance  of  the  egg  may 
have  decreased  upon  fertilization.  More  extensive  studies  will  be  required  to  re- 
solve this  matter.  However,  the  data,  whether  the  resistance  of  the  membrane  is 
constant  or  decreases,  differ  from  the  finding  that  the  membrane  resistance  of  acti- 
vated Fundulus  eggs  increases  4-  to  7-fold  (Kao,  1955).  The  difference  may  be 
explained  by  the  fact  that  in  Astcrias  the  egg  volume  and  surface  remain  nearly  con- 
stant after  fertilization,  whereas  the  activated  Fundulus  egg  shrinks  markedly.  The 
resultant  diminution  of  surface  may  therefore  lead  to  closer  packing  of  the  ion- 
permeable  units  of  the  plasma  membrane  (Kao.  Chambers  and  Chambers,  1954).  or 
the  effective  narrowing  of  pores  in  the  membrane. 

Cole  (1938)  reported  a  100%  rise  in  cortical  capacitance  of  fertilized  Arbacia 
and  Hippnnoc  eggs  and  Cole  and  Spencer  (1938)  report  300%  in  Arbacia.  In  the 
two  Astcrias  eggs  studied  before  and  after  fertilization,  the  calculated  membrane 
capacity  after  fertilization  remained  unaltered  in  one  and  decreased  in  the  other. 
As  noted  earlier,  the  accuracy  of  the  measurements  of  the  time  constant  was  not 
as  high  as  is  either  desirable  or  attainable  and  this  matter  must  be  left  open  subject 
to  future  work. 

SUMMARY 

1.  The  paper  describes  electrical  characteristics  of  the  egg  of  the  starfish  Astcrias 
forbesii  as  measured  with  a  microelectrode  penetrating  the  surface.     The  study  in- 
cluded the  effects  of  fertilizing  the  egg  while  the  latter  was  impaled  on  the  electrode. 

2.  It  has  been  confirmed  that  penetration  of  the  egg  membrane  cannot  be  indi- 
cated solely  by  the  seeming  visualization  of  the  microelectrode  at  the  center  of  the 
egg. 


174  A.  TYLER,  A.  MONROY,  C.  Y.  KAO  AND  H.  GRUNDFEST 

3.  A  method  involving  use  of  two  microelectrodes  is  described  for  ascertaining 
penetration  of  the  egg  surface.     One  internal  electrode  delivers  a  current  pulse  and 
the  other  records  the  time  course  of  the  resultant  membrane  IR  drop. 

4.  Contrary  to  the  reports  of  many  earlier  investigations  on  echinoderm  and 
other  eggs  a  potential  difference  is  found  upon  penetration  of  the  unfertilized  egg. 

5.  The  potential  difference  at  the  time  of  penetration  amounts  to  about  60  mv., 
inside  negative,  but  this  soon  decreases  to  lower  steady  values  ranging  from  •  -  10 
to  —  50  mv.  in  different  eggs. 

6.  Upon  insemination  of  the  impaled  egg  the  membrane  potential  abruptly  de- 
creases by  5  to  10  mv.  when  sperm  are  seen  to  have  reached  the  egg,  then  rises  dur- 
ing the  ensuing  1  to  21//>  minutes,  as  the  fertilization  membrane  is  raised,  reaching  a 
new  steady  value  5  to  20  mv.  greater  than  that  of  the  unfertilized  egg. 

7 .  The  possible  basis  of  these  changes  is  discussed. 

8.  The  internal  K+  of  unfertilized  Astcrias  eggs  is  from  17  to  21  times  higher 
than  that  of  sea  water.     The  sodium  determinations,  while  subject  to  larger  error, 
indicate  a  concentration  less  than  Sc/c  that  of  sea  water. 

9.  The  membrane  potential  of  either  unfertilized  or  fertilized  eggs  decreases 
when  the  external  KT  is  raised  and  returns  to  the  original  value  when  the  excess  K+ 
is  removed. 

10.  As  in  many  other  kinds  of  cells  the  potential  is  evidently  a  consequence  pri- 
marily of  the  high  internal  concentration  of.  and  permeability  to,  K+,  but  the  mag- 
nitude appears  less  than  predicted  by  the  Nernst  equation. 

11.  The  membrane  resistance  of  the  unfertilized  egg  averages  3100  ohm-cm.2, 
but  might  be  higher  on  the  assumption  of  possible  leaks  around  the  microelectrode. 

12.  The  measured  resistance  is  unchanged  after  fertilization,  but,  on  the  assump- 
tion of  the  formation  of  a  tighter  electrical  seal  the  actual  membrane  resistance  would 
be  lower. 

13.  The  membrane  capacity  is  of  the  order  of  0.5  juF/cm.-' 

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Vol.  Ill,  No.  2  October,  1956 

THE 

BIOLOGICAL  BULLETIN 

PUBLISHED  BY  THE  MARINE  BIOLOGICAL  LABORATORY 


RELATIONS  BETWEEN  METABOLISM  AND  MORPHOGENESIS 
DURING  REGENERATION  IN  TUBIFEX  TUBIFEX.     II. 

JANE  COLLIER  ANDERSON1 
Department  of  Zoology,  University  of  Missouri,  Columbia,  Missouri 

Analysis  of  the  relations  between  metabolism  and  morphogenesis  requires  that 
each  set  of  processes  be  separated  into  component  parts.  Metabolism  may  be  frac- 
tionated by  means  of  agents  of  which  the  effects  on  particular  enzyme  systems  are 
reasonably  well  known  and  the  relation  of  the  activity  of  such  systems  to  morpho- 
genesis may  then  be  tested.  In  the  annelid,  Tubifc.r  tubijcx,  morphogenesis  during 
posterior  regeneration  may  be  measured  fractionally  and  "rate  of  localization,"  "rate 
of  early  differentiation,"  and  "rate  of  later  differentiation"  expressed  quantitatively 
(Collier,  1947).  It  was  found  that  oxygen  consumption  and  loss  of  weight  by 
starving  worms  proceed  at  a  markedly  increased  rate  during  certain  stages  of  re- 
generation, and  that  rate  of  oxygen  consumption  was  correlated  with  "rate  of 
later  differentiation";  a  metabolic  (energetic)  cost  of  differentiation  was  hypothe- 
sized and  it  was  thought  possible  that  this  might  be  characterized  by  activity  of  par- 
ticular enzyme  systems.  The  present  report  concerns  the  effects  of  continuous 
poisoning  by  cyanide  and  by  iodoacetate  and  also  the  effects  of  high  oxygen  ten- 
sion, low  oxygen  tension,  and  complete  lack  of  oxygen  upon  morphogenesis  during 
posterior  regeneration  in  the  oligochaete  annelid,  Tubifc.v  tubifex  Mull. 

MATERIALS  AND  METHODS 

The  worms  were  handled  and  examined  as  described  earlier  (Collier,  1947). 
For  high  oxygen  tension,  gas  from  a  tank  was  bubbled  continuously  through  the 
water  in  which  the  worms  wrere  kept.  Presence  of  a  low  percentage  of  carbon 
dioxide  (about  5%)  was  found  to  have  no  effect  on  experimental  results.  For  low 
oxygen  tension,  nitrogen  or  hydrogen  was  bubbled  through  the  water  at  two-day 
intervals,  the  bottles  being  tightly  closed  between  treatments.  For  strictly  anaerobic 
conditions,  hydrogen  from  a  tank  was  first  freed  of  traces  of  oxygen  by  passing 
it  over  platinized  asbestos  heated  to  a  dull  red ;  then  it  was  bubbled  continuously 
through  wash  bottles  and  experimental  bottles  in  series.  That  the  continual  dis- 
turbance did  not  affect  regeneration  was  ascertained  by  using  a  control  set-up 
through  which  air  was  bubbled. 

1  Present  address :  care  of  Department  of  Physiology,  University  of  Illinois,  Urbana, 
Illinois. 

179 


180 


JANE  COLLIER  ANDERSON 


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FIGURE  1.  Effect  of  cyanide  on  rates  of  progress  through  various  stages  of  regeneration. 
Mean  deviation  within  each  group  of  worms  (thirty  individuals)  ranged  from  0.3  to  0.5  seg- 
ments per  worm  per  day,  increasing  w?ith  time. 


O2,  KCN,  IAA  ON  REGENERATION 


181 


"Rate  of  localization"  was  calculated  as  increase  in  total  number  of  segments  per 
worm  per  day;  "rate  of  early  differentiation"  as  increase  in  number  of  segments 
showing  some  cellular  differentiation  visible  in  vivo  under  low  power ;  and  "rate 
of  later  differentiation"  as  increase  in  number  of  segments  showing  setae. 

EFFECTS  OF  CYANIDE  ON  REGENERATION 

Over  300  worms  were  used  in  experiments  involving  continuous  poisoning  by 
potassium  cyanide.  A  group  of  thirty  worms  in  10~3  M  KCN  became  inactive  and 
showed  heavy  mortality  after  the  second  day.  The  last  survivors  formed  blastemae, 
but  no  localization  of  new  segments  occurred  in  nine  days.  Worms  in  10~4  M  KCN 
and  10~5  M  KCN  survived  well,  one  group  in  10~4  M  showing  60  per  cent  survival 
at  159  days.  A  few  individuals  showed  abnormal  regeneration:  one  double  tail 
and  seven  with  the  new  tail  at  an  angle.  Since  three  of  these  eight  were  in  a  con- 
trol group,  the  abnormalities  could  not  be  attributed  to  effects  of  cyanide. 


TABLE  I 
Effect  of  potassium  cyanide  on  progress  of  regeneration :  segments  per  worm  per  day 


Days 

Rate 

of  localization 

Rate  of  early 

differentiation 

Rate  of  later 

differentiation 

2-4 

4-7   7-10           10-15 

15-94 

2-4 

4-7    7-10 

10-15  15-94 

2-4 

4-7    7-10 

10-15  15-94 

Worms  in 

4.7 

2.4 

1.4 

0.2 

0 

2.0     2.2 

0.3 

0 

0 

1.4 

0.4 

10-s  M  KCN 

4.8 

2.4 

0 

2.0 

0 

0 

to 

\ 

\ 

\ 

tap  water 

1.1 

0.2 

2.3 

0.3 

1.4 

0.4 

Worms  in 

2.0 

1.9 

0.5 

0.3 

0 

0       1.1 

0.4 

0 

0 

0 

0.5 

ID-*  M  KCN 

2.4 

1.7 

0 

0 

0 

0 

to 

\ 

\ 

\ 

tap  water 

1.3 

0.3 

2.0 

0.4 

0 

0.5 

Worms  in 

tap  water 

3.0 

3.0 

1.5      -»0.8 

0.2 

0 

2.0     2.2 

->1.4             0.3 

0 

0 

1.3 

^1.9            0.4 

to 

\ 

\ 

\ 

10-s  M  KCN 

1.3 

0.2 

1.7             0.3 

2.1             0.4 

Data  on  rates  of  localization,  early  differentiation,  and  later  differentiation  are 
summarized  in  Figure  1.  During  the  first  six  days  the  worms  in  10~5  M  KCN 
showed  a  significantly  higher  rate  of  localization  than  controls,  while  worms  in  10~4 
M  KCN  showed  a  lower  rate.  Later  the  advantage  of  the  worms  in  10~5  M  KCN 
disappeared,  but  the  disadvantage  of  those  in  10~4  M  KCN  continued.  At  66  days 
the  worms  in  10~5  M  KCN  and  in  tap-water  had  practically  completed  regeneration : 
all  new  segments  were  in  later  stages  of  differentiation  and  had  grown  to  almost 
the  size  of  the  old  segments.  The  total  number  of  new  segments  was  almost  pre- 
cisely the  same  for  these  two  groups  (average  41  per  worm).  At  this  time  the 
worms  in  10"*  M  KCN  still  showed  segments  in  the  early  stages  of  formation  and 
the  average  total  number  of  new  segments  was  only  26  per  worm.  Growth  of  the 
new  segments  was  poor.  But  at  159  days  their  condition  was  fully  comparable 
with  that  of  the  others  at  66  days.  The  total  extent  and  perfection  of  regeneration 
were  unaffected  by  treatment,  but  rate  of  regeneration  was  markedly  affected. 

The  areas  under  the  rate  curves  at  the  top  of  Figure  1  represent  the  average  total 
number  of  segments  localized  per  worm.  For  tap  water  and  10~5  M  KCN,  these 
areas  between  two  days  and  nine  days  were  23  and  25,  respectively,  or  not  sig- 


182 


JANE  COLLIER  ANDERSON 


nificantly  different,  despite  the  early  rapid  rate  of  localization  in  dilute  cyanide.  It 
may  be  presumed  that  the  amount  of  cellular  material  available  for  localization  of 
segments  was  a  limiting  factor,  that  this  amount  was  unaffected  by  10~5  M  KCN 
and  that  the  narrowness  of  the  peak  of  rate  was  due  to  more  rapid  exhaustion  of  the 
material.  The  low  rate  of  localization  in  worms  in  10~4  M  KCN  and  the  fact  that 
the  area  under  the  curve  from  two  to  nine  days  is  less  than  half  of  that  under  the 
control  curve  suggests  that  availability  of  cellular  material  may  have  been  decreased 
in  10~4  M  KCN.  The  rapid  decline  to  a  very  low  rate  of  localization  strengthens 
this  suggestion. 

Since  rate  of  early  differentiation  during  a  particular  interval  of  time  should  be 
limited  by  the  number  of  segments  localized,  the  differences  in  rates  of  differentia- 
tion were  accounted  for  by  the  previous  differences  in  production  of  localized  seg- 
ments. It  appeared  probable  that  the  cyanide  affected  some  process  or  processes  oc- 
curring during  or  preceding  localization,  and  had  no  direct  effect  upon  later 
processes. 

TABLE  II 

Regeneration  in  low  oxygen 


Average  number  of  new  segments  per  worm 

Days  after  cutting 

Nitrogen 

Hydrogen 

Control  (air) 

2  to  7 

blastema 

blastema 

blastema 

10 

1.4 

1.5 

4.7 

13 

2.1 

2.5 

7.5  (setae) 

16 

3.7 

3.0 

9.8 

19 

5.0 

5.1 

11.7 

23 

6.0  (setae) 

13.8 

26 

7.0 

14.7 

Mean  deviation  was  about  ±1.0  segment  up  to  16  days,  =b  2  thereafter. 


An  experiment  was  set  up  to  test  this  idea.  Five  groups  of  thirty  worms  each 
were  cut  for  regeneration  and  two  groups  were  placed  in  10~5  M  KCN,  two  in  10~4 
M  KCN  and  one  in  tap  water.  At  the  end  of  seven  days  one  group  from  each 
cyanide  solution  was  transferred  to  tap  water.  Also,  at  the  end  of  ten  days  the 
group  in  tap  water  was  separated  worm  for  worm  into  two  comparable  groups  and 
one  of  these  transferred  to  10~5  M  KCN.  Data  (summarized  in  Table  I)  in  gen- 
eral confirm  the  supposition  that  cyanide  affects  localization  and  not  later  processes. 
Further,  it  is  suggested  that  this  effect  is  not  upon  mobilization  of  neoblasts,  which, 
according  to  Krecker  (1923)  and  Stone  (1932),  cease  their  metamorphosis  and 
migration  well  before  ten  days.  Rather  the  effect  must  be  upon  some  process 
more  directly  concerned  in  localization. 

EFFECTS  OF  Low  OXYGEN  TENSION 

If  high  oxygen  tension  acts  as  a  stimulus  to  regeneration  as  suggested  by  Earth's 
work  on  Tubularia  (1940),  regeneration  should  be  inhibited  or  retarded  by  low 
oxygen  tension,  while  high  oxygen  tension  should  accelerate  it.  Exactly  opposite 


O2,  KCN,  IAA  ON  REGENERATION 


183 


TABLE  III 

Observations  on  worms  kept  continuously  tinder  oxygen-free  atmosphere 


18  hours 

30  hour? 

42  hours 

20  intact  in  tap  water 

dark  red  inactive 

survival  about  30% 

survival  15% 

20  intact  in  lO'6  M  KCN 

dark  red  inactive 

survival  about  80% 

survival  45% 

20  regenerating  in  10~6 
M  KCN 

dark  red  inactive 

survival  about  95% 

survival  25% 

20  intact  in  lO"6  M 

dark  red  inactive 

no  survivor 

NalAc 

a  few  dead 

Controls  with  air  bubbled 

through  tap  water 
20  intact 
20  regenerating 

normal 

normal 

survival  100% 

results  would  be  expected  from  the  line  of  reasoning  that  in  low  oxygen  tension 
there  might  be  an  increase  in  glutathione  (cf.  Barren,  1951)  which,  as  found  by 
Coldwater  (1933),  can  increase  rate  of  regeneration  in  Tubifex.  One  might  then 
expect  high  oxygen  tension  to  retard  or  inhibit  regeneration. 

One  hundred  and  fifty  worms  were  used  in  two  experiments  with  atmospheres 
containing  less  than  4  per  cent  oxygen  (Table  II).  No  morphological  abnormali- 
ties appeared,  but  there  was  a  general  retardation  of  regenerative  processes. 

Results  of  an  experiment  on  survival  of  worms  under  more  strictly  anaerobic 
conditions  (Table  III)  showed  that  this  species  cannot  long  endure  complete  ab- 
sence of  oxygen.  Survival  was  significantly  briefer  in  presence  of  iodoacetate. 

TABLE  IV 

Effect  of  lack  of  oxygen  on  early  stages  of  regeneration.  (Each  day  access  to  air  was 
permitted  long  enough  to  allow  return  of  normal  color  and  activity) 


2  days 

4  days 

5  i  days 

7  days 

Oxygen-free 

Tap  water 

survival  7/30 

survival  0 

1/7  with  blas- 

tema 

10-6  M  KCN 

surv.  30/30 

30/30  with 

surv.  12/30 

survival  3/12 

24/30  with  bl. 

small  blast. 

no  blastema 

10-8  M  NalAc 

no  survivor 

Under  air 

30/30  with  blas- 

survival 30/30 

Tap  water 

tema 

aver.  19  new 

segments 

10-5  M  KCN 

15/15  with  blas- 

aver. 1  1  new 

survival  15/15 

tema 

segments 

10-8  M  NalAc 

15/15  with  blas- 

survival 15/15 

tema 

184  JANE  COLLIER  ANDERSON 

Controls  gave  assurance  that  no  materials  from  the  apparatus  or  wash  solutions  had 
been  responsible  for  destruction  of  the  experimental  worms. 

For  the  purposes  of  studying  regeneration  an  experiment  was  set  up  in  which 
survival  was  improved  by  allowing  a  short  period  of  access  to  air  once  a  day  (Table 
IV).  The  worms  did  produce  blastemae  at  the  usual  time  but  regeneration  pro- 
ceeded no  further  and  the  blastemae  disappeared.  It  appears  that  dilute  cyanide 
improved  survival  but  in  the  absence  of  oxygen  did  not  show  its  accelerating  action 
on  localization. 

The  effect  of  anaerobiosis  on  later  stages  of  regeneration  was  tested  using  worms 
which  had  regenerated  for  seven  days  under  normal  conditions  (Table  V).  The 
worms  kept  under  oxygen-free  atmosphere  (except  for  twenty  minutes  at  27  hours) 
showed  practically  no  progress  in  regeneration,  while  in  the  control  an  average  of 
3.6  new  segments  per  worm  had  been  localized,  5.7  had  undergone  early  differenti- 
ation, and  7.1  later  differentiation.  Regeneration  here  requires  the  presence  of 
oxygen. 

TABLE  V 
Effect  of  lack  of  oxygen  on  later  stages  of  regeneration 

0  hours  47  hours 

15  worms  under  oxygen-free  atmosphere  survival  100% 

Aver.  no.  segments  in  localization  7.0                             6.2 

Aver.  no.  segments  in  early  differentiation  11.2                           10.0 

Aver.  no.  segments  in  later  differentiation  0.6                             1.7 

Aver.  no.  segments  total  per  worm  18.8                           17.9 

15  worms  allowed  to  continue  under  air  (Control)  survival  100% 

Aver.  no.  segments  in  localization  7.0                             4.8 

Aver.  no.  segments  in  early  differentiation  11.3                             9.9 

Aver.  no.  segments  in  later  differentiation  0.6                             7.7 

Aver.  no.  segments  total  per  worm  18.9                           22.5 

Mean  deviation  was  about  ±  1.0  segment. 

EFFECTS  OF  HIGH  OXYGEN  TENSION 

Concurrent  with  the  experiments  with  low  oxygen,  two  groups  of  thirty  worms 
each  were  kept  under  an  atmosphere  of  95%  oxygen  and  5%  carbon  dioxide  and  a 
third  group  under  100%  oxygen.  Results  were  the  same  in  all  groups.  The 
worms  survived  well  for  several  days  and  formed  blastemae,  but  regeneration  pro- 
ceeded no  further  and  all  worms  had  died  by  ten  days.  To  test  whether  inhibition 
of  localization  by  oxygen  and  stimulation  of  localization  by  10~5  M  KCN  might  be 
based  on  opposite  effects  on  the  same  mechanism,  experiments  were  set  up  in  which 
worms  were  kept  in  1Q-5  M  KCN,  10'*  M  KCN,  and  10^3  M  KCN  under  an  atmos- 
phere of  pure  oxygen  (Table  VI).  The  10~5  M  KCN  partially  counteracted  the 
effects  of  oxygen  both  upon  survival  and  upon  regeneration;  10~4  M  KCN  was 
found  to  partially  counteract  the  effect  of  high  oxygen  upon  regeneration,  but  it 
did  not  even  partially  counteract  the  lethal  effect.  Accordingly,  high  oxygen  ten- 
sion had  an  effect  on  regeneration  independently  of  its  lethal  action  and,  far  from 
stimulating  regeneration  in  Tubifex,  very  high  oxygen  inhibits  it. 

EFFECTS  OF  IODOACETATE 

Since  experiments  in  which  worms  were  subjected  to  low  oxygen  tension  and  to 
complete  lack  of  oxygen  had  suggested  that  glycolysis  might  be  important  for 


O2)  KCN,  IAA  ON  REGENERATION 


185 


TABLE  VI 

Simultaneous  effects  of  high  oxygen  tension  and  cyanide  on  survival  (infractions') 
and  regeneration  (in  average  number  of  new  segments  per  worm} 


2  days 

4  days 

6  days 

9  days 

14  days 

Under  oxygen 
Tap  water 

30/30 

sm.  blastema 

23/30 
0  seg. 

4/30 
0  seg. 

no 
survivor 

lO-^  M  KCN 

30/30 
sm.  blastema 

30/30 
2  seg. 

28/30 
3  seg. 

21/30 
4  seg. 

no 
survivor 

10-«  M  KCN 

30/30 
sm.  blastema 

29/30 
1  seg. 

no 
survivor 

10-3  M  KCN 

no 
survivor 

Under  air 
Tap  water 

30/30 
blastema 

28/30 
4  seg. 

28/30 
13  seg. 

27/30 
23  seg. 

27/30 
31  seg. 

lO"6  M  KCN 

18/18 
blastema 

18/18 
6  seg. 

18/18 
18  seg. 

18/18 
25  seg. 

18/18 
33  seg. 

10-"  M  KCN 

15/15 
blastema 

12/15 
3  seg. 

10/15 
9  seg. 

10/15 
11  seg. 

10/15 
12  seg. 

ID"3  M  KCN 

29/30 
no  blastema 

no 
survivor 

survival  and  regeneration,  worms  were  allowed  to  regenerate  in  various  concen- 
trations of  sodium  iodoacetate :  1O3  M,  1O4  M.  1Q-5  M,  1O6  M,  1O7  M,  and  10'8  M 
NalAc.  Five  intact  worms  in  5  X  10~3  M  NalAc  showed  decreased  activity  after 
six  hours,  and  were  dead  at  thirty-six  hours.  In  the  other  concentrations  intact 
worms  survived  as  well  as  but  no  better  than  the  regenerating  worms.  The  group 
of  thirty  worms  in  1O3  M  solution  showed  high  mortality  after  three  days,  but  the 
few  survivors  maintained  normal  morphogenesis.  The  worms  in  10~4  M  solution 
showed  high  mortality  after  six  days,  but  six  of  the  thirty  worms  survived  for 
twenty-four  days  and  maintained  normal  regeneration.  Mortality  in  the  other 
groups  was  low.  Rates  of  progress  through  various  stages  of  regeneration  were 
very  nearly  the  same  for  all  concentrations  of  iodoacetate  (Fig.  2),  but  whereas  rate 
of  later  differentiation  in  tap  water  reached  a  peak  between  thirteen  and  seventeen 
days  after  cutting,  the  rate  of  later  differentiation  in  each  of  the  iodoacetate  solutions 

TABLE  VII 

Effect  of  dilute  iodoacetate  upon  rate  of  oxygen  consumption 


Sample 

Large  worms 
(over  4.5  cm.) 
Small  worms 
(under  2.5  cm.) 


Oxygen  consumption  in  cubic  millimeters  per 
milligram  of  worms  (wet  weight)  per  hour 


0.12  in  tap  water 
0.14  in  tap  water 


0.13  in  2  X  lO-8  M  NalAc 
0.13  in  2  X  10-6  Jl/NalAc 


186 


JANE  COLLIER  ANDERSON 


o 
Q 

<D 

O. 


o 
<u 

o     0 


o 

££ 


O   TAPWATER 
X   IO"8M    NoIAc 
+    IO"5M    NalAc 


10  13  17 

Days  after  cutting 


10          13  17 

Days  after  cutting 


30 


24 


I        3 


10          13  17 

Days  after  cutting 


24 


30 


FIGURE  2.     Effect  of  iodoacetate  on  rates  of  progress  through  various  stages  of  regeneration. 


02,  KCN,  IAA  ON  REGENERATION  187 

reached  its  peak  between  ten  and  thirteen  days  after  cutting.  lodoacetate  appeared 
to  accelerate  later  differentiation  without  affecting  earlier  processes.  Because  of  the 
range  of  concentrations  used,  and  because  the  extremely  dilute  solutions  used  here 
had  maximal  effect  on  differentiation,  it  may  be  presumed  that  iodoacetate  poisons 
some  process  (es).  inhibition  of  which  allows  further  activity  of  some  other  proc- 
ess (es)  in  a  system  of  multiple  pathways  of  hydrogen  and  electron  transfer  (cf.  Lip- 
mann,  1954).  The  inhibited  process  might  be  glycolysis,  while  the  reciprocally 
related  process  might  or  might  not  be  concerned  in  the  increased  oxygen  consump- 
tion previously  found  during  the  period  of  most  rapid  "rate  of  later  differentiation." 
A  determination  of  the  effect  of  sodium  iodoacetate  upon  oxygen  consumption  of 
normal  worms  was  made  (Table  VII).  It  is  clear  that  iodoacetate  had  no  sig- 
nificant effect  on  rate  of  oxygen  consumption. 

DISCUSSION 

It  has  been  held  that  differences  in  rate  of  metabolism  in  the  various  parts  of  an 
animal  may  be  the  basis  for  production  of  morphological  differences  (Child,  1940; 
Hyman,  1940;  Earth.  1938.  1940).  Much  of  the  supporting  evidence  conies  from 
experiments  on  regeneration  of  hydroids,  and  the  extremely  rapid  rate  of  regenera- 
tion here  makes  it  difficult  to  distinguish  between  a  factor  influencing  initiation  of 
regeneration  and  one  limiting  later  processes.  In  Tubifex,  slow  regeneration  per- 
mits sufficient  time  for  more  detailed  analysis.  Since  regeneration  here  is  initiated 
even  in  the  complete  absence  of  oxygen,  increased  oxygen  tension  in  the  tissues  at 
the  cut  surface  is  obviously  not  the  primary  stimulus  nor  is  it  even  a  necessary  con- 
dition. Instead  the  availability  of  oxygen  acts  as  a  limiting  factor  in  the  progress  of 
certain  later  processes  in  regeneration.  However,  the  concept  may  be  applied  to 
morphogenesis  during  regeneration  in  Tubifex  when  used  as  Lindahl  (1936)  ap- 
plied it  in  the  echinoderm  egg :  differences  in  rate  of  particular  fractions  of  metabo- 
lism may  be  the  basis  for  certain  initial  processes  in  morphogenesis. 

In  the  experiment  in  which  worms  with  partially  regenerated  tails  were  sub- 
jected to  lack  of  oxygen,  the  metabolism  which  supported  vital  processes  did  not  sup- 
port morphogenetic  processes.  Accordingly,  regeneration  must  depend  upon  ac- 
tivity of  some  aerobic  pathway.  The  fact  that  cyanide  affected  "rate  of  localization" 
indicates  a  cyanide-sensitive  system  important  during  localization.  The  fact  that 
cyanide  did  not  affect  rate,  extent  or  perfection  of  differentiation  indicates  that  the 
particular  system  has  little  or  no  importance  in  relation  to  differentiation.  Ac- 
cordingly, on  the  basis  of  cyanide-sensitivity  the  processes  supporting  "localization" 
and  "early  differentiation"  are  distinct.  Similarly,  on  the  basis  of  sensitivity  to 
iodoacetate  the  metabolic  processes  of  "early  differentiation"  and  of  "later  differenti- 
ation" are  distinct. 

During  the  period  in  regeneration  before  "later  differentiation"  appears,  oxygen 
consumption  was  found  to  be  only  slightly,  if  at  all,  above  normal  (Collier,  1947). 
However,  these  worms  lost  weight  almost  twice  as  rapidly  as  controls,  and  this  sug- 
gested an  energetic  cost  of  localization  which  was  not  reflected  in  oxygen  consump- 
tion. The  same  applied  to  a  possible  cost  of  "early  differentiation,"  but  "later 
differentiation"  was  found  associated  with  a  markedly  increased  consumption  of 
oxygen.  Determination  of  respiratory  sensitivity  to  cyanide  showed  that  the  in- 
crease was  cyanide-stable.  This  contrasts  with  the  findings  of  Bodine  and  Boell 


188  JANE  COLLIER  ANDERSON 

(1934)  for  grasshoppers  and  of  Sanborn  and  Williams  (1950)  for  Cecropia  moths, 
that  the  additional  oxygen  consumption  during  development  is  cyanide-sensitive 
although  the  respiration  during  diapause  is  entirely  cyanide-stable.  In  fact  it  ap- 
pears that  the  metabolic  mechanisms  of  morphogenesis  in  metamorphosing  insects 
(cf.  Williams,  1951)  can  hardly  be  compared  with  those  in  regenerating  Tubifex. 

The  presence  of  oxygen  was  found  to  be  essential  to  "localization"  but  high  oxy- 
gen tension  inhibited  it.  There  is  no  necessity  for  assuming  that  normal  oxygen 
tension  should  establish  optimal  conditions  for  localization.  Since  these  worms 
normally  live  partly  submerged  in  mud,  the  optimum  might  be  an  oxygen  tension 
lower  than  that  established  in  very  shallow  mudless  tap  water  under  air.  Fox  and 
Taylor  (1955)  found  this  true  for  survival  and  growth  of  young  worms  in  the 
laboratory. 

Respiration  as  measured  by  the  Warburg  method  was  entirely  stable  to  10~4  M 
and  10~5  M  KCN,  but  continuous  exposure  to  these  concentrations  of  cyanide  af- 
fected "rate  of  localization."  10'4  M  KCN  was  found  to  retard  while  lO^5  M  ac- 
celerated "localization."  Both  concentrations  counteracted  the  inhibitory  effect  of 
high  oxygen  tension. 

It  was  considered  that  the  accelerating  effect  of  the  more  dilute  cyanide  solution 
is  comparable  to  the  often  observed  and  seldom  explained  stimulation  of  various 
processes  by  other  inhibitors  in  extreme  dilution  (cf.  Commoner,  1940).  Since  in 
other  cases  the  stimulation  is  effective  upon  the  same  processes  which  are  inhibited 
by  higher  concentrations  of  the  poisons,  it  was  considered  that  the  two  concentra- 
tions of  cyanide  affected  the  same  process  in  "localization."  The  concentrations 
of  cyanide  which  activate  proteinases  in  vitro  are  at  least  fifty  times  higher  than  10~4 
M,  and  were  rapidly  lethal  to  the  worms  ( more  minutely  described  by  Hyman, 
1916).  Nevertheless  cyanide  here  may  have  been  effective  upon  the  reactions  of 
some  metalloprotein  other  than  those  of  the  cytochrome  system  or  of  the  haemo- 
globin in  the  blood  of  these  worms.  The  antagonism  of  high  oxygen  damage  by  cy- 
anide does  suggest  that  the  effects  of  high  oxygen  tension  and  of  cyanide  do  meet 
somewhere,  but  if  we  assume  that  cyanide  here  is  acting  as  an  oxidative  poison,  then 
the  cyanide-sensitive  system  cannot  be  responsible  for  any  large  proportion  of  the 
oxygen  consumption :  it  may  be  off  the  main  electron  transfer  pathway.  The 
lethality  of  high  oxygen  tension  also  suggests  an  autoxidizable  system  that  is  off  the 
main  pathway  (Gerschmann  ct  a/.,  1954).  Whatever  high  oxygen  affects,  whether 
protein  synthesis,  concentration  of  particular  normal  or  abnormal  components,  struc- 
tural integrity,  etc.,  it  was  at  least  partially  counteracted  in  Tubifex  by  cyanide. 

The  author  is  grateful  for  the  direction  and  encouragement  given  by  Dr.  Daniel 
Mazia,  for  the  kindlv  interest  of  Dr.  W.  C.  Curtis,  and  for  criticism  of  the  manu- 

j 

script  by  members  of  the  Department  of  Physiology,  University  of  Illinois. 

SUMMARY 

1.  Continuous  exposure  of  regenerating  Tubifc.r  tubife.r,  Mull,  to  cyanide  has 
been  found  to  affect  "rate  of  localization"  without  affecting  the  ultimate  extent  or 
perfection  of  localization  or  of  other  morphogenetic  processes. 

2.  Continuous  exposure  to  iodoacetate  has  been  found  to  increase  "rate  of  later 
differentiation"  without  having  other  effects  on  regeneration. 


CX,  KCN,  IAA  ON  REGENERATION  189 

3.  Low  oxygen  tension  was  found  to  retard  regenerative  processes  generally.     In 
complete  absence  of  oxygen,  blastema  formation  took  place  but  all  subsequent  proc- 
esses were  effectively  blocked. 

4.  High  oxygen  tension  blocked  morphogenesis  and  also  was  lethal  in  from  four 
to  eight  days.     Both  the  inhibition  and  the  lethal  effects  were  partially  relieved  by 
concurrent  treatment  with  cyanide. 

5.  It  is  concluded  that  the  availability  of  oxygen  limits  the  progress  of  later 
processes  in  morphogenesis  without  playing  any  necessary  part  in  the  initiation  of 
regeneration  in  Tubifex. 

6.  It  is  indicated  that  metabolic  processes  supporting  "localization,"  "early  dif- 
ferentiation," and  "later  differentiation"  are  at  least  partially  distinct  from  each 
other  and  from  the  metabolic  processes  essential  to  maintenance ;  that  energy  re- 
leased in  the  promotion  of  particular  morphogenetic  processes  must  be  released 
through  particular  enzyme  systems ;  and  that  such  specific  release  of  energy  is  es- 
sential to  the  progress  of  morphogenesis. 

LITERATURE  CITED 

BARROX,  E.  S.,  1951.     Thiol  groups  of  biological  importance.     Adv.  in  Enzymol,  2:  201-266. 
BARTH,  L.  G.,  1938.     Quantitative  studies  of  the  factors  governing  the  rate  of  regeneration  in 

Tubularia.     Biol.  Bull.,  74  :  155-177. 

BARTH,  L.  G.,  1940.     The  process  of  regeneration  in  hydroids.     Biol.  Rc:\,  15:  405-420. 
BODINE,  J.   H.,  AND  E.  J.   BOELL,   1934.     Respiratory   mechanism   of   normally   developing   and 

blocked  embryonic  cells   (Orthoptera).     /.  Cell.  Coin  p.  Physiol.,  5:  97-113. 
CHILD,   C.   M.,   1940.     Lithium  and  echinoderm  exogastrulation   with   a   review   of   the   physio- 
logical-gradient concept.     Physiol.  Zool.,  13  :  4—42. 
COLDWATER,  K.   B.,   1933.     The  effect  of  sulphydryl  compounds  upon  regenerative  growth.     /. 

Exp.  Zool,  65:  43-71. 
COLLIER,  JANE  G.,  1947.     Relations  between  metabolism  and  morphogenesis  during  regeneration 

in  Tubifex  tubifc.r.     I.  Biol.  Bull.,  92  :  167-177. 
COMMONER,  B.,  1940.     Cyanide  inhibition  as  a  means  of  elucidating  the  mechanisms  of  cellular 

respiration.     Biol.  Rev.,  15 :  168-201. 
Fox,  M.  H.,  AND  A.  E.  R.  TAYLOR,   1955.     The  tolerance  of  oxygen  by  aquatic  invertebrates. 

Proc.  Roy.  Soc.  London.  Scr.  B,  143:  214-225. 
GERSCHMANX,  REBECA,  D.  L.  GILBERT,  S.  W.  NYE,  P.  DWYER  AND  W.  O.  FEXN,  1954.     Oxygen 

poisoning  and  X-irradiation :  a  mechanism  in  common.     Science.  119:  623-626. 
HYMAN,  LIBBIE  H.,  1916.     An  analysis  of  the  process  of  regeneration  in  certain  microdrilous 

oligochaetes.     /.  Exp.  Zool.,  20:  99-163. 

HYMAN,  LIBBIE  H.,  1940.     Aspects  of  regeneration  in  annelids.     Amcr.  Nat.,  74:   513-527. 
KRECKER,  F.  H.,  1923.     Origin  and  activities  of  the  neoblasts  in  regeneration  of  microdrilous 

annelids.     /.  Exp.  Zool.,  37  :  27-46. 
LINDAHL,   P.   E.,   1936.     Zur  Kenntnis  der  physiologischen   Grundlagen   der   Determination  im 

Seeigelkeim.     Ada  Zool.,  17  :  179-365. 
LIPMANN,   F.,    1954.     Development   of   the   acetylation   problem,   a   personal    account.     Science, 

120:  855-865. 

SANBORX,  R.  C.,  AND  C.  M.  WILLIAMS,  1950.     The  cytochrome  system  in  the  Cecropia  silk- 
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33 :  579-588. 
STONE,  R.  G.,  1932.     The  effects  of  X-rays  on  regeneration  in  Tubifex  tuhifex.     /.  Morph.,  53: 

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THE  UPTAKE  OF  I131  BY  THE  THYROID  GLAND  OF  TURTLES 
AFTER  TREATMENT  WITH  THIOUREA  : 

SISTER  M.  CLAIRE  OF  THE  SAVIOR  BILEAU  2 
Department  of  Biology,  The  Catholic  University  of  America,  Washington,  D.  C. 

The  chemical  structure  and  relationships  to  goitrogenicity  of  several  hundred 
compounds  have  been  tested  by  a  number  of  investigators,  for  example :  the  Mack- 
enzies  (1943)  ;  Astwood,  Sullivan,  Bissell  and  Tyslowitz  (1943)  ;  Astwood  (1943)  ; 
Taurog,  Chaikoff  and  Franklin  (1945);  Astwood,  Bissell  and  Hughes  (1945); 
McGinty  and  Bywater  (1945)  and  VanderLaan  and  Bissell  (1946).  In  general 
the  active  substances  fall  into  one  of  three  classes :  1 )  thiourea  and  its  derivatives, 
2)  aniline  derivatives,  including  the  sulfonamides  and  other  aminobenzene  com- 
pounds, 3)  thiocyanates  and  organic  cyanides.  The  most  active  compounds  tested 
possess  a  thiourea  grouping  or  thioureylene  radical  — NH  •  CS  •  NH — .  Replacement 
of  the  hydrogens  of  thiourea  by  methyl  groups  increases  its  activity,  a  fact  which 
suggests  the  importance  of  the  thio  rather  than  the  mercapto  grouping  for  the  ac- 
tivity of  this  class  of  substances.  Extensive  reviews  concerning  antithyroid  agents 
have  been  published  by  Charipper  and  Gordon  (1947),  Astwood  (1949)  and  Comsa 
(1953). 

Vertebrates  of  every  class  have  been  tested  for  their  reaction  to  antithyroid 
drugs.  Such  studies,  however,  for  the  most  part,  have  dealt  with  mammals. 
Gordon,  Goldsmith  and  Charipper  (1943)  made  the  first  report  of  the  use  of  inhibi- 
tors on  the  thyroid  gland  of  cold-blooded  animals.  Since  then  a  number  of  in- 
vestigators have  studied  the  effects  of  goitrogenic  substances  on  poikilotherms. 
Lynn  and  Wachowski  (1951)  have  published  a  comprehensive  review  of  the  litera- 
ture dealing  with  the  thyroid  gland  and  its  functions  in  cold-blooded  vertebrates. 

Little  work  has  been  done  concerning  the  function  of  the  thyroid  in  reptiles. 
Ratzersdorfer,  Gordon  and  Charipper  (1949),  Adams  and  Craig  (1951)  and 
Fisher  (1953)  have  reported  the  effects  of  antithyroid  compounds  on  the  lizards. 
Naccarati  (1922)  described  the  normal  histology  and  gross  anatomy  of  the  thyroid 
gland  of  the  turtle  Em\s  curopa,  while  Evans  and  Hegre  (1940)  studied  seasonal 
changes  and  the  effects  of  pituitary  extract  on  the  thyroid  of  CJit-yscniys  picta  belli. 
Greenberg  (1948)  was  the  first  to  investigate  the  effects  of  thiourea  on  the  histology 
of  the  thyroid  gland  of  the  turtle.  In  her  work  immature  specimens  of  Pseudemys 
clcgans  were  used.  Since  then,  Adams  and  Craig  (1950)  and  Paynter  (1953) 
have  studied  the  thyroidal  response  to  goitrogens  in  Chryscmys  picta  picta.  Pastore 

1  A  contribution  from  the  Department  of  Biology,  The  Catholic  University  of  America, 
Washington,  D.  C.  This  paper  was  prepared  for  the  fulfillment  of  the  publication  requirement 
for  the  degree  of  Doctor  of  Philosophy  in  the  Graduate  School  of  Arts  and  Sciences  of  The 
Catholic  University  of  America,  Washington,  D.  C. 

The  author  is  deeply  indebted  to  Dr.   H.   E.   Wachowski,  for   suggesting  the  problem   as 
well  as  for  his  patient  guidance  and  encouragement,  and  to  Dr.  W.  G.   Lynn  and  Dr.   M.   P. 
Sarles  for  their  helpful  suggestions. 
-  Present  address :  Biology  Department,  Rivier  College,  Nashua,  New  Hampshire. 

190 


THIOUREA,  IIM  AND  TURTLE  THYROID  1^1 

(1950)  investigated  the  effects  of  thyroid-stimulating  and  thyroid-inhibiting  drugs 
upon  the  histology  of  the  thyroid  of  Clcinniys  inscnlpta  and  Graptemys  geographica. 
Dimond  (1954)  studied  the  reactions  of  developing  snapping  turtles,  Chclydra 
serpentina  serpcnlina.  to  thiourea. 

[nvestigators  who  have  worked  on  the  thyroid  gland  of  turtles  are  unanimous  in 
pointing  out,  as  an  explanation  for  the  observed  irregular  reactions  to  goitrogenic 
agents,  what  appears  to  be  an  inherent  variability.  This  variability  far  exceeds 
that  found  in  warm-blooded  vertebrates.  Uhlenhuth,  Schenthal,  Thompson,  Mech 
and  Algire  (1945)  working  with  the  newt,  Tritnrus  torosus,  claimed  that  such  a 
high  degree  of  variation  does  not  seem  explicable  on  the  basis  of  the  known  physio- 
logical roles  of  the  urodele  thyroid  and  must  be  attributed  to  what  might  be  called  a 
general  instability  of  the  endocrines  in  cold-blooded  vertebrates. 

Some  of  the  methods  used  to  acquire  a  more  thorough  knowledge  of  the  gland 
are  :  1 )  gravimetric  methods,  based  on  changes  in  thyroid  weight ;  2)  chemical  meth- 
ods, a  quantitative  as  well  as  a  qualitative  study  of  iodine  in  the  gland ;  3)  the  use  of 
radioisotope  technique  (radioactive  iodine  in  the  thyroid  increases  progressively 
with  increasing  dosage  of  TSH)  ;  4)  histological  methods,  which  study  mainly:  a) 
epithelial  height,  b)  staining  reactions  of  colloid  and  cells,  c)  position  and  shape  of 
nuclei  and  nuclear  volume;  5)  microhistometric  methods.  Uotila  and  Kannas 
(1952)  have  devised  a  linear  measurement  method,  which  permits  quantitative  de- 
terminations of  the  principal  components  of  the  thyroid  tissue.  The  method  ap- 
pears to  have  the  advantage  of  objectivity,  simplicity  and  economy  of  time.  This 
method  was  compared  with  the  planimetric  method  and  cell  height  measurement  by 
Tala  (1952)  and  found  to  give  an  accurate  picture  of  the  histological  activity  of  the 
thyroid  gland. 

The  use  of  radioactive  iodine  in  experimental  studies  on  the  thyroid  gland  has 
added  considerably  to  present-day  understanding  of  the  histophysiology  of  this 
organ.  The  early  studies  of  Hertz,  Roberts,  Means  and  Evans  (1940),  Hertz  and 
Roberts  (1(H1),  and  Hamilton  and  Soley  (1940),  in  which  radioactive  iodine  was 
used  for  the  first  time  in  thyroid  investigation,  ushered  in  a  technique  which  enabled 
more  precise  interpretation  of  the  relationship  between  iodine  metabolism,  the  thy- 
roid and  the  hypophysis. 

Tracer  studies,  using  radioiodine  after  treatment  with  thyroid-inhibiting  and 
thyroid-stimulating  substances,  are  perhaps  the  most  widely  applied  tools  in  thyroid 
investigation  today.  These  recent  developments  are  being  used  extensively  in 
studying  the  thyroid  function  in  mammals  and  are  now  being  employed  to  some  ex- 
tent on  cold-blooded  vertebrates.  However,  as  stated  above,  very  few  experiments 
have  been  performed  on  the  reptiles.  This  is  a  particularly  significant  gap  in  our 
knowledge,  in  view  of  the  fact  that  reptiles  are  considered  as  the  stem  of  the  birds 
and  mammals  in  the  vertebrate  scale,  and  as  the  only  cold-blooded  amniote.  One 
of  the  main  reasons  for  this  neglect  has  been  the  difficulty  in  performing  thyroidec- 
tomy  in  these  animals.  The  recent  development  of  an  effective  "chemical  thyroidec- 
tomy"  opens  new  opportunities  for  research  in  this  field.  Already,  some  attempts 
have  been  made  at  correlating  cell  height,  dry  weight  of  the  thyroid  and  the  histo- 
logical picture  with  the  uptake  of  radioiodine  in  cold-blooded  vertebrates  such  as 
salamanders  (Desmognathus  jnscns  (Rafinesque)  ;  Fisher,  1953)  and  in  the  turtle 
Chryscinys  picta  picta  by  Paynter  (1953).  The  results  obtained  in  these  experi- 
ments point  again  to  the  greatest  difficulty  encountered  in  the  study  of  thyroid 


192  SISTER  M.  CLAIRE  OF  THE  SAVIOR  BILEAU 

function  at  this  level,  namely,  the  astonishing  variability.  The  present  work  is  an 
attempt  at  investigating  some  of  the  factors  which  may  influence  this  great  variability 
in  the  function  of  the  thyroid  of  the  turtle  Chryscuiys  picta.  To  this  effect  a  study 
was  made  of  the  possible  correlations  between  radioiocline  uptake  and  colloid  level, 
percentage  epithelium,  cell  height  and  dry  weight  of  the  thyroid,  in  normal  and  in 
treated  animals ;  an  endeavor  was  also  made  to  determine  the  thyroid/serum  ratio, 
when  organic  binding  is  blocked  and  when  binding  is  permitted. 

MATERIALS  AND  METHODS 

Turtles  with  carapace  length  between  five  and  seven  inches  were  purchased 
from  The  Lemberger  Company,  Oshkosh,  Wisconsin  and  the  J.  R.  Schettle  Frog 
Farm,  Stillwater,  Minnesota. 

The  experimental  and  control  animals,  totaling  one  hundred  sixteen,  were  kept 
in  large  metal  tanks  which  were  arranged  so  that  the  animals  had  free  access  to 
running  water  or  dry  perches.  The  temperature  of  the  room  in  which  the  tanks 
were  located  was  kept  as  close  to  75°  F.  as  possible.  All  animals  used  in  this  series 
of  experiments  were  denied  food  during  the  term  of  treatment  since  the  amount  of 
iodine  in  the  food  could  not  have  been  controlled. 

The  experimental  animals  were  given  subcutaneous  injections  of  thiourea  three 
times  a  \veek.  The  dosage,  found  most  effective  by  Paynter  (1953)  for  these 
experiments,  was  0.25  cc./lOO  gr.  of  body  weight  of  a  0.1%  solution.  After  four, 
six  and  eight  weeks  of  treatment  the  animals  were  injected  with  a  tracer  dose  of 
three  microcuries  of  carrier-free  I131,  in  0.5  cc.  of  distilled  water.  The  untreated 
control  animals  received  the  same  dose  of  Ii:n.  Three  hours  after  administration 
of  the  radioactive  iodine  the  animals  were  sacrificed.  The  plastron  of  each  turtle 
was  removed,  blood  was  taken  from  one  of  the  large  vessels  leaving  the  heart  and 
allowed  to  clot.  Immediately  after  exsanguination,  the  thyroid  gland  was  removed 
and  cut  in  half.  The  right  half  was  cooled  by  placing  it  on  dry  ice.  The  left  half, 
in  27  of  the  experimental  and  20  of  the  control  animals,  was  immersed  in  iso- 
pentane,  cooled  by  liquid  nitrogen.  Afterward,  the  glands,  placed  in  individual 
tubes,  on  the  surface  of  degassed  paraffin,  were  transferred  to  the  drying  chamber 
of  a  freezing-drying  apparatus  (Altmann-Gersh  technique;  Gersh,  1932).  De- 
hydration was  carried  out  at  •-  40°  C.  and  continued  for  a  minimum  of  72  hours. 
The  system  was  then  gradually  brought  to  the  melting  point  of  paraffin  by  immersing 
the  drying  chamber  in  a  beaker  of  water  which  was  kept  at  constant  temperature  by 
placing  it  on  a  thermostatically  controlled  hot  plate.  The  vacuum  was  broken  only 
after  infiltration  was  completed. 

As  for  the  remainder,  42  experimental  and  27  control  animals,  the  left  half  of 
the  gland  was  placed  on  a  glass  slide  and  dried  at  37°  C.  to  determine  dry  weight. 
These  glands  were  recovered  by  soaking  in  0.025%  solution  of  trisodium  phosphate 
for  24  hours  and  in  10%  formalin  for  three  hours. 

The  right  half  of  the  gland,  for  each  animal,  was  placed  into  glass  homogenizers 
containing  6  ml.  of  ice-cold  distilled  water  and  one  mg.  of  Nal  as  carrier,  and 
homogenized  immediately.  The  homogenate  was  deproteinized  according  to  So- 
mogyi  (1945).  To  0.80  cc.  of  homogenate,  0.10  cc.  each  of  Ba(OH)2  and  ZnSO4 
were  added,  followed  by  vigorous  shaking,  and  centrifuging.  The  supernate  was 
removed  with  a  pipette,  three  drops  were  placed  on  each  of  three  glass  sides.  The 


THIOUREA,  I131  AND  TURTLE  THYROID  193 

same  was  done  with  the  precipitate  and  a  sample  of  the  homogenate  before  de- 
proteinization.     These  were  dried  at  37°  C. 

The  serum  was  deproteinized  by  diluting  a  0.05-ml.  sample  to  0.08  ml.  with  wa- 
ter containing  0.10(/o  Nal  and  adding  to  it  0.10  ml.  each  of  Ba(OH)2  and  ZnSO4 
as  above.  To  determine  the  I131  content  of  the  serum  an  aliquot  of  diluted  serum 
(  0.05  ml.  diluted  to  1.00  ml.  with  water  containing  0.10%  Nal)  was  dried  on  a  glass 
slide. 

Precipitable  and  non-precipitable  I131,  as  well  as  total  I131  in  both  gland  and 
serum,  were  measured  with  a  Geiger-Mueller  tube  (window  thickness  1.7  mg./ 
cm.~-)  placed  at  a  distance  of  4  mm.  from  the  object.  The  counts  were  also  deter- 
mined for  the  glands  of  both  series,  frozen-dried  and  oven-dried.  Three  determina- 
tions of  one  minute  duration  each  were  taken  and  averaged.  The  counts  were 
brought  up  to  the  value  on  the  day  of  injection  by  the  use  of  decay  factors  which 
were  based  on  the  eight  day  half-life  of  I131. 

The  glands  were  sectioned  at  5  micra  and  those  of  the  frozen-dried  series  were 
fixed  by  floating  on  10%  formalin  and  amphibian  Ringer's  solution  and  stained 
with  Gomori's  chrome  alum  hematoxylin  and  phloxin.  Three  sections  were  selected 
at  25%,  50%  and  75%  of  each  gland.  Film  strips  of  these  sections  were  made  and 
projected  on  white  paper  which  served  as  a  screen,  using  a  magnification  of  X  100. 
Two  lines,  intersecting  at  obtuse  angles  in  the  form  of  an  X,  were  drawn  in  ad- 
vance on  the  plane  to  which  the  image  was  to  be  projected.  The  image  of  the 
stained  specimen  was  positioned  so  that  its  center  fell  approximately  on  the  junc- 
tion of  the  intersected  lines.  The  outline  of  the  follicles  and  colloid  along  the  full 
length  of  the  two  lines  was  drawn.  The  segment  of  the  lines  covered  by  the  entire 
figure  was  then  measured  in  millimeters.  Similarly,  millimetric  lengths  of  the  seg- 
ments covered  by  epithelium  and  colloid  were  determined.  The  total  of  the  epi- 
thelium segments,  divided  by  the  whole  length  of  the  lines,  gave  the  percentage  of 
epithelium.  The  percentage  of  colloid  was  calculated  in  the  same  way.  This 
linear  measurement  method  of  determining  the  principal  components  of  the  thyroid 
gland  was  devised  by  Uotila  and  Kannas  (1952)  and  further  tested  by  Tala  (1952). 

OBSERVATIONS 

It  is  fairly  well  established  from  animal  experimentation  that  antithyroid  com- 
pounds of  the  thioureylene  type  owe  their  activity  to  their  property  of  preventing 
the  organic  binding  of  iodine,  or,  what  amounts  to  the  same  thing,  the  inhibition  of 
the  oxidation  of  iodide  to  iodine.  Under  the  influence  of  these  agents,  iodide  is 
still  able  to  concentrate  in  the  thyroid  gland,  but  it  remains  in  a  reduced,  ionic  state. 
As  a  result  of  treatment  with  thiourea  it  should  then  be  expected  that  radioiodine 
uptake  would  be  higher  in  the  experimental  animals  than  in  the  corresponding  con- 
trol groups.  This  fact  was  well  brought  out  in  the  course  of  the  study ;  in  all  cases 
the  counts  were  much  higher  in  the  treated  animals  than  they  were  in  the  untreated. 
Analysis  of  the  results  based  solely  on  length  of  treatment  were  much  too  variable 
to  draw  significant  conclusions.  Re-interpretation  of  these  results  on  the  basis  of 
seasonal  cycles  gave  a  more  valid  picture. 

A  total  of  72  animals  were  treated  with  thiourea;  52  of  them  received  18  sub- 
cutaneous injections  over  a  period  of  six  weeks  and  20  received  12  injections  over 
a  period  of  four  weeks.  Forty-seven  animals  were  used  as  controls  and  received 


194  SISTER  M.  CLAIRE  OF  THE  SAVIOR  BILEAU 

three  microcuries  of  radioiodine  at  the  same  time  as  the  treated  animals,  three  hours 
before  they  were  killed.  One  set  of  controls  was  examined  each  time  an  experi- 
mental group  was  run. 

An  analysis  of  the  results  from  two  different  points  of  view,  1 )  length  of  treat- 
ment and  2)  seasonal  factor,  follows. 

Length  of  treatment 

From  this  point  of  view,  the  outstanding  feature  throughout  the  entire  study 
is  the  great  variability  observed  in  control  groups  as  well  as  in  experimental  animals. 
Taking  the  various  correlations  specifically  the  following  can  be  reported. 

Six-week  series.  The  percentage  epithelium  was  lower  and  the  range  of  varia- 
tion in  percentage  epithelium  was  considerably  higher  in  experimental  animals. 
The  radioiodine  uptake  per  unit  epithelium  and  the  range  of  variation  in  uptake  were 
both  higher  in  the  treated  than  in  the  control  group.  The  coefficient  of  correlation 
between  the  per  cent  epithelium  and  radioiodine  uptake  was  --  0.38  for  the  experi- 
mental group  and  -  0.31  in  the  control  animals.  It  becomes  evident  from  the 
above  figures  that  the  correlations  between  per  cent  epithelium  and  uptake  were 
practically  nil  in  both  treated  and  untreated  groups. 

The  percentage  colloid,  radioiodine  uptake  per  unit  per  cent  colloid,  and  the 
range  of  variation  were  considerably  higher  in  the  treated  animals  than  in  the  control 
group.  The  coefficient  of  correlation  for  the  colloid  and  uptake  was  --  0.1  (>  in  the 
treated  animals,  while  it  was  --  0.69  in  the  control  group.  In  terms  of  colloid  up- 
take the  correlation  was  slightly  improved  in  the  treated  animals  but  remained  in- 
significant in  the  control  animals. 

Four-week  series.  There  was  no  significant  difference  between  the  epithelium 
percentage  and  colloid  level  of  experimental  and  control  animals.  The  radioiodine 
uptake  was  much  lower  than  in  the  six- week  group  and  the  range  of  variation  was 
greatly  reduced.  The  coefficients  of  correlation  were  as  follows  :  per  cent  epithelium 
and  uptake,  control  --  0.20,  experimental,  --  0.06;  unit  colloid  and  uptake,  control 
-0.10,  and  experimental  --0.27.  The  correlations  in  the  four-week  series  were 
considerably  improved  but  even  if  the  range  of  variation  was  reduced  it  was  still 
too  high  to  permit  significant  correlations. 

From  the  point  of  view  of  length  of  treatment  it  was  impossible  to  establish  sig- 
nificant correlations,  due  to  the  high  range  of  variation.  The  correlations  were 
poorest  in  the  six-week  series,  but  became  somewhat  improved  in  the  four-week 
series.  This  was  particularly  true  in  regard  to  colloid  level  and  iodine  uptake. 

Seasonal  factor 

During  the  course  of  this  study  the  influence  of  a  seasonal  factor  has  been  ob- 
served. Since  the  animals  had  been  obtained  from  the  supplier  at  three  different 
times  of  the  year,  the  three  series  therefore  varied  as  to  the  time  of  the  year  during 
which  the  experimental  work  was  done.  When  the  results  were  analyzed  in  terms 
of  seasonal  cyclic  activity,  the  correlation  between  epithelium  or  colloid  and  uptake 
was  greatly  improved  and  the  variability  was  decidedly  reduced,  especially  in  the  fall 
series.  The  first  series  was  carried  out  during  June  and  the  beginning  of  July. 
The  second  series  was  carried  out  during  November  and  the  last  series  during  the 
month  of  February  and  the  beginning  of  March.  As  a  result  of  such  spacing,  the 


THIOUREA.  i131  AND  TURTLE  THYROID 


195 


TABLE  I 
Radioiodine  uptake  per  itiilli^rum  dry  weight.     Difference  in  per  cent  of  controls 


Series 

Mean  count  per  milligram 

%  of  control  uptake 

Experimental 

Control 

Winter 
Summer 
Fall 

592.29 
985.34 
484.16 

527.01 
534.85 
378.55 

112.38% 
184.22% 
127.89% 

possibility  of  a  seasonal  factor  controlling  the  activity  of  the  thyroid  gland  in  the 
turtle  was  brought  out,  and  this  factor  appeared  to  exercise  its  influence  even  in  the 
treated  animals. 

Uptake  f>er  milligram  dry  weight.  In  the  determination  of  radioiodine  uptake, 
it  was  observed  that  1 )  the  counts  per  minute  per  milligram  dry  weight  were  higher 
in  the  treated  animals  than  in  the  corresponding  control  animals,  and  2)  the  counts 
for  both  experimental  and  control  animals  were  highest  during  the  summer  and 
lowest  in  the  fall  series.  This  last  point  is  a  strong  indication  of  the  influence  of  a 
seasonal  factor  and  is  in  agreement  with  Eggert  (1935)  who  reported  highest 
thyroid  activity  in  June,  in  the  case  of  hibernating  lizards,  and  lowest  activity  during 
December  and  January.  In  this  case  the  winter  group,  having  been  killed  at  the 
end  of  February  and  the  beginning  of  March,  wrould  correspond  to  the  resumption  of 
the  secretory  activity.  A  summary  of  the  results  in  per  cent  of  control  is  given  in 
Table  I. 

Thyroid/ serum  ratio.  Measurements  of  the  thyroid/serum  ratios  were  made 
when  organic  binding  was  blocked  with  thiourea  and  when  binding  was  permitted. 
Due  to  the  high  degree  of  individual  variation,  the  time  required  for  the  maximum 
uptake,  as  determined  by  Paynter  (1953),  was  found  to  vary  considerably  with 

TABLE  II 

Per  cent  epithelium.     Experiments  in  the  order  of  their  ranges  of  variation 


Range 

Series 

Seasons 

No.  of  cases 

Mean  % 
epithelium 

Maximum- 
minimum 

In  points 

In  per  cent 
of  mean 

Winter 

6  weeks 

12 

15.18 

18.33-11.44 

6.89 

45.38% 

Experimental 

Summer 

6  weeks 

11 

16.18 

20.90-10.27 

10.63 

65.69% 

Fall 

4  weeks 

18 

16.22 

18.18-14.26 

3.92 

24.16% 

Winter 

9 

17.01 

20.09-12.86 

7.23 

42.50% 

Control 

Fall 

13 

16.77 

19.84-12.70 

7.14 

42.57% 

196 


SISTER  M.  CLAIRE  OF  THE  SAVIOR  BILEAU 


each  animal.  Consequently  a  significant  ratio  could  not  be  established.  The  effects 
of  the  seasonal  cycle,  however,  could  still  be  observed.  In  taking  the  counts  per 
minute  for  the  homogenate  and  comparing  it  with  the  counts  for  the  serum  the 
following  results  were  obtained : 

1)  The  summer  group:  in  92', c  of  the  experimental  and  40%  of  the  control  ani- 
mals, the  thyroid  homogenate  had  a  higher  count  than  the  serum. 

2)  The  fall  group:  in  43' c  of  the  experimental  and  20%  of  the  control  animals, 
the  thyroid  homogenate  had  a  higher  count  than  the  serum. 

3  )  The  winter  group :  in  36f  <  of  the  experimental  and  16%  of  the  control  ani- 
mals, the  thyroid  homogenate  had  a  higher  count  than  the  serum. 

The  results  bring  out  very  clearly  the  influence  of  a  seasonal  cyclic  activity. 
The  values  obtained  for  the  summer  groups  are  almost  three  times  as  high  as  those 
for  the  winter  groups  and  twice  as  high  as  the  values  obtained  for  the  fall  series. 
These  differences  appear  to  be  directly  correlated  to  the  phase  of  activity  of  the 
thyroid  gland  in  hibernating  reptiles. 

TABLK  III 

Colloid  level.     Difference  between  ranges  of  controls  and  experimental 


Seasi  in 

Series 

Colloid  level 
Maximum-mini  ni  inn 

Range 

Difference  between  experimental 
and  control  ranges 

In  points 

In  c~c  of  con- 
trol range 

Winter 

Con  trnl 
Experimental 

75.50-63.12 
78.93-54.86 

12.38 
24.07 

+  11.69 

+94% 

Fall 

Control 
Experimental 

80.87-67.  5<> 
78.74-60.67 

13.30 
18.07 

+  4.77 

+35% 

I'ci-  cent  epithelium  and  iodine  uptake.  Two  six-week  series  were  carried  out, 
one  during  late  February  and  early  March  and  the  other  during  June  and  July,  and 
one  four- week  series  during  November.  The  range  of  variation  was  too  high  to 
establish  significant  correlations  between  the  per  cent  epithelium  and  radioiodine 
uptake,  but  the  influence  of  the  seasonal  factor  was  nevertheless  observed.  The  per 
cent  epithelium  in  the  treated  animals  was  higher  in  the  summer,  with  no  significant 
difference  in  percentage  for  the  fall  group,  but  decidedly  lower  for  the  winter.  In 
the  control  series,  comparisons  between  percentage  epithelium  or  colloid  level  are 
available  only  between  the  fall  and  winter  groups.  The  summer  control  animals 
were  not  used  for  measurements.  The  range  of  variation  in  the  three  series  gave  a 
definite  indication  of  a  .seasonal  cyclic  influence.  Table  II  lists  the  per  cent  epi- 
thelium with  the  range  of  variation  for  the  three  seasons  during  which  the  work  was 
carried  out. 

The  uptake  of  radioiodine  per  unit  per  cent  epithelium  was  considerably  higher 
during  the  summer  and  lowest  during  the  fall.  Here  again  the  results  seem  to  in- 
dicate that  the  state  of  activity  of  the  thyroid  gland  in  the  painted  turtle  corresponds 


THIOUREA,  I131  AND  TURTLE  THYROID 


197 


TABLF,  IV 

Radioiodine  uptake  per  unit  colloid.     Different  <•  befo 
ranges  of  centrals  and  ex  peri  mentals 


Sea-  in 

Series 

Radioactivity 

Maximum   minimum 

Range 

Difference  between  experimental 
and  control  ranges 

In  points 

In  %  of  con- 
trol range 

Winter 

Control 
Experimental 

122.74-10.88 
238.24-  6.58 

111.86 
231.86 

+  119.82 

+  107% 

Fall 

(  "(  ml  rul 
1  \perimental 

197.51-  4.59 
129.76-  6.58 

192.92 
123.18 

-   69.74 

•   36% 

rather  closely  to  the  seasonal  cycle  as  described  above  and  in  agreement  with  the  work 
of  Eggert  (  1935). 

The  counts  per  unit  epithelium  in  the  treated  animals  were  3S3.(>3  for  the  sum- 
mer, 335.79  for  the  winter  and  only  157.13  for  the  fall  groups.  Comparing  the 
counts  for  the  untreated  animals  the  same  general  cyclic  activity  could  be  observed. 
The  correlations,  even  though  somewhat  improved,  still  remained  insignificant  be- 
cause of  the  range  of  variation. 

Colloid  level.  The  influence  of  the  seasonal  factor  became  more  evident  in  the 
studies  on  correlation  between  colloid  level  and  radioiodine  uptake.  The  cyclic  pat- 
tern was  decidedly  in  accordance  with  the  various  phases  of  activity  described  for 
the  hibernating  reptiles.  The  mean  uptake  per  unit  colloid  level  was  79.91  for  the 
summer.  65.31  for  the  winter,  and  33.SC>  for  the  fall  group  in  the  treated  animals. 

The  best  evidence  for  this  factor  was  brought  out  in  the  closer  correlation  be- 

TABLE  V 

Colloid  level  experiments  in  the  order  of  their  ranges  of  variation 


Series 

Seasons 

No.    of   Ciisrs 

Mean  % 
colloid 

Maximum 
minimum 

Range 

In  points 

In  per  cent 
of  mean 

Winter 
6  weeks 

12 

69.46 

78.93-54.86 

24.07 

34.65% 

Experimental 

Summer 
6  weeks 

11 

75.95 

87.01-60.54 

26.47 

34.86% 

Fall 
4  weeks 

18 

72.76 

78.74-60.67 

18.07 

24.83% 

Control 

Winter 
Fall 

9 

13 

69.24 
71.81 

75.50-63.12 
80.87-67.59 

12.38 
13.30 

17.87% 
18.52% 

198 


SISTER  M.  CLAIRE  OF  THE  SAVIOR  BILEAU 


tween  the  colloid  level  and  the  uptake  of  radioiodine  in  the  fall  group.  The  range 
of  variation  is  at  its  lowest  in  both  colloid  level  and  range  of  uptake.  Tables  III 
and  IV  give  the  difference  between  the  ranges  of  variation  for  colloid  level  and  up- 
take in  per  cent  of  control  range.  It  is  to  be  observed  that  for  the  fall  group  the 
variation  in  colloid  level  for  the  experimental  group  was  35%  above  the  control,  as 
compared  with  94%  for  the  winter  series.  The  range  of  variation  of  iodine  uptake 
in  experimental  animals  for  the  fall  series  was  36%  below  the  controls  and  107%' 
above  in  the  winter  series. 

From  the  above  observations  it  was  possible  to  conclude  that  in  terms  of  length  of 
treatment,  the  range  of  variation  in  uptake  of  radioiodine,  either  per  unit  of  epi- 
thelium or  colloid  level,  was  much  too  high  to  establish  significant  correlations. 
When,  however,  the  results  were  analyzed  from  the  point  of  view  of  seasonal  cyclic 
activity  it  became  apparent  that  the  gland  was  under  the  influence  of  a  seasonal  fac- 
tor which  exercised  its  control  in  treated  as  well  as  in  untreated  animals.  More 


TABLE  VI 

Radioactivity  /unit  %  colloid.     Experiments  in  order  of  time  of  treatment 


Range 

Mean  radio- 

Maximum- 

Series 

Seasons 

No.  of  cases 

activity  per 

minimum 

unit  %  colloid 

In  points 

In  per  cent 
of  mean 

Winter 

6  weeks 

12 

65.31 

238.24-  6.58 

231.68 

354.74% 

Experimental 

Summer 

6  weeks 

11 

79.91 

141.83-18.69 

123.14 

154.09% 

Fall 

4  weeks 

18 

33.86 

129.76-  6.58 

123.18 

363.79% 

Winter 

9 

57.99 

122.74-10.88 

111.86 

192.89% 

Control 

Fall 

13 

40.61 

197.51-  4.59 

192.92 

475.05% 

complete  data  on  colloid  level  and  uptake  and  variation  are  given  in  Tables  V  and 
VI. 

DISCUSSION 

Antithyroid  compounds  may  inhibit  the  normal  function  of  the  thyroid  gland 
by  acting  directly  on  the  thyroid  itself  at  any  one  of  the  stages  of  hormone  produc- 
tion :  1 )  the  collection  of  iodide  from  the  circulation  ;  2  )  the  synthesis  of  thyroid  hor- 
mone;  3)  the  release  of  hormone  to  the  tissues. 

Thiocyanate  ions  exert  a  unique  effect  upon  the  thyroid  gland  shared  by  no 
other  substance  yet  known  (Astwood,  1949).  Animals  treated  with  this  sub- 
stance have  been  shown  to  be  unable  to  collect  iodide  from  the  circulation.  Other 
substances,  like  thiourea  and  thiouracil,  are  believed  to  block  an  enzyme  system, 
thereby  preventing  organic  synthesis  of  the  hormone,  but  having  no  effect  on  the 
"iodide  trap." 


THIOURKA,  P1  AND  TURTLE  THYKniI)  1  W 

Astwood  and  Bissell  (  1(<44)  found  that  in  rats  under  the  continuous  intluence 
of  thiouracil  the  iodine  content  of  the  thyroid  rapidly  falls  to  lo\v  levels  and  that  the 
thyroid  gland  simultaneously  enlarges.  Astwood  (1944-45)  showed  that  animals 
thus  depleted  of  iodine  are  still  able  to  concentrate  rapidly  considerable  quan- 
tities of  iodine  when  injected  with  potassium  iodide. 

D'Angelo,  Paschkis,  Cantarow,  Siegel  and  Riviero-Fontan  (1951)  have  ob- 
served that  despite  uniformly  decreased  radioiodine  uptake  with  chronic  propyl- 
thiouracil  treatment  the  total  radioactivity  in  the  thyroid  eventually  exceeds  normal 
when  sufficient  hyperplasia  has  occurred  to  offset  the  limited  uptake.  The  aug- 
mented radioiodine  collections  which  result  where  the  drug  is  withdrawn,  how- 
ever, are  greater  than  would  be  expected  from  hyperplasia  alone,  and  must  result  in 
part  from  increased  avidity  of  thyroid  tissue  for  the  radioactive  iodine.  The  aug- 
mented avidity  for  iodine  upon  withdrawal  of  the  drug  is  demonstrable  after  periods 
of  treatment  too  short  to  have  caused  hyperplasia,  although  it  increases  progres- 
sively with  longer  periods  of  treatment  and  consequently  with  thyroid  hyperplasia 
before  the  goitrogen  is  withdrawn. 

The  results  obtained  in  this  study  are  in  agreement  with  the  above  observations. 
The  uptake  of  radioiodine  was  considerably  higher  in  the  animals  in  which  binding 
was  blocked  as  compared  with  those  in  which  binding  was  permitted.  This  rapid 
uptake  of  iodine  by  the  thyroid  gland,  reaching  a  maximum  and  followed  by  a  dis- 
charge of  the  trapped  iodine,  varies  with  the  type  of  animals  used.  In  mammals, 
tor  example,  the  time  required  for  maximum  concentration  may  be  as  little  as  ten 
to  fifteen  minutes  as  reported  by  Hertz,  Roberts,  Means  and  Evans  (1940)  and 
Chaikoff  and  Taurog  (1949). 

In  cold-blooded  animals,  however,  great  variations  are  observed  in  the  reactions 
of  the  thyroid,  and  because  of  this  great  range  of  variability  the  maximum  uptake 
in  the  turtle  would  seem  to  be  controlled  by  a  factor  other  than  time  alone. 

In  accordance  with  Adams  and  Craig  (1950),  Paynter  (1953)  and  Dimond 
(  1(^54)  the  results  obtained  in  this  investigation  show  that  the  reactions  to  thiourea 
in  the  turtles  are  not  as  profound  as  those  reported  in  warm-blooded  animals,  and 
especially  in  mammals.  The  percentage  epithelium  was  slightly  lower  in  treated 
animals  than  in  control  animals,  while  the  colloid  was  higher  in  the  experimental 
than  in  the  control  animals.  This  could  be  explained  by  the  fact  that  the  experi- 
mental animals  were  injected  with  a  tracer  dose  of  I131  24  hours  after  the  last  treat- 
ment with  thiourea.  This  delay  would  allow  the  pituitary-thyroid  axis  to  be  re- 
stored to  normal  conditions,  and  consequently  after  a  period  of  increased  activity, 
the  thyroid  had  stored  a  considerable  amount  of  colloid. 

Seasonal  factor.  A  discussion  of  the  seasonal  physiology  of  any  vertebrate  im- 
mediately arouses  an  inquiry  concerning  the  behavior  of  its  endocrine  glands,  es- 
pecially that  of  the  thyroid  and  the  pituitary  because  of  the  reactions  to  temperature 
by  the  former  and  the  close  association  and  control  of  the  thyroid  function  by  the 
latter. 

In  general,  throughout  the  vertebrate  classes,  it  may  be  said  that  if  a  species  is 
inactive  (hibernating)  during  the  winter  months,  the  thvroid  is  inactive  at  this  time 
and  will  not  become  active  until  the  animal  resumes  activity.  Warm-blooded  ani- 
mals for  example,  which  are  active  all  winter,  have  thvroids  reported  to  be  more 
active  during  the  cold  season,  in  order  to  maintain  their  normal  body  temperature 
and  BMR.  The  same  animals  have  lower  rate  of  thyroid  activity  during  the  sum- 


200  SISTER  M.  CLAIRE  OF  THE  SAVIOR  BILEAU 

mer,  the  temperature  of  the  environment  tending  to  prevent  the  loss  of  body  tem- 
perature, thereby  lowering  the  energy  requirement  for  the  maintenance  of  a  normal 
I1,  MR. 

It  can  generally  be  said  that  the  reverse  is  true  in  cold-blooded  animals.  How- 
ever, the  seasonal  conditions  of  thyroids  in  cold-blooded  vertebrate  hibernators  are 
less  well  known.  Morgan  and  Fales  (1942)  reported  that  there  are  comparatively 
few  observations  on  the  full  seasonal  cycles  of  the  thyroid  of  amphibians  and  some  of 
these  are  conflicting.  Several  observers  are  in  agreement  concerning  the  seasonal 
condition  of  the  thyroids  in  various  species  of  frogs  and  toads,  all  of  which  are  hiber- 
nators. In  the  main  they  report  a  winter  phase  of  moderate  activity  and  a  summer 
phase  of  greatly  lowered  activity.  Burger  (194(>),  in  his  observations  on  seasonal 
conditions  of  the  thyroid  of  the  male  of  three  species  of  urodeles,  reports  variations 
between  animals  and  between  individual  follicles  of  the  same  gland.  The  three  spe- 
cies, however,  all  showed  a  similar  broad  cyclic  pattern  in  that  activity  was  highest 
in  the  spring,  lowest  during  the  summer  and  moderate  in  the  fall.  Similar  results 
have  been  reported  by  Miller  and  Robbins  (  1955)  for  the  urodele  amphibian 
Taricha  torosa  (Tritunts  torosiis). 

Although  few  in  number,  some  studies  of  the  seasonal  cycle  of  the  thyroid  of  rep- 
tiles have  been  made  on  hibernating  and  non-hibernating  species.  Eggert  (1935) 
studied  three  forms  of  European  Lacertas  and  reported  hibernation  beginning  from 
the  end  of  September  reaching  a  peak  in  December  and  January  when  the  gland  is  at 
its  most  reduced  activity.  Young  animals  resume  their  secretory  activity  in  Febru- 
ary and  attain  their  highest  activity  in  June.  Seasonal  variations  in  the  thyroid  of 
lizards  have  also  been  noted  by  Ratzersdorfer,  Gordon  and  Charipper  (1949)  in 
Anolis  carolincusis.  In  non-hibernating  Xantiisis  rit/ilis,  Miller  (1(>55)  has  ob- 
served that  the  cycle  is  closely  correlated  with  the  various  phases  of  the  life  history 
of  the  animal.  He  reports  the  lowest  thyroid  activity  for  the  fall  and  an  increase 
in  activity  during  the  winter.  This  increase  in  activity  during  the  winter  may  be 
related  to  the  fact  that  the  animals  are  active  and  feeding  during  the  coldest  months 
of  the  year.  The  influence  of  the  seasonal  factor  has  also  been  observed  in  the  cyclic 
activity  of  the  thyroid  in  turtles.  Evans  and  Hegre  (1940),  working  with  Chry- 
semys,  obtained  results  which  resembled  those  of  typical  hibernating  forms  even 
when  the  animals  were  fed  at  regular  intervals  and  kept  at  room  temperature  (70° 
F.)  throughout  the  fall  and  winter.  This  they  claim  would  indicate  that  the  thyroid 
gland  of  the  turtle  was  under  the  control  of  a  genetic  factor.  This  factor  exercises 
its  influence  independently  of  the  temperature  of  the  environment. 

During  the  course  of  this  study  the  influence  of  a  seasonal  factor  has  been  ob- 
served which  is  in  accordance  with  the  results  obtained  bv  l/hlenhuth,  Schenthal, 
Thompson  and  Zwilling  (1945);  Uhlenhuth,  Schenthal,  Thompson,  Mech  and 
Algire  (1945);  Evans  and  Hegre  (1940)  and  Greenberg  (1948).  The  experi- 
mental animals  had  been  treated  with  a  0.1%  solution  of  thiourea,  a  concentration 
claimed  to  be  most  effective  by  Paynter  (1953).  It  would  seem,  however,  that 
since  turtles  and  reptiles  in  general  appear  to  be  more  refractory  to  thiourea,  a 
higher  dose  would  produce  more  marked  results.  Despite  the  weak  responses  and 
the  great  range  of  individual  variation,  the  results  were  more  significant  when  in- 
terpreted in  terms  of  seasonal  activity. 

The  uptake  of  radioiodine  by  the  thyroid  gland  was  alwavs  considerably  higher 
tor  the  summer  groups,  treated  and  untreated,  whether  it  was  considered  from  the 


THIOUREA,  I131  AND  TURTLE  THYROID  201 

point  of  view  of  uptake  per  milligram  dry  weight ;  uptake  per  unit  epithelium,  unit 
colloid  or  thyroid/serum  ratio.  The  uptake  for  the  fall  and  winter  series  was  much 
lower  than  that  for  the  summer  series.  This  is  in  agreement  with  Eggert  (1935) 
who  reports  highest  thyroid  activity  in  June,  in  the  case  of  hibernating  lizards.  The 
November  series  then  would  be  nearing  the  most  reduced  thyroid  activity  which  in 
lizards  occurs  during  December  and  January.  The  winter  group,  killed  at  the  end 
of  February  and  the  beginning  of  March,  would  then  be  expected  to  show  resump- 
tion of  secretory  activity.  The  thyroid  gland  of  the  untreated  control  animals  was 
observed  to  have  a  lower  uptake  of  radioiodine  per  unit ;  however,  the  results  were 
shown  to  follow  the  same  general  pattern  as  the  treated  animals.  These  results 
gave  further  evidence  in  favor  of  the  seasonal  cyclic  activity. 

The  best  correlations  were  found  to  occur  in  the  fall  series.  The  epithelium  per- 
centages of  experimental  and  control  animals  were  more  closely  related  than  in  the 
other  series;  the  range  of  variation  in  per  cent  epithelium  was  lower  in  the  experi- 
mental animals  ;  the  correlation  between  epithelium  and  radioiodine  uptake  was  defi- 
nitely improved.  The  seasonal  influence,  however,  was  best  demonstrated  by  the 
better  degree  of  correlation  between  the  colloid  level  and  the  radioiodine  uptake. 
It  was  observed  that  the  colloid  level  was  lower  in  untreated  controls  than  in  treated 
thyroids.  This  may  be  due  to  the  avidity  of  the  treated  thyroids  for  the  iodine, 
since  the  injections  of  I131  were  given  24  hours  after  the  last  treatment  with  thiourea. 
Considering  the  fact  that  the  colloid  is  known  to  decrease  during  the  active  phase  of 
the  thyroid  gland,  it  is  not  surprising  that  a  lower  colloid  level  was  observed  in  the 
thyroids  of  controls  for  the  February-March  series  than  in  the  November  series, 
which  was  during  the  phase  of  decreasing  activity  and  colloid  storage. 

Since  the  turtles  were  kept  in  a  room  where  a  fairly  constant  temperature  was 
maintained  throughout  the  experiments,  it  would  seem  that  the  seasonal  factor  which 
regulates  the  cyclic  activity  of  the  thyroid  in  turtles  is  independent  of  the  environ- 
mental temperature  and  probably  is  genetic  in  nature. 

SUMMARY 

1.  The  painted  turtle,  Clir\scin\'s  picta    (Schneider),   was  treated   with  a  0.1 
per  cent  solution  of  thiourea  by  means  of  subcutaneous  injections,  then  injected 
with  radioiodine  in  order  to  determine  the  correlation  of  percentage  epithelium  and 
colloid  level  with  radioiodine  uptake.     The  data  obtained  in  this  study  were  analyzed 
statistically  in  terms  of  length  of  treatment  and  the  time  of  the  year  during  which 
the  work  was  carried  out. 

2.  From  the  point  of  view  of  length  of  treatment,  the  correlations  between  the 
uptake  of  I131  and  colloid  level  or  per  cent  epithelium  were  very  poor,  due  to  the 
high  degree  of  variability. 

3.  When  these  results  were  analyzed  from  the  point  of  view  of  seasonal  cyclic 
activity,  the  correlations  were  decidedly  improved. 

4.  The  best  correlations  were  obtained  in  the  fall  group,  where  the  percentage 
epithelium  of  experimental  and  control  animals  was  more  closely  related;  the  range 
of  variation  was  decidedly  lower,  and  the  correlation  between  colloid  level  and  up- 
take of  radioiodine  much  better  than  in  the  other  series. 

5.  Further  evidence  in  favor  of  the  seasonal  cyclic  activity  was  found  in  the 
uptake  per  milligram  dry  weight,  and  in  the  thyroid/serum  ratio. 


202  SISTER  M.  CLAIRE  OF  THE  SAVIOR  BILEAU 

6.  This  seasonal  factor  appears  to  be  genetic  in  nature  since  it  exerts  its  con- 
trol independently  of  the  environmental  temperature  and  the  effects  of  the  drug. 

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Psendeinys  clc<nins.     Master's  Dissertation,   Xew  York  University. 
HAMILTON,   T.  G.,  AND  M.  H.  SOLEV,  1940.     A  comparison  of  the  metabolism  of  iodine  and  of 

element  85  (eka-iodine).     Proc.  Nat.  Acad.  Sci.,  26:  483-489. 
HERTZ,  S.,  A.  ROBERTS,  J.  H.  MEANS  AND  R.  D.  EVANS,  1940.     Radioactive  iodine  as  an  indicator 

in  thyroid  physiology.     Iodine  collection  by  normal  and  hyperplastic  thyroids  in  rab- 
bits.    Amer.  J.  I'liysio/..  128:  565-576. 
HERTZ,  S.,  AND  A.   KOUKKTS,   1941.     Radioactive  iodine  as  an   indicator  in  thyroid  physiology. 

Endocrin..  29:  82-88. 


THIOUREA,  I131  AND  TURTLE  THYROID  203 

LYNX,  \V.  G.,  AND  H.  E.  WACHOWSKI,  1951.  Tlie  thyroid  gland  and  its  functions  in  cold- 
blooded vertebrates.  Quart.  Rev.  of  Biol.  26:  123-168. 

MACKENZIE,  C.  G.,  AND  J.  B.  MACKENZIE,  1943.  Effect  of  sulfonamides  and  tliionreas  on  the 
thyroid  gland  and  basal  metabolism.  Endocrin.,  32:  185-209. 

McGiNTY,  D.  A.,  AND  W.  G.  BYWATER,  1945.  Antithyroid  studies.  I.  The  goitrogenic  activity 
of  some  thioureas,  pyrimidines  and  miscellaneous  compounds.  ./.  Plmnn.  E.\-f>.  Tlierat1.. 
84:  342-357. 

MILLER,  MALCOLM  R.,  1955.  Cyclic  changes  in  the  thyroid  and  interrenal  glands  of  the  vivi- 
parous lizard  Xantitsia  vi<./ilis.  .-liuit.  A'<v.,  123:  19-31. 

MILLER,  MALCOLM  R.,  AND  MARILYN  E.  ROBBIXS,  1955.  Cyclic  changes  in  the  thyroid  and  in- 
terrenal glands  of  the  urodele  amphibian  Tariclut  torosa  (Trititn/s  torosus).  .hint. 
Rcc.,  122:  79-103. 

MORGAN,  A.  H.,  AND  C.  H.  FALES,  1942.  Seasonal  conditions  and  effect  of  low  temperature  in 
the  thyroid  glands  of  amphibians.  /.  Morph.,  71  :  357-384. 

NACCARATI,  SANTI,  1922.  Contribution  to  the  morphologic  study  of  the  thyreoid  gland  in 
Emys  ciirofcicn.  J.  Mar  ph.,  36:  279-297. 

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histology  of  the  turtle  thyroid.  Master's  Dissertation,  The  Catholic  University  of 
America,  \Yashington,  D.  C. 

PAYNTER,  O.  E.,  1953.  Thyroidal  response  to  goitrogens  by  the  painted  turtle,  as  indicated  by 
the  uptake  of  radioactive  iodine.  Unpublished  Doctor's  Dissertation,  The  Catholic  Uni- 
versity of  America,  \Yashington,  D.  C. 

RATZERSDORFER,  C.,  A.  S.,  GORDON  AND  H.  A.  CHARIPPER,  1949.  The  effects  of  thiourea  on  the 
thyroid  gland  and  molting  behavior  of  the  lizard,  .-I nulls  Ciirolincnsis.  J.  F..\-p.  Zool.. 
112:  13-27. 

SOMOGYI,  M.,  1945.     Determination  of  blood  sugar.     /.  Biol.  Clicin.,  160:  69-71. 

TALA,  PEKKA,  1952.  Histoquantitative  studies  on  the  effect  of  thyrotropin  and  thyroxin  on  the 
morphology  of  the  thyroid  gland,  with  special  reference  to  standardization  of  the  thyro- 
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1-100. 

TAUROG,  A.,  I.  L.  CHAIKOFF  AND  A.  L.  FRANKLIN,  1945.  The  structural  specificity  of  sulfanila- 
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VANDERLAAX,  W.  P.,  AND  A.  BISSELL,  1946.  The  influence  of  selected  goitrogenic  compounds 
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SOME  FACTORS  CONTROLLING  THE  INGESTION  OF  CARBO- 
HYDRATES BY  THE  BLOWFLY  ' 

V.  G.  DETHIER,  D.  R.  EVANS  AND  M.  V.  RHOADES 

Department  of  Bioloi/y  and  .Implied  Physics   Laboratory,   The  Joints   Hopkins   University, 

Baltimore  18,  Mil. 

Diet  selection  and  preference  are  commonly  evaluated  in  terms  of  quantity  of 
food  consumed;  however,  measurements  of  intake  alone  give  little  information  con- 
cerning the  degree  to  which  different  factors  participate  in  the  regulation  of  in- 
gestion.  It  is  clear  in  the  case  of  insects  that  a  sequential  contribution  by  various 
stimuli  governs  the  finding  of  food,  the  initiation  of  biting  or  sampling,  the  continu- 
ance of  feeding,  and  the  termination  of  feeding.  It  is  believed  by  some  (e.g., 
Dethier,  1953;  Fraenkel,  1953)  that  stimuli  which  initiate  sampling  and  which  drive 
continued  feeding  are  neither  necessarily  nor  invariably  correlated  with  nutritional 
values.  Other  workers,  notably  Kennedy  (1()53),  believe  that  there  is  an  im- 
portant causal  relationship  between  stimulating  and  nutritional  characteristics. 
The  present  study  is  intended  as  a  step  toward  the  ultimate  clarification  of  this 
problem. 

Carbohydrates  were  chosen  as  test  compounds  because  they  do  not  stimulate 
the  olfactory  sense  and  because  they  represent  all  possible  combinations  of  stimulat- 
ing effectiveness  and  nutritional  value.  There  are  sugars  which  are  stimulating 
but  non-nutritional,  stimulating  and  nutritional,  non-stimulating  but  nutritional,  and 
non-stimulating  and  non-nutritional.  Sugars  representing  these  categories  were 
employed  in  the  following  experiments:  (1)  preference-aversion  tests  in  which 
were  recorded  the  volumes  imbibed  by  tlies  given  a  choice  between  sugar  and  water 
or  between  one  sugar  and  another;  (2)  individual  feeding  tests  in  which  volume 
intake  was  measured  in  the  absence  of  a  choice  situation  ;  (  3  )  tests  of  the  sensitivity 
of  the  different  chemoreceptor  systems  to  stimulation;  (4)  measurements  of  the 
volume  intake  of  mixed  solutions;  (5)  longevity  tests  to  ascertain  the  nutritional 
value  of  the  various  sugars  at  different  concentration  levels. 

MATERIALS  AND  METHODS 

Preference-aversion  tests  were  conducted  according  to  the  procedure  of 
Dethier  and  Rhoades  (  1954).  In  essence,  the  tests  consisted  of  presenting  groups 
of  twenty  flies,  which  had  been  enclosed  in  one-quart  mason  jars,  with  the  choice  of 
drinking  from  either  or  both  of  two  J -shaped  volumetric  pipettes.  The  mean  per 
capita  fluid  intake  per  twenty-four  hours  was  calculated  from  the  total  volume  of 
fluid  taken  from  each  pipette  over  a  four-day  period.  In  two-choice  situations  of 
this  sort  the  intake  of  sugar  can  be  compared  with  that  of  water  or  of  any  other 
sugar  or  sugar  mixture. 

1  This  investigation  was  supported  by  a  grant  from  the  National  Science  Foundation. 

204 


1NGESTION  OF  CARBOHYDRATES  205 

In  order  to  ascertain  the  number  of  visits  which  were  made  to  each  pipette  and 
the  duration  of  each  visit,  the  original  apparatus  was  modified  as  follows.  A  silver 
wire  was  inserted  into  each  pipette  in  such  a  way  as  to  extend  the  entire  distance 
from  the  large  opening  to  a  point  just  one  millimeter  short  of  the  capillary  orifice. 
Silver-conducting  paint  ( DuPont  Silver  4916)  was  then  brushed  in  a  thin  line  from 
a  point  near  the  large  orifice  to  a  point  one  millimeter  short  of  the  capillary  orifice ; 
here  the  painted  line  was  extended  around  the  circumference  of  the  pipette  so  that 
a  fly  had  to  stand  on  the  paint  in  order  to  drink.  To  the  painted  line  near  the  large 
opening  was  soldered  a  silver  wire.  This  wire  and  the  wire  from  inside  the  pipette 
were  each  extended  to  the  terminals  of  a  Brush  BL907  amplifier  which  in  turn  was 
connected  to  a  BL202  recording  instrument.  Since  the  entire  apparatus  was  in- 
tentionally unshielded,  the  two  wires  acted  as  antennae  which  picked  up  60  cycle 
current  from  lights  and  various  motors  operating  in  the  laboratory.  Whenever  a 
tlv  attempted  to  drink  from  a  pipette,  it  closed  the  circuit  between  the  conducting 


Bini  i i ii 

• 


I 


FIGTKK  1.  Typical  example  of  automatically  recorded  periods  of  feeding.  The  thin  line 
represents  periods  of  feeding.  Note  that  the  fly  has  taken  one  long  drink  beginning  at  the  upper 
right  and  continuing  at  the  lower  left.  During  the  remainder  of  the  time  only  brief  samples 
were  taken.  Each  curved  line  represents  5  seconds. 

paint  on  which  it  was  standing  and  the  fluid  and  wire  within  the  pipette.  Since 
the  60  cycle  current  was  then  shorted  out,  the  period  of  drinking  appeared  on  the 
record  as  a  straight  line  instead  of  alternating  impulses  (  Fig.  1 ) .  The  authenticity 
of  records  obtained  by  this  method  was  confirmed  by  visual  observation.  At  the 
same  time  the  identity  of  the  drinker  was  noted. 

Finally,  the  fluid  intake  of  individual  flies  was  measured  by  direct  analyses  of 
sugar.  For  these  measurements  the  flies  were  fed  24  ±  2  hours  before  testing  on 
0.1  M  sucrose  and  then  received  neither  food  nor  water  until  the  experimental  in- 
gestion.  At  this  time  the  flies  \vere  mounted  on  waxed  sticks  and  individually  fed 
on  the  test  solutions.  Some  arbitrary  criterion  of  repletion  was  necessary  since  a 
fly  will  continue  alternately  to  extend  and  retract  its  proboscis  almost  indefinitely 
on  some  sugars,  all  the  while  taking  small  additional  amounts.  Repletion,  there- 
fore, was  defined  by  the  period  of  vigorous  proboscis  extension  and  active  uptake. 
Usually  a  fly  would  feed  continuously  and  actively  for  an  initial  prolonged  period  and 
then  perhaps  for  an  additional  shorter  period  when  its  labellar  hairs  were  brought 


206 


V.  G.  DETHIER,  D.  R.  EVANS  AND  M.  V.  RHOADES 


into  contact  with  the  solution.  This  period  of  active  feeding  was  usually  rather 
sharply  delineated,  as  indicated  by  the  agreement  of  duplicate  determinations  on 
different  groups  of  flies  treated  similarly.  The  standard  deviation  of  replicate  de- 
terminations of  volume  intake  ranged  between  0.377 /xl  for  1  AI  sucrose  and  1.34/xl 
for  1  A I  fucose. 

The  determination  of  quantity  ingested  was  accomplished  by  a  sensitive  spectro- 
photometric  reaction  for  carbohydrates  employing  anthrone  in  concentrated  sul- 
furic  acid  ( Dimler  ct  <?/.,  1952).  For  each  determination  the  abdomens  of  5-20 
Hies  were  ground,  immediately  after  feeding,  in  10  ml.  of  5c/o  trichloroacetic  acid. 
The  crop  and  intestine,  which  contain  the  ingested  sugar,  are  located  entirely  in  the 
abdomen  after  feeding.  Equally  large  groups  of  flies  similarly  treated,  but  fed 
nothing,  served  as  controls.  After  centrifugation,  aliquots  of  the  supernatant  were 
diluted  appropriately  to  produce  concentrations  from  30  to  200 /xg.  sugar  per  nil. 


^ 

Q 

—I 

0 

0.016 

FUCOSE      o-  0                                                                         A 

~ 

rr 

UJ 

0.014 

MANNOSE  A  A                                                                      '        \ 

~^r 

QL 

/                         i 

o 

SORBOSE*  •                                                           /                N    ^''      "A 

1 

of 

0012 

t+*                     N 

r^ 
Q. 

X 

/                                  X*                .                                                 \ 

0.010 

^r                                      /                                                                                          W 

r> 

CM 

^                 X                     ^ 

CO 

0.008 

/                   •  '                           ^         -• 

2 
0 

CL. 
UJ 

Q. 

0.006 

.  .,                                  /             x;^                                 \       i 

./       ,»-^                              v     i 

UJ 

_l 

0.004 

^•ii"  '  ;?V-~-A'                         •, 

o 

U. 

-•>--..'                                          i 

rr 

rr 

0.002 

—  •Cr*'                                                                                                       • 

UJ 

UJ 

^O  "^" 

> 

Q. 

0.000 

o* 

——  L—  —                                                             1                  1                    i                   III 

IxlO' 


I    2  346 


IxlO'4  IxlO'2 

MOLAR    CONCN. 

FIGURE  2.     Preference-aversion  curves  for  fucose,  sorbose,  and  mannose. 

One-nil,  samples  of  these  final  dilutions  were  employed  for  the  anthrone  reaction  and 
were  compared  spectrophotometrically  with  standards  of  the  several  sugars  fed. 
The  amount  of  sugar  in  the  fed  groups  in  excess  of  that  in  the  unfed  controls  was 
directly  convertible  to  volume  since  the  concentrations  of  the  solutions  employed 
for  feeding  were  known  precisely.  Longevity  was  measured  with  the  same  experi- 
mental set-up  employed  for  preference-aversion  testing. 

PREFERENCE- AVERSION  CURVES 

The  volume  intake,  as  compared  with  water,  was  measured  for  each  of  the  fol- 
lowing sugars  over  the  concentration  range  1  '.  '.  10  7  M  to  saturation:  fucose,  sor- 
bose, mannose.  The  results  are  summarized  in  Table  I  and  Figure  2. 

Fucose  is  a  methyl  pentose  which  is  rather  effectively  stimulating  for  the  tarsal 
chemoreceptors  (median  acceptance  threshold  ==  0.087  Al]  but  not  utilized  by  the 


INGESTION  OF  CARBOHYDRATES 


207 


TABLE  I 

Amount  of  solution  consumed  (ml. /fly/24  hrs.)  when  sugar  is  paired  with  water 


Molar  concen. 

Fucose 

Water 

Level  (%)  of  significance 
of  difference 

1.0 

0.0042 

0.0035 

— 

0.1 

0.0161 

0.0012 

0.1 

0.01 

00102 

0.0031 

0.1 

0.001 

0.0081 

0.0041 

0.1 

0.0001 

0.0037 

0.0022 

1.0 

0.00001 

0.0017 

0.0033 

— 

0.000001 

0.0008 

0.0007 

— 

0.0000001 

0.0000 

0.0019* 

0.1 

sorbose 

3.0 

0.0020 

0.0021 

2.0 

0.0075 

0.0036 

0.1 

1.0 

0.0074 

0.0027 

1.0 

0.1 

0.0072 

0.0036 

0.1 

0.01 

0.0076 

0.0050 

0.1 

0.001 

0.0045 

0.0030 

1.0 

0.0001 

0.0030 

0.0029 

— 

0.00001 

0.0031 

0.0012 

— 

0.000001 

0.0058 

0.0043 

— 

0.0000001 

0.0059 

0.0074* 

5 

man  nose 

6.0 

0.0097 

0.0024 

5 

4.0 

0.0106 

0.0016 

5 

2.0 

0.0124 

0.0011 

1.0 

1.0 

0.0132 

0.0023 

0.1 

0.1 

0.0111 

0.0022 

0.1 

0.01 

0.0074 

0.0032 

0.1 

0.001 

0.0036 

0.0046* 

5 

0.0001 

0.0043 

0.0054* 

5 

rhamnose 

1.0 

0.0068 

0.0004 

1.0 

0.1 

0.0067 

0.0005 

1.0 

lactose 

1.0 

0.0212 

0.0159 

1.0 

0.1 

0.0091 

0.0012 

1.0 

D-arabinose 

0.1 

0.0122 

0.0004 

0.1 

L-arabinose 

0.1 

0.0035 

0.0036 

— 

*  These  values  represent  the  concentration  range  where  water  is  taken  in  significantly  greater 
amounts  than  sugar. 


208  V.  G.  DETHIER,  D.  R.  EVANS  AND  M.  V.  RHOADES 

blowfly  Phormia  rcgina  (Hassett,  Dethier  and  Cans,  1950).  Sorbose,  a  hexose, 
also  stimulates  the  tarsal  chemoreceptors  (threshold  =  0.14  M)  although  it  is  not 
utilized.  Mannose,  a  hexose,  is  extremely  poor  in  stimulating  power  (tarsal 
threshold  =  7.59  M)  but  is  nutritionally  highly  effective. 

The  curves  describing  the  ingestion  of  the  three  sugars  are  substantially  similar 
to  those  obtained  by  Dethier  and  Rhoades  (1954)  with  the  nutritionally  adequate 
sugars  glucose  and  sucrose.  In  each  case  there  is  a  low  concentration  at  which  the 
sugar  is  not  distinguished  from  water  so  that  equal  amounts  of  solution  are  taken 
from  each  pipette.  Then,  as  the  concentration  is  increased,  a  point  is  reached  where 
more  sugar  than  water  is  imbibed.  This  point  represents  a  difference  threshold. 
It  occurs  at  a  lower  concentration  than  the  tarsal  acceptance  threshold  obtained  by 
standard  procedures.  As  the  concentration  is  further  increased  there  is  an  increase 
in  the  volume  of  solution  imbibed  until  a  maximum  intake  is  reached,  after  which 
there  is  a  marked  decrease.  A  cursory  examination  of  the  curves  reveals  no  rela- 
tion between  the  volume  intake  and  either  the  nutritional  value  or  the  relative 
stimulating  effectiveness.  Of  the  three  sugars,  the  maximum  intake  is  greatest  for 
fucose  and  least  for  sorbose.  None  is  consumed  in  as  great  quantities  as  glucose 
or  sucrose. 

Another  characteristic  of  these  curves  is  an  inversion  at  very  low  concentrations 
where  water  may  be  taken  in  preference  to  sugar.  With  fucose.  sorbose,  and  man- 
nose  the  inversion  occurs  at  1  X  10"7  M,  1  X  10'7  M,  and  1  X  W'3  --  1  X  10~4  M, 
respectively.  Bimodal  preference-aversion  relationships  of  sugars  were  first  noted 
by  Beck  (1956)  in  studies  of  the  larvae  of  the  European  corn  borer  (Pyransta  nubi- 
lalis  Hbn.).  A  re-examination  of  the  raw  data  of  Dethier  and  Rhoades  (1954)  re- 
veals similar  relationships.  The  meaning  of  rejection  at  low  concentrations  is  not 
at  all  clear. 

INDIVIDUAL  INTAKE 

When  measurements  were  made  of  the  volume  of  different  concentrations  of  su- 
gars imbibed  by  a  single  fly  at  one  feeding  (Table  II)  and  the  values  plotted  as  a 
function  of  the  concentration,  the  resulting  curves  differed  in  several  important  re- 
spects from  the  customary  preference-aversion  curves  ( Fig.  3 ) .  With  the  exception 
of  fucose  and  sucrose  there  was  no  evident  tendency  for  intake  to  decrease  at  high 
concentrations.  There  was,  however,  a  marked  tendency  for  intake  to  reach  a 
plateau.  On  the  other  hand,  regardless  of  the  procedure  employed  for  measuring 
intake,  the  weight  of  sugar  consumed  increased  throughout  the  entire  concentration 
range.  There  is  no  indication  that  the  flies  regulate  the  quantitative  intake  of 


sugar. 


In  comparing  individual  feeding  curves  with  preference-aversion  curves  based 
upon  four  days  of  feeding  the  further  difference  is  noted  that  the  volume  intake, 
while  approximately  the  same  in  both  experiments  at  high  concentrations,  at  low 
concentrations  is  much  smaller  when  measured  individually  than  when  measured  in 
a  two-choice  situation.  The  fact  that  one  experiment  involves  a  two-choice  situa- 
tion while  the  other  involves  no  choice  has  no  bearing  on  the  results  because  Dethier 
and  Rhoades  (1954)  have  shown  that  intake  is  the  same  in  one-choice  and  two- 
choice  situations.  It  seems  possible  to  explain  the  difference  on  the  basis  of  gusta- 
tory thresholds  and  behavior  as  affected  by  feeding.  Earlier  work  (rf.  Dethier  and 
Chadwick,  1948)  indicated  that  feeding  elevates  taste  thresholds,  and  it  seems 


INGESTION  OF  CARBOHYDRATES 


209 


reasonable  to  assume  that  the  greater  the  ingestion  of  sugar  the  longer  the  taste 
threshold  remains  elevated  (this  assumption  is  borne  out  by  experiments,  soon  to  be 
published,  on  the  determinants  of  taste  threshold  in  Phormia}.  Furthermore,  pres- 
ent data  show  that  in  general  the  volume  ingested  at  a  single  feeding  is  a  direct 
function  of  the  stimulating  effectiveness  of  the  test  solution.  Hence,  it  might  be  ex- 
pected that  in  preference-aversion  experiments,  after  once  feeding  on  1.0  or  2.0  M 

TABLE  II 

Amounts  of  various  sugars  ingested  at  a  single  feeding 


Sugar 

Molar 
concen- 
tration 

Number  of 
animals 

Mg./fly 

Ml./fly 
X  103 

Duration* 
(sec.) 

Rate  ml./ 
sec.  X  105 

Approximate 
viscosity 
(centipoises) 

Sucrose 

2.0 

20 

8.96 

13.0 

90 

14 

— 

1.0 

30 

4.78 

13.9 

47 

30 

—  • 

0.5 

10 

1.80 

10.5 

43 

24 

— 

0.25 

10 

0.440 

7.05 

36 

20 

— 

Glucose 

2.0 

15 

4.92 

13.7 

61 

26 

— 

1.0 

35 

2.27 

12.6 

44 

30 

—  • 

0.5 

15 

0.820 

9.11 

38 

25 

— 

Mannose 

4.0 

15 

6.49 

9.02 

51 

18 

— 

2.0 

15 

2.97 

8.25 

40 

21 

— 

1.0 

10 

1.12 

6.20 

38 

16 

—  - 

0.5 

10 

0.268 

2.98 

25 

12 

— 

Fucose 

1.0 

50 

0.843 

5.14 

30 

20 

— 

0.5 

15 

0.580 

7.08 

32 

35 

— 

Lactose 

1.0 

15 

0.903 

2.82 

18 

18 

— 

Sorbose 

3.0 

10 

1.93 

3.58 





— 

2.0 

20 

1.09 

3.04 

—  - 

— 

— 

1.0 

10 

0.168 

0.934 

— 

— 

— 

0.5 

6 

0.0481 

0.534 

— 

—  • 

— 

Sucrose 

1.0 

10 

4.62 

13.5 

54 

25 

2.75 

Sucrose 

l.CWin: 

Glycerol 

2.2 
5.4 

10 
10 

5.20 
2.86 

15.2 
8.34 

60 
50 

25 
17 

4.29 
7.79 

8.7 

4 

2.20 

6.45 

55 

12 

48.5 

*  Duration  times  were  recorded  for  fewer  flies  than  were  employed  in  ingestion  determinations. 

sugar,  the  fly  would  not  respond  to  the  solution  again  for  some  time  when  it  is  en- 
countered ;  and,  furthermore,  that  when  again  ingested  the  solution  will  be  taken  in 
far  lesser  quantities  as  a  result  of  the  partially  elevated  threshold.  Moreover,  the 
number  of  encounters  with  the  sugar  solution  is  markedly  reduced  with  flies  feeding 
on  1.0  or  2.0  M  sugar,  since  they  are  almost  completely  inactive  for  some  time  after 
ingestion  of  a  large  sugar  meal.  When  0.1  or  0.01  M  sugar  solutions  are  employed 
for  preference-aversion  tests,  the  post-ingestion  duration  of  threshold  elevation,  the 


210  V.  G.  DETHIER,  D.  R.  EVANS  AND  M.  V.  RHOADES 

period  of  quiescence,  and  the  interval  during  which  response  fails  upon  contact  with 
the  solution  are  all  shortened  relative  to  the  higher  concentrations.  The  frequency 
of  feeding  is  thereby  increased.  Thus  may  be  explained  the  discrepancy  of  a  higher 
daily  intake  of  0.1  M  than  1.0  M  sucrose,  although  at  a  single  feeding  much  more 
is  taken  of  the  higher  concentration. 

The  action  of  the  above  factors  is  again  seen  when  the  raw  data  of  the  preference- 
aversion  curves  of  Dethier  and  Rhoades  (1954)  are  analyzed  on  a  day-to-day  basis. 
It  was  found  that  curves  based  solely  on  the  first  24-hour  intake  were  displaced  to 
the  right,  that  is,  the  maximum  intake  occurred  at  very  high  concentrations.  For 
subsequent  24-hour  periods  the  intake  of  high  concentrations  drops  while  that  of 
low  concentrations  gradually  increases  (see  Dethier  and  Rhoades,  Fig.  2). 

The  expectation  of  more  frequent  feeding  on  0.1  M  than  1.0  M  sucrose  was  con- 
firmed by  automatic  recordings  of  preference-aversion  behavior.  During  the  first 
eighteen  hours  of  recording,  791  drinks  were  taken  from  0.1  M  sucrose  and  only 
236  from  1.0  M.  During  the  same  period  there  \vere  in  addition  1,336  tentative 
drinks  or  taste  samples  of  0.1  M  as  compared  with  898  of  1.0  M.  The  duration  of 
drinking  was  approximately  the  same  with  each  concentration ;  however,  the  vol- 
ume imbibed  per  drink  of  1.0  M  was  slightly  more  than  twice  that  of  0.1  M.  The 
rate  of  intake  was,  therefore,  greater  in  the  case  of  1.0  M.  It  was  also  noteworthy 
that  over  the  entire  18-hour  period  there  was  no  marked  decrease  in  the  number  of 
drinks  of  0.1  M  per  hour,  but  the  number  of  drinks  of  1.0  M  per  hour  had  decreased 
by  80%  at  the  end  of  12  hours.  The  number  had  reached  0  at  the  end  of  17  hours. 

SUGARS  PAIRED  WITH  EACH  OTHER 

In  all  of  the  foregoing  choice  experiments  the  test  sugar  was  paired  with  water. 
In  the  following  experiments  sugars  were  paired  with  other  sugars  at  many  differ- 
ent concentrations.  The  results  are  summarized  in  Table  III.  From  a  perusal  of 
these  data  it  may  be  seen  that  the  results  are  in  general  agreement  with  what  might 
have  been  expected  from  an  examination  of  Figure  2.  For  example,  it  might  have 
been  predicted  from  Figure  2  that  more  of  1.0  M  mannose  than  of  1.0  M  fucose 
would  be  ingested  because  the  curve  for  fucose  is  displaced  to  the  left  relative  to  the 
mannose  curve.  The  prediction  was  verified  when  the  two  solutions  were  actually 
paired  (Table  III).  Similarly,  the  relative  volumes  imbibed  in  other  two-choice 
tests  are  in  general  agreement  with  the  basic  preference-aversion  curves.  On  the 
other  hand,  the  absolute  volumes  are  not  the  same  in  the  two  types  of  experiments. 
Such  a  discrepancy  is  to  be  expected,  because  volume  intake  is  dependent  not  only 
on  the  concentration  of  the  test  solution  but  on  the  concentration  and  identity  of  all 
other  compounds  to  which  the  insect  is  simultaneously  exposed.  The  total  situation 
is  the  determinant.  For  example,  it  had  previously  been  found  by  Dethier  and 
Rhoades  that  the  less  preferred  of  two  sugars  in  a  paired  test  was  treated  as  though 
it  were  water  regardless  of  how  much  of  it  might  have  been  ingested  when  it  was 
presented  alone.  In  every  case  here,  with  the  exceptions  of  1  M  mannose  paired 
with  1  M  sorbose  and  0.5  M  mannose  paired  with  0.5  M  sorbose,  the  same  is  true. 
The  less  preferred  member  of  the  pair  is  ingested  at  approximately  the  same  level  as 
water  (cf.  Tables  I  and  III).  Consequently,  the  sum  of  the  two  volumes  ingested 
in  a  paired  test  is  generally  less  than  the  sum  of  volumes  of  each  sugar  which  would 
have  been  ingested  when  paired  with  water,  unless,  of  course,  the  less  preferred  is 


INGESTION  OF  CARBOHYDRATES 


211 


TABLE  III 

Volumes  (ml. /fly/24  hrs.)  ingested  when  different  sugars  are  paired  (preferred  sugar  underlined) 


No. 


Solutions  paired 


Significance  at 
1%  level 


5 
6 

7 


10 


11 


12 


13 


l.OM  mannose 
0.0077 

l.OM  mannose 
0.0112 


l.OM  mannose 
0.0121 


l.OM  mannose 
0.0154 

0.5M  mannose 
0.0113 


0.1M  mannose 
0.0017 

0.1M  mannose 
0.0065 

0.0 1M  mannose 
0.0074 


vs.     l.OM  fucose 
0.0007 

vs.     l.OM  sorbose 
0.0054 

vs.     0.1M  fucose 
0.0045 

vs.     0. 1 M  sorbose 
0.0014 

vs.     0.5M  sorbose 
0.008 

vs.     0.1M  fucose 
0.0138 

vs.     0. 1 M  sorbose 
0.0084 

vs.     0.0001  M  fucose 
0.0046 


0.001  M  mannose      vs.     0.0001  M  fucose 
0.0048  0.0059 


0.1M  fucose 
0.0129 


l.OM  fucose 
0.0000 

0.01  M  fucose 
0.00294 


vs.     0.1M  sorbose 
0.0026 

vs.     l.OM  sorbose 
0.0034 

vs.     0.01  M  sorbose 
0.00140 


0. 1M  D-arabinose     vs.     0.1M  L-arabinose 
0.0143  0.0043 


being  tested  at  a  concentration  at  which  it  is  not  normally  consumed  more  readily 
than  water.  In  this  last  case  the  total  consumption  in  the  paired  test  would  equal 
the  sum  of  the  two  sugars  tested  individually. 

In  previous  pairing  of  sucrose  with  glucose  and  sucrose  with  sucrose  the  volume 
intake  of  the  preferred  member  was  greater  than  in  sugar-water  pairs  when  the  con- 
centration in  question  fell  at  the  peak  of  the  preference-aversion  curve,  less  if  it  fell 
on  the  ascending  limb  (i.e.,  low  concentrations)  of  the  curve,  and  equal  if  on  the 


212  V.  G.  DETHIER,  D.  R.  EVANS  AND  M.  V.  RHOADES 

descending  limb.  In  the  tests  reported  here  the  volume  intake  of  the  preferred 
sugar  in  a  pair  generally  equalled  its  intake  when  paired  with  water  when  the  con- 
centration in  question  fell  at  the  peak  of  the  preference-aversion  curve. 

Both  sets  of  data  (Tables  I  and  III)  suggest  very  strongly  that  volume  intake 
is  under  sensory  control,  that  is,  that  the  stimulating  effectiveness  of  a  solution  de- 
termines how  much  of  it  will  be  imbibed.  Several  aspects  of  the  two-choice  data 
underline  the  importance  of  the  sensory  rather  than  the  nutritional  characteristic  of 
the  sugar  in  regulating  volume  intake.  Line  6  of  Table  III  indicates  a  preference 
for  0.1  M  fucose  (non-nutritional)  over  0.1  M  mannose  (nutritional).  This  result 
clearly  indicates  the  choice  of  a  stimulating  sugar  over  a  poorly  stimulating  one. 
The  choice  of  0.1  M  fucose  over  0.1  M  sorbose  (line  10),  both  sugars  being  non- 
nutritional,  reflects  the  superior  stimulating  effectiveness  of  fucose  at  this  level  of 
concentration.  The  relative  intake  of  two  sugars  at  concentrations  represented  on 
the  ascending  limbs  of  the  preference-aversion  curves  appears  to  be  sense-controlled, 
the  more  stimulating  sugar  always  being  preferred  (lines  4,  5,  6,  8,  10,  12).  This 
conclusion  is  in  agreement  with  the  findings  of  Dethier  and  Rhoades  (1954)  rela- 
tive to  the  intake  of  glucose  and  sucrose. 

When  comparisons  are  made  which  involve  concentrations  on  the  descending 
limbs  of  the  preference-aversion  curves,  stimulating  effectiveness  alone  is  apparently 
no  longer  the  sole  controlling  factor ;  hence,  comparisons  at  these  levels  are  more 
complex  (lines  1,  11).  For  example,  the  preference  for  1.0  M  mannose  over  1.0  M 
fucose  (line  1)  does  not  result  simply  from  the  superior  stimulating  effectiveness  of 
mannose,  for  indeed  fucose  is  the  more  stimulating;  instead,  the  preference  undoubt- 
edly reflects  a  negative  factor  causing  the  decline  in  fucose  intake  (rf.  Fig.  3)  as 
being  responsible  for  the  preference  of  mannose  in  the  two-choice  situation. 

ROLE  OF  SENSORY  SYSTEMS 

The  foregoing  results  clearly  implicate  the  sensory  systems.  There  are  three 
chemosensory  systems  (exclusive  of  olfaction)  definitely  known  to  be  involved  in 
the  feeding  behavior  of  Phormia;  namely,  the  tarsal  chemoreceptors,  the  labellar 
hairs,  and  the  interpseudotracheal  papillae  (Dethier,  1955).  The  first  two  men- 
tioned have  been  studied  to  a  greater  extent  than  the  papillae,  and  most  of  the  re- 
marks regarding  stimulating  effectiveness  in  the  foregoing  section  have  been  based 
on  information  so  derived.  However,  on  the  basis  of  these  studies  alone  mannose 
should  not  be  imbibed  at  all,  and  certainly  its  preference-aversion  curve  should  not 
fall  between  that  of  fucose  and  sorbose. 

The  difficulty  was  resolved  by  the  discovery  that  mannose,  while  poorly  stimu- 
lating to  tarsi  and  labellum,  was  an  effective  stimulus  for  the  papillae.  Its  effective- 
ness at  this  site  explains  quite  satisfactorily  other  difficulties  encountered  in  the 
foregoing  section.  Mannose  is  obviously  accepted  at  high  concentrations  in  pref- 
erence to  sorbose,  and  in  preference  to  water  because  of  its  stimulating  effect  on  the 
papillae.  Even  though  it  does  not  stimulate  the  tarsal  and  labellar  hairs,  except  at 
very  high  concentrations,  it  gains  access  to  the  papillae  as  a  result  of  the  fly's  ex- 
tending and  probing  with  its  proboscis  in  its  normal  exploratory  behavior  and  in  the 
course  of  ingesting  to  satisfy  its  need  for  water. 

The  discovery  of  the  stimulating  effectiveness  of  mannose  on  the  papillae  led  to 
a  series  of  tests  in  which  other  selected  sugars  were  applied  to  the  three  chemosen- 


INGESTION  OF  CARBOHYDRATES 


213 


cr 

LU 
Q. 


40 


36 


32 


2  28 


2    24 
O 

Q_ 

^ 

CO 

-Z- 

o 

O 

HJ     16 
O 


cr 

LU 


20 


I  2 


8 


I I 


0.01         0.25     0.50       1.0       2.0   3.04.06.0 
MOLAR    CONCN. 

FIGURE  3.  Comparison  of  ingestion  measured  by  single  feeding  and  by  preference-aversion 
intake  during  the  first  twenty-four  hours.  Solid  line,  preference-aversion ;  dotted  line,  single 
feeding.  O  =  sucrose,  •  =  glucose,  •  =  mannose,  A  —  fucose,  A  —  sorbose. 


214 


V.  G.  DETHIER,  D.  R.  EVANS  AND  M.  V.  RHOADES 


sory  systems.  The  results  are  given  in  Table  IV.  The  most  surprising  result  con- 
cerned L-arabinose.  which  was  found  to  act  as  a  repellent  to  the  papillae  even  though 
it  is  acceptable  in  terms  of  its  effect  on  tarsal  and  labellar  hairs.  This  characteristic 
of  L-arabinose  was  most  unexpected.  Clearly  it  stimulates  the  tarsal  and  labellar 
hairs,  as  a  result  of  which  the  fly  is  moved  to  extend  its  proboscis  and  commence 
feeding.  However,  as  soon  as  the  solution  comes  into  contact  with  the  papillae,  in- 
gestion  ceases  abruptly.  D-arabinose,  by  contrast,  is  acceptable  to  all  three  chemo- 
sensory  systems  and  is  consumed  in  appreciable  quantities  even  though  it  is  not 
utilized  (Table  I). 

RELATION  BETWEEN  INTAKE  AND  NUTRITIONAL  VALUE 

From  experiments  in  which  different  sugars  were  paired  there  were  already  in- 
dications that  the  stimulating  rather  than  the  nutritional  characteristic  of  a  sugar 
played  a  major  role  in  regulating  volume  intake  (cf.  line  6  of  Table  III).  The 
minor  importance  of  nutritional  factors,  at  least  under  experimental  conditions,  is 

TABLE  IV 

Effectiveness  of  selected  sugars  -in  stimulating  the  three  chemoreceptive  systems  of  Phormia 


Sugar 

Tarsal  threshold  (molar) 

Labellar  hairs 

Interpseudotracheal  papillae 

fucose 

0.087 

+ 

+ 

sorbose 

0.140 

-j- 

_|- 

mannose 

7.59 

— 

-j- 

D-arabinose 

0.144 

-f- 

4. 

L-arabinose 

0.536 

4- 

R 

D-xylose 
L-xylose 
rhamnose 

0.440 
0.337 

+ 

— 

ribose 

— 

— 

lactose 

— 

— 

— 

(+  stimulating,  —  non-stimulating  in  all  concentrations,  R  rejected) 

revealed  further  by  comparisons  of  the  results  of  preference  tests  with  sugar  mix- 
tures and  the  capacity  of  these  mixtures  for  sustaining  life.  Two  examples  serve 
to  illustrate  the  point,  the  behavior  of  flies  with  respect  to  glucose  and  D-arabinose 
and  with  respect  to  glucose  and  rhamnose. 

Glucose  alone  at  a  concentration  of  0.1  717  supported  life  for  14  days  (50% 
mortality)  ;  D-arabinose,  for  3.5  days;  a  mixture  containing  0.1  M  glucose  and  0.1 
M  D-arabinose,  for  5.5  days.  Survival  on  water  alone  averages  three  days  (cf. 
also  Hassett,  Dethier  and  Cans,  1950).  Yet  in  preference  tests  where  glucose  was 
paired  with  the  non-nutritional  mixture,  flies  consumed  greater  quantities  of  the  mix- 
ture. Similarly,  a  mixture  of  0.1  M  glucose  and  Q.I  M  rhamnose,  which  supported 
life  for  8  days  as  compared  to  3.5  days  for  rhamnose  alone  and  14  days  for  glucose 
alone,  was  consumed  in  greater  quantity  than  glucose  alone  in  a  paired  test  (Table 
VII).  Rhamnose  paired  with  water  was  preferred  slightly  (Table  I). 

From  these  results  it  would  appear  that  choices  were  made  solely  on  the  basis 
of  the  stimulating  effect.  There  is  no  indication  that  either  D-arabinose  or  rham- 


INGESTION  OF  CARBOHYDRATES  215 

nose  is  repellent,  since  each  is  in  fact  preferred  to  water.  While  neither  inter- 
feres with  the  stimulating  effect  of  glucose  on  sense  organs  (Table  V),  both  either 
are  toxic  or  block  glucose  utilization. 

INTAKE  OF  MIXTURES  OF  SUGARS 

Sometimes  the  acceptability  of  compounds  of  very  low  stimulating  power  cannot 
be  demonstrated  in  a  two-choice  test  with  water  or  by  simple  acceptance  threshold 
determinations.  Accordingly,  the  ruse  has  frequently  been  employed  of  mixing 
two  sugars  in  order  to  detect  suspected  additive  or  repellent  properties.  Kunze 
(1927)  and  von  Frisch  (1935),  for  example,  found  that  sugars  which  were  ac- 
ceptable to  the  honeybee  were  strictly  additive.  Unfortunately  the  technique  is  de- 
ceptively simple,  and  the  results  cannot  always  be  relied  upon  to  give  the  desired 
sensory  information  because  of  the  occurrence  of  two  phenomena  which  have  not 
been  given  due  consideration.  These  two  are  synergism  and  inhibition.  They  can 
be  demonstrated  most  easily  and  convincingly  by  measuring  tarsal  acceptance 
thresholds  to  sugars  and  sugar  mixtures.  They  also  occur  at  labellar  hairs.  Tests 
for  inhibition  and  synergism  have  not  been  made  with  interpseudotracheal  papillae, 

TABLE  V 

Examples  of  inhibition  revealed  by  ascertaining  the  effects  of  sugar  mixtures  on  tarsal  thresholds 
Sugar  Effect  Sugar  affected 

mannose  inhibits  fructose 

does  not  affect  glucose,  sucrose,  fucose,  maltose 

sorbose  inhibits  glucose,  fructose 

fucose  does  not  affect  glucose,  fructose 

rhamnose  does  not  affect  fucose,  glucose 

inhibits  fructose 

D-arabinose  does  not  affect  glucose 

mannitol  does  not  affect  fructose 

but  the  occurrence  of  the  phenomena  at  other  sites  indicates  that  an  additive  effect 
of  sugars  cannot  be  assumed  as  a  matter  of  course. 

For  example,  the  median  acceptance  threshold  for  fructose  is  0.0058 ;  for  glucose, 
0.132 ;  for  an  equimolar  mixture  of  the  two,  0.0078.  In  other  words,  the  concentra- 
tion at  which  the  mixture  is  stimulating  represents  0.0039  M  glucose  and  0.0039  M 
fructose.  Even  were  the  two  sugars  simply  additive,  they  would  not  be  expected 
to  stimulate  at  this  level.  The  fact  that  they  do  stimulate  implies  synergism.  Man- 
nose,  on  the  contrary,  when  added  to  fructose  inhibits  it,  that  is,  causes  a  ten-fold 
rise  in  the  fructose  threshold.  This  effect  is  not  due  to  repellence  because,  for 
Phormia,  mannose  is  preferred  to  water  in  all  concentrations  above  threshold. 
Furthermore,  mannose  has  no  effect  on  such  sugars  as  glucose,  sucrose,  or  maltose. 

The  results  of  threshold  tests  with  other  mixtures  are  summarized  in  Table  V. 
That  the  effects  observed  represent  inhibition  rather  than  repellence  is  further  con- 
firmed by  the  action  of  sorbose.  Sorbose  is  stimulating  in  its  own  right,  yet  it 
causes  an  increase  in  the  thresholds  of  glucose  and  fructose  when  mixed  with  them. 
Its  action  is  revealed  clearly  in  the  following  representative  results  (Table  VI) 
where  the  per  cent  response  of  a  sample  of  flies  to  various  concentrations  of  glucose 
and  of  sorbose  is  compared  to  their  response  to  a  series  of  solutions  which  contain 


216 


V.  G.  DETHIER,  D.  R.  EVANS  AND  M.  V.  RHOADES 


0.5  moles  of  sorbose.  In  this  case  the  stimulating  effect  of  the  mixture  at  low  glu- 
cose concentrations  stems  entirely  from  the  sorbose  which  is  present.  At  higher 
concentrations  of  glucose,  when  the  same  amount  of  sorbose  is  present,  there  is 
little  change  in  the  stimulating  effectiveness.  Not  only  do  the  two  sugars  fail  to 
add,  but  the  stimulating  effect  to  be  expected  of  the  high  concentrations  of  glucose  is 
absent.  When,  therefore,  a  smaller  volume  of  a  mixture  of  sugars  is  ingested  than 
of  either  of  the  constituents  alone,  the  result  cannot  always  be  ascribed  to  repellence, 
especially  when  both  constituents  can  be  shown  in  other  tests  to  be  preferred  to 
water. 

Galun  (1955)  has  reported  that  all  of  the  following  sugars  are  repellent  to 
Musca  donicstica:  D-xylose,  L-arabinose,  ribose,  rhamnose,  and  sorbose.  This 
conclusion  is  based,  however,  on  the  fact  that  the  addition  of  any  of  these  to  an 
acceptable  sugar  causes  a  lowering  of  intake.  Unless  the  sugars  can  be  shown  to 
have  a  repellent  effect  when  compared  with  water,  the  possibility  of  inhibition  can- 
not be  overlooked. 

In  the  present  studies  some  of  the  results  of  preference  tests  with  sugar  mixtures 
can  be  understood  in  terms  of  inhibition.  For  example,  the  volume  intake  of  a  mix- 


TABLE  VI 

Effect  of  sorbose  on  glucose  threshold 


Molar  concen.  of  glucose  solutions 

0.0625 

0.125 

0.25 

0.50 

1.0 

Per  cent  response 

0 

5 

15 

50 

80 

Molar  concen.  of  glucose  solutions  containing 

0.0625 

0.125 

0.25 

0.5 

1.0 

0.5  M  sorbose 

Per  cent  response 

45 

50 

60 

50 

65 

Molar  concen.  of  sorbose  solutions 

0.0625 

0.125 

0.25 

0.50 

1.0 

Per  cent  response 

5 

20 

45 

60 

80 

ture  of  mannose  and  glucose  is  greater  than  that  of  glucose  alone,  as  would  be  ex- 
pected (Table  VII).  In  contrast,  the  intake  of  a  mannose-fructose  mixture,  as 
compared  with  fructose  alone,  is  not  so  great  as  would  naturally  be  expected. 
Similarly  with  mixtures  containing  rhamnose  there  is  a  large  increase  in  volume 
intake  where  the  other  sugar  is  glucose  but  no  appreciable  increase  where  the  other 
sugar  is  fructose  or  sucrose  (Table  VII).  This  rinding  is  in  agreement  with  the 
threshold  data  (Table  V)  which  indicate  that  rhamnose  inhibits  fructose  but  not 
glucose. 

The  situation  with  regard  to  sorbose  is  not  so  clear  although  there  is  a  tendency 
for  the  intake  of  sorbose  mixtures  to  be  less  than  expected  on  a  purely  additive  basis. 
Such  a  result  would  agree  with  the  postulated  inhibitory  effect  of  sorbose  on  glu- 
cose and  fructose.  It  must  nevertheless  be  emphasized  that  the  effect  of  sugar  mix- 
tures on  the  papillae  is  not  known,  so  that  the  results  obtained  in  preference  tests 
of  mixtures  cannot  be  fully  interpreted  in  terms  of  demonstrated  inhibition  at  tarsal 
and  labellar  sites  alone. 

The  difference  between  repellence  and  inhibition,  at  least  with  Phormia,  is  a 
real  one.  Since  the  tarsal  and  labellar  hairs  of  Phormia  have  been  shown  to  con- 


INGESTION  OF  CARBOHYDRATES 


217 


sist  of  two  receptors,  one  of  which  mediates  rejection  and  one  of  which  mediates 
acceptance  (Dethier,  1955),  a  compound  which  is  repellent  might  he  expected  to 
stimulate  the  rejection  receptor  while  a  compound  which  is  an  inhibitor  might  be 
expected  to  prevent  stimulation  of  the  acceptance  receptor  by  interfering  with  the 
action  of  a  stimulating  compound  on  that  receptor. 

The  only  comparable  study  of  mixtures  on  another  insect  is  that  of  Wykes 
(1952),  who  measured  ingestion  of  single  sugars  and  mixtures  of  sugars  by  the 
honeybee.  Although  not  explicitly  stated,  the  experiment  tested  the  hypothesis 
that  the  volume  ingested  of  the  four  sugars  examined,  singly  and  in  mixtures,  was 
related  to  concentration  by  the  formula  V  —  a  +  C  where  V  is  volume  ingested 
at  concentration  C.  For  all  four  sugars,  then,  there  was  assumed  to  be  a  linear  re- 
lationship between  volume  ingested  and  concentration,  with  a  slope  of  unity  and  an 
intercept  depending  upon  the  sugar  involved.  Since,  however,  the  units  of  volume 


TABLE  VII 

Comparison  of  intake  of  mixed  solutions  with  that  of  water  or  single  sugars  in  a  two-choice  test 


Concentration  of  each  sugar  in  mixture 

Vol.  consumed 

ml.  /fly/24  hrs. 

Water  or  sugar 

Vol.  consumed 
ml.  /fly/24  hrs. 

0.05  M  fucose  and  0.05  M  sorbose 

0.0125 

water 

0.0019 

0.5  M  fucose  and  0.5  M  sorbose 

0.0116 

water 

0.0030 

0.05  M  fucose  and  0.05  M  mannose 

0.0213 

water 

0.0025 

0.5  M  mannose  and  0.5  M  sorbose 

0.0184 

water 

0.0018 

0.1  M  fructose  and  0.1  M  mannose 

0.0234 

0.1  M  fructose 

0.0130 

0.05  M  glucose  and  0.05  M  mannose 
0.1  M  glucose  and  0.1  M  mannose 
0.05  M  glucose  and  0.05  M  sorbose 
0.05  M  fructose  and  0.05  M  sorbose 

0.0090 
0.0228 
0.0099 
0.0220 

0.1  M  glucose 
0.1  M  glucose 
0.1  M  glucose 
0.05  M  fructose 

0.0030 
0.0090 
0.0162 
0.0160 

0.05  M  glucose  and  0.05  M  rhamnose 
0.1  M  glucose  and  0.1  M  rhamnose 
0.05  M  fructose  and  0.05  M  rhamnose 

0.0260 
0.0160 
0.0120 

0.05  M  glucose 
0.1  M  glucose 
0.05  M  fructose 

0.0130 
0.0070 
0.0130 

0.1  M  fructose  and  0.1  M  rhamnose 

0.0170 

0.1  M"  fructose 

0.0140 

0.05  M  sucrose  and  0.05  M  rhamnose 

0.0180 

0.05  M  sucrose 

0.0230 

0.1  M  glucose  and  0.1  M  D-arabinose 
0.05  M  glucose  and  0.05  M  D-arabinose 

0.0210 
0.0130 

0.1  M  glucose 
0.1  M  glucose 

0.0080 
0.0160 

employed  were  arbitrary  and  apparently  were  changed  from  one  concentration  to  the 
next,  this  hypothesis  was  not  tested  directly ;  it  was  implicitly  assumed  in  the  analysis 
of  ingestion  of  mixtures.  The  experiments  on  mixtures  consisted  of  measuring  the 
volume  ingested  of  a  solution  containing  equal  proportions  by  weight  of  two  to  four 
sugars  with  a  total  sugar  concentration  of  x%  and  testing  the  significance  of  the 
difference  between  this  value  and  the  average  of  the  volumes  ingested  of  each  of  the 
component  sugars  at  x%.  For  example,  the  volume  ingested  of  a  solution  con- 
taining 8.5%  sucrose  and  8.5%  glucose  was  compared  with  half  the  sum  of  the 
volumes  ingested  of  17%  glucose  and  17%  sucrose.  It  was  found,  rather  surpris- 
ingly, that  the  calculated  and  measured  figures  were  not  significantly  different; 
hence,  volume  and  concentration  are  linearly  related,  with  a  slope  of  unity  for  sucrose 
and  glucose  within  the  concentration  range  17.1  to  51.3%.  Similar  experiments 
indicate  that  the  same  relationship  is  true  for  maltose.  Furthermore,  with  one  ex- 


218  V.  G.  DETHIER,  D.  R.  EVANS  AND  M.  V.  RHOADES 

ception,  these  sugars  in  the  mixtures  tested  are  neatly  additive  in  their  effect  on  in- 
gestion.  The  one  exception  was  the  glucose-sucrose-fructose  mixture,  of  which 
more  was  ingested  than  was  predicted  (i.e.,  there  was  synergism).  This  may  reflect 
the  synergism  noted  above  on  the  tarsal  threshold  of  Phormia  for  a  mixture  of 
glucose  and  fructose. 

The  data  for  Phormia  relating  volume  and  concentration,  whether  for  intake  at  a 
single  feeding  or  preference-aversion  experiments,  never  present  so  simple  a  picture 
as  Wykes's  results.  The  only  similarity  may  be  the  striking  parallelism  (with  the 
exception  of  fucose)  of  volume  increase  from  low  to  the  optimum  concentrations  on 
a  semi-log  plot  of  ingestion  at  a  single  feeding  (Fig.  3).  Preference-aversion  ex- 
periments on  ingestion  of  mixtures  probably  are  not  comparable  to  ingestion  as 
measured  by  Wykes ;  clearly,  in  the  former  case  simple  additivity  of  sugars  in  a  mix- 
ture is  not  the  rule. 

THE  FEEDING  REACTION 

Initiation  of  feeding.  From  the  foregoing  experimental  facts  and  all  other  avail- 
able information  one  can  reconstruct,  at  least  in  part,  the  behavior  pattern  of  the 
normal  feeding  reaction  insofar  as  it  is  now  known. 

The  normal  pattern  consists  essentially  of  extension  of  the  proboscis,  spreading 
of  the  labellar  lobes,  sucking,  and  regurgitation.  Apparently  any  one  of  three  fac- 
tors may  initiate  proboscis  extension :  ( 1 )  olfactory  stimuli  operating  primarily 
through  the  antennae;  (2)  taste  and  possibly  tactile  stimuli  operating  through  the 
tarsal  receptors;  (3)  internal  factors  causing  extension  spontaneously.  In  the 
presence  of  vapors  of  an  attractive  nature  a  fly  will  extend  its  proboscis  (cf.  also 
Minnich,  1921).  If  the  antennae  are  amputated,  this  faculty  is  impaired.  Water 
(if  a  fly  is  thirsty)  or  specific  carbohydrates  can  stimulate  the  tarsi  with  a  resultant 
proboscis  extension.  In  the  absence  of  any  specific  external  stimuli  the  fly  will 
frequently  repeatedly  extend  its  proboscis  in  an  exploratory  manner. 

The  proboscis  having  been  extended  in  response  to  any  one  or  combination  of 
these  clues,  the  first  parts  which  come  into  contact  with  the  substrate  are  the  long 
hairs  of  the  aboral  labellar  surface.  If  the  stimulus  now  received  is  favorable,  the 
labellar  lobes  are  opened,  thus  presenting  the  oral  surface  to  the  food.  Sucking  then 
commences.  The  labellar  hairs,  therefore,  can  regulate  spreading  of  the  lobes  and 
sucking.  They  can  also  regulate  extension,  although  under  natural  conditions  it 
must  be  quite  unusual  for  the  hairs  of  the  retracted  proboscis  to  be  stimulated.  It 
could  \vell  be  that  in  the  event  of  the  omission  of  an  initial  step  in  the  normal  se- 
quence of  stimulation,  e.g.,  stimulation  of  the  labellar  hairs  before  the  proboscis  is 
extended,  the  hairs  trigger  the  missing  step,  in  this  case  extension,  before  initiating 
the  remaining  steps.  Control  of  the  hairs  over  sucking  is  easily  demonstrated.  If, 
in  a  fastened  fly,  a  drop  of  liquid  just  at  the  threshold  of  rejection  is  placed  on  the 
open  labellum,  it  remains  undisturbed,  and  the  fly  regurgitates  into  it.  Surface  ten- 
sion prevents  the  fly  from  closing  the  labellum,  and  the  feet  cannot  be  employed  to 
remove  the  drop  because  they  are  fastened.  If  now  a  single  labellar  hair  is  stimu- 
lated with  a  concentrated  sugar  solution  (e.g.,  1  M  sucrose),  the  drop,  diluted  with 
regurgitated  fluids,  is  immediately  swallowed. 

Having  opened  the  labellar  lobes  and  commenced  swallowing,  the  fly  would  no 
longer  be  in  complete  sensory  control  of  the  situation  were  it  not  for  the  interpseudo- 
tracheal  papillae.  Once  the  labellar  lobes  are  opened  the  majority  of  the  aboral 


INGESTION  OF  CARBOHYDRATES  219 

hairs  are  no  longer  in  contact  with  the  solution.  Even  if  they  had  been,  the  speed 
with  which  they  adapt  would  certainly  prevent  a  continual  input  from  sugar  stimu- 
lation from  reaching  the  central  nervous  system.  There  is  ample  evidence  that  the 
papillae  supply  this  defect. 

Feeding  can  be  monitored  at  four  levels.  If  an  odorous  component  of  food 
attains  a  repellent  level  of  concentration,  feeding  may  be  inhibited  although  ordi- 
narily feeding  will  not  have  commenced  under  these  conditions.  Secondly,  if  the 
tarsal  receptors  are  stimulated  by  unacceptable  compounds,  feeding  is  ordinarily 
stopped  and  the  proboscis  withdrawn.  This  reaction  is,  of  course,  the  basis  of  all 
measurements  of  tarsal  rejection  thresholds.  Thirdly,  if  the  labellar  hairs  are 
affected  by  adverse  stimuli,  feeding  stops.  Fourthly,  if  the  papillae  are  stimulated 
by  unacceptable  compounds,  feeding  is  terminated. 

As  might  be  expected,  these  various  levels  of  control  are  finely  balanced.  The 
coordination  of  sensory  input  from  all  of  the  receptor  systems  involved  is  extremely 
important  for  the  proper  accomplishment  of  feeding.  Consider,  for  example,  the 
relation  between  tarsal  receptors  and  those  on  the  mouthparts.  Normally  a  fly  will 
not  commence  feeding  on  a  solution  which  has  first  been  rejected  by  the  tarsi.  How- 
ever, if  arrangements  are  made  to  stimulate  tarsi  and  mouthparts  simultaneously  with 
different  solutions,  the  tightness  of  control  of  each  system  over  feeding  can  be 
assessed.  Application  of  sugar,  however  concentrated,  on  the  tarsi  will  not  cause 
feeding  if  a  critical  concentration  of  NaCl  is  placed  on  the  labellum ;  but  a  low  con- 
centration of  NaCl  can  be  found  which  will  be  imbibed  when  the  tarsi  are  stimulated 
with  sugar,  even  though  this  salt  is  refused  in  the  absence  of  tarsal  stimulation. 
Conversely,  concentrated  NaCl  on  the  tarsi  will  not  prevent  imbibition  of  sucrose 
applied  to  the  labellum.  The  mouthparts,  as  might  be  expected,  exert  a  tighter 
control. 

On  the  mouthparts  themselves  the  actions  of  the  labellar  hairs  and  interpseudo- 
tracheal  papillae  are  usually  coordinated.  Experimentally  either  can  be  stimulated 
alone.  The  papillae  alone  are  stimulated  by  inserting  a  micropipette  between  the 
closed  labellar  lobes  or  by  rendering  the  hairs  inoperative  through  waxing.  The 
papillae  are  extremely  sensitive  to  NaCl,  and  the  application  of  salt  by  pipette  causes 
an  immediate  cessation  of  feeding.  However,  it  is  sometimes  possible  to  force  salt 
imbibition  by  simultaneous  stimulation  of  labellar  hairs  with  concentrated  sucrose. 
Swallowing  is  accomplished  with  great  hesitation  on  the  part  of  the  fly  if  the  salt 
solution  is  at  all  concentrated.  Conversely,  if  the  hairs  are  stimulated  with  NaCl 
while  the  papillae  are  stimulated  with  sucrose,  feeding  can  be  stopped,  albeit  some- 
what slowly  and  temporarily.  From  the  results  of  these  two  experiments  it  would 
appear  that  the  papillae  exercise  tighter  control  over  actual  feeding  than  do  the 
labellar  hairs.  The  behavior  of  the  fly  toward  L-arabinose  confirms  this.  The 
hierarchy  of  command  over  sucking  in  ascending  order  is  tarsi,  labellar  hairs,  inter- 
pseudotracheal  papillae.  For  proboscis  extension  and  spreading  of  the  labellar 
lobes,  it  is  tarsi,  labellar  hairs.  Stimulation  of  the  papillae  seldom  causes  proboscis 
extension  or  spreading  of  the  lobes  so  that  by  means  of  a  micropipette  a  fly  can  be 
induced  to  feed  without  extending  its  proboscis  or  expanding  the  labellum.  In  every 
case  mentioned  above  the  relative  concentrations  of  the  opposing  stimuli  are  ex- 
tremely critical  insofar  as  the  nature  of  the  final  response  is  concerned. 

Control  of  volume  intake.  Although  the  various  chemoreceptors  generally  work 
in  harmony  to  regulate  the  economy  of  feeding  response,  the  imbibition  of  liquids  is 


220  V.  G.  DETHIER,  D.  R.  EVANS  AND  M.  V.  RHOADES 

only  the  beginning  of  a  longer  and  more  complex  chain  of  events.  Once  the  insect 
has  begun  to  feed,  it  obviously  does  not  continue  indefinitely.  Assuming  that  the 
substance  being  eaten  or  drunk  is  an  acceptable  one  and  that  its  stimulating  effect 
(odor  or  taste)  initiated  feeding,  what  are  the  factors  which  ensure  continuance  of 
feeding  and  control  of  volume  intake  ?  It  seems  unlikely  that  the  initial  stimulation 
is  alone  sufficient  to  supply  momentum  for  continued  feeding  without  itself  con- 
tinuing, or,  in  other  words,  that  feeding  once  started  continues  automatically  until 
shut  off.  It  is  more  probable  that  there  is  an  additional  factor  which  drives  con- 
tinuous feeding  and  another  which  terminates  it. 

Odorous  foods  not  only  can  supply  the  initial  stimulus  but  can  also  continue  to 
stimulate  for  the  duration  of  feeding.  With  odorless  foods  such  as  sugars,  uninter- 
rupted stimulation  is  also  possible.  If  the  fly  is  standing  in  sugar,  the  tarsal  re- 
ceptors can  supply  a  continuous  sensory  input  to  the  central  nervous  system  until 
they  become  adapted.  The  principal  stimulation  from  the  mouthparts  during  feed- 
ing originates  at  the  interpseudotracheal  papillae  because  most  of  the  labellar  hairs 
are  no  longer  in  contact  with  the  solution  once  the  lobes  have  been  spread.  Even 
if  the  labellar  hairs  were  in  contact  with  the  sugar,  they  adapt  very  rapidly.  An  ex- 
periment can  be  designed  to  show  that,  in  the  absence  of  any  stimulation  except  that 
from  the  labellar  hairs,  complete  adaptation  of  these  hairs  brings  an  end  to  feeding. 
For  example,  a  fly  which  is  not  thirsty  can  be  made  to  drink  water  if  one  or  more 
of  the  labellar  hairs  are  stimulated  with  sugar.  Adaptation  of  the  hair  or  hairs  be- 
ing stimulated  causes  feeding  to  cease,  whereupon  stimulation  of  different  hairs 
which  are  still  sensitive  results  in  resumption  of  swallowing.  From  this  result  it 
would  appear  that  a  continual  sensory  input  is  indeed  essential  to  uninterrupted 
feeding.  Even  stimulation  of  the  tarsal  receptors  can  drive  feeding,  and  one  way 
to  force  flies  to  imbibe  non-stimulating  fluids  ( i.e.,  those  which  are  neither  acceptable 
nor  repellent)  is  to  apply  sucrose  to  the  legs.  For  many  of  the  insects  in  which  feed- 
ing reactions  have  been  studied  the  prerequisite  of  sensory  input  is  the  rule  (cf. 
Dethier,  1953). 

Under  natural  circumstances  a  fly  does  not  feed  to  full  capacity  upon  first  con- 
tact with  an  acceptable  food  but  rather  takes  repeated  samples.  This  behavior  is 
graphically  demonstrated  by  automatic  recording  (Fig.  1).  In  this  way  each  new 
extension  of  the  proboscis  places  the  labellar  hairs  again  in  contact  with  the  solu- 
tion for  fresh  stimulation  which  imparts  renewed  impetus  to  feeding.  At  some 
point  in  the  proceedings,  however,  feeding  finally  ceases  ;  a  definite  quantity  has  been 
consumed.  This  volume  is  not  constant  but  depends  upon  the  hunger  state  of  the 
fly,  the  nature  of  the  food,  and  its  concentration.  Clearly  neither  gut  capacity  nor 
carbohydrate  requirements  immediately  controls  volume  intake  (cf.  also  Dethier 
and  Rhoades,  1954).  Thus,  under  normal  conditions  intake  may  cease  long  before 
the  gut  is  fully  extended.  Furthermore,  an  isolated  head  does  not  drink  equal 
amounts  of  all  sugars.  It  takes  in,  for  example,  less  sorbose  than  fucose  and  less 
fucose  than  sucrose,  indicating  control  by  structures  of  head  alone. 

An  explanation  which  conforms  most  closely  to  the  facts  as  now  known  is  that 
intake  is  shut  off  by  sensory  adaptation.  As  an  examination  of  Table  II  will  re- 
veal, the  rate  of  imbibition  and  the  duration  of  feeding  increase  with  increasing 
concentration — up  to  a  point.  Since  rate  does  increase  with  concentration  and 
since  maximum  rates  for  different  sugars  are  greatest  for  the  more  stimulating  ones, 


INGESTION  OF  CARBOHYDRATES  221 

there  is  reason  to  conclude  that  rate  of  intake  is  related  to  sensory  input.  It  is 
highly  probable,  therefore,  that  the  relationship  prevails  over  the  entire  concentra- 
tion range  but  that  at  a  certain  point  (where  measured  rate  declines)  some  negative 
factor  intervenes.  Since  gram  intake  never  declines  nor  becomes  constant,  the  nega- 
tive factor  cannot  be  the  amount  of  sugar  or  excessive  or  repellent  stimulation.  The 
cause  must  be  sought  in  some  other  characteristic  of  the  solutions.  Increase  in 
viscosity  at  high  concentrations  is  one  limiting  factor.  Measurements  of  rates  of 
intake  of  a  series  of  glycerol  solutions  of  1  M  sucrose  showed  that  rate  decreases 
sharply  with  relatively  small  increases  in  viscosity.  This  finding  is  in  agreement 
with  the  results  which  Betts  (1929)  had  obtained  in  experiments  with  honeybees, 
where  rate  of  intake  declined  sharply  as  concentrations  of  sugar  exceeded  50%  by 
weight.  Betts  concluded  that  viscosity  was  the  limiting  factor  in  this  concentration 
range.  At  lower  concentrations,  however,  she  observed  little  change  in  rate  with 
change  in  either  viscosity  or  concentration.  For  the  honeybee,  temperature  appears 
to  exercise  greater  control  over  rate  of  intake  than  concentration  does. 

From  the  fact  that  duration  of  feeding  increases  with  concentration  one  may  in- 
fer that  adaptation  is  one  factor  bringing  an  end  to  feeding.  This  inference  is  in  ac- 
cord with  observed  increases  in  adaptation  time  with  increased  concentration 
(Dethier,  1952).  Additional  evidence  in  support  of  this  view  derives  from  the  ob- 
servation that  a  fly  which  has  ceased  to  feed  on  a  given  concentration  may  be  induced 
to  continue  on  a  higher  one  and  that  a  fly  which  has  been  feeding  on  a  high  con- 
centration refuses  to  continue  feeding  on  a  lower  one.  In  this  respect  isolated  heads 
behave  similarly.  If  the  inference  is  correct,  it  would  appear  that  flies  adapt  most 
quickly  to  fucose  and  less  quickly  to  mannose,  glucose,  and  sucrose,  respectively, 
because  this  is  the  inverse  order  of  duration  of  feeding. 

Although  the  immediate  cessation  of  imbibition  can  be  explained  in  terms  of 
adaptation,  peripheral  and  central,  and  there  is  no  evidence  of  action  by  internal  fac- 
tors at  this  point,  it  is  almost  certain  that  subsequent  intake  at  various  times  after 
feeding  to  repletion  is  regulated  by  internal  factors.  These  factors  have  been 
investigated  and  will  be  discussed  in  a  latter  communication. 

SUMMARY 

1.  The  ingestion  of  sucrose,  glucose,  fucose,  sorbose,  mannose,  and  lactose  by 
the  blowfly  Phormia  rcgina  was  studied  by  means  of  preference-aversion  tests  con- 
ducted for  four-day  periods ;  individual  feeding  tests ;  measurements  of  the  sensi- 
tivity of  the  different  chemoreceptor  systems ;  measurements  of  volume  intake  of 
mixed  solutions ;  and  longevity  tests. 

2.  The  preference-aversion  curves  for  all  sugars  studied  indicated  an  increase  in 
volume  intake  with  increasing  concentration  up  to  an  optimum  point,  after  which 
there  was  a  decrease  in  intake.     At  very  low  concentrations  water  was  preferred  to 
sugar. 

3.  Volume  intake  measured  by  individual  feeding  tests  did  not  exhibit  a  pro- 
nounced decline  at  high  concentrations.     The  difference  between  this  finding  and 
the  one  noted  above  resulted  from  the  fact  that  flies  ingested  a  maximum  volume 
of  concentrated  solutions  during  the  first  visits  to  the  pipette  and  then  gradually 
ceased  feeding  altogether,  while  their  ingestion  of  less  concentrated  solutions  con- 


V.  G.  DETHIER,  D.  R.  EVANS  AND  M.  V.  RHOADES 

tinned  repeatedly  over  the  entire  test  period.     In  all  experiments  the  weight  of 
sugar  taken  increased  over  the  entire  concentration  range. 

4.  There  is  no  relation  between  the  amount  of  sugar  taken  and  its  nutritive  value. 

5.  Volume  intake  is  under  sensory  control.     The  coordinated  actions  of  three 
principal  chemosensory  systems  regulate  the  complete  feeding  reaction.     The  in- 
take of  mixed  solutions  depends  upon  the  stimulating  effectiveness  of  the  mixture 
and  whether  or  not  any  of  the  components  exhibit  synergism  or  inhibition.     Some 
sugars  show  inhibition  but  no  repellence. 

6.  The  initiation  of  the  feeding  reaction  is  under  sensory  control.     Continuance 
of  feeding  is  dependent  upon  continuous  sensory  input.     The  rate  of  imbibition  in- 
creases with  concentration  until  viscosity  begins  to  exert  a  restraining  effect.     The 
termination  of  feeding  may  be  brought  about  by  adaptation. 

LITERATURE  CITED 

BECK,  S.  B.,  1956.  A  bimodal  response  to  dietary  sugars  by  an  insect.  Biol.  Bull.,  110:  219- 
228. 

BETTS,  A.  D.,  1929.  Das  Aufnahmevermogen  der  Bienen  beim  Zuckerwasserfiittern.  Arch. 
Biencnkunde,  10:  301-309. 

DETHIER,  V.  G.,  1952.  Adaptation  to  chemical  stimulation  of  the  tarsal  receptors  of  the  blow- 
fly. Biol  Bull,  103:  178-189. 

DETHIER,  V.  G.,  1953.  Host  plant  perception  in  phytophagous  insects.  Trans.  IXth  Internal. 
Congress  Ento.,  2:  81-89,  Amsterdam. 

DETHIER,  V.  G.,  1955.  The  physiology  and  histology  of  the  contact  chemoreceptors  of  the  blow- 
fly. Quart.  Rev.  Biol.,  30:  348-371. 

DETHIER,  V.  G.,  AND  L.  E.  CHADWICK,  1948.  Chemoreception  in  insects.  Physiol.  Rev.,  28: 
220-254. 

DETHIER,  V.  G.,  AND  M.  V.  RHOADES,  1954.  Sugar  preference-aversion  functions  for  the  blow- 
fly. /.  Exp.  Zool,  126:  177-204. 

DIMLER,  R.  J.,  W.  C.  SCHAEFER,  C.  S.  WISE  AND  C.  E.  RIST,  1952.  Quantitative  paper  chro- 
matography  of  D-glucose  and  its  oligosaccharides.  Anal.  Chcm.,  24:  1411-1414. 

FRAENKEL,  G.,  1953.  The  nutritional  value  of  green  plants  for  insects.  Trans.  IXth  Internat. 
Congress  Ento.,  2  :  90-100,  Amsterdam. 

VON  FRISCH,  K.,  1935.  t)ber  den  Geschmackssinn  der  Biene.  Zcitschr.  f.  vcrgl.  Physiol,  21  : 
1-156. 

GALUN,  R.,  1955.  Physiological  responses  of  three  nutritionally  diverse  dipterous  insects  to  se- 
lected carbohydrates.  Dissertation,  University  of  Illinois,  Urbana. 

HASSETT,  C.  C,  V.  G.  DETHIER  AND  J.  GANS,  1950.  A  comparison  of  nutritive  values  and 
taste  thresholds  of  carbohydrates  for  the  blowfly.  Biol  Bull,  99:  446-453. 

KENNEDY,  J.  S.,  1953.  Host  plant  selection  in  Aphididae.  Trans.  IXth  Internat.  Congress 
Ento.,  2:  106-113,  Amsterdam. 

KUNZE,  G.,  1927.  Einige  Versuche  iiber  den  Geschmackssinn  der  Honigbiene.  Zool  Jahrb., 
Abt.  Zool  Physiol,  44:  287-314. 

MINNICH,  D.  E.,  1921.  An  experimental  study  of  the  tarsal  chemoreceptors  of  two  nymphalid 
butterflies.  /.  Exp.  Zool,  33:  173-203. 

WYKES,  G.  R.,  1952.  The  preference  of  honeybees  for  solutions  of  various  sugars  which  occur 
in  nectar.  /.  Exp.  Biol,  29:  511-519. 


THE  CULTURE  OF  BRINE  ALGAE1 

AARON  GIBOR 

Hopkins  Marine  Station  of  Stanford  University,  Pacific  Grove,  California 

The  biological  productivity  of  the  solar  evaporation  ponds  used  in  salt  manu- 
facture is  apparent  even  to  the  casual  observer :  they  are  a  deep  green  or  rich  red. 
The  ecological  association  in  these  ponds  was  the  subject  of  several  investigations 
(Peirce,  1914;  Baas-Becking,  1928;  Carpelan,  1953).  Carpelan  concluded  that  the 
productivity  of  the  ponds,  per  unit  area,  was  comparable  to  that  of  the  ocean  in  its 
richer  spots.  But  the  ponds,  being  only  half  a  meter  deep,  have  a  much  more  con- 
centrated plankton  community  than  does  the  productive  zone  of  the  ocean,  which 
is  10  to  15  meters  deep. 

With  the  increased  interest  in  recent  years  in  mass  culturing  of  algae  for  food 
production,  the  possibility  of  utilizing  these  evaporation  ponds  for  such  purposes  has 
been  considered.  With  this  in  mind,  a  laboratory  study  was  undertaken  on  the 
cultivation  of  several  of  the  unicellular  algae  which  thrive  in  the  ponds. 

Pure  cultures  were  isolated  by  the  procedures  described  by  Pringsheim  (1946) 
with  the  added  advantage  of  utilizing  antibiotics,  as  suggested  by  Spencer  (1952). 
The  following  five  algae  were  studied : 

Stichococcus  sp. 
Platymonas  sp. 
Dunaliclla  viridis 
Dunaliclla  salina 
Stephanoptera  gracilis 

These  were  isolated  from  the  ponds  of  the  Leslie  Salt  Company,  on  San  Fran- 
cisco Bay.  The  author  is  indebted  to  this  company  for  many  privileges  during  the 
course  of  the  study. 

SALINITY  TOLERANCE 

The  determination  of  the  range  of  salinity  tolerated  by  the  different  algae  under 
study  is  of  primary  importance.  For  this  purpose  several  strains  of  the  algae  were 
inoculated  into  solutions  of  artificial  sea  water  of  varying  concentrations. 

Stichococcus  strains  numbers  1,  2  and  3,  Platymonas  strains  5  and  7,  Dunaliclla 
viridis  strains  6,  8  and  9,  Dunaliclla  salina  strain  10,  and  Stephanoptera  gracilis 
were  studied.  The  strain  numbers  refer  to  the  evaporation  ponds  from  which  they 
were  isolated.  Pond  number  1  contained  San  Francisco  Bay  water  (concentration 
less  than  sea  water).  The  successive  ponds  contained  increasing  concentrations 
of  brine  to  NaCl  saturation  in  pond  number  10. 

Artificial  sea  water  was  prepared  after  Pringsheim  (1946).  Multiples  of  the  sea 
water  formula  were  used  for  preparing  the  desired  concentrations.  Concentra- 

1  Study  partly  supported  by  a  grant  from  the  National  Science  Foundation  to  Stanford 
University. 

223 


224 


AARON  GIBOR 


tions  up  to  ten-fold  sea  water  were  prepared ;  the  solutions  of  concentration  of  X  5 
and  above  were  saturated  with  respect  to  the  CaSO4,  excess  solid  being  filtered  off. 
Dilutions  of  sea  water  to  %,  %„,  as  well  as  fresh  water,  were  also  used.  All  solu- 
tions were  equally  enriched  with  50  mg.  KNCX,  10  mg.  KH2PO4  and  Hutner's  trace 
elements  mixture  (Hutner  et  a!.,  1950).  Five-mi,  portions  of  the  various  solutions 
were  pipetted  into  test  tubes  and  equally  inoculated.  The  test  tubes  were  kept  in 
an  inclined  position  under  continuous  illumination.  Light  was  provided  by  several 
fluorescent  lights  suspended  above  the  test  tubes;  its  intensity  was  about  150  foot 
candles. 

Growth  was  estimated  by  cell  counts  after  16  days  of  culture.  At  this  time  the 
tubes  in  which  no  growth  had  taken  place  were  re-inoculated  with  0.1  ml.  of  the 
culture  which  was  nearest  to  them  in  concentration.  Thus,  if  a  culture  in  the  tubes 
of  X  7  sea  water  did  not  grow,  it  was  inoculated  with  the  growing  X  6  culture.  The 

TABLE  I 
Relative  growth  of  algal  strains  isolated  from  different  salinities  in  media  of  different  concentrations 


Strain  : 

Medium  Concentrations 

0 

1/10 

1/2 

1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

Stichococcus  sp. 
1. 
2. 
3. 

30% 
T 

74% 
46% 
T 

170% 

135% 
90% 

100% 
100% 
100% 

NT 

15% 
4(1', 

NT 

NT 

Platymonas  sp. 
5. 
7. 

77% 

77% 
50% 

90% 
100% 

100%, 

10(1'  , 

90% 

125% 

47% 
230%, 

NT 
60% 

NT 

Dunaliella  viridis 
6. 
8. 
9. 

63% 

NT 

82% 
34% 
T 

240%, 
? 
60% 

100% 
100% 
100% 

100%, 

225% 
210% 

95% 
320% 
400%, 

85% 
230% 

320% 

50% 
125% 
310% 

26% 
100% 
120% 

NT 
18% 
40% 

T 
T 

T 

T 

Dunaliella  salina 
10. 

T 

100% 

100% 

160%, 

220% 

215% 

185%, 

145% 

80% 

60% 

55% 

35% 

Stephanoptera  gracilis 
11. 

T 

12% 

100% 

120% 

135% 

67% 

30% 

T 

T 

T 

(Concentrations  in  multiples  of  sea  water;  T  and  NT  refer  to  trainability  or  non-trainability  of  the  algae  for  growth  in 
the  specific  concentrations.  Growth  in  sea  water  taken  as  base  value  for  all  strains  except  Stephanoptera  gracilis  in 
which  X  2  sea  water  was  taken  as  base  value.) 

results  of  these  experiments  are  given  in  Table  I.  The  results  are  expressed  as 
per  cent  of  the  growth  in  sea  water,  except  for  the  values  for  Stephanoptera  gracilis, 
which  are  expressed  as  per  cent  age  of  X  2  sea  water  medium.  The  results  of  the 
secondary  inoculation  are  expressed  as  T  (for  trainable)  and  NT  (for  not  trainable). 
From  Table  I  it  is  apparent  that  the  Stichococcus  strains  under  study  are  pri- 
marily brackish  water  organisms.  The  Platymonas  sp.  appear  to  be  more  resistant 
to  salt,  although  they  too  did  not  grow  in  the  highly  concentrated  brines.  The  last 
three  algae,  all  of  the  Polyblepharidaceae  family,  tolerate  a  wide  range  of  salinity. 
Of  these,  Stephanoptera  gracilis  did  not  grow  well  at  concentrations  below  X  2  sea 
water. 

TEMPERATURE 

To  determine  the  temperature  range  for  the  organisms  under  study,  the  fol- 
lowing procedure  was  adopted.     Sea  water  was  used  for  Dunaliella  viridis,  Platy- 


CULTURE  OF  BRINE  ALGAE 


225 


monas  sp.  and  Stichococcus  sp. ;  Dunaliclla  salina  and  Stephanoptera  gracilis  were 
cultured  in  X  2  sea  water.  Media  were  enriched  with  nitrate,  phosphate  and 
Hutner's  trace  elements.  Portions  of  sterile  media  were  inoculated  with  the  above 
mentioned  cultures,  five-mi,  portions  being  pipetted  into  sterile  test  tubes. 

Cultures  were  incubated  in  thermostatically  controlled  incubators,  with  light 
provided  from  two  15-watt  fluorescent  lamps.  The  test  tubes  were  set  on  an  in- 
clined rack  at  a  distance  of  20  centimeters  from  the  light  source.  Light  intensity  at 
the  culture  tube  level  was  above  100  foot  candles.  After  10  days  of  continuous  il- 
lumination, growth  was  estimated  in  the  cultures  by  cell  count.  The  results  of  the 
counts  are  given  in  Table  II.  The  range  of  temperatures  recorded  by  Carpelan 
(1953)  for  the  evaporation  ponds  of  the  same  region  was  from  8°  to  20°  in  the 
winter,  and  15°  to  30°  in  the  summer. 


pH  EFFECT 

The  effect  of  pH  on  the  growth  of  the  algae  was  investigated  with  the  aid  of 
media  buffered  with  "Tris"  (1,  3-propendiol-2-amino-2-hydroxymethyl).     The  pH 

TABLE  II 

Effect  of  temperature  on  growth  of  brine  algae 
(Cells  per  cubic  millimeter) 


Temperature  °  C. 

Dunaliella  salina 

Stephanoptera 
gracilis 

Dunaliella  viridis 

Platymonas  sp. 

Stichococcus  sp. 

8-10° 

20 

90 

2,700 

120 

4,200 

14-16° 

200 

560 

36,000 

340 

24,000 

24-26° 

160 

640 

1,100 

47,000 

30° 

450 

1,800 

35,000 

1,000 

40,000 

35° 

25 

40 

no  growth 

1,100 

16,000 

range  between  7  and  9  was  investigated  since  this  is  the  most  common  value  for  the 
sea  water  brines.  Culture  media  were  prepared  as  before  except  for  the  addition 
of  0.4  M  "Tris"  buffer ;  pH  was  adjusted  to  7.2,  8.0  or  9.0.  Five-mi,  portions  of  the 
media  were  pipetted  into  test  tubes  and  equally  inoculated.  The  test  tubes  were  kept 
inclined  under  continuous  illumination.  Cell  counts  were  made  after  9  days  of 
growth.  The  results  are  recorded  in  Table  III. 

As  seen  from  the  table  only  Stichococcus  sp.  and  Platymonas  sp.  show  a  definite 
preference  for  higher  pH  values.  The  other  three  algae  do  not  seem  to  be  very 
sensitive  to  pH  changes  in  the  range  studied. 

PHOSPHATE  CONCENTRATION 

The  optimum  range  of  phosphate  concentration  for  the  algae  under  study  was 
determined  in  an  experiment  in  which  5  ml.  of  inoculated  media  in  test  tubes  were 
enriched  with  graded  quantities  of  phosphate.  The  media  were  buffered  to  pH  8 
with  .04  M  "Tris"  buffer.  Nitrate  and  micronutrients  were  supplied  as  in  the 
previous  experiments.  Cell  counts  were  made  after  10  days  of  cultivation  under 
continuous  illumination.  The  results  are  tabulated  in  Table  IV.  The  optimum 


226 


AARON  GIBOR 


TABLE  III 

Effect  of  pH  on  growth  of  brine  algae 


Original  pH 

Final  pH 

Cells/mm.3 

Dunaliella  salina 

7.2 

7.5 

770 

8.0 

8.1 

1,300 

9.0 

9.1 

1,380 

Stephanoptera  gracilis 

7.2 

7.8 

1,100 

8.0 

8.1 

740 

9.0 

9.2 

760 

Dunaliella  viridis 

7.2 

7.6 

24,000 

8.0 

8.0 

15,000 

9.0 

9.2 

20,000 

Stichococcus  sp. 

7.2 

7.4 

16,000 

8.0 

7.9 

25,000 

9.0 

9.2 

69,000 

Platymonas  sp. 

7.2 

7.5 

570 

8.0 

8.0 

1,000 

9.0 

9.2 

2,270 

range  as  seen  in  the  table  is  in  the  same  range  of  concentration  which  is  recom- 
mended for  use  with  several  other  marine  algae  (Koch,  1953;  Ketchum  and  Red- 
field,  1938;Kylin,  1943). 

NITROGEN  SUPPLY 

The  growth  of  the  algae  on  nitrate  or  ammonium  as  the  nitrogen  source  was  in- 
vestigated. These  are  easily  available  forms  of  fixed  nitrogen  for  mass  cultivation 
of  plants. 

Media  were  prepared  as  before,  buffered  with  "Tris"  to  pH  8.1.  Phosphate  and 
trace  elements  were  supplied.  Potassium  nitrate  was  added  in  concentrations  of 
50  mg./lOO  ml.  NH4C1  was  added  in  concentrations  of  26.5  mg./lOO  ml.  Cell 
counts  were  done  after  two  weeks  of  culture  under  continuous  illumination.  In 
Table  V  the  results  of  this  experiment  are  summarized. 

TABLE  IV 

Effect  of  phosphate  concentration  on  growth  of  brine  algae 
(Cells  per  cubic  millimeter) 


KH2PO<  (micro- 
gram/ml.) 

Dunaliella  salina 

Slephanoptera 
gracilis 

Dunaliella  viridis 

Platymonas  sp. 

Stichococcus  sp. 

0 

340 

280 

1,300 

270 

8,500 

20 

450 

630 

3,000 

520 

8,800 

40 

710 

600 

2,800 

830 

12,700 

100 

540 

450 

2,400 

700 

13,700 

200 

420 

300 

1,400 

730 

13,500 

500 

200 

190 

1,200 

560 

12,300 

CULTURE  OF  BRINE  ALGAE 


227 


In  another  experiment,  summarized  in  Table  VI,  using  an  unbuffered  medium 
and  higher  concentrations  of  nitrate  and  ammonium  (%  nig.  N/ml.),  the  superi- 
ority of  nitrate  over  ammonium  for  growth  of  all  the  investigated  algae  was  clearly 
demonstrated.  In  this  experiment  urea  was  also  studied ;  it  was  found  to  be  a 
superior  nitrogen  source  only  for  Dunaliella  salina. 

The  ability  of  the  algae  to  grow  when  supplied  with  organic  nitrogenous  sub- 
stances as  sole  nitrogen  source  was  also  investigated.  Media  were  prepared  en- 
riched with  phosphate  and  micro-nutrients.  The  nitrogen  source  was  added  to  a 

TABLE  V 

Nitrate  and  ammonium  as  nitrogen  sources  for  brine  algae 
in  media  buffered  to  pH  8.1 
(Cells  per  cubic  millimeter) 


Dunaliella  salina 

Stephanofitera 
gracilis 

Dunaliella  viridis 

Platymonas  sp. 

Stichococcus  sp. 

NO3 
NH4 

1,700 
1,900 

1,500 
2,500 

4,700 
13,000 

6,000 
6,300 

48,000 
28,000 

final  concentration  of  50  micrograms  N/ml.  The  media  were  buffered  with  "Tris" 
to  either  7.5  or  9.0.  Cell  counts  were  made  after  12  days  of  culture,  the  results 
being  tabulated  in  Table  VII. 

Contrary  to  expectation,  uric  acid,  which  is  the  main  nitrogen  form  in  bird  ex- 
crement, does  not  serve  as  a  good  nitrogen  source  for  Stcphanoptcra  gracilis  al- 
though this  alga  is  usually  found  to  bloom  in  high  tide  pools  on  rocks  which  are 
coated  with  bird  excrement.  Platymonas,  which  also  blooms  under  similar  condi- 

TABLE  VI 

Effect  of  nitrogen  source  on  growth  of  brine  algae 
(Number  of  cells  per  cubic  millimeter  and  final  pH  values  indicated) 


Dunaliella  salina 

Stephanoptera 

gracilis 

Dunaliella  viridis 

Platymonas  sp. 

Stichococcus  sp. 

Nitrogen 

Cells 

pH 

Cells 

pH 

Cells 

pH 

Cells 

pH 

Cells 

PH 

N03 

1,000 

8.2 

1,700 

8.9 

39,000 

8.5 

2,000 

9.5 

20,000 

9.3 

NH4 

600 

6.4 

700 

5.0 

6,000 

5.5 

400 

6.5 

10,000 

6.9 

Urea 

1,400 

7.8 

500 

7.5 

6,000 

7.5 

300 

8.3 

8,000 

7.8 

tions,  can  utilize  uric  acid.  Platymonas  alone  was  found  to  grow  on  all  the  organic 
nitrogenous  substances  studied ;  this  might  be  a  clue  to  an  observation  that  this  alga 
appeared  to  coat  glassware  in  which  animal  or  plant  materials  are  kept  under 
running  sea  water  in  the  laboratory.  Stichococcus  sp.  prefers  nitrate  to  uric  acid 
and  asparagin;  this  is  in  contrast  to  the  strains  studied  by  Ryther  (1954)  which  ap- 
parently grow  better  on  uric  acid  and  asparagin  than  on  nitrate. 

The  experiment  was  performed  at  two  pH  values  with  the  hope  of  gaining  infor- 
mation on  the  relative  availability  to  the  cells  of  the  different  ionic  forms  of  the  ex- 


228 


AARON  GIBOR 


TABLE  VII 

Effect  of  various  nitrogen  sources  on  growth  of  brine  algae 
(Number  of  cells  per  cubic  millimeter,  and  final  pH  values  indicated) 


Nitrogen  source 

Original 
pH 

Dunaliella 

salina 

Stephana  ptera 
gracilis 

Dunaliflla 
viridis 

Platymonas  sp. 

Stichococcus  sp. 

Cells 

pH 

Cells 

pH 

Cells 

pH 

Cells 

pH 

Cells 

pH 

Nitrate 

7.5 
9 

870 
1,700 

7.8 
9.0 

1,370 
380 

7.9 
8.9 

25,000 
39,000 

7.8 
9.1 

530 

1,100 

7.8 
9.1 

28,000 
70,000 

7.7 
9.1 

Uric  acid 

7.5 
9 

880 
1,100 

7.5 
8.7 

0 
0 

3,000 
4,500 

7.5 
8.7 

530 
1,300 

7.6 
8.9 

1  1  ,000 
17,000 

7.6 
8.8 

dl-Aspartic  acid 

7.5 
9 

0 
0 

0 
0 

0 
0 

130 
200 

7.3 
8.6 

0 
0 

Glutamic  acid 

7.5 
9 

970 
530 

7.9 
8.9 

530 

270 

7.8 
8.8 

0 
0 

500 
500 

7.7 
8.9 

0 
0 

Asparagine 

7.5 
9 

210 
210 

7.6 
8.6 

120 
110 

7.6 

8.7 

0 
0 

470 
1,200 

7.7 
8.9 

8,000 
8,000 

Glycine 

7.5 
9 

0 
0 

0 
0 

0 
0 

1,000 
1,200 

7.7 
8.9 

0 
0 

amined  substances.  The  growth  on  uric  acid  was  found  in  general  to  be  pH- 
dependent  in  much  the  same  way  as  growth  on  nitrate.  On  the  other  hand  growth 
on  glutamic  acid  seems  to  show  a  reverse  pH  response  in  the  case  of  Dunaliella 
salina. 

ORGANIC  NUTRIENTS 

The  ability  of  the  brine  algae  to  grow  in  the  dark  on  several  organic  substances, 
as  well  as  the  effect  of  these  substances  on  the  growth  in  light,  was  investigated. 
Two  organic  energy  sources  were  investigated,  glucose  and  acetate.  The  effect  of 

TABLE  VIII 

Effect  of  organic  substances  on  growth  of  brine  algae 
(Cells  per  mm.3) 


Medium 

Dunaliella 
salina 

Slephanoptera 
gracilis 

Dunaliella 
viridis 

Platymonas 
sp. 

Slichococcus 
sp. 

1.  Mineral  onlv 

700 

970 

6,250 

630 

27,000 

2.  Glucose 

670 

980 

11,300 

430 

26,000 

3.  Acetate 

580 

870 

6,200 

770 

— 

4.  YE*  Glucose 

700 

750 

9,200 

420 

16,000 

5.  YE  Acetate 

780 

720 

7,200 

970 

— 

6.  BH**  Glucose 

1,300 

1,070 

11,500 

700 

20,000 

7.  BH  Acetate 

670 

900 

9,500 

920 



*  Yeast  extract. 

**  Brain-heart  infusion. 


CULTURE  OF  BRINE  ALGAE  229 

these  was  studied  alone  and  in  conjunction  with  complex  mixtures  such  as  yeast  ex- 
tract (YE)  or  brain-heart  infusion  (BH).  The  media  were  enriched  with  nitrate, 
phosphate  and  trace  elements,  and  strongly  buffered  to  pH  8.1  with  0.1  M  "Tris" 
buffer.  Glucose  or  acetate  were  used  to  final  concentration  of  %%,  brain-heart  in- 
fusion to  .02^  and  yeast  extract  to  .01%.  Five-mi,  portions  of  the  inoculated  media 
were  pipetted  into  test  tubes.  Two  equal  sets  of  test  tubes  were  maintained,  one 
under  continuous  illumination  and  the  other  in  the  dark.  The  illuminated  tubes 
were  counted  after  7  days  of  culture.  The  results  are  given  in  Table  VIII.  No 
growth  was  detectable  in  the  tubes  which  were  kept  in  the  dark  for  three  weeks. 

The  inhibition  of  growth  of  Stichococcus  by  the  acetate  is  remarkable.  The  in- 
hibitory effect  of  undissociated  acetate  on  Chlorclla  is  discussed  by  Myers  (1951). 
In  our  case,  however,  the  concentration  of  undissociated  acetate  is  very  low  (about 
.0003  M).  Dunaliella  viridis  seems  to  be  stimulated  by  glucose,  while  Platymonas 
is  stimulated  by  acetate. 

SUMMARY 

1.  The  conditions  which  the  brine  algae  require  for  growth  were  found  to  be 
relatively  simple.     The  high  temperatures  in  the  pond  waters  during  the  summer 
are  well  within  the  tolerance  range  of  these  organisms. 

2.  Simple  nitrogenous  substances  and  no  organic  supplements  are  required.     No 
need  for  organic  growth  factors  could  be  demonstrated  under  the  conditions  of 
cultivation  used.     Sea  water,  especially  as  it  concentrates  by  evaporation,  probably 
contains  most  of  the  required  trace  elements  in  sufficient  quantity.     It  is  possible 
that  for  mass  cultivation  of  very  dense  algal  suspensions,  supplements  of  the  micro- 
nutrients  will  be  required. 

LITERATURE  CITED 

BAAS-BECKING,   L.   G.   M.,    1928.     On  organisms   living  in   concentrated   brine.     Tijdsch.   Ned. 

Dicrk.  Vcr.,  scr.  3,  I   (1)  :  6-9. 

CARPELAN,  L.  H.,   1953.     The  hydrobiology  of  the  Alviso  Salt  Ponds.     Thesis,   Stanford  Uni- 
versity. 
HUTNER,  S.  H.,  L.  PROVASOLI,  A.  SCHATZ  AND  C.  P.  HASKINS,  1950.     Some  approaches  to  the 

study  of  the  role  of  metals  in  the  metabolism  of  micro-organisms.     Proc.  Amer.  Philos. 

Soc.,  94:  152-170. 
KETCHUM,  B.  H.,  AND  A.  C.  REDFIELD,   1938.     A  method  for  maintaining  a  continuous  supply 

of  marine  diatoms  by  culture.     Biol.  Bull.,  75  :   165-169. 
KOCH,   W.,    1953.     Untersuchungen  an   Massenkulturen   von   Porphyridhim    cruentum    Naegeli. 

Arch.  Mikrobiol,  18:  232-241. 
KYLIN,  H.,  1943.     Uber  die  Ernahrung  von   Ulva  Lactuca.     Kungl.  Fysiogr.  Sdllsk.  i  Lund, 

Fork.,  13  (21)  :  1-13. 

MYERS,  J.,  1951.     Physiology  of  the  algae.     Ann.  Rev.  Microbiol.,  5:  157-180. 
PEIRCE,   G.   J.,    1914.     The   behavior   of   certain   micro-organisms    in    brine.     The    Salton    Sea. 

Carnegie  Inst.  Washington  Pub.,  193  :  49-70. 

PRINGSHEIM,  E.  C,  1946.     Pure  cultures  of  algae.     University  Press,  Cambridge. 
RYTHER,  J.   H.,   1954.     Ecology  of  phytoplankton  blooms   in   Moriches   Bay   and   Great   South 

Bay,  Long  Island,  New  York.     Biol.  Bull,  106 :   198-209. 
SPENCER,  C.  P.,  1952.     On  the  use  of  antibiotics  for  isolating  bacteria-free  cultures  of  marine 

phytoplankton  organisms.     /.  Mar.  Biol.  Assoc.,  31  :  97-106. 


SOME  ECOLOGICAL  RELATIONSHIPS  BETWEEN  PHYTO-  AND 

ZOOPLANKTON  1 

AARON  GIBOR 
Hopkins  Marine  Station  of  Stanford  University,  Pacific  Grove,  California 

The  possibility  of  increasing  human  food  resources  by  cultivation  of  unicellular 
algae  is  being  rather  widely  investigated  now  (cf.  Burlew,  1953).  Two  main  ap- 
proaches are  considered :  closed  system  cultivation  of  a  pure  algal  culture  under 
optimum  growth  conditions,  and  an  open  system,  utilizing  ponds.  The  closed 
system  has  the  advantage  of  providing  the  maximal  rate  of  photosynthesis  and  crop 
yield ;  however,  it  is  expensive  to  install  and  maintain.  The  open  system  does  not 
require  elaborate  installations  and  has  the  added  advantage  of  utilizing  ponds  which 
may  be  constructed  for  a  different  purpose,  algae  being  a  by-product.  Of  such 
systems  suitable  for  algal  cultivation,  sewage  oxidation  ponds  are  being  studied. 
The  growth  of  algae  in  these  ponds  is  beneficial  to  the  oxidation  process,  and  the 
harvested  algae  save  much  of  the  nitrogen  wastes  and  minerals  which  are  otherwise 
poured  into  the  sea  (Gotaas  ct  a!..  1954). 

The  solar  evaporation  ponds  of  the  salt  industry  might  also  be  utilized  for  this 
purpose.  The  natural  productivity  of  these  ponds  per  unit  area  approaches  that 
of  the  open  ocean  ( Carpelan,  1953),  the  difference  being  that  the  ponds  are  only 
one-half  meter  deep  while  the  productive  zone  of  the  ocean  is  ten  or  more  meters 
deep.  This  gives  a  very  dense  standing  crop. 

One  of  the  economically  limiting  factors  in  mass  cultivation  of  algae  is  their 
harvesting.  In  dense  algal  suspensions  the  volume  of  cells  is  still  onlyva  fraction  of 
the  total  volume.  To  aid  in  the  harvesting,  suggested  procedures  have  been  sedi- 
mentation by  slow  settling  (Smith,  1953)  or  flocculation  of  cells  by  added  alum 
(Gotaas  ct  a!.,  1954). 

Another  possibility  is  utilizing  grazing  animals  for  the  purpose  of  harvesting 
the  algal  cells.  Raising  fish  in  ponds  is  an  old  practice,  the  growth  of  plankton  be- 
ing accelerated  by  various  fertilizers.  However  no  attempt  is  usually  made  to  main- 
tain a  maximal  rate  of  production  of  the  primary  food,  the  algae. 

In  a  preliminary  investigation  of  the  possibilities  of  the  utilization  of  the  sea 
water  evaporation  ponds  of  the  salt  industry,  several  unicellular  algae  which  grow 
in  this  environment  were  studied  (Gibor,  1956).  We  considered  the  possibilities  of 
converting  the  algal  crop  to  an  animal  crop  by  feeding  a  zooplankton  grazer. 

One  of  the  important  grazers  in  the  evaporation  ponds  is  the  brine  shrimp 
Artcinia.  This  organism  is  easily  maintained  in  the  laboratory;  its  "eggs"  (cysts) 
are  readily  available  and  can  be  kept  in  the  laboratory  for  many  years  without  losing 
their  ability  to  hatch.  We  attempted  to  study  the  nutritive  value  to  Arteinia  of  sev- 
eral of  the  unicellular  green  algae  which  were  isolated  from  the  brines  of  the  evapo- 
ration ponds.  The  algae  used  were : 

1  Study  partly  supported  by  a  grant  from  the  National  Science  Foundation  to  Stanford 
University. 

230 


ECOLOGICAL  RELATIONSHIPS  OF  PLANKTON  231 

Dunaliella  viridis 
Dunaliella  salina 
Stephanoptera  gracilis 
Platymonas  sp. 
Stichococcus  sp. 

Dunaliclla  salina  and  Stephanoptera  gracilis  were  cultured  in  sea  water  evaporated 
to  half  its  original  volume ;  the  other  three  species  were  cultivated  in  sea  water. 

The  feeding  experiments  were  carried  out  as  follows.  Artcmia  cysts  had  been 
collected  two  years  earlier  from  salt  ponds  of  the  Leslie  Salt  Company,  and  kept  in  a 
closed  jar  in  the  laboratory.  Such  cysts  were  suspended  in  water  and  centrifuged 
(in  a  clinical  centrifuge)  for  several  minutes.  The  light  cysts  were  decanted  off 
as  suggested  by  Dempster  (1953).  The  heavy  cysts  were  re-suspended  in  merthio- 
late  solution  (1  :  1000,  in  water)  for  10  minutes,  then  centrifuged.  The  merthiolate 
solution  was  decanted  off  and  the  cysts  washed  four  times  with  sterile  sea  water 
to  get  rid  of  the  merthiolate.  Finally  the  sterile  cysts  were  transferred  into  a  one- 
liter  Erlenmeyer  flask  containing  sterile  sea  water  and  allowed  to  hatch. 

The  sterility  of  the  cysts  and  larvae  was  determined  by  suspending  a  fraction  of 
the  sterilized  cysts  in  sea  water  enriched  with  2%  yeast  extract-glucose  solution  and 

TABLE  I 

Size  of  Artemia  salina  fed  on  different  brine  algae  for  six  days 
(average  length  exclusive  of  caudal  f  urea) 

Alga  Sterile  culture  Non-sterile  culture 

Stephanoptera  gracilis  2.3  mm.  2.8  mm. 

Dunaliella  viridis  2.1mm.  2.8mm. 

D.  salina  1.5  mm.  2.3  mm. 

Platymonas  sp.  1.5  mm.  2.0  mm. 

Stichococcus  sp.  0.7  mm.  0.4  mm. 

None  (unfed)  0.2  mm.  0.2  mm. 

incubating  for  several  weeks.  To  further  verify  their  sterility  a  dense  cyst  sus- 
pension (%  ml.  eggs  in  one  ml.  enriched  sea  water)  was  also  incubated.  The  auto- 
lysing  cysts  should  supply  additional  growth  requirements  for  contaminating  bac- 
teria. Both  these  tests  were  negative  and  indicated  the  adequacy  of  the  sterilization 
method.  The  larvae  were  transferred  when  needed  with  a  sterile  Pasteur  pipette  into 
a  test  tube  for  addition  to  the  algal  culture.  Ten  to  20  larvae  were  introduced  into 
one  liter  of  a  dense  algal  culture  in  a  two-liter  Fernbach  flask.  The  culture  wras 
kept  under  continuous  illumination,  with  continuously  bubbling  air.  After  6  days 
the  cultures  were  still  green,  indicating  that  until  this  time  the  quantity  of  food  was 
not  limiting  the  growth  of  the  animals.  At  this  stage  the  Artemia  were  harvested  by 
passing  the  whole  solution  through  a  fine  plankton  net.  The  animals  were  fixed 
in  0.5%  formalin  in  sea  water.  The  control  larvae  in  sterile  sea  water  were  dead 
of  starvation  at  this  time. 

The  results  of  the  feeding  experiment  are  shown  in  Table  I. 

Dunaliclla  viridis  and  Stephanoptera  gracilis  appear  to  be  superior  as  foods  to 
Dunaliella  salina  and  Platymonas  sp.,  while  Stichococcus  sp.,  is  evidently  a  poor 
nutrient  for  Artemia.  Consistent  results  were  obtained  in  a  second  experiment  ex- 


232  AARON  GIBOR 

cept  that  growth  on  Dnnaliella  salina  in  this  case  was  as  good  as  on  the  other  two 
Polyblepharidaceae. 

For  ecological  purposes  it  seemed  advisable  to  find  whether  these  results  also 
hold  under  non-sterile  conditions.  The  apparent  superiority  of  the  Polyblephari- 
daceae might,  for  example,  be  due  to  the  absence  of  a  rigid  cellulose  wall ;  bacteria 
(in  the  Artcmia  gut)  might  aid  in  the  digestion  of  the  cellulose  wall  of  Platymonas 
and  Stichococcns.  An  experiment  therefore  was  conducted  under  conditions  identi- 
cal to  the  first  except  for  the  use  of  unsterilized  Artemia  larvae.  These  results 
(Table  I)  show  an  improved  growth  in  all  cultures  except  Stichococcus  sp.  with 
the  Polyblepharidaceae  still  showing  better  growth.  Bond  (1933)  found  that 
Platymonas  sp.  was  slightly  superior  to  Dnnaliella  viridis  as  food  for  Artcmia.  His 
criterion  was  the  time  in  which  Artcmia  reached  the  mating  stage :  on  Platymonas 
sp.  the  required  time  was  28  to  29  days,  on  Dnnaliella  viridis  31  days.  However 
in  one  of  our  experiments  non-sterile  Artcmia,  growing  on  Dnnaliella  viridis,  were 
found  copulating  after  13  days. 

To  investigate  whether  Stichococcus  is  producing  an  inhibitor  to  the  growth  of 
Artcmia,  a  mixed  culture  of  Stichococcus  sp.  and  Dnnaliella  viridis  was  inoculated 
with  Artcmia  larvae.  Good  development  of  the  larvae  showed  that  no  inhibitor  was 
produced. 

Microscopic  observation  established  that  the  Artemia  larvae  do  ingest  Sticho- 
coccns; the  deficient  growth  on  this  alga  is  thus  not  due  to  the  inability  of  the  ani- 
mal to  filter  and  ingest  the  smaller  cells  of  this  genus. 

On  the  basis  of  the  estimation  of  the  Stichococcns  crop  and  the  population  of 
Artemia  in  the  evaporation  ponds  Carpelan  (1953)  concluded  that  Artemia  utilizes 
only  a  small  fraction  of  the  crop  of  Sticlwcoccns.  Our  results  based  on  laboratory 
tests  corroborate  this  opinion. 

The  observations  made  in  the  experiment  on  the  nutritive  value  of  algae  for 
Artemia  aid  in  understanding  the  ecological  relationships  in  the  series  of  evaporation 
ponds. 

One  of  the  striking  facts  observed  in  the  salt  ponds  is  the  predominance  of 
Sticliococcns  in  brines  of  relatively  low  salinity  (to  about  three-fold  sea  water). 
Both  Platymonas  sp.  and  Dnnaliella  I'iridis  can  be  isolated  from  such  low  salinity 
brines,  and  both  algae  grow  well  in  these  concentrations.  However,  they  are  always 
overgrown  by  Stichococcus. 

The  finding  that  Artcmia  does  not  grow  on  Stichococcus  suggested  that  the  ani- 
mal might  act  as  a  differential  filter,  ingesting  the  algae  on  which  it  grows  well  and 
leaving  those  on  which  it  can  not  grow.  The  possibility  of  a  differential  mechani- 
cal effect  was  eliminated  by  observations  on  starved  Artcmia  put  into  a  Stichococcus 
suspension.  As  mentioned  above,  the  animals  fill  their  gut  with  this  alga  in  a  few 
minutes. 

To  determine  whether  live  cells  survive  in  the  fecal  pellets,  sterile  Artemia  were 
fed  on  a  pure  culture  of  Stichococcns.  After  feeding  for  several  hours  the  animals 
were  washed  by  transferring  them  into  a  corner  of  a  Petri  dish  containing  sterile 
sea  water.  Use  was  made  of  the  positive  phototropic  response  of  the  Artcmia.  The 
fast  swimming  animals  were  collected  from  the  opposite,  light  side  of  the  dish  and 
transferred  to  a  second  dish  for  repeated  washing.  After  4-5  such  washings  the 
animals  were  transferred  into  a  depression  slide  containing  sterile  sea  water  and  left 
for  several  hours.  Fecal  pellets  accumulated  in  the  depression  slide.  Single  pel- 


ECOLOGICAL  RELATIONSHIPS  OF  PLANKTON  233 

lets  were  picked  with  a  sterile  Pasteur  pipette  and  transferred  through  a  series  of 
sterile  sea  water  droplets.  The  washed  pellets  were  finally  inoculated  into  test  tubes 
containing  several  milliliters  of  sea  water  enriched  with  minerals,  and  kept  under 
continuous  illumination. 

Test  tubes  in  which  growth  of  algae  occurred  were  examined  microscopically 
to  determine  whether  we  were  dealing  with  the  same  algae  as  fed  to  the  animal. 
Ten  test  tubes  so  treated  were  found  to  contain  growing  Stichococcus  cultures. 
Clearly  some  cells  survived  ingestion.  An  ecological  advantage  for  one  algal  spe- 
cies over  another  might  be  established  by  even  a  slight  difference  in  such  ease  of 
digestibility  in  the  gut  of  a  non-differentiating  filter  feeder. 

In  order  to  investigate  this  possibility  in  a  mixed  algal  population  the  following 
experiment  was  performed.  Young  growing  cultures  of  Stichococcus,  Dunaliclla 
viridis  and  a  mixture  of  both  algae  were  divided  into  two  equal  portions  of  10  ml. 
each  in  50-ml.  Erlenmeyer  flasks.  Into  one  flask  of  each  pair  about  12  Arteniia 
larvae  were  added.  The  flasks  were  kept  under  continuous  illumination. 

After  ten  days  the  following  results  were  recorded :  the  Stichococcus  flasks  were 
both  equally  green,  and  growing.  No  appreciable  growth  of  the  larvae  had  oc- 
curred although  some  were  still  alive.  The  Dunaliclla  viridis  cultures  were  entirely 
different.  The  flask  without  the  animals  was  bright  green,  while  the  flask  con- 
taining the  animals  was  completely  clear,  and  with  the  larvae  growing  well.  The 
results  of  the  mixed  cultures  were  striking.  The  flask  without  the  animals  was 
deep  green  and  growth  of  both  algae  was  obvious.  (The  presence  of  Dunaliella 
viridis  was  easily  ascertainable  without  a  microscope  since  motile  cells  accumulated 
on  the  illuminated  side  of  the  flask. )  The  flask  with  the  animals  was  not  as  green 
as  the  control  flask  and  no  obvious  population  of  Dunaliclla  viridis  could  be  seen  by 
superficial  observation.  The  animals  in  this  culture  were  alive  and  growing,  but 
they  were  not  as  large  as  the  animals  grown  on  the  pure  Dunaliclla  viridis  culture. 
In  the  mixed  culture  without  animals  microscopic  examination  revealed  the  pres- 
ence of  a  large  number  of  both  Stichococcus  and  Dunaliclla  cells.  In  the  flask  con- 
taining the  animals,  very  few  Dunaliclla  cells  could  be  seen  among  the  many 
Stichococcus  cells.  Later  observations  on  the  flasks,  three  weeks  after  the  begin- 
ning of  the  experiment,  revealed  that  the  well  developed  Artemia,  which  had  eaten 
and  cleared  the  Dunaliclla  viridis,  were  dead,  apparently  of  starvation.  There  was 
an  indication  of  fresh  growth  of  Dunaliclla  viridis.  The  animals  were  also  dead  in 
the  dense  Stichococcus  sp.  culture.  On  the  mixed  culture  several  living  individuals 
were  seen.  However  they  were  not  as  well  developed  as  the  animals  which  grow  on 
the  pure  Dunaliclla  viridis  culture. 

A  similar  experiment  was  performed  with  a  different  zooplankton  organism,  the 
copepod,  TigriopHs.  The  results  were  similar  to  those  with  Arteniia.  These  ani- 
mals could  not  utilize  the  SticJwcoccus  cells  available  to  them ;  Dunaliella  viridis 
cells,  on  the  other  hand,  were  readily  consumed.  The  ecological  implications  of 
these  observations  are  of  considerable  importance.  In  standard  oceanographic  ob- 
servations all  the  phytoplankton  is  considered  as  available  food  to  the  zooplankton. 
The  curious  phenomenon,  often  observed,  of  the  scarcity  of  zooplankton  in  waters 
rich  in  phytoplankton,  and  vice-versa,  was  explained  as  due  either  to  overgrazing 
(Harvey  et  al.,  1935)  or  to  animal  exclusion  by  production  of  inhibitors  (Ryther, 
1954).  The  present  study  suggests  the  possibility  that  certain  phytoplankton  or- 
ganisms are  not  suitable  food  for  some  planktonic  grazers. 


234  AARON  GIBOR 

SUMMARY 

1.  Several  planktonic  algae  from  the  brines  of  the  sea  water  evaporation  ponds 
were  fed  to  the  brine  shrimp  Art  curia.     They  were  found  to  differ  in  their  nutritive 
value  to  this  filter-feeding  animal.     One  of  these  algae,  Stichococcns  sp.,  could  not 
be  utilized  by  the  animal  as  a  food  source. 

2.  Controlled  experiments  of  the  effect  of  filter-feeding  Artemia  and  Tigriopus 
on  a  mixed  population  of  two  unicellular  algae  indicate  that  the  animals  are  capable 
of  acting  as  differential  grazers.     The  heavy  bloom  of  Stichococcus  in  the  evapora- 
tion ponds  could  be  due  to  the  effect  of  preferential  digestion  of  competing  algae  by 
grazing  animals. 

LITERATURE  CITED 

BOND,  R.  M.,  1933.  A  contribution  to  the  study  of  the  natural  food  cycle  in  aquatic  environ- 
ments with  particular  consideration  of  micro-organisms  and  dissolved  organic  matter. 
Bull.  Bint/ham  Occanogr.  Coll.,  4  (IV)  :  1-89. 

BURLEW,  J.  S.,  1953.  Algal  culture  from  laboratory  to  pilot  plant.  Carnegie  hist.  Washington 
Pub.,  600,  357  pp. 

CARPELAN,  L.  H.,  1953.  The  hydrobiology  of  the  Alviso  salt  ponds.  Thesis,  Stanford  Uni- 
versity. 

DEMPSTER,  R.  P.,  1953.  The  use  of  larval  and  adult  brine  shrimp  in  aquarium  fish  culture. 
Calif.  Fish  and  Game,  39 :  355-364. 

GIBOR,  A.,  1956.     The  culture  of  brine  algae.     Biol.  Bull,  111:  223-229. 

GOTAAS,  H.  B.,  W.  J.  OSWALD  AND  H.  F.  LUDWIG,  1954.  Photosynthetic  reclamation  of  or- 
ganic wastes.  Scl.  Monthly,  79 :  368-378. 

HARVEY,  H.  W.,  L.  N.  H.  COOPER,  M.  V.  LEBOUR  AND  F.  S.  RUSSELL,  1935.  Plankton  produc- 
tion and  its  control.  /.  Mar.  Biol.  Assoc.  20:  407-441. 

RYTHER,  J.  H.,  1954.     Inhibitory  effect  of  algae  on  Daphnia.     Ecology,  35  :  522-533. 

SMITH,  J.  H.  C.,  1953.  Cultivation  of  Chlorclla  in  a  vertical  sedimentation  tube.  Carnegie  hist. 
Washington  Pub.,  600 :  143-152. 


FURTHER  OBSERVATIONS  OF  HOMING  TERNS 

TIMOTHY  H.  GOLDSMITH  AND  DONALD  R.  GRIFFIN 
Biological  Laboratories,  Harvard  University,  Cambridge  38,  Massachusetts 

Among  the  most  promising  recent  experiments  in  the  field  of  bird  orientation 
have  been  those  of  Kramer  (1952,  1953)  and  Matthews  (1953a,  1953b)  in  which 
pigeons  and  Manx  shearwaters  released  in  unfamiliar  territory  oriented  approxi- 
mately towards  home  while  still  within  view  of  the  release  point.  This  ability  to 
choose  the  homeward  direction  within  a  few  minutes  after  release  seems  to  be  lost 
when  the  sun  is  obscured  by  clouds.  We  have  recently  reported  homing  experi- 
ments in  which  common  terns  (Sterna  hinmdo)  showed  a  directional  orientation  by 
tending  to  fly  southeast  whether  the  home  direction  was  northeast,  south,  or  south- 
west (Griffin  and  Goldsmith.  1955).  In  these  experiments  terns  from  breeding 
colonies  in  Massachusetts  and  Maine  were  removed  to  inland  areas,  released  singly, 
and  observed  with  binoculars  for  as  long  as  possible  from  two  points  about  half  a 
mile  apart.  The  simultaneous  cross  bearings  thus  obtained  allowed  us  to  recon- 
struct the  first  few  minutes,  and  the  first  one  to  two  miles,  of  the  birds'  flight.  The 
terns  showed  a  consistent  tendency  to  head  approximately  southeast  when  the  sun 
was  visible ;  but  with  heavy  cloud  cover  they  seemed  to  scatter  at  random.  The 
southeasterly  headings  were  independent  of  the  home  direction,  of  the  wind  direc- 
tion, of  the  time  of  day,  and  (except  for  bodies  of  water)  of  local  topography. 
Even  though  common  terns  do  not  seem  to  be  as  skillful  navigators  as  the  pigeons 
studied  by  Kramer  and  Matthews,  the  results  are  of  interest  because  they  add  to  the 
growing  body  of  evidence  pointing  to  the  sun  as  an  important  factor  in  the  orienta- 
tion of  birds. 

Matthews  (1955)  has  questioned  whether  these  results  do  in  fact  represent  a 
directional  tendency  on  the  part  of  the  terns  rather  than  "a  crude  form  of  home- 
ward orientation."  We  therefore  wished  to  extend  our  previous  observations  by 
adding  a  release  point  at  which  home  would  lie  in  the  opposite  direction  from 
southeast.  A  further  reason  for  this  additional  experiment  was  our  suggestion  that 
the  habit  of  flying  southeast  when  suddenly  released  in  unfamiliar  inland  territory 
might  be  an  advantageous  one  for  terns  nesting  along  the  eastern  coast  of  the  United 
States  since  it  would  bring  them  quickly  back  to  the  coast.  Both  considerations  in- 
dicated an  experiment  to  compare  the  initial  headings  of  terns  nesting  in  the  Great 
Lakes  region  with  those  from  the  Atlantic  coast. 

METHODS 

The  release  point  selected  for  this  experiment  was  the  airport  at  Cortland,  New 
York,  about  midway  between  Cape  Cod  and  Detroit.  This  airport  is  about  12 
miles  from  the  nearest  lake  of  any  size  (Skaneateles),  it  has  little  traffic,  and  the  ter- 
rain affords  a  relatively  clear  view  in  all  directions.  Thirty-two  terns  were  cap- 
tured late  on  the  afternoon  of  June  8,  1955  at  a  nesting  colony  at  Metropolitan 

235 


236 


TIMOTHY  H.  GOLDSMITH  AND  DONALD  R.  GRIFFIN 


Beach,  Michigan  (15  miles  north  of  Detroit  on  the  shore  of  Lake  St.  Clair)  ;  on  the 
morning  of  June  9,  nine  others  were  caught  on  Penikese  Island,  Massachusetts. 
Both  groups  were  transported  by  automobile  in  boxes  covered  so  as  to  prevent  the 
birds  from  observing  their  surroundings,  and  all  were  released  on  June  10  between 
9:30  A.M.  and  6:30  P.M.  E.S.T.,  after  24—48  hours  in  captivity.  The  methods  of 
capture  and  handling  were  the  same  as  in  our  previous  experiments.  The  terns 
were  again  observed  through  binoculars  mounted  on  tripods  equipped  with  alidades 
so  that  bearings  could  be  noted  by  an  assistant.  Three  observers  participated  in 
this  experiment,  so  that  the  bearings  were  taken  from  the  three  corners  of  a  tri- 
angle with  legs  approximately  0.4,  0.6,  and  0.8  mile  in  length — the  eastern  corner 
being  the  release  point.  The  terns  from  the  two  colonies  were  released  in  an  ir- 


N 


W 


N 


H 


MICH. 


MASS. 


0° 


lOCf 


200° 


300° 


FIGURE  1.  Graphic  comparison  of  the  initial  headings  of  25  common  terns  from  colonies 
in  Massachusetts  and  Michigan  released  the  same  day  at  Cortland,  New  York.  Homeward 
direction  is  indicated  by  an  "H",  mean  heading  of  each  group  by  an  unmarked  vertical  line. 


regular  sequence,  and  each  bird  was  set  free  only  after  the  previous  one  had  been 
lost  from  view  by  all  three  observers.  No  attempt  was  made  to  check  the  homing 
performance  by  subsequent  observations  of  the  nests,  since  earlier  studies  had 
shown  that  the  homing  times  of  terns  are  too  long  to  contribute  any  useful  informa- 
tion about  the  route  flown  (Griffin,  1943). 

We  should  like  to  express  our  gratitude  to  R.  Gibbs,  W.  Jablonski,  Mrs.  B. 
Johnston,  W.  Nickell,  A.  Novick,  R.  Payne,  and  R.  Risebrough  whose  help  in  cap- 
turing and  observing  the  birds  made  this  experiment  possible,  as  well  as  to  the 
Office  of  Naval  Research  which  provided  financial  support  through  a  research  con- 
tract with  Harvard  University. 


FURTHER  OBSERVATIONS  OF  HOMING  TERNS 


237 


OBSERVATIONS  OF  INITIAL  HEADINGS 

All  of  the  terns  were  released  under  clear  or  partly  cloudy  skies  when  the  posi- 
tion of  the  sun  was  evident,  though  the  sun  itself  was  sometimes  temporarily  hid- 
den behind  a  cumulus  cloud.  The  day  was  warm,  and  there  were  numerous  up- 
drafts  on  which  the  terns  tended  to  climb  and  soar.  We  thus  lost  sight  of  some 
birds  at  heights  of  several  hundred  feet  above  the  ground  before  they  had  indicated  a 
definite  heading  away  from  the  release  point.  Unknown  to  us  at  the  time  the  re- 
leases were  begun,  there  was  also  a  small  pond  1.2  miles  to  the  southwest  of  the 
release  point  which  attracted  some  of  the  terns. 


FIGURE  2.  Sample  flight  paths  of  eight  common  terns.  Solid  circles  and  triangles  stand 
for  crossbearings,  open  circles  and  triangles  for  single  bearings.  Directions  of  the  home  col- 
onies are  shown  by  the  arrows  in  the  box  (H)  at  the  left.  Positions  of  the  observers  are  rep- 
resented by  the  corners  of  the  large  triangle.  Contour  interval  is  100  feet.  See  the  text  for  a 
discussion. 

The  results  are  summarized  in  Figure  1,  in  which  the  points  of  the  compass  are 
"unrolled"  on  the  horizontal  axis ;  each  bird  is  represented  by  a  vertical  rectangle. 
Only  those  birds  are  included  that  gave  a  distinct  heading ;  the  nine  attracted  to 
the  pond  and  the  seven  lost  while  still  circling  less  than  3000  feet  from  the  release 
point  have  been  omitted.  Home  direction  is  indicated  by  a  capital  "H"  and  a  verti- 
cal line,  the  mean  heading  by  an  unmarked  vertical  line.  The  graph  shows  that  the 
birds,  from  the  Michigan  colony  at  least,  tended  to  head  in  a  southeasterly  direction. 
The  average  of  the  19  Michigan  terns  was  139°  (40^237°),  while  the  average  head- 
ing for  the  6  Massachusetts  terns  was  93°  (64—130°).  In  previous  experiments 


TIMOTHY  H.  GOLDSMITH  AND  DONALD  R.  GRIFFIN 

with  terns  from  coastal  colonies  the  average  initial  headings  were  140°,  142°,  142°, 
and  149°  when  the  home  directions  were  115°,  115°,  44°,  and  211°,  respectively. 
The  Michigan  terns  released  at  Cortland  thus  showed  nearly  the  same  average 
heading  as  the  coastal  terns  studied  in  previous  seasons.  The  more  easterly  head- 
ings of  the  Massachusetts  terns  released  at  Cortland  have  no  obvious  explanation, 
but  there  were  too  few  birds  involved  to  render  the  difference  between  the  two 
groups  significant.  While  the  individual  headings  varied  more  widely  at  Cortland 
than  in  our  previous  experiments,  the  distribution  is  by  no  means  random,  nor  do 
the  average  headings  differ  significantly  from  our  previous  results. 

Figure  2  is  a  selection  of  eight  sample  flight  paths  plotted  from  the  simultaneous 
cross  bearings  and  superimposed  on  a  topographic  map  of  the  area.  The  portions 
of  the  lines  marked  by  open  circles  and  triangles  represent  parts  of  the  path  for 
which  only  one  observer  had  the  bird  in  view  and  are  extrapolations  of  the  earlier 
part  of  the  flight  (solid  circles  and  triangles)  for  which  there  were  cross  bearings. 
For  clarity,  some  of  the  first  cross  bearings  from  each  flight  path  have  been  omitted. 
It  is  from  such  flight  paths  as  these  that  Figure  1  was  constructed.  Birds  num- 
bered 5,  6,  7,  and  8  (triangles)  are  from  the  Michigan  colony,  which  lies  about  335 
miles  to  the  west.  Numbers  5  and  8  represent  the  extremes  of  the  distribution ; 
numbers  6  and  7,  as  well  as  the  other  fifteen  from  Figure  1,  lie  between  these  two. 
Similarly  numbers  1,  2,  3,  and  4  (circles)  are  terns  from  the  Massachusetts  colony, 
about  290  miles  to  the  ESE,  and  numbers  1  and  4  are  the  extremes  of  the  Massachu- 
setts distribution.  This  figure  shows  that  the  topography  had  no  obvious  influence, 
although  certain  of  the  birds  (not  shown  in  Figure  2)  were  attracted  to  a  pond  to 
the  southwest.  For  example,  note  that  number  7  flew  over  a  hill  which  rises  to 
over  300  feet  above  the  release  point  while  number  2  flew  over  the  city  of  Cortland. 
While  the  birds  could  certainly  see  terrain  which  lies  beyond  the  boundaries  of  this 
map,  reference  to  a  smaller  scale  map  shows  no  topographical  features  which  ex- 
plain the  southeast  headings. 

DISCUSSION 

This  experiment  demonstrates  that  the  tendency  to  fly  in  a  southeasterly  direction 
when  first  released  in  unknown,  inland  territory  is  not  confined  to  common  terns 
nesting  along  the  coast ;  it  was  equally  evident  in  terns  from  a  colony  in  the  Great 
Lakes  area  even  though  for  these  birds  home  lay  almost  due  west.  The  same  di- 
rectional tendency  has  thus  been  observed  when  the  home  direction  was  northeast, 
east-southeast,  south,  southwest  and  west.  Austin  (1953),  who  has  analyzed  the 
migration  route  of  this  species  on  the  basis  of  banding  returns,  has  shown  that  both 
the  Great  Lakes  and  New  England  tern  populations  have  a  pronounced  southeast 
component  in  the  fall  migration  route.  But  whether  this  is  relevant  in  the  case  of 
birds  removed  from  their  nesting  grounds  during  the  breeding  season  is  open  to 
question. 

Arnould-Taylor  and  Malewski  (1955)  have  recently  suggested  that  topographic 
cues  have  been  responsible  for  most  of  the  results  obtained  by  observing  initial  head- 
ings of  homing  birds.  That  topography  may  be  the  dominating  influence  under 
certain  conditions  has  been  firmly  established;  for  example,  Griffin  (1952)  re- 
ported that  in  a  series  of  airplane  observations  of  homing  pigeons,  certain  of  the 
birds  followed  roads,  railroads,  and  lake  shores.  We  have  pointed  out  that  in  these 
experiments  with  common  terns,  local  bodies  of  water  attract  many  birds ;  but, 


FURTHER  OBSERVATIONS  OF  HOMING  TERNS  239 

when  precautions  are  taken  to  avoid  release  points  with  ponds  in  the  vicinity,  the 
terns  tend  to  fly  approximately  southeast.  Because  it  is  so  difficult  to  explain  such 
observations  on  the  basis  of  topographic  cues,  it  is  perhaps  too  soon  to  abandon  all 
thought  that  birds  employ  some  more  refined  method  of  navigation. 

SUMMARY 

1.  Our  observations  of  the  initial  flight  directions  of  common  terns  released  in 
unfamiliar  territory  have  been  extended  to  include  birds  from  the  Great  Lakes 
region  as  well  as  from  the  New  England  coast.     When  terns  from  both  populations 
were  released  on  the  same  day  at  Cortland,  New  York,  both  groups  showed  a 
tendency  to  head  approximately  southeast. 

2.  The  first  mile  or  two  of  the  terns'  flight  paths  were  plotted  on  a  topographic 
map  of  the  area.     Aside  from  the  fact  that  a  small  pond  attracted  some  of  the  birds, 
topography  did  not  offer  any  apparent  explanation  of  their  headings,  and  a  few 
persisted  on  a  southeasterly  course  over  moderately  high  hills. 

LITERATURE  CITED 

ARNOULD-TAYLOR,  W.  E.,  AND  A.  M.  MALEWSKI,  1955.     The  factor  of  topographical  cues  in 

bird  homing  experiments.     Ecology,  36:  641-646. 
AUSTIN,   O.  L.,   1953.     The  migration  of  the  common  tern    (Sterna   hirundo)    in  the   western 

hemisphere.     Bird  Banding,  24 :  39-55. 
GRIFFIN,    D.    R.,    1943.     Homing    experiments    with    herring    gulls    and    common    terns.     Bird 

Banding,  14 :  7-33. 
GRIFFIN,  D.  R.,  1952.     Airplane  observations  of  homing  pigeons.     Bull.  Mus.  Comp.  Zoo/.,  107  : 

411-440. 
GRIFFIN,  D.  R.,  AND  T.  H.  GOLDSMITH,  1955.     Initial  flight  directions  of  homing  birds.    Biol. 

Bull,  108:  264-276. 

KRAMER,  G.,  1952.     Experiments  on  bird  orientation.     Ibis,  94 :  265-285. 
KRAMER,    G.,    1953.     Wird   die    Sonnenhohe   bei    der    Heimfindeorientierung    verwertet?     /.    /. 

Ornithol,  94:  201-219. 

MATTHEWS,  G.  V.  T.,  1953a.     Sun  navigation  in  homing  pigeons.    /.  Exp.  Biol.,  30 :  243-267. 
MATTHEWS,  G.  V.  T.,  1953b.     Navigation  in  the  Manx  shearwater.     /.  Exp.  Biol.,  30:  370-396. 
MATTHEWS,  G.  V.  T.,  1955.     Bird  navigation.     Cambridge  University  Press,  England. 


MODIFICATION  OF  X-RAY  INJURY  TO  HYDRA  LITTORALIS  x 

BY  POST-IRRADIATION  TREATMENT  WITH  MAGNESIUM 

SULFATE  AND  GLUTATHIONE  2 

HELEN  D.  PARK 

National  Institute  of  Arthritis  and  Metabolic  Diseases,  National  Institutes  of  Health,  Public 
Health  Service,  U.  S.  Department  of  Health,  Education,  and  Welfare, 

Bethesda,  Maryland 

Very  few  studies  have  been  reported  on  the  damaging  effects  of  ionizing  radia- 
tions on  Hydra.  Zawarsin  (1929),  Strelin  (1929)  and  Evlakhova  (1946),  how- 
ever, studied  the  effect  of  sublethal  doses  of  x-rays  and  found  that  inhibition  of  bud- 
ding and  regeneration  varied  with  the  dose  of  radiation  used.  Daniel  and  Park 
(1951,  1953)  reported  a  toxic  effect  of  x-ray-treated  media  on  Hydra  tentacles  and 
(1954)  direct  x-ray  damage  leading  to  death  in  24  hours. 

A  number  of  investigators  (Barren  ct  al.,  1949;  Bellack  and  Krebs,  1951 ;  Chap- 
man and  Cronkite,  1950;  Chapman  ct  al.,  1950;  Patt  et  al.,  1950;  Bacq,  1951 ;  Cron- 
kite  et  al.,  1951)  demonstrated  that  glutathione  modifies  some  of  the  biological  ef- 
fects of  ionizing  radiations.  In  general,  protection  resulted  only  if  the  glutathione 
was  given  before  irradiation.  Similarly,  in  most  cases  cysteine  has  to  be  present  at 
the  time  of  irradiation  in  order  to  exert  a  protective  effect  (see  Patt,  1953). 
Barren  and  co-workers,  however,  found  that  when  glutathione  was  added  to 
aqueous  solutions  of  succinoxidase  after  irradiation,  the  enzyme  was  partially  reac- 
tivated. Patt  ct  al.  (1952)  reported  protection  to  mammalian  thymocytes  when 
cysteine  was  added  immediately  after  irradiation. 

Daniel  and  Park  (1954)  showed  that  when  hydras  given  25,000  r  were  placed 
immediately  in  a  dilute  solution  of  salts  containing  either  MgSO4  or  MgCl2,  about 
twice  as  many  survived  24  hours  as  were  living  in  the  same  salt  solution  without 
Mg++.  In  view  of  this  result,  and  of  the  few  cases  reporting  modification  of  x-ray 
damage  by  post-irradiation  treatment  with  sulfhydryl  compounds,  the  present  stud- 
ies were  made  on  the  effects  of  continuous  post-irradiation  treatment  with  MgSO4 
plus  glutathione  on  survival  and  on  budding  of  hydras. 

MATERIALS  AND  METHODS 

The  hydras  used  in  the  present  studies  were  from  a  clone  culture  grown  in  the 
laboratory  at  a  room  temperature  of  25°  ±  1.5°  C.  The  cultures  were  kept  in  a 
standard  salt  solution  of  1.7  X  1Q-3  M  NaCl,  5.4  X  10"5  M  KC1  and  3.3  X  10~4  M 
CaCL  in  double-distilled  water  (the  second  distillation  being  from  glass).  This 
solution  contained  the  same  salts  and  in  approximately  the  same  concentration  as 

1  Kindly  identified  by  Dr.  Libbie  H.  Hyman,  American  Museum  of  Natural  History,  New 
York,  N.  Y. 

2  These  data  are  from  a  thesis  submitted  to  the  Graduate  Council  of  the  George  Washington 
University  by  Helen  D.  Park  in  partial  fulfillment  of  the  requirements  for  the  degree  of  Doctor 
of  Philosophy. 

240 


MODIFICATION  OF  X-RAY  INJURY  241 

that  used  by  Daniel  and  Park  (1954),  and  will  hereafter  be  referred  to  as  "stand- 
ard saline."  The  hydras  were  fed  newly  hatched  brine  shrimp  daily  and  were 
washed  and  changed  to  fresh  standard  saline  one  hour  after  each  feeding.  They 
were  transferred  to  clean  dishes  once  a  week.  Under  these  conditions  the  hydras 
reproduced  asexually  by  budding. 

For  all  experiments,  hydras  of  equal  size,  without  buds,  were  selected  from  the 
stock  cultures  before  the  daily  feeding  and  washed  in  standard  saline  before  treat- 
ment. The  hydras  were  irradiated  in  a  Pyrex  glass  dish  containing  50  ml.  of 
standard  saline  which  gave  a  solution  depth  of  17  mm.  The  hydras  immediately 
were  washed  with  standard  saline,  then  within  10  minutes  were  placed  in  the  solu- 
tions to  be  studied.  Except  while  the  hydras  were  under  observation  the  dishes 
were  kept  in  moist  chambers. 

Irradiation  factors  were  50  kv  constant  potential,  50  ma  beryllium  window  tube ; 
2700  r  per  minute.  Dose  determinations  were  made  by  the  method  of  Andrews  and 
Shore  (1950).  An  aluminum  plate  0.020  inch  thick  served  as  an  x-ray  filter  and 
as  a  dish  cover.  Water  cooling  of  the  irradiation  dish  kept  the  temperature  of  the 
contents  within  2°  C.  of  the  temperature  of  the  laboratory. 

RESULTS 

Survival  experiments 

In  order  to  compare  the  effect  of  glutathione  with  that  of  MgSO4.  equal  numbers 
of  hydras  exposed  to  25,000  r  were  placed  in  (1)  standard  saline;  and  (2)  5.0  X 
10-*  M  MgSO4,  (3)  1.0  X  10-5  M  glutathione,  and  (4)  5.0  X  10~4  M  MgSO,  plus 
1.0  X  10~5  M  glutathione,  each  in  standard  saline.  Non-irradiated  controls  were 
also  treated  with  the  four  solutions.  The  MgSO4  concentration  was  within  the 
range  previously  found  by  Daniel  and  Park  to  protect  hydras  exposed  to  25,000  r. 
Within  this  range  the  protective  effect  of  MgSO4  was  not  a  function  of  the  ionic 
strength  of  the  solutions.  The  concentration  of  glutathione  had  previously  been 
shown  to  modify  a  toxic  effect  of  irradiated  water  on  hydra  tentacles.  The  ani- 
mals were  left  in  their  respective  solutions  24  hours,  at  which  time  the  survivors 
were  counted.  Five  complete  experiments,  each  consisting  of  10  animals  per  group, 
were  carried  out  in  a  period  of  30  days. 

TABLE  I 

Effect  of  post-irradiation  exposure  to  MgSO*  and  glutathione  on  survival  of  hydras  after  25,000  r. 

Fifty  hydras  in  each  treatment  group 

Number  alive  after  24  hours 
Treatment  Irradiated  Non-irradiated 

Standard  saline  12±2  50 

5.0  X  10-W  MgSO4  in  standard 

saline  30±4  50 

1.0  X  10-8Af  glutathione  in 

standard  saline  18±4  50 

5.0  X  10-W  MgSO4  +  1.0  X  10~5 

M  glutathione  in  standard  saline  21±4  50 

Standard  error  estimated  from  variation  among  5  experiments. 


242  HELEN  D.  PARK 

As  shown  in  Table  I  none  of  the  non-irradiated  hydras  died.  All  of  the  irradi- 
ated groups  showed  by  chi  square  test  significantly  3  fewer  survivors  than  their  con- 
trols. The  only  statistically  significant  differences  among  the  irradiated  groups 
were  between  the  hydras  in  MgSO4  and  those  in  saline,  and  between  those  in  MgSO4 
and  those  in  glutathione.  There  is  not  sufficient  statistical  evidence  to  assert  defi- 
nitely that  either  glutathione  or  the  combined  treatment  had  a  protective  effect 
against  the  radiation.  The  present  results  confirm  the  conclusion  of  Daniel  and 
Park  that  MgSO4  had  a  specific  protective  effect  against  the  radiation. 

Budding  experiments 

Hydras  reproduce  asexually  by  the  formation  of  buds  which  constrict  from  the 
parent  as  adult  hydras.  The  process  involves  increase  in  the  mass  of  protoplasm, 
cell  division  and  differentiation.  In  the  stock  cultures  maintained  in  this  laboratory, 
the  development  of  a  bud,  from  the  time  it  is  first  recognizable  until  its  separation 
from  the  parent,  takes  from  two  to  four  days. 

The  effects  of  continuous  post-irradiation  exposure  to  MgSO4  and  glutathione 
on  budding  were  studied  using  4500  r,  a  dose  the  author  had  previously  found  to  be 
approximately  one-third  that  necessary  to  inhibit  bud  production  completely  for  10 
days.  Forty  irradiated  and  40  non-irradiated  hydras  in  groups  of  10  \vere  put  in 
the  standard  saline,  MgSO4  saline,  glutathione  saline  and  MgSO4  +  glutathione 
saline  solutions  previously  described.  Beginning  on  the  first  day  after  irradiation, 
the  hydras  were  fed  daily.  All  descendants  derived  from  the  original  10  hydras 
in  each  group  were  kept  with  the  parents.  Each  day  for  11  days  adults  and  at- 
tached buds  were  counted  as  separate  individuals  and  all  were  transferred  to  fresh 
solutions  in  clean  dishes.  Five  experiments,  each  including  all  of  the  treatment 
groups,  were  carried  out  at  intervals  over  a  period  of  two  months. 

For  all  analyses  4  of  the  data,  statistical  significance  or  the  lack  thereof  was  de- 
termined by  comparing  an  average  effect  over  the  five  replicate  experiments  with 
the  variation  of  this  effect  among  the  five  experiments. 

Figure  1  shows,  from  days  zero  through  eleven,  the  average  number  of  adults 
plus  buds  present  per  experiment  in  each  treatment  group.  Among  the  non- 
irradiated  hydras,  those  in  MgSO4  and  MgSO4  +  glutathione  produced  significantly 
greater  numbers  of  descendents  by  the  end  of  1 1  days  than  those  in  standard  saline 
or  glutathione.  Since  neither  of  the  other  two  intergroup  differences  among  non- 
irradiated  hydras  was  significant,  it  seems  probable  that  during  the  combined  ex- 
posure it  was  MgSO4  which  caused  the  increase  in  budding. 

Comparison  of  the  groups  in  saline  alone  shows  that  4500  r  depressed  signifi- 
cantly the  budding  rate.  The  data  for  the  irradiated  hydras  in  standard  saline  sug- 
gest that  the  normal  budding  rate  was  regained  by  day  nine,  but  since  a  parabola 
does  not  fit  the  points  better  than  a  straight  line,  the  break  in  the  curve  may  be 
fortuitous. 

The  budding  rate  of  the  irradiated  hydras  in  MgSO4  wras  not  significantly 
greater  than  that  of  their  irradiated  controls.  On  the  other  hand,  the  irradiated 
hydras  in  glutathione  produced  buds  at  a  significantly  greater  rate  than  the  irradiated 

3  The  .05  level  of  probability  was  used  throughout  the  present  work. 

4  The  author  wishes  to  thank  Mr.  Jerome  Cornfield  of  the  National  Institutes  of  Health  for 
his  help  in  analyzing  the  data  statistically. 


MODIFICATION  OF  X-RAY  INJURY 


243 


controls.  The  irradiated  hydras  in  MgSO4  +  glutathione  produced  buds  faster 
than  the  irradiated  controls  or  the  irradiated  hydras  in  MgSO4  or  the  irradiated 
hydras  in  glutathione,  and  at  the  same  rate  as  the  non-irradiated  hydras  in  standard 
saline.  It  can  be  concluded,  therefore,  that  under  the  conditions  of  these  experi- 
ments, continuous  post-irradiation  exposure  to  MgSO4  +  glutathione  restored  the 
budding  rate  of  irradiated  hydras  to  that  of  non-irradiated  hydras  in  standard  saline. 
In  addition  to  showing  the  budding  rates  of  all  the  groups,  Figure  1  shows,  on  the 


UJ 


cc 

UJ 
CL 
X 

UJ 

ID 

cc 
o 


UJ 

(/) 

UJ 
DC 
CL 


Q 

>- 
I 


cc 

UJ 

CD 


100 


+  Mg 

+  GSH 

CONTROL 

+  Mg  +  GSH 

+  Mg 

+  GSH 

CONTROL 


2  3 

DAYS 


4 
AFTER 


56789 
START    OF    TREATMENT 


FIGURE  1.     Effect  of  post-irradiation  treatment  with  MgSO4  and  glutathione  on  budding  of  hy- 
dras after  4500  r.     Treatment  solutions  were  made  in  standard  saline. 


average,  the  time  at  which  all  10  of  the  hydras  in  each  group  initiated  their  first 
buds  (i.e.,  when  20  adults  and  buds  were  present  in  each  group).  The  data  for  the 
individual  experiments  show  that  all  50  of  the  non-irradiated  hydras  in  standard 
saline  initiated  their  first  buds  by  day  six.  The  total  number  of  irradiated  hydras 
in  each  treatment  group  that  had  initiated  their  first  buds  by  the  time  all  first  buds 
appeared  in  the  non-irradiated  standard  saline  group  was :  standard  saline  17, 
MgSO4  39,  glutathione  35,  and  MgSO4  +  glutathione  47.  It  can  be  concluded  that 
one  of  the  effects  of  the  radiation  was  to  delay  the  time  of  appearance  of  first  buds. 


244 


HELEN  D.  PARK 


Magnesium  sulfate,  glutathione,  and  MgSO4  +  glutathione  reduced  the  severity  of 
the  radiation  effect,  but  only  the  combined  treatment  shortened  the  time  of  first  bud 
initiation  to  that  of  the  non-irradiated,  saline  controls. 

Since  the  irradiated  hydras  in  MgSO4  +  glutathione  produced  buds  faster  than 
the  irradiated  hydras  in  MgSO4  alone  or  glutathione  alone,  the  effect  of  concentra- 
tion of  the  two  agents  on  budding  after  irradiation  was  studied  in  order  to  ascertain 
whether  the  greater  effect  produced  by  the  two  agents  together  is  valid  when  related 
to  optimal  effects  of  each  when  used  separately.  Accordingly,  hydras  were  irradi- 
ated with  4500  r  and  placed  in  groups  of  five  in  solutions  in  which  both  MgSO4  and 
glutathione  concentrations  were  varied  from  %  to  16  times  those  used  in  the  pre- 
ceding experiments.  The  hydras  were  counted  on  the  eleventh  day  after  irradiation. 

Table  II  shows  the  mean  number  of  hydras  present  on  the  eleventh  day  in  each 
treatment  group.  The  results  presented  in  column  1  show  that  up  to  a  concentra- 
tion of  8.0  X  10"3  mole  per  liter,  MgSO4  did  not  modify  the  inhibiting  effect  of 
4500  r  of  x-rays.  The  results  shown  in  line  1  of  the  table  indicate  that  glutathione  in 

TABLE  II 

Effect  of  concentration  of  MgSOt  and  glutathione  on  budding  of  hydras  after  4500  r. 
Mean  number  of  individuals  present  on  llth  day  post-irradiation  per  5  hydras  treated 


Moles  per  liter  of  glutathione 

Moles  per  liter 

of  MgS04 

0.0 

5.0  X  ID-6 

1.0  X  10-5 

2.0  X  lO-5 

4.0  X  ID"5 

8.0  X  10~5 

1.6  X  10-* 

0.0 

5.0(5) 

6.5(4) 

9.0(4) 

6.8(4) 

10.0(4) 

5.0(3) 

0.3(3) 

2.5X10-" 

5.5(4) 

13.0(3) 

11.3(3) 

12.3(3) 

11.0(3) 

6.0(3) 

2.0(3) 

5.0X10-" 

5.0(5) 

9.3(3) 

13.0(2) 

17.3(3) 

19.0(3) 

8.3(3) 

4.0(3) 

i.oxio-3 

5.2(4) 

9.3(3) 

12.0(3) 

10.3(3) 

13.0(3) 

1.7(3) 

2.0  X10-3 

5.4(5) 

12.0(3) 

22.0(4) 

5.0(3) 

4.0X10-3 

5.7(3) 

10.3(3) 

14.0(1) 

10.0(3) 

1.0(1) 

8.0  X10-3 

5.0(3) 

6.0(2) 

6.0(2) 

4.0(2) 

Figures  in  parentheses  =  number  of  groups  of  hydras  treated.  The  mean  number  of  individuals 
present  on  the  llth  day  in  5  groups  of  5  non-irradiated  hydras  in  standard  saline  was  12.0.  No 
concentration  tests  were  run  on  non-irradiated  hydras. 

concentrations  between  5.0  X  10~6  and  4.0  X  10  5  mole  per  liter  reduced  the  inhibi- 
tory effect  of  the  radiation  on  bud  production;  1.6  X  10~4  M  glutathione  was  highly 
toxic.  Radiation  was  probably  not  a  factor  in  this  toxicity  as  five  non-irradiated 
hydras  placed  in  this  solution  were  dead  five  days  later.  The  data  show  that  the 
optimal  concentrations  of  the  two  agents  when  supplied  together  were  in  the  range 
of  2.0  X  10-5  to  4.0  X  lO'5  M  glutathione  and  5.0  X  1Q-*  to  2.0  X  10"3  M  MgSO4. 
In  addition  they  show  that  combined  treatment  within  these  ranges  resulted  in 
greater  bud  production  than  at  optimal  concentrations  of  either  agent  used  separately. 
Furthermore,  the  amount  of  budding  that  took  place  during  exposure  to  optimal 
concentrations  of  both  agents  together  was  as  great  as  that  of  the  non-irradiated  hy- 
dras in  standard  saline. 

DISCUSSION 

At  first  glance  the  effects  of  MgSO4,  glutathione  and  combined  treatment,  after 
the  two  radiation  exposures  employed,  appear  to  be  anomalous.     Since  the  two  sets 


MODIFICATION  OF  X-RAY  INJURY  245 

of  results  are  expressed  in  different  units — number  surviving  out  of  total  number 
treated,  and  rate  of  increase  in  numbers  of  hydras  present — they  cannot  be  compared 
statistically.  Taking  the  apparent  discrepancies  at  face  value,  however,  it  seems 
reasonable  that  a  particular  agent  might  be  more  effective  against  a  mild  cellular 
damage  which  would  partially  inhibit  budding  than  against  a  more  drastic  injury 
leading  to  death  in  24  hours,  or  that  another  agent  might  be  more  effective  in 
keeping  an  animal  alive  for  24  hours  than  in  maintaining  it  in  a  reproductive  state 
for  a  period  of  10  days. 

The  mechanism  of  the  stimulating  action  of  MgSO4  on  budding  of  non-irradiated 
hydras  is  not  known.  However,  this  effect  is  perhaps  not  surprising  in  view  of  the 
fact  that  MgSO4  has  been  shown  to  affect  growth  in  many  organisms  as  widely 
separated  phylogenetically  as  bacteria  (Webb,  1953),  protozoa  (Mast  and  Pace, 
1939),  and  mammals  (Kruse  et  al.,  1932) .  Since  the  addition  of  increasing  amounts 
of  MgSO4  did  not  increase  the  amount  of  budding  of  irradiated  hydras,  we  may 
conclude  that  lack  of  MgSO4  was  not  the  factor  which  limited  budding  after  ir- 
radiation. 

Mechanisms  of  radiation  protection  have  been  considered  in  reviews  by  Ord  and 
Stocken  (1953)  and  by  Patt  (1953).  One  of  the  theories  of  protection  by  sulfhy- 
dryl  compounds  is  that  there  is  a  competition  by  -— SH  groups  for  free  radicals 
formed  from  water  in  an  irradiated  solution.  Since,  in  the  study  reported  here,  the 
hydras  were  not  irradiated  in  the  presence  of  glutathione,  and  were  washed  im- 
mediately after  irradiation  and  at  least  ten  minutes  elapsed  between  the  end  of 
irradiation  and  beginning  treatment  with  glutathione,  the  effect  on  budding  would 
seem  to  have  been  due  to  some  mechanism  other  than  a  competition  of  — SH 
groups  for  free  radicals  within  or  at  the  surface  of  the  hydra  cells. 

It  is  not  known  whether  hydras  need  an  external  source  of  glutathione  for  bud- 
ding. If  they  do,  it  is  possible  that  the  reason  glutathione  did  not  stimulate  the 
budding  of  the  non-irradiated  hydras  was  because  they  were  already  getting  a  suffi- 
cient amount  for  budding  in  their  normal  intake  of  food.  If  hydras  do  not  need  an 
external  source  of  glutathione  for  budding,  stimulation  would  not  occur  on  the  ad- 
dition of  glutathione  to  the  medium. 

It  was  not  practicable  to  determine  the  amount  of  food  eaten  by  any  of  the 
hydras.  However,  if  the  irradiated  hydras  ate  less  food  than  the  controls,  the  rate 
of  budding  would  be  reduced  from  that  of  the  controls.  The  effect  of  glutathione 
in  increasing  the  budding  rate  of  irradiated  hydras  might  thus  have  been  due  to  the 
fact  that  this  agent  stimulates  mouth  opening  (Loomis,  1955)  which  in  turn  might 
permit  the  hydras  to  consume  more  food.  A  second  possibility  is  that  the  require- 
ment of  irradiated  hydras  for  glutathione  or  some  part  of  the  molecule  may  be 
greater  than  that  of  non-irradiated  hydras,  e.g.,  because  of  the  reconstitution  of  in- 
jured regions.  Thus  the  requirement  might  not  be  met  even  with  normal  food 
intake,  causing  a  decreased  budding  rate ;  addition  of  glutathione  to  the  medium 
might  satisfy  the  greater  requirement  and  increase  the  budding  rate  over  that  of  the 
irradiated  controls. 

The  fact  that  MgSO4  stimulated  budding  of  non-irradiated  hydras  that  were 
presumably  getting  an  adequate  amount  of  glutathione  through  their  normal  intake 
of  food,  and  the  fact  that  none  of  the  concentrations  of  MgSO4  used  after  irradia- 
tion had  a  significantly  stimulating  effect  unless  added  glutathione  was  present, 


246  HELEN  D.  PARK 

suggest  the  possibility  that  in  all  hydras,  stimulation  of  budding  by  MgSO4  depends 
on  the  presence  of  an  adequate  level  of  glutathione  or  sulfhydryl  in  the  hydra  tissues. 

SUMMARY 

1.  Hydras  were  left  for  24  hours  in  solutions  of  MgSO4,  glutathione  and  MgSO4 
+  glutathione  after  exposure  to  25,000  r  x-rays.     Only  the  hydras  in  MgSO4  alone 
were  significantly  protected  against  the  effects  of  the  radiation. 

2.  Hydras  were  exposed  continuously  to  MgSO4,  glutathione,  and  MgSO4  + 
glutathione  solutions  after  4500  r.     The  rates  of  budding  in  each  solution  were  de- 
termined.    It  was  found  that : 

(a)  Forty-five  hundred  r  inhibited  the  budding  of  hydras  significantly. 

(b)  Magnesium  sulfate  and  MgSO4  +  glutathione  stimulated  budding  of  non- 
irradiated  hydras  while  glutathione  alone  did  not. 

(c)  Magnesium  sulfate  alone  did  not  significantly  modify  the  inhibitory  effect 
of  the  radiation  on  budding. 

(d)  Glutathione  alone  partially  reversed  the  inhibitory  effects  of  the  x-rays. 

(e)  Within  a  fairly  wide  range  of  concentrations  of  MgSO4  and  glutathione,  the 
two  agents  together  restored  the  budding  rate  of  irradiated  hydras  to  that 
of  the  non-irradiated  animals  in  standard  saline. 

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THE  MORPHOLOGY  AND  LIFE-HISTORY  OF  THE  DIGENETIC 
TREMATODE,  AZYGIA  SEBAGO  WARD,  1910  * 

HORACE  W.  STUNKARD  2 

New  York  University,  Neiu  York,  N.  Y .,  and  The  Marine  Biological  Laboratory, 

Woods  Hole,  Massachusetts 

The  genus  Asygia  was  erected  by  Looss  (1899)  to  contain  Fasciola  tercticollis 
Rudolphi,  1802  (=  Fasciola  lucii  Mueller,  1776,  renamed).  The  worms  were  from 
the  stomach  of  Esox  lucius.  According  to  Dawes  (1946),  this  species,  Asygia  lucii 
(Mueller,  1776)  Lithe,  1909,  infects  a  number  of  different  salmonid  fishes,  and  other 
species  of  Asygia  described  from  Europe  are  identical  with  it.  The  species  has 
been  reported  in  North  America  as  Distoina  tcrcticollc  by  Leidy  (1851)  from 
Esox  rcticulatus;  by  Stafford  (1904)  from  Esox  lucius,  Lota  maculosa  and 
Ameiurus  nigricans;  and  as  Asygia  lucii  by  Cooper  (1915)  from  Lucius  lucius 
(=  Esox  lucius),  Lucius  masquinongy  (  =  Esox  masquinongy},  Liopcrca  sp.,  and 
immature  specimens  presumably  of  the  same  species  were  found  in  Salvclinus 
naiiiayciisJi  and  Micropterus  dolomicu. 

Meanwhile,  other  species  of  Asygia  were  described  in  the  United  States  and 
Canada.  Leidy  (1851)  described  Distoina  longum  on  the  basis  of  six  specimens 
from  the  stomach  of  Esox  cstor  Lesueur,  1818  (the  American  pike),  collected  near 
Cleveland,  Ohio,  and  received  from  Professor  Spencer  F.  Baird.  The  worms  meas- 
ured 30  to  76  mm.  (3  inches)  in  length  and  as  much  as  1.6  mm.  in  breadth;  the 
maximum  diameter  of  the  oral  sucker  was  1.27  mm.  and  of  the  acetabulum  1.06  mm. 
Measurements  given  by  Leidy  for  specimens  from  the  stomach  of  E.  rcticulatus, 
which  he  identified  as  Distoina  tcrcticollc  Rudolphi,  were :  length  up  to  17  mm. ; 
width,  1.06  mm.;  oral  sucker,  0.52  mm.;  and  acetabulum  0.7  mm.  There  is  some 
confusion  here  since  Manter  (1926)  (p.  66)  reported,  "Leidy's  Dist.  tcreticollc 
(from  Esox  rcticulatus)  also  was  compared  with  them  (specimens  of  D.  longum 
from  the  Leidy  and  Cooper  collections),  and  in  the  single  specimen  available  in  the 
Leidy  collection,  the  oral  sucker,  contrary  to  Leidy's  description,  was  found  to  be 
slightly  larger  than  the  acetabulum."  Stafford  (1904)  erected  the  genus  Mcgadi- 
stomum  to  contain  specimens  from  Esox  masquinongy  which  he  regarded  as  identi- 
cal with  Distoina  longum  of  Leidy  and  distinct  from  Asygia  tercticollis.  Specimens 
of  Mcgadistonium  longum  (Leidy,  1851 )  measured  up  to  5  inches  in  length  when 
fully  extended  and  up  to  3  mm.  in  breadth,  whereas  those  identified  as  A.  tercticollis 
measured  12  mm.  in  length  and  1  mm.  in  width.  Stafford  reported  that  the  largest 
specimens  of  A.  tereticollis  were  smaller  than  immature  specimens  of  M.  longum. 
Furthermore,  he  described  worms  from  the  stomachs  of  Lota  maculosa  and  of 
Stisostedion  vitrewn  as  members  of  a  new  genus  and  species,  Mimodistomum 
angusticaudum. 

1  Research  supported  in  part  under  ONR  Contract  No.  Nonr-1497    (00). 

2  Present  address :  The  American  Museum  of  Natural  History,  New  York,  N.  Y. 

248 


LIFE-CYCLE  OF  AZYGIA  SEBAGO  249 

Marshall  and  Gilbert  (1905)  described  Asygia  loossi  from  the  large-mouth  bass, 
Micropterus  salmoides;  the  pike,  Lucius  Indus;  and  the  bowfin,  Anna  calva.  The 
worms  contained  only  a  few  eggs  and  obviously  were  not  fully  mature.  They 
measured  5  to  7  mm.  in  length,  0.5  mm.  in  width ;  the  acetabulum  was  near  the 
middle  and  the  gonads  in  the  caudal  one-sixth  of  the  body. 

Ward  (1910)  described  Asygia  scbago  from  Sahno  scbago  taken  at  Lake  Sebago, 
Maine.  All  of  seven  fishes  examined  were  infected ;  the  worms  measured  up  to  10 
mm.  in  length  and  from  0.7  to  1.0  mm.  in  width.  From  the  magnification  given, 
the  figured  specimen  was  about  6  mm.  long  and  0.8  mm.  wide.  The  average  diam- 
eter of  the  oral  sucker  was  given  as  0.68  mm.  and  the  acetabulum  was  "distinctly 
smaller."  Specimens  presumed  to  belong  to  the  same  species  were  found  in  other 
fishes  of  Lake  Sebago.  Two  worms  were  removed  from  the  stomach  of  a  single 
specimen  of  Perca  flavcsccns  and  measurements  were  given  for  one  of  them.  It 
was  4.08  mm.  long,  0.77  mm.  wide ;  the  oral  sucker  measured  0.51  by  0.57  mm.  and 
the  acetabulum  0.35  by  0.40  mm.  Four  of  nine  eels,  AnguiUa  chrysypa  (=  A. 
rostrata)  were  infected  with  an  average  of  three  worms  per  fish.  No  descriptive 
data  were  given,  so  presumably  they  conformed  to  the  specific  diagnosis.  Eleven  of 
twelve  young  Esox  reticulatus  were  heavily  infected ;  as  many  as  80  worms  were 
found  in  a  single  host.  These  parasites  were  more  slender  and  measured  10  to  18 
mm.  in  length.  It  may  be  doubted  whether  they  are  conspecific  with  the  shorter, 
more  robust  worms  from  the  other  hosts.  Ward  reported  that  smelt,  Osmcrus 
mordax,  were  eaten  by  the  larger  fishes ;  the  parasites  were  found  also  in  the 
stomachs  of  smelt,  although  in  this  host  the  worms  were  usually  smaller  and  sexually 
immature.  He  noted  that  the  specimens  identified  by  Stafford  (1904)  as  Asygia 
tereticolle  are  smaller  (12  mm.  long  and  1  mm.  wide)  than  the  European  species 
and  expressed  the  belief  that  they  may  have  been  A.  sebago. 

Goldberger  (1911)  recognized  A.  loossi  as  a  valid  species  and  did  not  mention 
A.  sebago,  as  Ward's  account  was  probably  not  available  when  he  wrote  his  paper. 
He  reported  on  specimens  collected  from  Amia  calva  taken  in  Indiana  lakes ;  certain 
of  the  worms  were  identified  as  A.  lucii,  and  others  were  described  as  members  of 
two  new  species,  Asygia  bulbosa  and  Asygia  acuminata.  Also,  he  described  worms 
from  the  stomach  of  the  rock  bass,  Ambloplites  ruprestris,  as  members  of  a  new 
genus  and  species,  Hassallins  hassalli. 

Odhner  (1911)  erected  the  family  Azygiidae  to  contain  Asygia,  Otodistouiwn, 
Lcuceruthrus,  and  Ptychogonimus.  He  stated  that  in  the  genus  Asygia,  measure- 
ments of  eggs  and  extent  of  vitellaria  have  little  value  for  specific  determination. 
He  declared  that  Mcgadistomwn  longuin  (Leidy,  1851)  Stafford,  1904  and  Mimo- 
distonmm  angusticaudum  Stafford,  1904  are  members  of  the  genus  Asygia  and  the 
two  generic  names  were  relegated  to  synonymy.  He  suggested  the  probable 
identity  of  A.  tcreticollis  of  America  with  A.  lucii  of  Europe.  He  criticized  Gold- 
berger's  work,  suppressed  Hassallins  as  a  synonym  of  Asygia,  and  expressed  the 
belief  that  A.  angusticauda,  A.  loossi,  A.  acuminata,  and  A.  bulbosa  are  members  of 
a  single  species. 

As  noted,  Cooper  (1915)  described  worms  which  he  identified  as  A.  lucii  from 
the  pike,  Lucius  lucius;  the  muskellunge,  Lucius  masquinongy;  Lucioperca  sp. ;  and 
immature  specimens  were  recovered  from  Salvelinus  namaycush  and  Micropterus 
doloinicu.  He  stated  that  all  the  worms  from  the  muskellunge  are  identical  with 
Stafford's  Megadistomum  longuin  (Leidy)  and  the  smallest  one  with  eggs  was  8 


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LIFE-CYCLE  OF  AZYGIA  SEBAGO  251 

mm.  long.  Cooper  noted  the  variable  size  of  worms  at  the  time  of  egg  production. 
The  smallest  gravid  specimen  from  the  pike  was  6  mm.  long,  but  another  from  the 
pike,  14  mm.  long,  was  less  mature  than  the  one  6  mm.  long;  others  6  to  14  mm.  in 
length  were  fully  gravid.  All  the  worms  from  the  trout,  6\  namaycush,  and  the 
black  bass,  M.  doloinicit,  including  the  largest  one,  11  mm.  long,  were  immature. 
Other  young  and  immature  specimens  from  the  stomach  of  Perca  flavescens  were 
regarded  as  possible  members  of  this  species.  Worms  from  the  pickerel  (not 
named)  resembled  A.  angusticaudum  (Stafford,  1904)  but  were  too  contracted  to 
permit  positive  identification,  and  others  from  the  pike  had  a  large,  globose  ex- 
cretory vesicle,  described  by  Goldberger  as  characteristic  of  A.  bulbosa,  but  Cooper 
stated  that  the  shape  of  the  excretory  vesicle  as  well  as  the  length,  extent,  and 
"breaking"  of  the  vitellaria  are  so  variable  as  to  be  of  little  use  in  the  delineation  of 
species.  Cooper  recognized  the  validity  of  A.  acwninata,  since  9  specimens  from 
the  stomach  of  Amia  calva  agreed  substantially  with  Goldberger's  description  of 
this  species. 

Ward  (1918)  stated  (p.  392),  "Despite  many  records  of  its  occurrence,  the 
common  European  A.  lucii  (=  A.  tcrcticollc)  has  not  been  found  in  North  America. 
Several  species  peculiar  to  this  continent  occur  in  Amia  calva,  Micropterus  sal- 
moid  cs  and  dolomieu,  Esox  Lucius  and  rcticulatus,  Ambloplites  ruprcstris,  Salve- 
linns  naiuaycush,  Liopcrca,  Lota  lota,  and  Sahno  sebago." 

Manter  (1926)  gave  a  systematic  review  of  the  family  Azygiidae;  he  agreed 
with  Ward  in  regarding  the  American  specimens  as  specifically  distinct  from  those 
of  Europe  but  admitted  (p.  57)  that  "Asygia  is  the  only  genus  of  the  family  show- 
ing taxonomic  confusion  in  its  species."  Accepting  the  statements  of  Odhner  and 
Ward,  he  distinguished  Asygia  longa  from  A.  lucii  on  the  extent  of  the  vitellaria, 
which  in  the  European  species  are  reported  not  to  extend  behind  the  testes,  and  on 
the  shape  of  the  pharynx,  which  in  A.  lucii  is  reportedly  cylindrical  and  twice  as 
long  as  wide.  After  detailed  study  and  tabular  comparison  of  morphological  fea- 
tures, Manter  recognized  only  three  species  of  Asygia  in  North  America,  vis.,  A. 
longa  (Leidy,  1851),  A.  angusticanda  (Stafford,  1904),  and  A.  acuminata  Gold- 
berger, 1911.  Manter  confirmed  the  suspicion  of  Odhner  (1911)  that  Asygia 
loossi  is  identical  with  Mimodistomum  angusticaudum  Stafford,  1904.  As  syno- 

PLATE  i 

FIGURE  1.  Asygia  lucii,  from  Esox  Indus ;  specimen  collected  and  identified  by  Prof.  M. 
Braun,  Konigsberg,  7  July  1902;  23  mm.  long,  ventral  view;  U.  S.  National  Museum,  Helmin- 
thological  Collection  No.  3359. 

FIGURE  2.  Asygia  lucii,  from  Amia  calva;  20  mm.  long,  ventral  view,  (Ward  Collection) 
U.  S.  N.  M.,  Helminth.  Coll.  No.  51,403. 

FIGURE  3.  Asygia  longa,  from  Esox  rcticulatus,  identified  by  Albert  Hassall ;  12.4  mm. 
long;  U.  S.  N.  M.,  Helm.  Coll.  No.  49. 

FIGURE  4.  Asygia  longa,  from  Esox  nigcr;  5.2  mm.  long,  collected  1955  by  Paul  Krupa, 
southern  New  Hampshire. 

FIGURE  5.  Asygia  longa,  from  Esox  nigcr;  19  mm.  long,  collected  1955  by  Paul  Krupa, 
southern  New  Hampshire. 

FIGURE  6.  Asygia  angusticanda  (type  of  Asygia  loossi,  Marshall  and  Gilbert,  1905),  from 
Micropterus  salmoides;  4.88  mm.  long,  ventral  view ;  U.  S.  N.  M.,  Helm.  Coll.  No.  10,679. 

FIGURE  7.  Asygia  angusticanda,  from  Stisostedion  vitreum;  10.5  mm.  long ;  ventral  view, 
(Ward  Collection)  taken  by  H.  W.  Manter  4  April  1926,  Rock  River,  Illinois ;  U.  S.  N.  M., 
Helm.  Coll.  No.  51,402. 


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LIFE-CYCLE  OF  AZYGIA  SEBAGO  253 

nyms  of  A.  longa  (Leidy),  Manter  listed:  Distomwn  longum  Leidy,  1851;  Disto- 
mum  tereticolle  of  Leidy,  1851 ;  Mcgadistomum  longum  (Leidy)  of  Stafford,  1904; 
Asygia  tereticolle  of  Stafford,  1904;  Asygia  sebago  Ward,  1910;  Asygia  bulbosa 
Goldberger,  1911 ;  Hassallius  Iiassalli  Goldberger,  1911 ;  and  Asygia  lucii  of  Cooper, 
1915.  He  discussed  the  problems  of  specific  determination,  noted  the  bundles  of 
longitudinal  muscles  which  traverse  the  parenchyma  and  quoted  Leuckart's  de- 
scription of  them,  and  stated  that  in  such  elongate  and  powerfully  muscled  trema- 
todes,  contractions  not  only  alter  the  general  shape  of  body  but  the  form  and  rela- 
tive position  of  internal  organs.  Concerning  differences  in  size  and  sexual  ma- 
turity, he  observed  that  in  the  related  species,  Otodistomum  cestoides,  specimens 
increase  six  to  seven  times  in  size  after  attainment  of  sexual  maturity.  This  fact 
was  used  to  justify  the  inclusion  in  a  single  species,  A.  longa,  of  gravid  specimens 
3.9  mm.  long  which  had  been  described  as  A.  bulbosa,  and  others  which  measured 
up  to  3  inches  in  length  and  had  been  described  as  A.  longum.  It  is  true  that  these 
specimens  were  from  different  host  species  and  worms  grow  larger  in  larger  hosts, 
but  it  is  doubtful  whether  host  influences  can  produce  such  extreme  range  in  size 
within  a  single  species.  Manter's  description  of  A.  longa  was  based  largely  on 
worms  which  Ward  had  described  as  A.  sebago  and  which  Manter  regarded  as 
identical  with  A.  longa.  The  specific  features  of  A.  sebago  were  not  clearly  de- 
fined ;  there  is  uncertainty  concerning  the  species,  since  there  is  strong  probability 
that  material  of  more  than  one  species  was  included  in  the  specific  diagnosis.  Ac- 
cording to  Manter  who  studied  the  Ward  collection  (p.  64),  "A.  sebago  averages 
about  6  to  8  mm.  in  length.  Specimens  were  found  as  small  as  1  mm.  and  no  ova 
were  present  in  forms  2.85  mm.  long.  ...  Of  the  other  Azygia  species,  A.  bulbosa 
Goldberger  is  most  evidently  identical  with  A.  sebago.  Type  material  of  both  spe- 
cies was  studied.  .  .  .  The  original  type  material  of  Hassallius  Iiassalli  was  also 
examined  for  comparison.  ...  In  fact,  after  allowance  is  made  for  body  contrac- 
tion, this  form  can  not  be  distinguished  from  the  other  common  American  forms  as 
represented  by  A.  sebago  and  A.  bulbosa." 

Van  Cleave  and  Mueller  (1934)  remarked  on  the  variability  in  fundamental 
characters,  such  as  the  anterior  and  posterior  limits  of  the  vitellaria  and  the  position 
of  the  gonads,  in  the  genus  Azygia.  They  endorsed  the  action  of  Manter  in  re- 
ducing the  number  of  species  in  North  America  and  went  even  further  in  reducing 
A.  aciiminata  to  synonymy  with  A.  longa.  They  noted  that  Manter  had  listed  A. 
bulbosa  as  a  synonym  of  A.  longa,  and  since  they  regarded  A.  acuminata  and  A. 

PLATE  n 

FIGURE  8.  Asygia  sebago,  from  Perca  ftavescens,  Sebago  Lake,  Maine,  1907,  4.26  mm.  long, 
ventral  view,  (Ward  Collection)  ;  U.  S.  N.  M.,  Helm.  Coll.  No.  51,401. 

FIGURE  9.  Azygia  acuminata,  from  Amia  calva,  Indiana,  type  of  Goldberger,  1911,  6.6  mm. 
long,  ventral  view;  U.  S.  N.  M.,  Helm.  Coll.  No.  10,500. 

FIGURE  10.  Asygia  sebago,  from  Anguilla  rostrata,  Falmouth,  Mass.,  1955,  flattened  speci- 
men, 12.5  mm.  long,  ventral  view. 

FIGURE  11.  Asygia  bulbosa,  from  Amia  calva,  Indiana,  type  of  Goldberger,  1911,  8.6  mm. 
long,  ventral  view ;  U.  S.  N.  M.,  Helm.  Coll.  No.  10502. 

FIGURE  12.  Asygia  sebago,  from  Anguilla  rostrata,  Falmouth,  Mass.,  1954,  immature 
specimen,  2.66  mm.  long,  ventral  view. 

FIGURE  13.  Asygia  sebago,  from  Anguilla  rostrata,  Falmouth,  Mass.,  1955,  young  specimen 
with  46  eggs  in  the  initial  one-half  of  the  uterus,  5.3  mm.  long,  ventral  view. 


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LIFE-CYCLE  OF  AZYGIA  SEBAGO  255 

bidbosa  as  synonyms,  A.  acitininata  should  also  become  a  synonym  of  A.  longa.  The 
reasoning  is  sound  if  the  postulates  are  correct,  which  now  appears  doubtful.  The 
specimen  shown  in  their  Figure  9(5)  which  is  referred  to  A.  angusticauda  and  the 
one  Figure  9  (7)  referred  to  A.  longa  are  so  similar  that  they  are  probably  con- 
specific  and  they  may  not  belong  to  either  A.  angusticauda  or  A.  longa.  They 
closely  resemble  the  worms  from  the  eel,  identified  in  this  paper  as  A.  sebago. 

In  other  surveys  of  trematode  parasites  of  fishes,  A.  angusticauda  and  A.  longa 
have  been  reported  in  eastern  North  America  but  A.  longa  may  not  extend  into 
the  area  of  Lake  Huron  and  northern  Wisconsin  and  neither  species  has  been  found 
in  the  fishes  of  western  Canada.  Lyster  (1939)  reported  A.  longa  from  Esox  lucius 
and  Anguilla  rostrata  in  Canada.  The  single  worm  from  E.  lucius  is  probably  a 
young  specimen  of  A.  longa,  but  those  from  eels  are  very  different.  He  stated 
that  some  of  them  could  be  assigned  to  A.  angusticauda  and  specimen  No.  1  in  his 
table,  which  is  4.8  mm.  wide  at  the  acetabulum,  is  probably  A.  angusticauda.  The 
others,  which  are  the  same  length  as  the  one  from  E.  lucius  but  are  twice  as  wide, 
are  very  similar  to  those  from  eels  on  Cape  Cod.  Miller  (1940)  reported  A. 
angusticauda  from  Stizostedion  vitrcmn  and  Micropterus  dolomicu  in  the  central 
St.  Lawrence  watershed.  Miller  (1941)  restudied  the  collection  of  Stafford.  He 
found  a  specimen  from  the  muskellunge  which  he  identified  as  Megadistomum 
long  um;  it  was  18.5  mm.  long,  1.2  mm.  wide,  and  there  are  no  eggs  in  the  uterus. 
Another  specimen,  from  Lota  inaculosa  and  identified  as  A.  tcreticolle,  is  6.5  mm. 
long,  0.5  mm.  wide,  and  contains  eggs.  If  these  worms  belong  to  A.  longa,  as 
stated,  it  is  difficult  to  explain  the  sexual  maturity  of  the  smaller  individual.  In 
the  Stafford  collection  Miller  found  two  mature  and  several  juvenile  specimens  of 
Mimodistoinuni  angusticauduin.  One  of  the  mature  specimens,  7.25  mm.  long 
and  1.65  mm.  wide,  shown  in  his  Figure  13,  is  typical,  with  the  acetabulum  near 
the  middle  and  the  gonads  in  the  posterior  one-sixth  of  the  body.  This  account  of 
the  original  Stafford  specimens  definitely  relegates  Asygia  loossi  Marshall  and 
Gilbert,  1905  to  synonymy  with  A.  angusticauda  (Stafford,  1904).  Choquette 
(1951)  reported  both  A.  longa  and  A.  angusticauda  from  the  muskellunge,  Esox  m. 
tnasquinongy,  in  the  St.  Lawrence  watershed.  Meanwhile.  Bangham  (1944)  ex- 
amined 1,330  fishes,  representing  38  different  species,  from  40  different  locations  in 
northern  Wisconsin.  He  did  not  find  A.  longa,  but  A.  angusticauda  was  present  in 
12  species  of  fish.  Bangham  and  Venard  (1946)  examined  676  fishes,  belonging 
to  22  species,  from  Algonquin  Park  lakes.  Worms  from  Anguilla  rostrata  were 

PLATE  in 

FIGURE  14.  Dugcsia  tigrinum.,  7  mm.  long,  experimental  infection,  two  juvenile  Asygia 
sebago  in  the  pharyngeal  pockets. 

FIGURE  15.     Asygia   sebago,  0.81    mm.   long,   natural   infection,   from    pharyngeal   cavity   of 
D.  tigrinum,  juvenile  worm  found  by  J.  Louis  Bouchard. 

FIGURE  16.  Asygia  sebago,  juvenile  worm  from  D.  tigrinum,  1.17  mm.  long,  natural  in- 
fection, specimen  from  J.  Louis  Bouchard. 

FIGURE  17.     Asygia  sebago,  cercaria,  naturally  emerged,  a  fixed  and  stained  specimen. 

FIGURE  18.  Asygia  sebago,  cercaria,  from  a  crushed  snail,  larva  not  entirely  mature  and 
only  partially  enclosed  in  the  enlarged,  basal  end  of  the  tail,  furci  shriveled,  a  fixed  and  stained 
specimen. 

FIGURE  19.     Asygia  sebago,  miracidium  in  egg,  from  sketches  made  of  living  larvae. 

FIGURE  20.  Asygia  sebago,  redia  in  which  the  pharynx  is  recognizable;  the  body  of  the 
cercaria  is  0.8  mm.  long,  the  furci  0.18  mm.  long;  fixed  and  stained  specimen. 


256  HORACE  W.  STUNKARD 

identified  as  A.  longa;  others  from  Microptcrus  dolomicu,  Pcrca  flavcsccns  and 
Lepowis  gibbosus  were  identified  as  A.  angusticauda.  Bangham  and  Adams 
(1954)  did  not  find  Azygia  in  the  examination  of  5456  fishes,  belonging  to  36  dif- 
ferent species,  taken  in  the  Columbia,  Fraser  and  other  rivers  of  western  Canada. 
In  a  survey  of  parasites  from  1667  fishes,  representing  53  species,  from  Lake  Huron 
and  Manitoulin  Island,  Bangham  (1955)  found  A.  angusticauda  in  the  northern 
channel  catfish,  Ictalurus  I.  lacustris.  This  parasite  obviously  can  infect  a  large 
number  of  species  of  fish. 

Knowledge  of  the  life-history  of  azygiid  trematodes  dates  from  the  publication 
by  Szidat  (1932)  on  the  developmental  cycle  of  Azygia  lucii,  a  common  parasite  in 
the  stomachs  of  salmonid  fishes,  especially  species  of  Esox,  in  Europe.  Szidat  found 
that  the  large,  furcocercous,  cystocercous  larva,  Cercaria  mirabilis  Braun  from  Lyin- 
naea  palustris,  when  fed  to  young  pike,  Eso.v  Indus,  developed  in  ten  days  into  adult 
Azygia  lucii.  He  recalled  the  statement  of  Looss  (1894),  that  when  small  pike  are 
eaten  by  larger  ones,  the  azygiid  parasites  leave  the  stomach  of  the  ingested  fish 
and  establish  themselves  on  the  stomach  of  the  preditor;  and  stated  (p.  501), 
"tiberdies  sind  altere  Hechte  keine  Planktonfresser  mehr,  so  class  fiir  sie  die 
Ubertragung  auf  dem  zuletzt  geschilderten  Wege  den  vorherrschenden  Modus 
darstellen  wird,  und  die  jugendlichen  Hechte  demnach  biologisch  doch  als  Zwischen- 
oder  Hilfswirte  zu  werten  sind."  Szidat  reported  that  other  small  fishes  also  ingest 
the  cercariae  and  may  serve  as  transport  hosts,  but  in  these  species  the  parasites  do 
not  develop  to  sexual  maturity.  He  found  juvenile  A.  lucii  in  the  stomachs  of 
small  predacious  fishes  belonging  to  the  genera  Pcrca,  Lucioperca,  and  Gastcrostcns. 
Szidat  described  the  cercaria-producing  generation  as  a  redia,  which  lacks  a  diges- 
tive tract  but  in  which  the  pharynx  persists  as  an  organ  for  ingesting  fragments  of 
the  digestive  gland  of  the  snail  host  and  also  as  a  birth  pore.  He  traced  the  de- 
velopment of  the  cercariae  and  noted  their  resemblance  to  those  of  the  strigeids  and 
schistosomes.  The  cercariae  are  not  encysted  in  the  snail  host.  The  body  of  the 
cercaria  sits  in  a  narrow  depression  at  the  anterior  end  of  the  flattened  tail-stem. 
The  cercariae  mature  in  the  haemocoele  of  the  snail  and  emerge  into  the  mantle 
cavity.  In  water,  the  proximal  portion  of  the  tail  begins  to  swell  and  the  body  of 
the  larva,  anchored  in  the  base  of  the  depression  by  the  tubule  of  the  excretory  sys- 
tem, is  enveloped  by  the  base  of  the  tail  and  enclosed  in  it.  Szidat  also  described 
Cercaria  splendens,  believed  to  represent  a  second  species  of  Azygia,  but  the  adult 
stage  and  final  hosts  were  not  discovered. 

The  achievement  of  Szidat  in  working  out  the  life-cycle  of  A.  lucii  disclosed  that 
the  furcocercous,  cystocercous  larvae  of  the  Mirabilis  type,  originally  regarded  by 
Leuckart  as  free-swimming  sporocysts  and  shown  by  Braun  (1891)  to  be  cercariae 
when  he  described  Cercaria  mirabilis,  are  developmental  stages  of  azygiid  trema- 
todes. The  first  member  of  the  group  was  found  by  Wright  in  a  fresh-water 
aquarium  and  described  (1885)  as  a  free-swimming  sporocyst.  Ward  (1916) 
named  the  species  Cercaria  zvrighti  and  described  a  second  species,  Cercaria  an- 
choroidcs,  collected  in  top  and  bottom  tow  every  day  from  July  25  to  August  5, 
1893.  in  Lake  St.  Clair,  Michigan.  Subsequent  investigators  have  reported  other 
members  of  the  Mirabilis  group ;  sixteen  species  have  been  described,  but  some  of 
them  are  identical.  Several  of  the  named  species  were  described  from  immature 
stages,  taken  from  crushed  snails,  and  can  not  be  identified  with  certainty.  Others 
were  described  from  free-swimming  cercariae  and  the  hosts  are  unknown.  Certain 


LIFE-CYCLE  OF  AZYGIA  SEBAGO  257 

of  them  have  proved  to  be  larvae  of  species  in  the  genus  Protcromctra,  erected  by 
Horsfall  (1933)  to  contain  Cere  aria  macrostoma.  Faust,  1918.  Reviews  of  the 
cystocercous  cercariae  were  published  by  Horsfall  (1934),  Smith  (1936)  and 
Dickerman  (1946).  Those  with  forked  tails  were  designated  as  furcocystocercous 
by  Le  Zotte  (1954)  who  showed  that  members  of  the  family  Bivesiculidae  also  have 
larvae  of  this  type. 

The  second  report  on  the  life-cycle  of  azygiid  trematodes  was  given  by  Stunkard 
(1950).  Larval  distomes  had  been  referred  to  him  for  identification  in  the  winter 
of  1949-1950  by  Mr.  J.  Louis  Bouchard,  then  a  graduate  student  at  the  University 
of  Oklahoma.  The  worms  had  been  found  in  planarians,  Dugesia  tigrinum,  re- 
ceived from  the  Marine  Biological  Laboratory,  Woods  Hole,  Massachusetts.  The 
structure  of  the  larvae  indicated  that  they  were  azygiids  and  study  of  the  life-cycle 
was  begun  at  the  Marine  Biological  Laboratory  in  the  summer  of  1950.  Records 
of  the  Supply  Department  of  the  M.  B.  L.  showed  that  the  planarians  sent  to  the 
University  of  Oklahoma  had  been  collected  in  Morse's  Pond  in  Falmouth.  Thirty- 
eight  D.  tigrinum  were  collected  there  on  July  10,  1950  and  a  larval  trematode, 
identical  with  the  specimens  sent  by  Mr.  Bouchard,  was  found  in  the  pharyngeal 
pockets  of  two  of  them.  Eight  additional  worms  of  natural  infection  were  found  in 
120  D.  tigrinum  examined.  To  discover  the  first  intermediate  host,  different  spe- 
cies of  mollusks  were  collected  from  Morse's  Pond  and  isolated.  Furcocercous 
cercariae  of  the  azygiid  type  emerged  from  nine  of  246  Amnicola  limosa.  Four 
planarians,  examined  under  the  microscope  and  known  to  be  uninfected,  were  placed 
in  a  finger-bowl  with  three  specimens  of  A.  limosa  which  were  shedding  these  cer- 
cariae. After  six  days  exposure,  one  to  four  larvae  were  found  in  the  pharyngeal 
cavities  of  each  of  the  planarians.  These  larvae  were  identical  with  those  sent  by 
Mr.  Bouchard.  The  tails,  in  the  bases  of  which  the  bodies  of  the  cercariae  for- 
merly were  enclosed,  had  completely  disappeared.  Other  planarians  were  subse- 
quently placed  in  dishes  with  infected  A.  limosa  and  larvae  found  in  their  pharyngeal 
pockets.  The  larvae  may  persist  for  several  weeks  in  D.  tigrinum,  but  they  do  not 
encyst  or  grow7  and  it  is  apparent  that  the  planarians  serve  merely  as  paratenic  or 
transport  hosts.  Attempts  to  feed  the  cercariae  to  goldfish  and  small  perch  were 
not  successful ;  the  fish  would  not  take  the  larvae  and  when  introduced  into  their 
mouths,  the  cercariae  were  expelled.  Planarians  infected  with  cercariae  were  fed, 
but  the  results  were  uncertain  and  the  short  period  in  which  the  work  could  be  con- 
ducted, led  to  no  further  information  at  that  time.  It  was  clear  that  the  larvae 
belonged  to  a  species  of  Azygia,  but  specific  determination  could  not  be  established. 

Sillman  (1953a)  reported  that  in  the  vicinity  of  Ann  Arbor,  Michigan,  the  mud 
pickerel,  Esox  vermiculatus,  and  the  bowfin,  Amia  calva,  harbor  Azygia  longa. 
Eggs  of  the  trematode,  containing  mature  miracidia,  were  fed  to  both  wild  and 
laboratory-raised  Amnicola  limosa.  Cercariae  producing  rediae  were  found  after 
21  days  and  cercariae  emerged  42  days  after  infection.  Cercariae  fed  to  Esox 
vermiculatus  developed  in  20-30  days  into  egg-bearing  worms.  Two  of  13,500 
Amnicola  limosa  were  found  naturally  infected  with  cercariae  which  appeared  iden- 
tical with  those  in  experimentally  infected  snails. 

In  a  thesis  submitted  for  the  Ph.D.  degree  at  the  University  of  Michigan,  Sill- 
man (1953b)  gave  further  information.  He  stated  that  two  species  of  Azygia  are 
present  in  the  Ann  Arbor  area.  One  species,  which  he  identified  as  A.  longa,  oc- 
curs in  both  Esox  vermiculatus  and  Amia  calva.  The  other  species,  which  he  iden- 


258  HORACE  W.  STUNKARD 

tified  as  A.  acuminata,  was  found  only  in  Auiia  cak'a.  Worms  assigned  to  A.  longa 
were  somewhat  longer,  more  slender  and  the  suckers  were  slightly  smaller  than 
those  of  A.  acuminata,  but  the  measurements  of  worms  and  organs  overlapped. 
According  to  Sillman,  the  collecting  ducts  of  the  excretory  system  branch  from  the 
vesicle  behind  the  testes  in  A.  longa  and  between  the  testes  in  A.  acuminata.  Al- 
though there  was  much  variation,  the  average  size  of  eggs  in  A.  longa  was  55  by  31 
microns  whereas  that  of  A.  acuminata  was  69  by  38  microns.  Furthermore,  speci- 
mens of  Amnicola  Ihnosa  did  not  become  infected  when  fed  eggs  of  A.  acuminata. 

Investigation  of  the  life-cycle  and  development  of  Asygia-  has  been  continued  at 
the  Marine  Biological  Laboratory,  Woods  Hole,  Mass.,  during  the  summer  months 
since  1950.  An  abstract  of  the  results  was  presented  (Stunkard,  1955).  Infected 
snails  were  found  each  year  and  the  morphology  of  the  young  distome,  especially 
the  details  of  the  excretory  system,  was  studied.  Hundreds  of  fishes,  including 
Eso.v  niger,  Pcrca  flavcsccns,  M  or  one  aniericanus,  Micropterus  salinoidcs,  Microp- 
tcnts  dolomicn,  and  others,  were  examined  in  the  attempt  to  find  the  sexually  ma- 
ture stage  of  the  parasite.  The  first  to  be  discovered,  a  small,  immature  specimen 
of  Asygia  (Fig.  12)  was  found  in  the  stomach  of  an  eel,  Anguilla  rostraia,  late  in 
the  summer  of  1954.  During  the  summer  of  1955,  42  eels  were  examined;  10  of 
them  were  infected  and  many  fully  mature  worms  were  collected.  Continued  ex- 
amination of  other  fishes,  especially  the  pickerel,  Esox  niger,  from  the  same  ponds 
where  the  infected  eels  were  taken,  has  not  disclosed  infection  by  members  of  the 
genus  Asygia,  and  it  appears  that  the  eel  is  the  natural  and  possibly  the  only  host 
for  the  species  in  the  Woods  Hole  region.  The  larger  ponds  in  the  area  are  under 
the  control  of  the  Division  of  Fisheries  and  Game,  Bureau  of  Wildlife  Research  and 
Management  of  the  State  of  Massachusetts,  and  many  of  them  have  been  stocked 
with  game  fishes  from  time  to  time.  Through  the  kind  cooperation  of  Mr.  Russell 
Cookingham,  a  large  number  of  fishes,  belonging  to  various  species,  were  provided 
during  the  summer  of  1955,  when  certain  of  these  ponds  were  inspected  to  deter- 
mine their  productivity. 

Specific  determination  of  the  parasites  from  the  eel  has  proved  difficult.  De- 
scriptions are  wholly  unsatisfactory  and  accordingly,  specimens  of  Azyc/ict  in  the 
U.  S.  National  Museum  were  borrowed  through  the  kindness  of  Dr.  E.  W.  Price 
and  Mr.  Allen  Mclntosh.  The  material  consisted  of  6  specimens  in  alcohol  (bottle 
M  248-D),  the  type  specimens  of  Distoininn  longiun  Leidy,  and  other  specimens 
mounted  on  slides  and  bearing  the  following  labels,  U.  S.  National  Museum, 
Helminthological  Collection : 

No.  49.  Distoiiinni  longitm  from  Eso.v  reticnlatiis,  determined  by  Albert  Has- 

sall;  1  slide.     (Plate  I,  Fig.  3.) 
No.  3359.       Asygia  lucii  from  Eso.v  Indus,  collected  and  determined  by  Professor 

M.  Braun,  7  July  1902,  Konigsberg,  Germany;   1   slide.      (Plate  I, 

Fig.  1.) 
No.   10500.     Azygia  acuminata  from  Anna   cak'a,  type  and  paratypes ;  •     slides. 

(Plate  II,  Fig.  9.) 
No.   10502.     Azygia    bitlbosa   from    A  mi  a   cak'a,   type    and    paratypes;    3    slides. 

(Plate  II,  Fig.  11.) 
No.   10679.     Azygia  loossi  from  Micropterus  salmoides,  cotypes ;  3  slides.      (Plate 

I,  Fig.  6.) 


LIFE-CYCLE  OF  AZYGIA  SEBAGO  259 

No.  51399.     Azygia  scbago  from  Saluio  scbago,  5.2  mm.  long,  1  slide,  H.  B.  Ward 

collection. 
No.  51401.     Asygia  scbago  from  Pcrca  flavcsccns,  H.  B.  Ward  collection;  2  slides. 

(Plate  II,  Fig.  8.) 
No.  51403.     Azygia  scbago  from  Anna  calva,  20  mm.  long,  1  slide,  H.  B.  Ward 

collection.      (Plate  I,  Fig.  2.) 
No.  51402.     Azygia  angusticauda  from  Stizostedion  vitreum,  collected  by  H.  W. 

Manter,  4  April   1926,  Rock  River,  Illinois,  2  slides,  H.  B.  Ward 

collection.     (Plate  I,  Fig.  7.) 

Examination  of  the  specimen  of  Asygia  lucii,  No.  3359  in  the  U.  S.  National 
Museum,  invalidates  the  criteria  used  by  Ward  and  Manter  to  distinguish  between 
A.  longa  and  A.  lucii.  In  this  specimen  (Fig.  1)  which  measures  23  mm.  in  length, 
collected  and  identified  by  Professor  M.  Braun,  the  pharynx  is  not  twice  as  long  as 
broad ;  in  fact,  the  organ  measures  0.80  mm.  long  and  0.60  mm.  wide.  Further- 
more, the  vitellaria  extend  far  behind  the  posterior  testis ;  the  follicles  on  the  left 
side  about  one-half  the  distance  from  the  testis  to  the  end  of  the  body.  In  the  Ward 
collection  there  is  a  specimen  from  Amia  calva  (Fig.  2)  which  measures  20  mm.  in 
length  and  which  resembles  the  European  specimen  so  closely  that  I  am  disposed 
to  regard  the  two  as  specifically  identical.  Leidy,  Stafford  and  Cooper  all  reported 
the  finding  of  A.  lucii  and  it  appears  that  this  species  does  occur  in  North  America. 
Eso.v  Indus,  the  type  host,  is  circumpolar  in  range,  and  the  distribution  of  its  para- 
sites may  be  expected  to  parallel  that  of  the  host.  The  dispersal  of  fishes  in  the 
northern  hemisphere  following  the  last  glacial  period  has  been  traced  by  Walters 
(1955). 

Although  the  criteria  used  by  Ward  and  Manter  to  distinguish  A.  longa  from 
A.  lucii  are  inadequate,  the  two  forms  are  probably  distinct.  About  100  specimens 
collected  by  Mr.  Paul  Krupa  from  Eso.v  nigcr  in  southern  New  Hampshire  during 
the  summer  of  1955  are  so  similar  to  the  six  worms  in  alcohol,  now  in  the  U.  S. 
National  Museum,  which  constitute  the  original  material  of  the  species  described 
by  Leidy  (1851)  as  D.  longnm,  that  they  must  be  regarded  as  identical.  A  repre- 
sentative example  from  the  Krupa  collection  is  shown  (Fig.  5)  and  a  smaller  one 
(Fig.  4).  These  worms  are  very  slender.  The  Krupa  specimens  were  dropped 
in  cold  Duboscq-Brasil  fluid  and  fixed  without  narcotization  or  pressure.  Ovifer- 
ous  specimens  vary  from  4  to  26  mm.  in  length  and  1.1  mm.  is  the  greatest  width. 
The  width  does  not  increase  very  much  as  the  worms  grow  in  length.  Comparison 
of  Figure  5  with  that  of  A.  lucii  (Fig.  1)  portrays  what  are  believed  to  be  specific 
differences.  Further  evidence  that  A.  longa  is  distinct  from  A.  lucii  is  afforded  by 
comparison  of  the  cercariae.  Cercaria  mirabilis  Braun,  1891,  shown  by  Szidat 
(1932)  to  be  the  larval  stage  of  A.  lucii,  is  very  different  from  the  cercaria  described 
by  Sillman  as  the  larval  stage  of  A.  longa.  Moreover,  in  Europe  A.  lucii  uses  a 
pulmonate  snail,  Lymnaea  palusiris  corrus,  as  the  first  intermediate  host,  whereas 
according  to  Sillman,  the  asexual  stages  of  A.  longa  occur  in  the  pectinibranchiate 
snail,  Amnicola  limosa. 

Recognition  of  two  distinct  species,  A.  lucii  and  A.  longa,  may  resolve  certain 
difficulties  and  clear  up  confusion  in  the  literature.  The  worms  from  Eso.v  mas- 
quinongy  which  Stafford  (1904)  described  as  Megadistomum  longurn  (Leidy) 
measured  up  to  five  inches  in  length  when  extended  and  probably  were  not  iden- 


260  HORACE  W.  STUNKARD 

tical  with  A.  longa  of  Leicly.  Stafford  reported  a  specimen  18  mm.  long  which 
contained  no  eggs.  Cooper  (1915)  identified  specimens  from  E.  masquinongy, 
which  he  regarded  as  identical  with  those  of  Stafford,  and  others  from  E.  Indus, 
as  Azygia  litcii.  His  specimens  from  the  muskelltmge  measured  21  to  48  mm.  in 
length  and  1.40  to  2.40  mm.  in  width,  whereas  those  from  the  pike  were  14  to  20 
mm.  in  length  and  0.74  to  1.42  mm.  in  width.  Comparison  of  the  small  worms  from 
E.  masquinongy  with  worms  from  E.  Indus  led  Cooper  to  regard  them  as  con- 
specific.  But  he  was  unable  to  account  for  the  variable  size  at  which  eggs  are  pro- 
duced in  different  individuals.  He  reported  that  a  specimen  14  mm.  long  from 
E.  Indus  was  less  mature  than  another  6  mm.  long  from  the  same  host  species,  and 
that  worms  from  the  trout  and  small-mouthed  black  bass  were  all  immature  al- 
though one  from  6\  namaycush  was  11  mm.  long.  Discussing  the  effect  of  season 
on  sexual  maturity,  Manter  (1926)  wrote  (p.  67.)  ".  .  .  it  is  certain  that  what  is 
evidently  the  same  species  does  not  attain  sexual  maturity  at  the  same  time  in  dif- 
ferent hosts  in  which  it  occurs.  Thus,  while  average  sized  forms  are  producing  eggs 
in  such  hosts  as  pike,  pickerel,  and  salmon,  specimens  fully  as  large  are  still  sexually 
immature  in  such  hosts  as  smelt,  trout,  small  mouthed  black  bass,  and  perch." 
Admittedly,  members  of  a  trematode  species  attain  a  greater  size  in  a  larger  host 
species,  and  E.  masquinongy  is  much  larger  than  E.  Indus,  but  present  information 
strongly  indicates  that  A.  longa  is  distinct  from  A.  ludi,  if,  indeed,  the  large  Ameri- 
can species  is  actually  A.  ludi  of  European  fishes. 

All  previous  authors  have  agreed  on  the  identity  of  A.  angusticauda  (Stafford, 
1904)  and  A.  loossi  Marshall  and  Gilbert,  1905.  A  cotype  specimen  of  A.  loossi 
(U.  S.  Nat.  Mus.,  10,679),  shown  in  Figure  6,  is  4.88  mm.  long  and  is  obviously 
young,  with  only  a  few  eggs  in  the  uterus.  A  fully  mature,  gravid  specimen  (U.  S. 
Nat.  Mus.,  No.  51,402)  from  the  walleye,  Stizostcdion  ritrciuu,  collected  by  Manter 
in  1926,  which  measures  10.5  mm.  in  length,  is  shown  in  Figure  7.  In  both,  the 
acetabulum  is  near  the  middle  and  the  gonads  are  situated  in  the  caudal  one-sixth 
of  the  body.  The  distinctness  of  this  species  appears  to  be  well  established. 

The  specimens  of  Azygia  found  in  the  eel  at  Woods  Hole  are  clearly  distinct 
from  A.  angusticauda  and,  as  noted,  are  probably  distinct  from  A.  longa.  Speci- 
mens of  A.  longa  are  slender  and  much  elongate ;  those  from  the  eel  are  shorter  and 
more  robust.  The  worms  collected  by  Mr.  Krupa  from  Eso.v  nigcr  in  New  Hamp- 
shire and  identified  as  A.  longa  remained  well  extended  when  dropped  into  Duboscq- 
Brasil  killing  fluid,  whereas  those  from  the  eel  contracted  strongly  with  the  result 
that  the  length  was  only  6  to  8  mm.,  less  than  one-half  that  of  A.  longa.  Accord- 
ingly, most  of  the  worms  from  the  eel  were  killed  and  fixed  under  pressure,  which 
resulted  in  longer,  wider,  and  flatter  specimens.  The  size  of  the  suckers  increased 
as  a  result  of  the  compression  but  comparison  of  Figures  10  and  13,  which  were 
made  from  one  of  the  largest  and  one  of  the  smallest  oviferous  specimens,  with 
Figures  5  and  4,  of  comparable  specimens  of  A.  longa,  portrays  differences  between 
the  two  forms  which  are  believed  to  be  specific. 

Whereas  the  worms  from  the  eel  differ  distinctly  from  those  identified  as  A. 
longa,  they  agree  almost  completely  with  Goldberger's  description  of  A.  acuminata 
and  agree  almost  as  well  with  the  descriptions  of  A.  scbago  as  given  by  Ward  and 
Manter.  Certain  worms  from  the  eel  are  very  similar  to  specimens  in  the  Ward 
collection  labelled  A.  scbago.  It  is  probable  that  Ward  had  more  than  one  species 
and  that  his  description  of  A.  sebago  was  based  on  specimens  of  both  A.  longa  and 


LIFE-CYCLE  OF  AZYGIA  SEBAGO  261 

A.  sebago.  The  worm  from  the  Ward  collection  which  bears  the  U.  S.  Nat.  Mtis., 
No.  51,401,  shown  in  Figure  8,  is  clearly  A.  sebago,  and  the  worm  on  U.  S.  Nat. 
Mus.,  No.  51,403,  from  Amia  calva  shown  in  Figure  2,  is  so  like  A.  liicii  (cf.  Fig. 
1),  that  the  two  might  be  regarded  as  specifically  identical.  Other  specimens  of 
A.  sebago  agree  so  completely  with  Goldberger's  description  of  A.  acuminata  (com- 
pare Figs.  8  and  9),  that  I  am  inclined  to  regard  them  as  identical.  Since  Ward 
probably  confused  two  species  in  his  description  of  A.  sebago,  the  removal  of  the 
elongate  specimens  leaves  the  description  virtually  the  same  as  that  of  A.  acuminata. 
Specific  determination  may  be  impossible  on  the  basis  of  adult  morphology  alone 
and  knowledge  of  life-cycles  and  larval  stages  may  be  required  to  finally  solve  the 
problem.  Why  the  species  occurs  only  in  Anguilla  rostrata  in  the  Woods  Hole  area 
is  quite  unknown.  The  larval  stages  are  relatively  abundant  in  the  snails  of  the 
region,  but  sexually  mature  worms  have  so  far  been  found  only  in  the  eel.  The 
chain  pickerel,  Eso.v  nigcr,  is  common  in  these  ponds  where  it  has  been  introduced 
in  stocking  operations.  Since  the  worms  develop  in  eels  in  ponds  where  pickerel, 
perch,  bass  and  other  fishes  are  not  infected,  it  appears  either  that  the  ecological 
conditions  and  food-chain  lead  to  the  infection  of  eels  rather  than  other  fishes  or 
else  the  other  fishes  do  not  retain  the  parasites.  In  the  latter  event,  a  separate  spe- 
cies must  be  involved. 

When  worms  were  removed  from  the  stomachs  of  eels  and  placed  in  pond  water, 
the  eggs  in  the  terminal  coils  of  the  uterus  were  extruded  in  a  string  of  mucus. 
These  eggs  appeared  to  be  fully  embryonated  and  the  miracidium  was  studied  in 
the  egg.  Although  active,  the  larvae  did  not  emerge  in  water  and  hatching  oc- 
curred only  after  the  eggs  were  ingested  by  the  snail  host.  Empty  shells  were  re- 
covered in  the  feces  of  Amnicola  limosa  that  had  eaten  the  eggs.  Some  of  these 
snails  were  found  later  to  be  infected  but  since  they  had  been  collected  from  loca- 
tions where  previous  exposure  to  infection  was  liable,  it  would  be  difficult  if  not 
impossible  to  distinguish  between  a  natural  infection  acquired  before  collection  and 
an  experimental  one.  But  the  snails  laid  eggs  in  the  finger  bowls  and  young 
laboratory-raised  specimens  wrere  fed  eggs  of  the  parasite.  These  small  snails  be- 
came infected  and  although  emerged  cercariae  were  not  obtained  before  the  end  of 
the  summer,  the  developmental  stages  in  these  experimental  infections  were  indis- 
tinguishable from  comparable  stages  in  natural  infections.  In  nature,  the  eggs  of 
the  parasite  are  passed  in  mucous  material  from  the  intestines  of  eels  and  settle  on 
vegetation  and  on  the  slimy  surfaces  of  submerged  rocks  and  sticks.  The  snails 
rasp  these  surfaces  for  the  diatoms  which  form  a  major  constituent  of  their  food 
and  incidentally  ingest  the  eggs.  The  larvae  remain  alive  for  long  periods  and 
since  the  eggs  do  not  hatch  until  they  are  eaten,  the  probability  of  reaching  a  suit- 
able host  and  continuing  the  life-cycle  is  much  enhanced.  The  larvae  emerge  in  the 
intestine  of  the  snail  and  bore  through  the  wall  to  reach  the  haemocoele,  where  they 
become  sporocysts.  Young  sporocysts  have  been  found  adjacent  to  the  intestinal 
wall  two  weeks  after  eggs  of  the  parasite  were  added  to  the  finger  bowl  with  the 
young  snails.  Older  infections  with  rediae  and  developing  cercariae  were  found 
later,  which  definitely  link  the  experimental  and  natural  infections.  However,  the 
rate  of  development  of  the  parasites  and  the  degree  of  maturity  of  the  infection  are 
not  regarded  as  significant.  It  is  common  knowledge  that  asexual  stages  of  di- 
genetic  trematodes  persist  but  fail  to  grow  or  reproduce  if  the  hosts  are  not  fed. 
Thus,  infections  overwinter  in  a  quiescent  stage  in  mollusks  that  are  dormant  or  in 


262  HORACE  W.  STUNKARD 

which  metabolism  is  reduced  to  a  low  level.  In  the  present  instance,  although  va- 
rious methods,  including  those  recommended  by  Moore  ct  al.  (1953)  and  by  Sand- 
ground  and  Moore  (1955)  for  the  rearing  of  related  snails,  were  employed,  it  was 
obvious  that  the  snails,  although  most  of  them  remained  alive,  were  not  properly 
nourished,  did  not  grow  normally,  and  the  tissues  had  the  atrophic  appearance 
typical  of  inanition. 

Cercariae  from  natural  infections  were  snapped  up  by  guppies  and  by  small 
bluegill  sunfish,  Lcponris  inacrochints,  2  to  4  cm.  in  length.  The  young  worms 
were  recovered  from  the  stomachs  of  these  sunfish  two  and  three  weeks  after  they 
were  eaten,  but  there  was  very  little  development  of  the  parasites.  These  small 
fishes  also  ate  planarians,  Dugesia  tigrinuui;  so  in  nature  the  fishes  could  contract 
the  infection  by  eating  either  the  cercariae  or  infected  planarians.  The  tails  of  the 
cercariae  cease  to  beat  after  about  48  hours  and  they  would  then  not  be  attractive 
to  fishes;  moreover  the  larvae  die  during  the  next  48  hours.  As  stated  earlier,  the 
young  worms  live  for  weeks  in  the  pharyngeal  pockets  of  D.  tigrinwn  and  this  ac- 
cessory method  of  employing  an  additional  paratenic  or  transfer  host  enhances  the 
likelihood  of  survival  and  aids  in  the  completion  of  the  life-cycle.  The  cercariae 
are  probably  not  eaten  by  eels  which  are  at  the  end  of  the  food-chain  that  leads  to 
their  infection. 

DESCRIPTION  OF  STAGES  IN  THE  LIFE-CYCLE 
Adult 

The  worms  are  only  slightly  flattened,  almost  cylindrical,  with  rounded  ends 
and  enormously  developed  musculature.  Because  of  the  ability  to  extend  and  re- 
tract the  entire  body  or  particular  regions  to  an  extraordinary  degree,  measure- 
ments of  length  and  width  and  location  of  individual  organs  have  limited  signifi- 
cance. A  specimen  may  extend  to  four  or  five  times  its  length  when  contracted, 
and  contraction  of  different  regions  can  make  distances  between  organs  so  variable 
that  measurements  may  be  very  misleading.  Ward  (1918)  wrote  (p.  392), 
"Azygia  is  a  powerfully  muscular  type  and  is  usually  much  distorted  in  the  process 
of  preservation  so  that  a  lot  of  specimens  taken  from  the  same  host  at  the  same 
time  present  marked  external  differences  in  the  preserved  condition.  Such  ex- 
treme specimens  have  been  the  basis  for  various  new  genera,  e.g.,  Mcgadlstomnm 
of  Leidy  and  Stafford,  Mimodistomum  of  Leidy  (sic)  and  Hassallins  of  Golcl- 
berger.  This  same  factor  has  lead  to  the  separation  of  too  many  as  species." 
Oviferous  specimens  from  the  eel,  fixed  by  the  shaking  method  of  Looss,  are  3  to 
9  mm.  long  and  when  fixed  under  pressure  measure  4  to  12.5  mm.  in  length.  Be- 
cause of  the  variations  caused  by  muscular  contractions  on  the  shape  of  the  body 
and  location  of  organs,  dimensions  of  the  suckers  and  gonads  provide  the  most 
reliable  morphological  data,  but  these  organs  appear  larger  in  specimens  that  have 
been  fixed  under  heavy  pressure.  Egg  sizes  vary  too  much  to  provide  reliable 
specific  criteria.  The  worms  continue  to  grow  after  sexual  maturity.  A  large  one 
and  a  small  one  are  shown  in  Figures  10  and  13;  both  were  fixed  under  pressure 
and  are  therefore  comparable.  Measurements  in  millimeters  of  the  larger  one  are : 
length,  12.5  ;  width,  2.2 ;  oral  sucker,  0.96;  acetabulum,  0.8;  pharynx,  0.36  long  and 
0.32  wide ;  ovary,  0.54  by  0.23 ;  anterior  testis,  0.5  by  0.33 ;  posterior  testis,  0.5  by 
0.4.  Corresponding  measurements  of  the  smaller  worm  are:  length,  5.3;  width 


LIFE-CYCLE  OF  AZYGIA  SEBAGO  263 

0.65  ;  oral  sucker,  0.41  ;  acetabulum,  0.34;  pharynx,  0.19  long  and  0.16  wide;  ovary, 
0.195  by  0.12;  anterior  testis,  0.195  by  0.143;  posterior  testis,  0.24  by  0.16.  The 
eggs,  alive,  averaged  0.06  by  0.034  mm. ;  under  oil  immersion  and  slight  pressure, 
to  study  the  miracidium,  they  were  slightly  larger ;  in  fixed  and  stained  worms  they 
were  smaller,  and  averaged  0.055  by  0.030  mm.  In  such  mounted  specimens  the 
eggs  are  usually  collapsed  and  distorted. 

Miracidium 

The  miracidium  of  Azygia  lucli  was  described  by  von  Nordmann  (1832), 
Schauinsland  (1883)  and  Looss  (1894)  and  that  of  Azygia  acuminata  (possibly  a 
synonym  of  A.  sebago)  by  Manter  (1926).  The  miracidium  of  the  worms  identi- 
fied as  A.  sebago,  studied  alive  in  the  egg  (Fig.  19)  and  in  stained  sections  of  gravid 
worms,  is  similar  to  that  of  related  genera  in  the  family  Azygiidae,  as  reviewed  by 
Manter  (1926).  Like  the  others,  it  lacks  cilia  and  is  provided  with  bristle  plates 
or  plaques.  It  almost  fills  the  egg-shell ;  the  anterior  end  may  be  protruded  as  a 
conical  papilla  on  which  the  ducts  of  the  secretory  cells  open.  Radiating  from  this 
area,  there  are  five  plates  or  plaques  that  bear  fine  bristles  arranged  in  a  chevron- 
like  pattern.  The  anterior  ends  of  the  plates  are  separated  by  short  intervals,  which 
become  wider  posteriorly.  The  plaques  extend  backward  about  one-third  of  the 
length  of  the  larva ;  the  bristles  on  the  anterior  portions  are  larger  and  longer  than 
those  more  posteriad.  From  a  naked  area  at  the  posterior  end  of  the  larva,  four 
bristle-bearing  bands  extend  forward  past  the  middle  of  the  body.  The  bands  are 
equidistant  from  each  other  and  both  the  anterior  and  posterior  ones  manifest  a 
spiral  tendency,  but  this  aspect  may  be  the  result  of  rotation  of  the  larva  within  the 
shell.  The  appearance  of  the  miracidium  is  almost  identical  with  that  of  Protcro- 
metra  macrostoma  as  reported  by  Hussey  (1945).  Hussey  described  a  structure, 
designated  by  earlier  authors  as  a  "primitive  gut",  with  four  nuclei  arranged  in  a 
linear  series.  In  A.  sebago,  the  corresponding  structure,  which  is  glandular  and 
probably  serves  in  penetration,  consists  of  four  cells  which  lie  side  by  side  rather 
than  in  linear  series.  These  cells  are  disposed  as  reported  by  Manter  (1926)  for 
the  miracidia  of  Otodistomum  cestoidcs,  Otodistomum  vcliponun  and  Asygia  acu- 
minata. Manter  reviewed  previous  accounts  and  presented  a  strong  argument  that 
the  organ  is  not  a  primitive  gut,  but  a  group  of  unicellular  glands.  Immediately 
posterior  to  the  glandular  organ  there  is  a  bilobed  "brain"  and  the  region  behind  it 
contains  several  large,  germinal  cells.  On  either  side,  near  the  middle  of  the  body, 
there  is  a  single  flame  cell  from  which  an  excretory  tubule  leads  caudad,  but  the 
ducts  were  not  traced  to  the  pores. 

Asexual  generations 

The  youngest  sporocyst  was  recovered  from  a  loose  network  of  connective  tissue 
on  the  somatic  side  of  the  intestinal  wall  of  a  laboratory-raised  snail  that  had  been 
exposed  12  days  previously.  It  was  oval,  0.094  by  0.062  mm.,  with  no  lumen; 
it  contained  germinal  cells  but  no  germ-balls  (embryos).  Other  larger  sporocysts 
were  found  in  older  infections;  one,  0.126  by  0.08  mm.,  contained  germinal  cells 
and  6  small  germ-balls;  another,  0.189  by  0.12  mm.,  contained  germinal  cells  and 
9  germ-balls  of  varying  sizes.  In  a  snail  killed  one  month  after  exposure,  the 
mother  sporocyst  could  not  be  recognized  but  there  were  26  rediae  scattered  about 


264  HORACE  W.  STUNKARD 

in  the  haemocoele.  The  smallest  was  0.25  by  0.18  mm.,  and  in  addition  to  germinal 
cells  it  had  four  small  spherical  to  oval  germ-balls,  0.02  to  0.04  mm.  in  diameter. 
A  redia  with  larger  germ-balls  but  no  recognizable  cercariae  measured  0.57  mm. 
long  and  0.18  mm.  wide;  the  pharynx  was  0.08  mm.  in  diameter  and  there  was  a 
sac-like  gut,  0.11  mm.  long  and  0.032  mm.  wide.  The  largest  redia  was  1.3  by 
0.3  mm.  and  in  addition  to  smaller  embryos,  it  contained  two  cercariae,  one  of  which 
was  more  than  half-grown  and  had  small  furci.  Whether  or  not  there  is  a  second 
generation  of  rediae  was  not  determined. 

The  cercaria-producing  generation  of  species  in  the  genus  Azygia  was  recog- 
nized by  Szidat  (1932)  as  redial,  although  the  pharynx  undergoes  reduction  to  a 
mere  vestige  and  the  intestine  completely  disintegrates.  As  noted  by  Szidat  in  A. 
lucii,  the  pharynx,  which  he  termed  "rudimentary",  serves  for  the  ingestion  of  bits 
of  the  digestive  gland  of  the  host  and  persists  as  a  birth-pore  through  which  the 
cercariae  emerge.  The  small  rediae  are  vermiform  and  very  active ;  the  pharyngeal 
end  may  be  inrolled  and  then  everted,  while  the  opposite  end  may  be  protruded  as 
a  pointed,  tail-like  structure.  Older  rediae  may  extend  to  a  length  of  3  mm.  and 
on  contraction  of  the  circular  muscles,  present  an  annulate  appearance.  On  con- 
traction of  the  longitudinal  muscles  they  become  oval  and  about  1  mm.  in  width. 
The  one  shown  in  Figure  20  is  bent  and  as  mounted  measures  1.12  by  0.325  mm.; 
in  it  the  pharynx  is  still  distinct.  The  older,  larger,  rediae  have  little  mobility  but 
pulsations  of  one  and  sometimes  two  can  occasionally  be  seen  through  the  shell  of 
an  infected  snail.  The  number  of  cercariae  in  a  redia  is  small;  often  there  is  only 
one  and  rarely  are  there  more  than  three  recognizable  cercariae ;  other  individuals 
are  still  in  the  germ-ball  stage,  together  with  a  few  germinal  cells  attached  to  the 
body  wall,  chiefly  at  the  posterior  end  of  the  redia.  Apparently  the  development 
of  one  cercaria  restrains  the  development  of  others.  An  infected  snail  may  liberate 
one  or  two  cercariae  each  day  for  a  few  days  and  then  none  for  a  week  or  more. 
The  large  size  of  the  cercariae  is  correlated  with  the  slow  development  and  the 
small  number  produced. 

Cercaria 

Developing  cercariae  are  typical  furcocercous  larvae.  As  the  embryo  reaches 
a  length  of  approximately  0.25  mm.,  a  constriction  appears  and  gradually  separates 
the  posterior  one-fourth  to  one-third  of  the  larva  as  an  oval,  tail-rudiment.  At 
about  this  stage,  the  oral  sucker  is  faintly  outlined.  When  the  larva  has  reached 
a  length  of  0.4  to  0.5  mm.,  the  suckers  are  distinct,  the  acetabulum  is  in  the  posterior 
half  of  the  body,  the  tail  is  about  three-eighths  of  the  total  length,  and  the  furcal 
buds  are  beginning  to  appear.  As  development  proceeds,  the  tail  increases  in  length 
more  rapidly  than  the  body ;  its  basal  portion,  about  one-sixth  of  its  length,  begins 
to  enlarge  and  by  the  time  the  gonads  are  recognizable,  the  anterior  end  of  the  tail 
forms  a  cup-like  ring  (Fig.  20),  at  the  base  of  which  the  constricted  caudal  end  of 
the  distome  is  continuous  with  the  tissues  of  the  tail.  The  cercariae  complete  their 
growth  in  the  rediae  and  emerge  into  the  haemocoele  of  the  snail.  While  studying 
the  excretory  pattern  of  a  redia  which  was  under  some  pressure,  an  immature  cer- 
caria emerged,  tail  first,  through  the  old  pharyngeal  opening.  The  cercariae  mature 
in  the  haemal  sinuses  of  the  snail,  especially  the  branchial  sinus,  and  emerge  through 
the  respiratory  opening.  During  growth,  the  basal  portion  of  the  tail  is  much  en- 


LIFE-CYCLE  OF  AZYGIA  SEBAGO  265 

larged  by  the  accumulation  of  spongy,  fibre-elastic,  alveolar  tissue  which,  when  the 
cercaria  emerges  from  the  snail,  absorbs  water  and  expands  rapidly.  As  a  result, 
this  portion  of  the  tail  extends  forward,  encapsulating  the  body  of  the  cercaria.  If 
infected  snails  are  crushed  and  immature  larvae  are  liberated  into  water,  the  base 
of  the  tail  is  unable  to  completely  engulf  the  body  of  the  larva  (Fig.  18). 

Mature,  normally  emerged  cercariae  measure  1.8  to  2.3  mm.  in  length.  The 
expanded,  basal  portion  of  the  tail  is  flattened,  0.5  to  0.75  mm.  in  width,  and  slightly 
more  in  length.  The  stem  of  the  tail,  that  portion  from  the  spongy,  rigid,  basal 
part  to  the  furci,  is  1.0  to  1.5  mm.  in  length  and  0.26  to  0.46  mm.  in  width.  It 
tapers  slightly  from  the  basal  to  the  distal  end.  It  is  distinctly  flattened  and  set  at 
right  angles  to  the  dorso  ventrally  flattened  body  of  the  larva,  so  that  when  looking 
at  the  flat  aspect  of  the  tail,  the  body  appears  in  lateral  view  (Fig.  17).  This  stem 
portion  of  the  tail  consists  of  two  bands  of  longitudinal  muscles,  one  on  each  of  the 
flat  surfaces.  These  muscles  are  attached  at  one  end  to  the  rigid,  spongy  portion 
of  the  tail  and  at  the  other  end  to  the  bases  of  the  furci.  The  furci  are  flattened, 
0.55  to  0.90  mm.  in  length  and  0.20  to  0.28  mm.  in  width.  Normally  they  are  held 
almost  at  right  angles  to  the  tail  stem,  whose  muscle  bands  contract  alternately,  so 
that  the  flapping  of  the  tail  from  side  to  side  produces  a  sculling  effect  that  pulls 
the  larva  through  the  wrater.  After  the  beat  of  the  tail  is  unable  to  lift  the  larva 
from  the  bottom,  it  continues  for  a  day  or  two  and  this  flapping  motion  makes  the 
larva  an  attractive  lure  for  small  fishes  and  perhaps  other  predators.  The  basal 
end  of  the  tail  becomes  sticky  and  may  lightly  attach  the  larva  to  the  substratum. 
How  the  larvae  reach  the  pharyngeal  cavity  of  the  planarians  is  not  clear.  The 
body  is  firmly  enclosed  in  the  chamber  at  the  anterior  end  of  the  tail  and  could  be 
liberated  only  by  dissolution  of  the  tail.  According  to  Hyman  (1951,  p.  107), 
"The  triclads  do  not  swallow  their  food  whole  but  suck  it  in  by  peristaltic  action 
of  the  protruded  pharynx."  and  (p.  199),  "The  Turbellaria  are  as  a  class  carniv- 
orous. .  .  .  Favorite  items  of  food  of  the  smaller  species  are  rotifers,  copepods, 
cladocerans,  nematodes,  annelid  worms,  etc.,".  Perhaps  the  planarian  seizes  the 
larva,  and  as  the  tail  is  sucked  in  and  digested,  the  young  worm  is  liberated  and 
attaches  to  the  external  surface  of  the  pharynx,  whence  it  is  carried  into  the  cavity 
when  the  pharynx  is  retracted. 

The  tail  bears  many  papillae,  scattered  somewhat  irregularly  over  the  surface 
except  for  the  distal  three-fourths  of  the  furci.  Each  is  about  0.05  mm.  in  diameter, 
0.025  mm.  tall,  and  is  surmounted  by  a  recurved  hook,  0.012  to  0.015  mm.  in  length. 
The  tail  also  has  many  opaque  patches,  which  on  higher  magnification  are  seen  to 
consist  of  minute  spherules.  The  excretory  system  of  the  larval  body  is  continuous 
with  that  of  the  tail  and  the  constricted  caudal  end  of  the  body  contains  the  common 
excretory  canal  which  traverses  the  stem  of  the  tail,  bifurcates  at  the  bases  of  the 
furci,  and  the  resulting  tubules  open  at  the  tips  of  the  furci.  The  pattern  of  flame 
cells  in  the  tail  was  not  resolved. 

The  morphology  of  the  young  worm,  released  from  the  chamber  in  the  tail,  is 
typically  azygiid  (Figs.  15,  16).  The  cuticula  is  unarmed  but  the  preacetabular 
region  bears  many  papillae  and  a  bristle  has  been  observed  at  the  tip  of  certain  of 
them.  There  are  at  least  a  dozen  papillae,  0.018  to  0.020  mm.  in  diameter,  around 
the  anterior  end  of  the  worm.  Living  specimens  vary  from  0.7  to  1.3  mm.  in  length 
and  0.16  to  0.28  mm.  in  width.  The  acetabulum  varies  from  0.10  to  0.13  mm., 
and  the  oral  sucker,  0.11  to  0.14  mm.,  in  diameter.  The  pharynx  measures  0.05 


266  HORACE  W.  STUNKARD 

to  0.07  mm.  in  length  and  usually  slightly  less  in  width.  The  digestive  ceca  are 
filled  with  yellow  material,  derived  from  the  digestive  gland  of  the  snail.  The  ex- 
cretory system  is  complex  but  has  been  worked  out  completely.  The  pore  is  ter- 
minal and  a  common  duct  leads  forward  almost  to  the  level  of  the  testes.  The 
posterior  one-half  of  this  duct  may  expand  to  form  a  bladder-like  enlargement,  or 
if  the  pore  is  blocked  and  fluid  accumulates,  the  enlargement  may  extend  farther 
forward.  Behind  the  testes  the  common  duct  divides,  forming  two  ducts  which 
pass  forward,  median  to  the  digestive  ceca.  As  the  ceca  turn  mediad  to  join  the 
pharynx,  the  excretory  ducts  pass  below  them  and  continue  on  either  side  of  the 
oral  sucker  almost  to  the  anterior  end  of  the  body.  There  is,  however,  no  connec- 
tion between  the  ducts  of  the  two  sides.  Anterolateral  to  the  oral  sucker,  the  duct 
of  each  side  doubles  backward  and  continues  posteriad,  giving  off  eleven  branches. 
Each  branch  divides  three  times,  forming  two  primary,  four  secondary  and  eight 
tertiary  branches.  Each  tertiary  branch  receives  the  capillaries  from  four  flame 
cells.  The  flame  cell  formula  accordingly  is  2  (11  X  32)  or  704  flame  cells  in  the 
body.  This  observation  is  in  agreement  with  that  of  Looss  (1894)  who  described 
the  same  pattern  in  Azygia  tcrcticolle  (--A.  Incii).  He  regarded  the  ascending 
portions  of  the  excretory  system  as  parts  of  the  excretory  vesicle  and  the  descend- 
ing limb  with  its  branches  as  the  collecting  ducts.  He  suggested  the  possibility  of 
variation  in  the  number  of  branches  and  of  anastomoses  between  collecting  ducts ; 
however,  I  have  found  a  constant  number  of  branches  and  the  apparent  anastomoses 
can  be  resolved  as  places  where  one  duct  crosses  another.  Counting  backward 
from  the  anterior  end  of  the  body,  the  first  side  branch  is  located  at  the  level  of  the 
oral  sucker ;  the  second  is  at  the  level  of  the  bifurcation  of  the  digestive  tract,  i.e., 
the  posterior  end  of  the  pharynx ;  the  third  branch  is  anterior  to  the  acetabulum ; 
the  fourth  is  at  the  middle  of  the  acetabulum ;  the  fifth  is  at  the  level  of  the  posterior 
end  of  the  acetabulum;  the  sixth  and  seventh  are  close  together  a  short  distance 
behind  the  acetabulum ;  the  eighth,  ninth  and  tenth  are  almost  equally  spaced ;  while 
the  eleventh  and  last,  which  is  the  terminal  group  of  the  recurrent  limb,  is  distrib- 
uted to  the  extreme  posterior  end  of  the  body  around  the  excretory  bladder.  The 
reproductive  organs  are  represented  by  groups  of  deeply  staining  cells,  shown  in 
Figures  15,  16  and  17. 

SUMMARY 

A  chronological  account  of  the  genus  Azygia  discloses  discordant  observations 
and  divergent  opinions.  Dawes  (1946)  recognized  only  a  single  species,  A,  Incii, 
in  Europe.  In  it  he  included  A.  robitsta  Odhner,  1911,  which  reaches  a  length  of 
47  mm.  and  Ptychogonimus  rolgcnsis  von  Linstow,  1907,  which  measures  5  to  6 
mm.  in  length  and  had  been  transferred  to  Azygia  as  a  valid  species  by  Odhner 
(1911).  In  America  several  species  have  been  described,  but  there  is  no  agreement 
on  the  number  that  are  distinct  and  valid.  In  fact,  there  is  no  adequate  information 
on  the  extent  of  variation  that  occurs  in  a  natural  species,  and  consequently  on  the 
features  that  can  be  relied  on  to  distinguished  between  species.  This  situation  is 
not  peculiar  to  Asygia,  but  obtains  in  many  genera.  It  is  the  natural  result  of  de- 
velopment by  members  of  a  parasitic  species  in  different  hosts,  invertebrate  and 
vertebrate,  often  of  different  taxonomic  groups,  which  differ  in  their  nutritional  and 
other  physiological  conditions,  and  accordingly  influence  the  development  and 


LIFE-CYCLE  OF  AZYGIA  SEBAGO  267 

morphological  features  of  the  parasite.  Until  the  life-cycle  is  known  and  the  vari- 
ation that  normally  occurs  in  each  possihle  host  is  measured,  the  precise  limits  of 
specificity  will  remain  uncertain.  Comparison  of  specimens  and  descriptions  indi- 
cates that  A.  lucii  may  be  endemic  in  North  America,  that  possibly  it  is  distinct  from 
A.  longa  (Leidy),  that  A.  angusticanda  (Stafford)  is  a  valid  species,  and  that  the 
species  described  by  Goldberger  (1911)  may  be  identical  with  A.  sebago  Ward. 
Information  concerning  the  life-history  of  species  in  the  genus  Azygia  is  meager. 
Szidat  (1932)  showed  that  Ccrcaria  mirabilis  Braun  is  the  larva  of  A.  lucii.  He 
described  a  second  larva,  Ccrcaria  splcndens,  presumably  another  species  of  Azygia, 
but  the  adult  stage  remains  unknown.  Sillman  (1953a)  reported  the  life-cycle  of 
a  species  that  he  identified  as  A.  longa  and  the  present  paper  presents  data  on  the 
morphology  and  life-history  of  a  species  believed  to  be  A.  sebago.  Stages  in  the 
cycle  are  described  and  figured. 

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TWINNING  AND  REPRODUCTION  OF  TWINS  IN  PELMATO- 

HYDRA  OLIGACTIS 

C.  L.  TURNER 

Department  of  Biological  Sciences,  Northwestern   University,  Ei'anston.  Illinois 

This  paper  is  written  to  extend  the  observations  of  the  writer  (Turner,  1950; 
1952)  on  the  reproductive  potential  in  clones  of  Pelmatohydra  oligactis  and  the  regu- 
lation of  spontaneous  structural  anomalies  in  the  same  species.  A  review  of  relevant 
literature  and  certain  conclusions  drawn  from  the  literature  are  contained  in  the 
two  articles  and  they  will  not  be  repeated  here  except  for  a  few  citations  bearing  di- 
rectly upon  the  subjects  of  longitudinal  fission  and  regulation. 

Hyman  (1928)  states  (p.  73)  "that  fission  in  hydra  is  not  a  normal  method 
of  asexual  reproduction  but  a  mode  of  regulation  of  previously  existing  abnormali- 
ties." The  writer  (Turner,  1952)  observed  (p.  107)  that  "the  bifurcated  condi- 
tion of  the  apex  which  initiates  longitudinal  fission  originates  most  commonly,  and 
possibly  exclusively,  in  buds  which  are  in  the  latter  stages  of  development  and  are 
still  attached  to  the  parent."  Chang,  Hsieh  and  Liu  (1952)  found  that  fission  oc- 
curred in  buds  in  a  ratio  of  about  one  in  a  thousand.  The  writer  has  found  since 
1952  in  large  mass  cultures  and  also  in  isolated  strains  that  twinning  in  buds  is 
fairly  common  and  that  true  twinning  originates  only  in  buds.  Conditions  re- 
sembling twinning  have  been  discovered  in  mass  cultures  but  when  these  specimens 
are  followed  through  day  by  day  some  of  them  have  proved  to  be  cases  of  actual 
twinning  but  others  have  proved  to  be  specimens  undergoing  regulation.  The  com- 
plexes are  often  resolved  by  fusion  of  the  members,  absorption  of  one  member  by  the 
other  or  one  member  forms  a  new  foot  and  separate  from  the  other. 

The  term  "twinning"  is  used  here  advisedly  and  has  most  of  the  features  com- 
mon to  monozygotic  twinning  in  higher  forms.  In  both  hydra  and  higher  forms  an 
embryonic  mass,  produced  asexually  as  a  bud  in  hydra,  divides  and  two  individuals 
proceed  separately  to  develop  into  whole  equal  animals.  The  separation  of  the  two 
individuals  may  be  incomplete  in  higher  forms  and  produce  monstrosities  in  various 
degrees.  In  hydra,  individuals  will  become  separated  eventually  because  they  have 
retained  to  a  remarkable  degree  the  capacity  for  regulation  during  which  multiple 
complexes  are  reduced  to  simple  individuals  with  the  characteristics  of  the  parent 
animal.  In  hydra  the  initiation  of  twinning  may  occur  at  any  stage  of  bud  develop- 
ment but  it  is  not  completed  by  the  time  of  detachment  from  the  parent.  Another 
process  comparable  to  twinning  in  its  end  result  is  double  budding.  In  this  process 
two  buds  in  precisely  the  same  stage  of  development  arise  at  the  same  time  in  the 
budding  zone  of  the  parent  as  separate  masses,  usually  on  opposite  sides  of  the  body 
stalk,  and  after  developing  at  the  same  rate  they  become  detached  at  the  same  time. 
At  no  stage  are  they  connected  and  they  differ  from  the  ordinary  bud  in  that  ordi- 
nary buds  arise  in  a  sequence,  differ  from  each  other  in  stages  of  development  and 
become  detached  from  the  parent  at  different  times. 

269 


270  C.  L.  TURNER 

MATERIALS  AND  METHODS 

Specimens  were  separated  from  a  large  mass  culture  and  were  placed  individu- 
ally in  bottles  of  about  100  cc.  capacity.  They  were  fed  with  Entomostraca  daily 
and  the  water  in  which  each  lived  was  replaced  daily  with  water  from  the  large  tank 
in  which  the  mass  culture  was  maintained.  Daily  records  were  kept  of  each  speci- 
men concerning  its  behavior,  its  reproduction  and  its  periods  of  physiological 
depression. 

In  order  to  secure  an  accurate  determination  of  the  extent  of  twinning  in  the 
members  of  the  mass  culture,  single  non-twinning  specimens  were  separated  from 
the  mass  culture  and  a  record  was  kept  of  all  of  the  young  produced  by  budding. 
The  record  of  each  was  followed  for  20  to  30  days  and  the  proportion  of  twinning 
to  non-twinning  buds  was  determined  from  the  totals.  The  process  was  repeated 
three  times  at  two-month  intervals. 

When  twinning  buds  were  discovered  they  were  isolated  after  they  had  become 
detached  from  the  parents  and  daily  records  were  kept  of  buds  produced  by  the 
single  undivided  portions  and  by  the  bifurcated  portions  of  the  twin.  The  total 
number  of  buds  produced  by  the  complex  was  secured  in  this  way  and  a  comparison 
could  be  made  between  twinning  individuals  and  single  individuals  of  the  rate  of 
bud  production.  The  proportion  of  single  to  twinning  buds  was  obtained  and  could 
be  compared  to  the  production  of  single  and  twinning  buds  in  control  specimens 
which  had  no  previous  history  of  twinning.  The  possibility  that  certain  strains  had 
a  tendency  to  produce  a  high  proportion  of  twinning  buds  could  be  examined  from 
the  results. 

PROPORTION  OF  TWINNING  TO  NON-TWINNING  INDIVIDUALS 

Any  estimate  of  the  proportion  of  twinning  to  non-twinning  individuals  based 
upon  bifurcated  specimens  taken  from  a  large  mass  culture  would  be  invalid  because 
a  part  of  the  individuals  in  the  bifurcated  state  would  not  be  cases  of  genuine  twin- 
ning. Some  of  them  would  be  mature  individuals  in  which  a  bifurcation  had  arisen 
at  the  apex  during  or  immediately  after  a  period  of  depression.  By  the  selection  of 
healthy  single  individuals  and  the  recording  of  each  bud  which  arose  in  these  indi- 
viduals, it  was  possible  to  know  whether  any  suspected  case  of  twinning  was  real 
and,  if  it  was  real,  to  know  also  the  place  of  its  origin  and  its  subsequent  history. 
Twenty-five  single  individuals  selected  from  the  large  mass  culture  were  given  the 
most  favorable  conditions  for  reproduction  and  within  a  period  of  20  to  30  days 
they  produced  a  total  of  705  buds  of  which  14  were  twins.  The  twinning  buds 
represented  1.98  +%  of  the  total.  Two  months  later  11  single  individuals,  selected 
from  the  same  mass  culture  and  maintained  in  the  same  way,  produced  274  buds  of 
which  4  (or  1.45  +%)  were  twins.  The  operation  was  repeated  two  months  later 
and  12  specimens  produced  299  buds  of  which  6  (or  2.01%)  were  twins.  In  the 
three  samples,  48  isolated  individuals  produced  1278  buds  of  which  24  or  1.87  +% 
were  twin  buds. 

Twenty  of  the  twin  buds  were  isolated  when  they  were  detached  from  the  parent 
and  a  record  was  kept  of  the  production  of  buds  by  each  specimen  for  13  to  22  days. 
A  total  of  523  buds  was  produced  of  which  19  (3.63  +%)  were  twin  buds.  During 
this  time  ten  of  the  twinning  specimens  produced  only  single  buds  but  one  of  them 
produced  three  twinning  buds  (Fig.  3)  and  several  produced  two  twinning  buds 


TWINNING  IN  HYDRA  271 

(Figs.  1  and  2).  Since  the  reproducing  individuals  derived  from  twinning  buds 
produced  nearly  twice  as  many  twinning  buds  as  individuals  taken  at  random  from 
the  mass  culture,  it  may  be  inferred  that  an  inherited  tendency  for  bud  twinning 
existed  in  the  strains  showing  unusual  bud  twinning. 

ORIGIN  AND  STRUCTURE  OF  TWINS 

Twinning  has  been  observed  in  buds  at  various  stages  of  development.  In  the 
earliest  cases  the  bud  develops  a  bilobed  condition  almost  from  the  moment  of  its 
origin  as  a  bud  (Fig.  17).  Each  lobe  then  elongates  and  differentiates  (Figs.  4 
and  5)  and  by  the  time  of  detachment  from  the  parent  the  two  members  of  the  twin 
are  separate  except  at  the  base  (Fig.  6).  If  the  specimen  indicated  in  Figure  6 
were  to  be  found  in  a  mass  culture  without  its  previous  history  being  known,  it 
might  be  interpreted  as  a  case  of  apico-basal  fission  but  it  is  actually  one  in  which 
no  fission  is  involved  up  to  the  time  of  its  detachment.  Rather,  two  separate  por- 
tions of  the  original  single  bud  have  undergone  parallel  development.  After  de- 
tachment the  twinning  individual  undergoes  fission  and  the  two  members  are  sepa- 
rated. It  may  be  stated  that  twinning  gives  rise  to  an  anomalous  state  which  is 
resolved  by  regulation  (fission). 

A  bud  may  develop  as  a  single  unit  for  some  time  and  then  give  rise  to  dual 
masses  at  the  apex  (Fig.  7).  The  basal  single  portion  elongates  somewhat  and  at 
the  same  time  the  members  of  the  divided  portion  will  elongate  and  differentiate. 
After  detachment  from  the  parent  (Fig.  8)  fission  of  the  remaining  common  stalk 
and  the  base  occurs.  Regulation  (fission)  is  involved  to  a  greater  extent  than  it 
is  in  cases  of  very  early  bud-twinning. 

Twinning  occurs  at  a  late  stage  of  bud  development  in  some  cases  (Fig.  9)  and 
when  the  bud  is  detached  from  the  parent  only  the  apical  portion  of  the  bud  is 
bifurcated.  The  regulation  process  (fission)  occupies  a  longer  part  of  the  entire 
period  during  which  two  complete  individuals  are  formed  from  a  single  bud. 

A  bud  occasionally  gives  rise  to  three  instead  of  two  units.  In  the  case  illustrated 
in  Figure  11  three  hypostomes,  each  with  a  circle  of  tentacles,  arose  in  a  late  stage 
of  bud  development.  Before  the  bud  was  detached  the  hypostome  of  one  unit  was 
absorbed  by  another  but  the  tentacles  of  the  absorbed  unit  remained  intact.  When 
the  bud  was  detached  it  resembled  the  one  shown  in  Figure  10  except  that  one  of 
the  terminal  units  possessed  supernumerary  tentacles.  In  this  case  three  processes 
of  regulation  would  be  involved  before  two  normal  single  individuals  were  formed 
from  the  complex  bud.  Absorption  of  the  hypostome  occurs  before  bud  detach- 
ment. Apico-basal  fission  would  separate  the  two  members  after  detachment  of  the 
complex  from  the  parent,  and  still  later  fusion  of  tentacles  would  occur  in  the  indi- 
vidual having  supernumerary  tentacles  until  the  normal  number  of  tentacles  was 
produced. 

Secondary  twinning,  i.e.,  twinning  in  one  or  both  members  of  a  specimen  which 
is  itself  a  twin,  has  been  observed  a  number  of  times.  In  Figure  12  a  single  speci- 
ment  is  illustrated  together  with  a  twinning  bud.  The  primary  twinning  occurred 
when  the  bud  was  half  developed  and  the  two  members  of  the  twin  bud  proceeded 
to  elongate  and  differentiate.  At  a  late  stage  in  differentiation  one  of  the  mem- 
bers divided  at  the  apex.  When  the  bud  was  detached  it  consisted  of  a  common 
stalk  and  foot  and  a  stalk  divided  in  its  terminal  half.  Also  one  member  of  the  di- 


272 


C.  L.  TURNER 


1   1 

1  1  1 

T 

M  M   M   1  1  1  1   1  1      • 

rfl 

R 

n 

^J                     rj 

^           ^^-^                                      *•  T 

T 

n 

1  1  1 

T 

II       M   II   1   1   M 

5                   IO                  15                  20 
AFTER    DETACHMENT  


FIGURE  I 


1  1  M  1 

1  1 

0  5  10  15 

DAYS    AFTER     DETACHMENT^ 

FIGURE  2 


n 

--Or              PI      - 

r 

r  1  1 

1  1  r  n  M  • 

r    1 

i 

-r^IlN. 

r  ~ 
j  

rfl 

1  r  • 

rllii 

p  ] 

1  1  • 

• 

0                   5 

DAYS  AFTEt 

10                 IS 

?    DFTACHMFNT 

20 

FIGURE  3 

FIGURE  1.  Reproduction  by  budding  in  twinning  specimen  shown  in  Figure  10.  Period 
covers  time  from  detachment  from  parent  to  time  of  separation  of  twins  by  apico-basal  fission. 
Fission  was  completed  in  24  days.  Squares  indicate  new  buds  per  day.  Reproductive  record 
of  single  portion  shown  at  left,  of  divided  portions,  in  I  and  II.  T  indicates  twinning  in  a  bud. 


TWINNING  IN  HYDRA  273 

vided  portion  had  two  hypostomes.  In  the  member  which  was  divided  at  the  end, 
fusion  occurred  and  within  several  days  a  single  hypostome  was  present,  surrounded 
by  eight  tentacles.  Fission  separated  the  two  primary  members  eighteen  days  after 
detachment  of  the  twinned  bud  from  the  parent.  Figures  15,  16  and  17  illustrate 
cases  in  which  twinning  buds  had  become  detached  from  the  parent  and  were  ac- 
tively reproducing.  Fission  which  will  separate  the  primary  twins  is  in  progress. 
A  new  bud  appearing  on  one  of  the  stalks  of  the  specimen  (Fig.  17)  is  twinning  in 
an  early  stage  producing  a  case  of  secondary  twinning.  Similar  situations  are 
illustrated  in  Figures  15  and  16  except  that  in  both  cases  fission  has  divided  the  de- 
tached primary  twin  down  through  the  budding  zone  and  the  secondary  twinning 
buds  are  arising  from  divided  instead  of  the  common  undivided  parts  of  the  stalk. 
A  remarkable  case  is  illustrated  in  Figure  18.  The  complex  of  four  members  arose 
as  a  single  bud  which  twinned  at  an  early  stage.  After  a  short  period  of  elongation 
of  the  two  members  each  member  twinned  again.  Elongation  of  each  of  the  four 
members  occurred  and  each  differentiated  with  a  full  complement  of  tentacles  and  a 
hypostome.  After  the  complex  was  detached  from  the  parent  the  primary  division 
was  completed  by  fission  and  some  days  later  a  secondary  fission  separated  the  mem- 
bers of  the  secondary  twins.  No  fusion  of  any  kind  occurred.  It  appears  in  com- 
paring the  cases  shown  in  Figures  9,  10  and  18  that  secondary  twinning  is  likely  to 
be  effective  and  to  produce  separate  individuals  if  it  occurs  early  and  the  members 
are  able  to  grow  and  to  differentiate  a  full  complement  of  normal  parts,  but  if  the 
twinning  occurs  very  late  one  member  is  likely  to  be  absorbed  by  the  other. 

Complexes  are  encountered  occasionally  which  involve  at  the  same  time  multi- 
ple twinning  and  other  unusual  features.  They  present  a  complicated  appearance 
and  the  units  can  be  accounted  for  only  if  a  complete  record  has  been  kept  from 
the  time  of  origin  of  the  individual  producing  the  complex.  A  case  in  point  is  the 
complex  illustrated  in  Figure  16,  and  the  history  of  this  case  is  as  follows.  A  bud 
arose  from  a  normal  single  hydra  and  proceeded  to  twin  when  half  developed.  The 
twinning  bud  became  detached  from  the  parent  and  began  to  produce  buds  of  its 
own  in  the  common  stalk  almost  immediately.  Fission  of  the  twinned  individual 
was  carried  basally  and  passed  through  the  budding  zone.  At  the  moment  il- 
lustrated in  Figure  16,  I  and  II  represent  the  divided  portions  of  the  individual  de- 
scribed. Portion  I  has  three  buds,  A,  B  and  C.  Bud  C  is  going  through  the  un- 
usual process  of  giving  rise  to  a  new  bud  before  it  has  become  detached  from  the 
parent.  Portion  II  is  giving  rise  to  buds  A',  B'  and  C'.  Bud  C  twinned  at  an 
early  stage  and  has  not  become  detached.  The  subsequent  history  of  the  complex 
involved  further  reproduction  and  a  resolution  into  single  normal  units.  Portions 
I  and  II  gave  rise  to  new  buds  while  they  were  being  separated  by  fission  and 
thereafter  appeared  as  normal  single  budding  individuals.  Bud  C  became  detached 
within  a  few  hours  and  became  a  single  reproducing  individual.  Twinning  bud  C' 

The  notch  in  the  broken  line  at  the  right  indicates  the  time  of  separation  by  fission  of  the  mem- 
bers of  the  twin. 

FIGURE  2.  Reproduction  by  budding  in  twinning  specimen  divided  for  half  of  its  length 
when  detached  from  parent  as  in  Figure  8.  Fission  complete  on  16th  day.  Symbols  as  in 
Figure  1. 

FIGURE  3.  Reproductive  record  in  twinning  specimen  divided  for  %  of  its  length  at  time 
of  detachment  from  parent.  Reproductive  period  extends  from  time  of  detachment  of  twinned 
bud  from  parent  (0  days)  to  completion  of  fission  (10  days)  and  in  addition,  10  days  after  com- 
pletion of  fission.  Symbols  as  in  Figure  1. 


274 


C.  L.  TURNER 


15 


13 


16 


17 

FIGURES  4-18. 


8 


10 


12 


18 


TWINNING  IN  HYDRA  275 

became  detached  in  several  hours  and  nine  days  later  fission  had  separated  the 
members  of  the  twin.  In  the  meantime  twinning  bud  C  was  budding  off  new  in- 
dividuals. A  total  of  eighteen  days  were  required  from  the  time  of  the  appearance 
of  the  original  bud  for  the  formation  of  the  complex  and  for  its  resolution  into 
single  individuals. 

REPRODUCTION  IN  TWINS 

Reproduction  in  twins  was  studied  for  the  purpose  of  determining  whether  the 
process  of  twinning  and  subsequent  regulation  interfered  with,  or  in  any  way  af- 
fected, the  process  of  reproduction  and  also  to  compare  the  over-all  production  of 
new  individuals  in  twins  with  that  of  single  individuals.  Three  cases  were  selected 
for  illustration  on  the  basis  of  the  degree  of  apico-basal  division  in  the  twins  at  the 
time  of  the  detachment  from  the  parent. 

In  case  1  (Fig.  1)  the  twinning  bud  was  detached  from  the  parent  when  the 
apical  part  was  divided  for  approximately  one-third  of  the  entire  length  of  the 
bud.  The  budding  zone  in  the  common  stalk  was  not  divided  at  the  time  of  de- 
tachment and  three  individual  buds  were  produced  within  the  first  four  days  on 
the  common  stalk.  Buds  produced  by  the  common  stalk  are  shown  by  blocks  in  the 
middle  line  of  the  three  lines  of  blocks.  On  the  fourth  day  after  detachment,  fission 

FIGURE  4.  Single  parent  with  bud  which  twinned  at  an  early  stage  and  developed  as  two 
individuals  separated  except  for  basal  %  at  time  of  detachment. 

FIGURE  5.  Single  parent  with  bud  which  twinned  at  an  early  stage  and  was  almost  com- 
pletely divided  at  time  of  detachment  from  parent. 

FIGURE  6.     Bud  shown  in  Figure  4  after  detachment  from  parent. 

FIGURE  7.     Young  bud  which  twinned  when  half  developed. 

FIGURE  8.     Bud  shown  in  Figure  7  after  detachment  from  parent. 

FIGURE  9.  Bud  which  twinned  at  a  late  stage  of  development.  Supernumerary  tentacles 
and  fusing  pairs  of  tentacles  in  parent  indicate  that  parent  is  an  incompletely  regulated  speci- 
men in  which  two  apical  units  have  fused.  Bud  was  divided  for  %  of  its  length  when  detached. 

FIGURE  10.  Bud  shown  in  Figure  9  after  detachment  from  parent.  Apico-basal  fission  was 
completed  in  24  days.  See  Figure  1. 

FIGURE  11.  Multiple  division  of  the  apical  end  of  a  bud.  After  detachment  one  of  the  hy- 
postomes  fused  with  the  nearest  hypostome.  The  two  remaining  units  were  separated  by  apico- 
basal  fission. 

FIGURE  12.  Twinning  occurred  at  an  intermediate  stage  of  bud  development  and  secondary 
twinning  occurred  late  in  the  development  of  one  of  the  units.  The  secondary  twinning  was 
reduced  by  absorption  of  one  member  by  the  other. 

FIGURE  13.  Budding  in  the  undivided  portion  of  a  specimen  which  twinned  as  a  bud  and  is 
undergoing  apico-basal  fission.  See  Figure  2. 

FIGURE  14.  Same  specimen  as  in  Figure  13.  Apico-basal  fission  has  proceeded  down  to  the 
budding  zone. 

FIGURE  15.  Same  specimen  as  in  Figure  13.  Apico-basal  fission  has  proceeded  through 
the  budding  zone  down  to  the  stalk.  One  of  the  new  buds  is  a  twin. 

FIGURE  16.  Specimen  8  days  after  it  was  detached  from  parent  as  a  twinned  bud  divided  for 
about  %  of  its  length.  Apico-basal  fission  has  proceeded  downward  through  the  budding  zone. 
Member  I  has  three  buds,  A,  B  and  C.  C,  not  yet  detached,  is  producing  a  bud.  Member  II 
has  produced  three  buds,  A',  B'  and  C'.  C'  is  a  twinning  bud.  Specimen  produced  43  buds 
before  fission  was  completed  in  21  days. 

FIGURE  17.  Specimen  which  arose  as  a  twinned  bud  and  is  now  producing  a  twinning  bud. 
Divided  members  of  the  new  bud  have  unusual  arrangement  along  apico-basal  axis  of  parent. 

FIGURE  18.  Complete  double  twin.  Specimen  twinned  in  an  early  bud  stage  and  each 
member  twinned  at  a  later  stage  before  the  four-member  complex  was  detached  from  the  parent. 


276  C.  L.  TURNER 

had  carried  down  to  the  hudding  zone  and  two  new  buds  appeared  on  the  common 
stalk  and  two  upon  one  of  the  divided  portions  above  the  point  of  function  with  the 
other  member.  On  the  fifth  day  after  detachment  another  bud  appeared  on  the 
same  member.  On  the  sixth  day  one  new  bud  appeared  upon  the  common  stalk 
and  one  each  on  the  divided  members.  Thereafter  no  new  buds  appeared  upon  the 
common  stalk  and  it  is  apparent  that  fission  had  carried  down  through  the  budding 
zone  and  that  the  time  for  its  passage  through  the  budding  zone  was  three  to  four 
days.  The  time  of  the  complete  separation  of  the  members  of  the  twin  was  23  days 
after  the  twinned  bud  had  become  detached  from  the  parent.  The  time  of  separa- 
tion is  indicated  by  the  notch  in  the  broken  line  at  the  right  of  the  figure.  During 
the  process  of  fission,  during  which  the  members  were  separated  from  each  other, 
reproduction  continued  in  each  member  at  approximately  the  same  rate  as  it  would 
have  occurred  in  a  single  individual. 

Reproduction  in  a  bud  which  had  twinned  somewhat  earlier  than  the  one  shown 
in  Figure  1  is  represented  in  Figure  2.  When  the  bud  was  detached  from  the 
parent  it  was  divided  apically  for  about  one-half  of  its  length,  presenting  the  ap- 
pearance of  the  specimen  shown  in  Figure  8.  The  division  point  was  within  the 
budding  zone  and  each  member  began  to  form  new  buds  the  day  after  the  twin  was 
detached  from  the  parent.  Fission  carried  down  through  the  budding  zone  within 
three  days  during  which  time  the  common  stalk  produced  two  buds.  The  mem- 
bers of  the  twin  were  separated  from  each  other  16  days  after  the  twinned  bud  was 
detached  from  the  parent  and  each  produced  buds  continually  during  the  regula- 
tory process  of  fission. 

A  twin-bud  specimen  which  was  divided  for  about  seven-eighths  of  its  length 
when  detached  from  the  parent  reproduced  as  shown  in  Figure  3.  The  specimen 
(Fig.  6)  had  twinned  at  an  early  stage  and  separate  and  complete  budding  zones 
were  represented  in  each  member.  Each  member  began  to  give  off  new  buds  as 
soon  as  the  specimen  was  detached  from  the  parent  and  continued  to  do  so  for  the 
ten  days  required  for  fission  to  separate  the  members.  The  reproductive  history 
of  each  member  for  ten  days  after  separation  is  shown  at  the  right  in  the  diagram. 
It  will  be  noted  that  the  rate  of  bud  production  in  each  member  was  about  equal  and 
that  the  rate  is  the  same  whether  the  members  were  attached  to  each  other  or 
separated. 

It  is  apparent  from  the  results  that  the  process  of  twinning,  and  of  fission  which 
separates  the  members  of  a  twinned  individual  after  its  detachment  from  the  parent, 
do  not  affect  the  process  of  reproduction  by  budding.  It  may  be  added  that  there 
is  no  interference  with  the  formation  and  maturing  of  spermaries.  Buds,  whether 
single  or  twinning,  become  detached  from  the  parent  within  48  hours  after  their  first 
appearance  if  the  parent  is  not  in  a  state  of  depression,  and  spermaries  are  not 
formed  until  later.  However,  spermaries  have  been  observed  in  both  members  of 
twinned  specimens  during  the  process  of  fission  which  later  separates  the  members 
of  the  twin. 

DURATION  OF  FISSION 

Fission,  as  the  term  is  used  here,  refers  to  the  process  moving  in  the  apico-basal 
axis  by  which  the  members  of  a  twinned  individual  are  separated.  It  occurs  after 
the  bud  has  been  detached  from  the  parent.  The  rate  at  which  fission  proceeds  is 


TWINNING  IN  HYDRA  277 

quite  variable  and  the  duration  of  the  process  depends  upon  the  degree  of  initial 
separation  of  the  members  at  the  time  of  detachment  from  the  parent  and  upon  the 
rate  at  which  it  proceeds.  Four  individuals  divided  at  the  apical  end  for  a  distance 
of  one-fifth  of  the  total  length  required,  respectively,  17  days,  27  days,  38  days  and 
51  days  for  complete  separation.  The  process  moved  through  the  apical  third 
rapidly,  and  proceeded  through  the  budding  zone  in  three  to  four  days.  Fission 
moved  at  a  slower  rate  through  the  body  stalk  at  the  basal  end  and  lagged  greatly 
in  the  region  of  the  foot.  In  an  extreme  case  20  days  were  required  for  separa- 
tion of  the  foot  after  fission  had  carried  down  to  that  point.  Two  specimens,  each 
separated  for  one-half  of  the  total  length  at  detachment,  required  23  days  for  the 
completion  of  fission.  Three  specimens  divided  almost  to  the  base  at  the  time  of 
detachment  required,  respectively,  8,  9  and  1 1  days  for  complete  separation. 

SUMMARY 

1.  In  the  specimens  observed  in  pedigreed  cultures,  genuine  twins  arose  only 
in  buds. 

2.  Fission,  regarded  as  a  separate  regulatory  process  in  which  twinning  com- 
plexes are  resolved  into  single  individuals,  occurs  after  the  twinning  buds  have  be- 
come detached  from  the  parent. 

3.  In  pedigreed  cultures  specimens  arising  as  single  buds  produced  1278  buds  of 
which  24  were  twin  buds.     Specimens  arising  as  twinning  buds  produced  523  buds 
of  which  19  were  twin  buds. 

4.  Twinning  may  occur  in  a  bud  at  any  stage  of  development.     An  early  twin- 
ning bud  is  deeply  divided  at  the  time  of  detachment  from  the  parent  and  a  late 
twinning  bud  is  divided  only  at  the  apex. 

5.  Multiple  twinning  occurs  occasionally  in  which  one  or  both  members  of  a 
twin  bud  undergo   secondary  twinning  before   detachment   of  the   complex   from 
the  parent. 

6.  Bud  production  by  a  specimen  arising  as  a  twin  bud  is  equal  to  that  of  a  single 
individual  as  long  as  the  budding  zone  is  undivided.     Bud  production  is  doubled  as 
the  budding  zone  is  divided  by  fission. 

7.  Completion  of  fission  of  a  twin  bud  requires  usually  from  8  to  27  days  but 
may  take  as  long  as  51  days  in  a  depressed  specimen.     Fission  proceeds  rapidly  at 
the  apical  end,  passes  through  the  budding  zone  in  three  or  four  days  and  is  retarded 
most  at  the  basal  end  of  the  body  stalk  and  the  foot. 

LITERATURE  CITED 

CHANG,  JOSEPH  T.,  H.  H.  HSIEH  AND  D.  P.  Liu,  1952.     Observations  on  hydra,  with  special 

reference  to  abnormal  forms  and  bud  formation.     Physiol.  Zool,  25  :   1-10. 
HYMAN,   LIBBIE   H.,    1928.     Miscellaneous   observations   in    Hydra,    with    special    reference   to 

reproduction.     Biol.  Bull,  54:  65-109. 
TURNER,  C.  L.,  1950.     The  reproductive  potential  of  a  single  clone  of  Pelmatohydra  oligactis. 

Biol.  Bull.,  99 :  285-299. 
TURNER,   C.   L.,   1952.     The   regulation  of   spontaneous   structural   anomalies   in   Pelmatohydra 

oligactis.    Biol.  Bull.,  103:  104-119. 


PROPERTIES  OF  THE  CONNECTIVE  TISSUE  SHEATH  OF  THE 
COCKROACH  ABDOMINAL  NERVE  CORD  l- 2 

B.  M.  TWAROG3  AND  K.  D.  ROEDER 
Department  of  Biology,  Tufts  University,  Mcdjord,  Massachusetts 

Hoyle  (1952,  1953)  has  drawn  attention  to  the  continuous  sheath  which  sur- 
rounds nerve  fibers  and  ganglia  of  Locusta  and  other  insects.  He  has  described  the 
structure  of  this  sheath  and  demonstrated  that  its  effectiveness  as  a  diffusion  bar- 
rier enables  the  nerves  of  Locusta  to  function  normally  despite  wide  variations  in 
the  ionic  composition  of  the  surrounding  fluid.  His  valuable  work  indicates  basic 
similarity  in  the  membrane  properties  of  insect  nerve  and  nerve  of  vertebrates  and 
of  invertebrates  other  than  insects.  In  the  present  study  of  the  ventral  nerve  cord 
of  the  roach,  Hoyle's  conclusions  are  confirmed.  Normally  sheathed  and  de- 
sheathed  cords  were  compared  with  respect  to  interference  with  nervous  function 
by  variation  in  total  salt  concentration,  sodium  deficiency,  excess  potassium  ions 
and  acetylcholine.  Certain  structural  details  were  studied  histologically. 

MATERIALS  AND  TECHNIQUES 

Adult  male  specimens  of  Pcriplancta  amcricana  have  minimal  fatty  deposits 
about  the  cord  and  were  therefore  used.  For  observations  of  the  effects  of  ions 
on  axonic  conduction  in  the  ventral  cord,  the  head  was  crushed,  and  the  cockroach 
was  pinned,  ventral  side  up,  on  a  cork  platform,  with  the  legs  taped  down.  Test 
solutions  were  applied  and  conduction  examined  in  a  segment  of  nerve  cord  com- 
prising the  fourth  abdominal  ganglion  and  the  connectives  between  the  fourth  and 
fifth  ganglia.  Cuticle  was  removed  over  this  region  and  a  thin  paraffin  sheet  was 
placed  beneath  the  test  segment  (Fig.  la).  Drainage  arranged  from  below  the 
paraffin  minimized  mixture  of  hemolymph  with  the  test  solutions,  which  were  per- 
fused over  the  segment  lying  on  the  paraffin.  In  some  experiments,  it  was  pos- 
sible to  avoid  cutting  any  large  nerves  or  tracheal  branches  by  locating  the  paraffin 
entirely  under  the  connectives.  Silver-silver  chloride  hook  electrodes  (Roeder, 
1946)  were  placed  below  the  cord  and  moved  over  the  test  area  so  that  localized 
axonic  block  could  be  detected  by  changes  in  form  of  the  compound  action  po- 
tential in  the  giant  fibers  (Fig.  Ib). 

Action  potentials  conducted  into  or  through  the  test  area  could  be  elicited  either 
directly  via  stimuli  from  a  pair  of  silver  electrodes  (Fig.  la,  Sx)  inserted  under  the 
nerve  cord  through  a  small  cuticular  opening  near  the  first  abdominal  ganglion,  or 
transynaptically  via  stimuli  from  a  similar  pair  (SL.)  inserted  into  the  base  of  a 
cercus.  An  uninterrupted  sequence  of  square  pulses  (0.5  per  second;  0.2  msec. 

1  The  work  described  in  this  paper  was  done  under  contract  between  the  Medical  Division, 
Chemical  Corps,  U.  S.  Army  and  Tufts  University.  Under  the  terms  of  this  contract,  the 
Chemical  Corps  neither  restricts  nor  is  responsible  for  the  opinions  or  conclusions  of  the  authors. 

-  The  authors  wish  to  acknowledge  the  able  assistance  of  Miss  Janice  Green  and  Mr. 
Edward  L.  Raymond  in  the  histological  work. 

3  Present  address :  The  Biological  Laboratories,  Harvard  University,  Cambridge  38,  Mass. 

278 


COCKROACH  NERVE  SHEATH 


279 


S 


la 


NORMAL  SALINE 


SODIUM  DEFICIENT 
SALINE 

Ib 

FIGURE  1.  (a)  Si,  stimulating  electrode  pair  on  cord  near  first  abdominal  ganglion ;  S,, 
stimulating  electrode  pair  on  cereal  nerve ;  p,  paraffin  sheet ;  r,  recording  electrode  pair ;  A,  B, 
C,  sites  in  test  area  at  which  action  potentials  were  recorded,  (b)  Records  of  action  potentials 
at  sites  A,  B,  and  C ;  stimulation  at  Si  in  a  preparation  desheathed  at  B.  In  sodium-deficient 
saline,  the  spikes  are  larger,  due  to  decreased  shunting  by  electrolyte.  Localized  block  in  the 
desheathed  region  between  electrodes  is  indicated  by  the  monophasic  spike  at  B. 

duration)  was  applied  through  Sj  except  for  hrief  periods  when  ascending  conduc- 
tion was  checked  through  stimulation  at  SL,.  In  normal  or  potassium-free  saline, 
this  preparation  responded  uniformly  well  for  many  hours.  "Normal"  saline  re- 
fers to  Hoyle's  (1953)  basic  mixture  for  Locusta,  which  proved  most  satisfactory 
in  our  experiments.1  High  potassium  and  potassium-free  salines  were  made  up  as 
detailed  by  Hoyle. 


MvCl 

NaCl 

Call, 


10  mM./L. 

130  mM./L. 

2  mM./L. 


MgCL 

NaH..PO4 

NaHCO3 


2  mM./L. 
6  mM./L. 
4  mM./L. 


280 


B.  M.  TWAROG  AND  K.  D.  ROEDER 


The  last  abdominal  ganglion,  exposed  as  described  by  Roeder,  Kennedy  and 
Samson  (1947),  was  the  test  object  in  acetylcholine  studies.  Electrical  stimuli 
were  applied  to  the  cereal  nerve  at  low  frequency  (0.5/sec.)  and  the  postsynaptic 
response  was  led  off  near  the  fifth  ganglion.  The  last  ganglion  was  continuously 
perfused  with  saline,  to  which  acetylcholine  wras  added  for  tests. 

The  recording  system  consisted  of  a  Grass  P-4  preamplifier  and  a  Dumont 
304-A  oscilloscope.  Responses  were  photographed  with  a  Dumont  oscillograph  rec- 
ord camera,  type  297.  Square  pulses  were  delivered  from  a  Grass  S-2  stimulator. 

Methods  were  developed  for  desheathing  ganglia  and  connectives  in  the  above 
preparations.  These  operations  were  most  conveniently  performed  under  a  dis- 
secting binocular  at  a  magnification  of  80  X,  using  two  pairs  of  fine-ground  watch- 
maker's forceps.  Lowering  the  saline  level  briefly  caused  a  barely  perceptible 


FIGURE  2.  Cereal  nerve  stimulated.  Postsynaptic  responses  recorded  from  abdominal  cord 
near  fifth  ganglion,  (a)  In  normal  saline;  last  abdominal  ganglion  normally  sheathed,  (b) 
After  10  minutes  in  acetylcholine  10~2  M ;  normally  sheathed,  (c)  After  one  minute  in  acetyl- 
choline 1CT2  AI ;  desheathed.  (d)  After  three  minutes  in  acetylcholine  10""  AI ;  desheathed. 
(e)  After  four  minutes  in  acetylcholine  10~2  M;  desheathed.  (f)  After  washing  in  normal 
saline ;  desheathed. 

crinkling  of  the  sheath  about  the  ganglion.  The  sheath  was  lifted,  torn,  and  gently 
pulled  away  from  the  entire  dorsal  surface  of  the  ganglion.  The  saline  level  was 
rapidly  adjusted  to  prevent  drying  of  the  desheathed  ganglion.  This  procedure  in 
no  way  altered  the  character  of  the  postsynaptic  response  (see  Fig.  2).  Desheath- 
ing the  connectives  was  more  difficult  since  conduction  failed  if  even  a  brief  drying 
occurred,  and  visualization  of  the  sheath  under  saline  took  some  practice.  Stretch- 
ing the  desheathed  connective  had  to  be  avoided.  The  sheath,  once  torn,  could  be 
rolled  back  along  the  connective,  which  it  enveloped  in  stocking-like  fashion.  The 
sheath  is  quite  elastic  and  constriction  of  the  cord  by  the  rolled-up  sheath  had  to 
be  avoided  by  proper  tearing.  The  stripping,  successfully  accomplished,  did  not 
alter  the  nerve  action  potential.  In  normal  saline,  desheathed  preparations  re- 
sponded without  change  for  hours. 

The  posterior  portion  of  the  abdominal  cord,  containing  the  fifth  and  last  ab- 
dominal ganglia,  was  examined  in  6-micron  serial  sections.  A  mercuric  chloride- 
acetic  acid  mixture  provided  most  satisfactory  fixation.  Ester  wax  (Steedman, 


COCKROACH  NERVE  SHEATH 


281 


1947),  with  increased  paraffin  content,  proved  an  excellent  embedding  medium. 
Conventional  staining  techniques  included :  Mallory's  triple  stain,  Masson's  tri- 
chrome,  Gomori's  chromium-hematoxylin-phloxin  (Gomori,  1941)  and  Holme's 
silver  as  modified  by  Batham  and  Pantin  (1951).  Desheathed  specimens  were 
fixed  after  physiological  tests. 

A  series  of  cords  \vas  stained  by  Laidlaw's  method  as  described  by  Krnjevic 
(1954)  after  the  fifth  ganglion  and  one  half  of  the  connective  between  fifth  and 
sixth  ganglia  had  been  desheathed.  The  living  cord  was  placed  in  0.5%  silver 
nitrate  and  observed  under  strong  illumination.  One  of  this  series  was  fixed,  sec- 
tioned and  counterstained  with  Mallory's  (Fig.  3b). 


FIGURE  3.  (a)  Normally  sheathed  last  abdominal  ganglion  stained  with  Masson's  trichrome. 
Note  outer  homogeneous  layer  and  inner  nucleated  granular  layer  of  sheath,  (b)  Normally 
sheathed  last  abdominal  ganglion  after  silver  nitrate  treatment.  Note  adherence  of  inner  and 
and  outer  sheath  layers  in  region  of  shrinkage.  Note  silver  granules  in  sheath,  absence  of  gran- 
ules in  interior  of  ganglion.  Counterstained  with  Mallory's  triple  stain. 


OBSERVATIONS 


Osmotic  changes 


The  intact,  normally  sheathed  cord  was  not  observed  to  swell  or  shrink  and 
nervous  conduction  was  unaffected  when  total  solute  concentration  was  reduced  to 
the  equivalent  of  0.140  M  NaCl  by  dilution  or  increased  to  0.180  M  by  adding  NaCl 
or  sucrose  to  normal  Hoyle's  saline  (0.156  M).  Desheathing  in  the  hypotonic 
(0.140  M)  saline  resulted  in  gross  swelling  and  functional  impairment.  Synaptic 
conduction  failed  totally.  Desheathing  in  the  saline  hypertonic  to  Hoyle's  saline 
(0.180  M)  resulted  in  no  immediate  structural  or  functional  alternation. 


282 


B.  M.  TWAROG  AND  K.  D.  ROEDER 


High  potassium  saline 

In  Table  I  are  listed  typical  times  for  total  conduction  block  on  perfusing  a  pre- 
viously untreated  cord  with  0.180  A I  KC1  or  Hoyle's  balanced  saline  containing  140 
mM/L.  K+.  Recovery  was  followed  in  K+-  free  saline. 

Blocking  was  more  rapid  in  0.180  M  KC1.  This  could  well  be  attributed  to  the 
absence  of  sodium  and  other  ions  rather  than  higher  K+  concentration.  Desheath- 
ing  obviously  reduced  recovery  time  as  well  as  blocking  time.  The  presence  of  the 
cut  ends  of  small  nerves  (severed  in  dissecting  the  fourth  ganglion)  did  not  alter 
the  blocking  time  appreciably. 

TABLE  I 

Effect  of  potassium  ions  on  impulse  conduction  in  intact  and  desheathed  cords 

A.     Intact  segment 


Solution 

Blocking  time 

Recovery  time 

Type  preparation 

0.180  M  K+ 

12  min. 

10  min. 

Connective  onlv 

0.180  .17  K+ 
0.180  M  K+ 

15  min. 
18  min. 

10  min. 
20  min. 

Ganglion  included 
Ganglion  included 

140  mM./L.  K+ 
140  mM./L.  K+ 

30  min. 
22  min. 

25  min. 
33  min. 

Ganglion  included 
Ganglion  included 

B.     Test  segment  desheathed  at  B  (see  Fig.  la) 


Solution 

Blocking  time 

Recovery  time 

Type  preparation 

0.180  .!/  K+ 

10  sees. 

4  min. 

Ganglion  included 

140  mM./L.  K+ 
140  mM./L.  K+ 

60  sees. 
90  sees. 

2  min. 
2  min. 

Ganglion  included 
Ganglion  included 

Repeated  applications  of  high  K"  solutions  to  intact  cord  segments  led  to  in- 
creasingly rapid  block  and  delay  in  recovery.  This  effect  was  not  seen  in  de- 
sheathed nerve. 

Sodium-deficient  saline 

Substitution  of  sucrose  for  the  sodium  of  the  normal  saline  did  not  affect  the 
normally  sheathed  segment  in  experiments  continued  several  hours,  although  this 
caused  conduction  block  in  a  stripped  preparation  within  30  seconds  (Fig.  lb). 
Recovery  was  complete  within  two  minutes  in  normal  saline. 

Acetylcholine 

Treatment  of  the  intact  ganglion  with  extremelv  high  concentrations  of  acetyl- 
choline (10-  M  )  did  not  alter  synaptic  responses  (Fig.  2  a,  h),  as  had  been  re- 
ported by  Roeder  (1948).  However,  effects  of  10~-  M  acetylcholine  on  synaptic 
function  in  the  desheathed  ganglion  were  usually  rapid  and  pronounced.  In  two  of 
a  series  of  seventeen  experiments,  only  a  moderate  decrease  in  synaptic  response 


COCKROACH  NERVE  SHEATH  283 

was  noted.  In  all  others,  within  one  to  five  minutes  bursts  of  asynchronous  action 
potentials  were  followed  by  synaptic  depression  and  block.  Figure  2  shows  the 
rapid  and  easily  reversible  block  of  a  ganglion  desheathed  in  10~2  M  acetylcholine. 
The  lowest  effective  concentration  of  acetylcholine  was  between  3  and  5  X  1O:;  M 
in  the  uneserinized  desheathed  ganglion.  After  eserine,  acetylcholine  between  10~4 
M  and  10~3  M  blocked  the  synapse.  No  effect  on  axonic  conduction  was  noted. 

Sheath  structure 

Since  the  foregoing  experiments  demonstrated  the  functional  importance  of  the 
sheath,  an  attempt  was  made  to  examine  its  mechanical  properties  and  structure. 

It  was  extremely  difficult  to  penetrate  the  sheath  of  the  intact  nerve  cord  with 
any  object.  Even  a  finely  tapered  capillary  microelectrode  merely  dimpled  the 
surface  and  broke.  After  desheathing,  penetration  of  the  cord  and  individual 
neurons  presented  no  difficulty.  During  dissection,  the  sheath  felt  tough  and  elas- 
tic. As  has  been  mentioned,  in  some  dissections  the  sheath  severely  constricted  the 
cord.  The  almost  explosive  bulging-out  of  nerve  substance  through  a  small  hole 
made  in  the  sheath  when  the  preparation  was  immersed  in  hypotonic  saline  strik- 
ingly illustrated  the  mechanical  resistance  the  sheath  offers  to  swelling.  Injected 
air  bubbles  were  trapped  beneath  it.  Histologically,  the  sheath  was  continuous  and 
clearly  double-layered.  The  outer,  almost  homogeneous  layer,  two  to  five  micra  in 
thickness,  stained  deeply  with  aniline  blue.  Occasional  nuclei  were  evident  in  this 
layer,  possibly  representing  fibroblasts.  The  inner  layer  consisted  of  granular 
squamous  epithelial  cells,  one  to  three  micra  thick.  The  flattened  nuclei  of  the 
inner  layer  were  prominent  in  both  cross  and  coronal  sections  (Fig.  3a).  (The 
outer  layer  referred  to  above  corresponds  to  the  homogeneous,  non-cellular,  neural 
lamella  described  by  Scharrer  (1939)  and  Hoyle  (1952).  the  inner  layer  to  the 
thin,  continuous  cytoplasmic  cylinder  which  Scharrer  terms  the  perineurium  and 
Hoyle  terms  the  perilemma.)  These  layers  were  closely  adherent  to  one  another 
and"  pulled  away  together  from  the  nerve  substance  when  the  preparation  was  de- 
sheathed or  when  shrinkage  occurred  in  fixation  (Fig.  3b).  No  other  continuous 
structures  lay  external  to  these  layers  in  our  intact  preparations.  These  two  layers 
were  always  absent  in  areas  functionally  desheathed. 

It  should  be  mentioned  that  within  the  nerve  cord  itself,  fibrous  investments, 
almost  capsular  in  appearance  in  silver-stained  sections,  but  probably  discontinuous, 
surrounded  certain  large  cell  bodies  and  groups  of  cell  bodies.  These  fibrous  in- 
vestments may  correspond  to  the  glial  elements  described  by  Scharrer  (1939). 
The  giant  axons  were  individually  encased  in  a  delicate  (one  micron  in  thickness) 
sheath-like  structure  throughout  their  length. 

When  a  freshly-dissected,  unfixed  cord  was  observed  during  soaking  in  silver 
nitrate,  the  normally  sheathed  areas  showed  at  first  a  fine  network  of  black  lines, 
then  became  stippled  and  blotched  in  appearance  and  finally  almost  uniformly  black. 
The  blackening  was  close  to  the  surface  and  the  deep  black  layer  could  be  lifted  off 
by  desheathing.  Desheathed  cord  soaked  in  silver  nitrate  did  not  blacken  super- 
ficially, but  individual  neurons  could  be  observed  to  stain  black  in  these  prepara- 
tions. Sections  of  the  intact  last  abdominal  ganglion,  treated  while  fresh  with  silver 
nitrate,  showed  black  granules  concentrated  in  the  surface  layer  corresponding  to 


284  B.  M.  TWAROG  AND  K.  D.  ROEDER 

the  sheath  (Fig.  3b),  while  the  desheathed  fifth  ganglion,  after  similar  treatment, 
showed  granules  distributed  throughout. 

DISCUSSION  AND  CONCLUSIONS 

The  sheath  surrounding  the  nerve  cord  of  Periplancta  clearly  limits  diffusion 
of  ions,  as  Hoyle  (1953)  showed  in  Locusta.  However,  in  efficiency  of  sheath 
function  as  measured  by  total  time  required  for  block  in  excess  potassium,  the  intact 
Periplancta  cord  is  more  comparable  to  the  amphibian  sciatic  nerve,  which  will  block 
in  13-20  minutes  in  isotonic  KC1  (Lorente  de  No,  1947;  Feng  and  Liu,  1949)  than 
to  Locusta  nerve,  which  resists  block  as  long  as  four  hours  in  saline  containing 
140  mM  potassium,  provided  the  tracheal  supply  is  undamaged.  Hoyle  pointed  out 
the  importance  of  adequate  oxygenation  in  maintaining  sheath  function.  It  may  be 
that  in  our  preparation,  since  abdominal  movements  were  severely  restricted,  oxy- 
genation through  the  tracheal  supply  was  insufficient  to  maintain  sheath  function 
although  efforts  were  made  to  avoid  all  damage  to  the  tracheal  supply. 

The  retardation  of  potassium  diffusion  is  a  two-way  function  of  the  sheath, 
judging  by  the  large  decrease  in  recovery  time  as  well  as  blocking  time  in  desheathed 
cord.  The  cumulative  effect  of  repeated  high  potassium  solutions,  as  well  as  the 
appearance  of  the  silver  nitrate-treated  cord  (Fig.  3b),  suggests  that  ions  penetrate 
the  sheath  readily  but  are  accumulated  there.  Shanes  (1954)  showed  with  tracer 
techniques  that  the  sheath  of  the  frog  sciatic  nerve  is  entirely  responsible  for  re- 
tardation of  ion  diffusion  ovitward  as  well  as  inward. 

Desheathed  Periplancta  cord  is  soon  blocked  by  sodium  deficiency,  although 
conduction  in  the  intact  cord  is  unaffected  for  long  periods  in  sodium-free  saline. 
This,  then,  is  direct  evidence  that  insect  neurons  are  similar  to  the  neurons  of  other 
groups  not  only  in  potassium  sensitivity  but  in  their  susceptibility  to  inactivation 
by  sodium  deficiency,  and  that  it  is  the  sheath  which  masks  this  susceptibility  just 
as  does  the  sheath  which  invests  vertebrate  nerve  (Huxley  and  Stampfli,  1951). 

Although  much  pharmacological  evidence  suggests  the  cholinergic  nature  of  the 
synapse  between  cereal  nerves  and  giant  fibers  in  the  last  abdominal  ganglion  of 
Periplancta,  not  even  very  high  acetylcholine  concentrations  affect  the  intact  gan- 
glion. This  total  lack  of  effect  is  clearly  due  to  restriction  of  acetylcholine  pene- 
tration by  the  sheath.  However,  effective  concentrations  of  acetylcholine  are  high 
even  in  desheathed  preparations  so  that  the  synaptic  specificity  of  the  acetylcholine 
action  is  still  in  question.  (See  Twarog  and  Roeder,  1957  for  further  data.)  The 
sheath-like  structure  which  invests  the  giant  fibers  may  represent  another  barrier 
to  ion  diffusion,  or  the  synaptic  terminations  of  the  cereal  nerves  may  be  other- 
wise "protected." 

Water  diffuses  through  the  sheath  quite  readily,  as  is  evident  from  the  imme- 
diate swelling-out  of  nerve  substance  which  occurs  when  a  small  portion  of  the 
sheath  is  removed  while  the  cord  is  soaking  in  hypotonic  saline.  It  is  obvious  that 
the  physical  restraint  exerted  by  the  sheath  in  preventing  swelling  limits  total  water 
uptake  and  preserves  structural  integrity.  Lorente  de  No  (1952),  Shanes  (1953) 
and  Krnjevic  (1954)  have  emphasized  the  possibility  that  the  mechanical  rigidity 
of  the  vertebrate  sheath  may  serve  an  osmoregulatory  function.  The  fluctuations 
in  hemolymph  water  in  insects  are  often  very  great  (Mellanby,  1939),  and  an  osmo- 
regulatory function  of  the  sheath  may  be  as  important  as  its  role  in  salt  regulation. 


COCKROACH  NERVE  SHEATH  285 

The  actual  site  of  ion  regulation  by  the  sheath  is  in  one  or  both  of  the  two 
layers  described  above,  which  were  also  described  by  Hoyle  (1952)  in  Locnsta. 
Huxley  and  Stampfli  (1951)  suggested  that  ion  regulation  by  the  frog  sheath  is  a 
function  of  the  innermost  epithelial  layer  first  described  by  Ranvier  in  1876  rather 
than  the  loose  connective  tissue  of  the  epineurium.  Krnjevic  (1954)  showed  con- 
clusively that  this  is  true.  Within  the  epineurium  he  described  the  two-layered 
perineurium  :  an  inner  continuous  layer  of  squamous  epithelium,  4-6  micra  thick, 
with  an  external  layer  of  comparatively  undifferentiated  connective  tissue.  In  some 
regions  of  the  sciatic  nerve  additional  cellular  layers  were  seen,  but  these  were  not 
continuous  over  the  entire  nerve.  He  succeeded  in  showing  that  the  silver  granules 
are  most  concentrated  in  the  epithelial  layer.  The  analogy  in  structure  between  the 
insect  sheath  and  the  frog  perineurium  is  rather  striking.  It  is  likely  that  it  is  the 
squamous  epithelial  layer  which  fulfills  the  important  regulatory  function  in  the 
insect.  Krnjevic  (1954)  has  discussed  the  importance  of  epithelial  layers  in  regu- 
lation of  diffusion  through  capillary  walls  and  through  the  connective  tissue  sheaths 
which  surround  nervous  structures  in  vertebrates.  The  inner,  epithelial  layer  of 
the  sheath  may  well  bear  some  physiological  and  structural  resemblance  to  the  so- 
called  blood-brain  barrier. 

It  must  be  mentioned  here  that  the  desheathing  technique,  in  addition  to  having 
utility  as  a  method  of  studying  this  interesting  ion-regulating  structure  itself,  pre- 
sents advantages  in  investigating  insect  nervous  function.  It  has  been  employed  by 
Twarog  and  Roeder  (1957)  in  pharmacological  studies  to  avoid  total  failure  of 
drug  penetration  or  insufficiently  rapid  penetration  to  the  site  of  action.  Perhaps 
more  important  is  the  fact  that  it  makes  possible  routine  exploration  of  the  insect 
nervous  system  with  microelectrode  techniques.  Resting  cell  membrane  potentials 
of  50  to  70  MV  have  been  easily  measured  and  sustained.  Cell  action  potentials 
have  been  obtained  but  no  systematic  study  has  yet  been  made. 

SUMMARY 

1.  A  method  is  described  for  removing  portions  of  the  connective  tissue  sheath 
which  invests  the  nervous  system  of  the  cockroach,  making  possible  studies  of  sheath 
function  and  of  basic  physiological  properties  of  the  insect  neuron. 

2.  This  sheath  restricts  diffusion  of  potassium  and  sodium  ions  and  of  acetyl- 
choline  from  the  surrounding  fluid  to  the  interior  of  the  cord. 

3.  The  ability  of  the  sheath  to  restrict  swelling  suggests  a  possible  osmoregula- 
tory  function. 

4.  The  functional  sheath  consists  of  an  inner  continuous  squamous  epithelial 
layer  and  an  outer,  almost  homogeneous  connective  tissue  layer. 

5.  This  study  confirms  the  conclusions  of  Hoyle  (1952,  1953)  with  respect  to 
ion  regulation  by  the  insect  sheath,  and  indicates  close  functional  and  structural 
parallels  in  the  sheaths  investing  insect  and  vertebrate  nervous  tissue  as  well  as  in 
the  basic  properties  of  the  nervous  tissues  of  insects  and  vertebrates. 

LITERATURE  CITED 

BATHAM,  E.  J.,  AND  C.  F.  A.  PANTIN,  1951.  The  organization  of  the  muscular  system  of 
Metridium  senile,  L.  Quart.  J.  Micr.  Sci.,  92 :  27-54. 

FENG,  T.  P.,  AND  Y.  M.  Liu,  1949.  The  connective  tissue  sheath  of  the  nerve  as  effective  dif- 
fusion barrier.  /.  Cell.  Comp.  Physiol.,  34 :  1-16. 


286  B.  M.  TWAROG  AND  K.  D.  ROEDER 

GOMORI,  G.,  1941.     Observations  with  differential  stains  on  human  islets  of  Langerhans.     Amer. 

J.  Path.,  17 :  395^06. 
HOYLE,  G.,   1952.     High  blood  potassium  in  insects  in   relation  to  nerve  conduction.     Nature, 

169:  281-282. 

HOYLE,  G.,  1953.     Potassium  ions  and  insect  nerve  muscle.     /.  E.vp.  Biol.,  30:  121-135. 
HUXLEY,  A.  F.,  AND  R.  STAMPFLI,  1951.     Effect  of  potassium  and  sodium  on  resting  and  action 

potentials  of  single  myelinated  nerve  fibers.     /.  Physiol.,  112:  496-508. 
KRNJEVIC,  K.,  1954.     The  connective  tissue  of  the  frog  sciatic  nerve.     Quart.  J.  Exp.  Physiol., 

39:  55-72. 
LORENTE  DE  No,  R.,  1947.     A  study  of  nerve  physiology.     Studies  from  the  Rockefeller  Institute, 

131 :  1-496. 
LORENTE  DE  No,  R.,   1952.     Observations  on  the  properties   of  the  epineurium  of  frog   nerve. 

Cold  Spring  Harbor  Symposium  Quant.  Biol.,  17:  299-315. 
MELLANBY,  K.,  1939.     The  functions  of  insect  blood.     Biol.  Rev.,  14 :  243-260. 
ROEDER,   K.   D.,    1946.     Fine-tapered   silver   electrodes    for    physiological    work.     Science,    104 : 

425-426. 

ROEDER,  K.  D.,  1948.     The  effect  of  anticholinesterases  and  related  substances  on  nervous  ac- 
tivity in  the  cockroach.     Bull.  Johns  Hopkins  Hospital,  83  :  587-599. 
ROEDER,  K.  D.,  N.  K.  KENNEDY  AND  E.  A.  SAMSON,  1947.     Synaptic  conduction  to  giant  fibers 

of  the  cockroach  and  the  action  of  anticholinesterases.     /.  Neiirophysiol.,  10:   1-10. 
SCHARRER,  B.  C.  J.,  1939.     The  differentiation  between  neuroglia  and  connective  tissue  sheath 

in  the  cockroach  (Pcriplancta  am  eric  ana) .     J.  Comp.  Neural.,  70:  77-88. 
SHANES,  A.  M.,  1953.     Effects  of  sheath  removal  on  bullfrog  nerve.     /.  Cell.  Comp.  Physiol., 

41:  305-311. 
SHANES,  A.   M.,   1954.     Sodium  exchange  through  the  epineurium   of  the  bullfrog   sciatic.     /. 

Cell.  Comp.  Physiol.,  43:  99-105. 
STEEDMAN,   H.   F.,   1947.     Ester   wax :   a  new   embedding  medium.     Quart.   J.   Micr.   Sci.,  88 : 

123-133. 
TWAROG,  B.  M.,  AND  K.  D.  ROEDER,  1957.     Pharmacological  observations  on  the  desheathed  last 

abdominal  ganglion  of  the  cockroach.     Ann.  Ent.  Soc.  Amer.     (in  press). 


ABSTRACTS  OF  PAPERS  PRESENTED  AT 
THE  MARINE  BIOLOGICAL  LABORATORY 

1956 

ABSTRACTS  OF  SEMINAR  PAPERS 

JULY  10,  1956 
Amphibian  yolk:  chemistry  and  ultrastructure.     PAUL  R.  GROSS. 

The  amphibian  yolk  platelet  is  a  complex  structure,  both  chemically  and  physically.  In- 
tact washed  platelets  contain  protein,  lipid,  non-protein  nitrogen,  organic  non-protein  phos- 
phorus, and  pentose,  probably  in  PNA.  DNA  is  absent,  as  shown  by  a  negative  diphenylamine 
reaction.  The  protein  of  the  platelets  is  made  up  of  at  least  three  macromolecular  species, 
probably  more.  Sedimentation  experiments  at  a  variety  of  pH  and  ionic  strength  levels  show 
that  the  main  component,  vitellin,  accounts  for  more  than  85%  of  the  protein,  while  the  re- 
mainder is  in  the  form  of  two  apparently  polydisperse  populations  of  particles.  The  vitellin 
sediments  at  about  12  S. 

Further  kinetic  studies  of  the  reaction  in  which  the  yolk  platelets  are  lysed  by  low  con- 
centrations of  Ca++  strengthen  the  hypothesis  that  this  reaction  is  mediated  by  a  Ca-activated 
enzyme  system  which  resides  within  the  platelet.  As  the  concentration  of  Ca  is  lowered,  the 
dissolution  reaction  undergoes  a  change  of  kinetic  order  from  one  to  zero.  Such  a  situation 
would  not  be  expected  were  the  solubilization  a  result  of  simple  salting  in,  as  is  indeed  the  case 
in  uniunivalent  salts  at  ionic  strengths  greater  than  0.4.  The  Ca-dissolution  reaction  also  has 
an  optimum  over-all  ionic  strength  which  lies  far  below  the  minimum  value  for  dissolution  by 
uniunivalent  salts. 

The  platelets  are  solids  in  water,  and  can  be  broken  with  microneedles  into  insoluble  frag- 
ments. As  the  ionic  strength  is  raised,  the  platelets  are  seen  to  swell  and  assume  a  more  iso- 
diametric  shape,  and  at  this  point,  preceding  complete  disruption,  they  are  seen  to  be  sur- 
rounded by  a  membrane.  We  have  been  able  to  photograph  this  "ghost"-like  structure  with 
the  electron  microscope. 

Amphibian  yolk:  the  phosphoprotcin  phosphatase  system.     SYLVAN   NASS. 

The  "PPP'ase"  activity  of  yolk  platelets  isolated  from  ovarian  frog  eggs  was  studied  under 
a  variety  of  experimental  conditions.  The  pH  optimum  for  platelets  incubated  in  NaCl  of  ionic 
strength  between  0.1  and  0.4  was  4.9.  The  temperature  optimum  for  90-minute  incubations  was 
45°  C.  In  systems  to  which  CaCL  (/a  =  0.25)  had  been  added,  the  pH  optimum  was  4.3,  while 
the  temperature  optimum  was  at  37°  C.  Relative  to  the  uniuvalent  salt,  CaCl,  acts  in  these 
systems  as  an  inhibitor  of  P  release.  The  Ca++  solubilizes  the  platelets,  but  no  correlation  be- 
tween this  process  and  P  release  was  found. 

Differences  in  PPP'ase  activity  between  platelets  incubated  in  NaCl  at  /i  =  0.1  and  /j.  =  0.4 
can  be  directly  related  to  the  structural  integrity  of  the  platelet.  At  low  ionic  strength,  the 
platelets  usually  remain  intact  during  the  incubation  periods  at  the  chosen  temperatures  and  pH. 
Increase  in  ionic  strength  causes  rapid  dissolution  of  the  platelets.  Variations  in  temperature, 
pH,  and  time  of  incubation  also  determine  the  solubility  of  these  structures.  In  all  systems  in 
which  solubilization  occurs  (no  Ca  present),  there  is  an  increase  in  P  release  over  that  in  con- 
trol systems  with  platelets  intact. 

Kinetic  studies  show  that  the  P  release  from  intact  platelets  at  any  time  is  approximately 
half  of  that  from  platelets  solubilized  at  the  outset  of  an  experiment.  If  then  the  intact  platelets 

287 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

are  dissolved  by  any  means  other  than  adding  Ca++,  a  sharp  rise  in  the  (Pim,rK. )  is  obtained, 
bringing  this  value  to  that  in  the  system  solubilized  at  zero  time.  This  may  be  interpreted  in 
terms  of  a  membrane-like  diffusion  barrier  investing  the  platelet  which  retards  release  into 
the  bulk  phase  of  the  hydrolyzed  phosphate. 

The  relation  of  gonad  hormones  to  ^-irradiation  sensitivity  in  mice.     JOAN  WOLFF 
AND  ROBERTS  RUGH. 

A  single  injection  of  estradiol  benzoate  was  made  I. P.  10  days  before  whole  body  x-irradia- 
tion  exposure  to  an  LD/50/30  day  level  of  49  normal  intact  male  CF,  mice  and  41  mice  which 
had  been  castrated  by  surgical  excision.  Fifty  other  castrates  and  fifty  intact  mice,  all  of  the 
same  age,  received  no  injections.  Lethality  data  were  collected  for  a  period  of  30  days  follow- 
ing the  irradiation.  When  the  percentage  survival  is  plotted  against  days  post-irradiation,  it  is 
found  that  the  intact  male  controls,  which  received  no  hormone  injection,  all  died  by  the  17th 
day.  At  30  days  post-irradiation  some  38%  of  the  castrated  males  were  still  alive,  indicating 
an  adverse  effect  of  the  presence  of  the  male  hormone,  testosterone,  in  the  controls.  Maximum 
survival  was  found  in  the  castrated  males  which  had  also  been  injected  with  estradiol  benzoate. 
The  normal  intact  males  which  had  been  injected  with  this  hormone  showed  a  survival  value 
almost  as  good,  namely  63.3%.  Thus  the  injected  female  hormone  must  have  counteracted 
almost  entirely  the  adverse  effects  of  the  male  hormone.  The  differential  in  survival  of  male 
and  female  mice,  when  exposed  to  whole  body  penetrating  x-irradiation,  seems  to  be  due  in 
part  to  the  gonadal  hormones  present.  Intact  males,  with  the  normal  quota  of  testosterone, 
have  less  survival  value  than  do  castrate  males.  Further,  castrate  males  injected  with  the 
female  hormone,  eat  radiole  benzoate,  exhibit  maximum  survival,  approaching  that  of  the 
intact  female. 

GENERAL  SCIENTIFIC  MEETINGS 
AUGUST  20-22,  1956 

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 

Experimental  hypothermia  and  carbon  dio.vidc  production  in  the  white  rat.     C. 
LLOYD  CLAFF,  FREDERICK  N.  SUDAK  AND  NAOMI  R.  STONE. 

A  technique  was  described  whereby  carbon  dioxide  production  could  be  measured  in 
conscious  rats  utilizing  barium  hydroxide,  N-butyl  alcohol  and  thymolphthaline  indicator.  The 
efficacy  of  this  method  was  tested  wit^h  a  carbon  dioxide  generator.  Carbon  dioxide  up  to 
seven  times  the  output  of  a  normal  rat  was  generated,  and  100%  of  this  amount  was  collected. 

Twelve-hour  fasted  male  white  rats  were  injected  with  Thorazine ;  2,4-Dichlorophenoxy- 
acetic  acid;  and  a  combination  of  the  two  drugs.  Studies  at  room  temperature  (24°  C.)  re- 
vealed that  a  combination  of  Thorazine  and  "2,4  D."  decreased  the  rectal  temperature  of  a 
white  rat  from  36.3°  C.  to  31.8°  C.  with  a  diminution  in  carbon  dioxide  production  of  30%  in 
five  hours.  This  was  in  marked  contrast  to  the  temperature-carbon  dioxide  relationship  in 
animals  which  received  only  Thorazine.  The  rectal  temperatures  of  these  rats  were  reduced 
6.0°  C.  in  seven  hours  with  an  increase  of  33%  in  carbon  dioxide  output. 

One  and  one  half  hours  after  injection,  animals  were  subjected  to  2.0°  C.  Twenty  minutes 
later,  CO2  and  temperature  measurements  were  taken.  Normal  rats  increased  their  CO...  output 
136%,  and  maintained  normal  body  temperatures.  Animals  receiving  "2,4  D."  and  Thorazine 
in  combination  showed  no  significant  change  from  normal  output  in  their  carbon  dioxide,  and  a 
decrease  in  rectal  temperature  of  6.5°  C.  Thorazine-injected  rats  increased  their  CO2  output 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY          289 

91%  and  rectal  temperature  dropped  5.5°  C.  Animals  injected  with  "2,4  D."  could  only  increase 
CO2  output  to  54%.  A  drop  of  3.0°  C.  in  rectal  temperature  occurred.  All  animals  survived, 
and  normal  behavior  patterns  were  observed  within  24  hours. 

The  possibility  of  inducing  hypothermia  by  interfering  with  the  temperature  regulating 
mechanism  of  a  homoeothermic  animal  by  means  of  drugs  was  discussed. 

Sperm  entry  in  Hydroides  hc.vagonus  (Annelida)  and  Saccoglossus  kowalevskii 
(Enteropncusta) .  ARTHUR  L.  COLWIN,  LAURA  HUNTER  COLWIN  AND  DEL- 
BERT  E.  PHILPOTT. 

The  authors  have  demonstrated  previously  (1954,  1955,  1956)  that  in  a  number  of  species 
the  acrosome  filament  of  the  spermatozoon  penetrates  into  the  egg  proper.  The  present  spe- 
cies were  selected  for  electron  microscope  studies  because  their  eggs  are  essentially  jellyless  and 
their  spermatozoa  make  direct  contact  with  the  egg  membranes.  Moderate  polyspermy  was 
induced  in  order  to  increase  the  chances  of  finding  examples  of  sperm-egg  association  in  thin 
sections.  It  is  not  established  whether  or  not  the  spermatozoa  here  described  were  the  specific 
ones  which  initiated  the  activation  of  the  eggs  studied. 

In  Hydroides,  electron  microscope  photographs  show  that  at  the  earliest  contact  between 
spermatozoon  and  egg  the  acrosome  touches  the  thin,  hexagonally  patterned,  layer  which  sur- 
rounds the  much  thicker  vitelline  membrane.  Subsequently  this  thin  outer  layer  seemingly  rises 
up  around  the  sperm  head,  even  before  a  major  part  of  the  sperm  head  has  entered  the  mem- 
brane. The  vitelline  membrane,  too,  appears  to  rise  up  around  the  sperm  head,  thickening  in 
this  vicinity.  Near  the  acrosomal  region  of  spermatozoa  which  appear  to  have  breached  the 
vitelline  membrane  completely,  the  perivitelline  space  is  almost  twice  as  wide  as  elsewhere. 
The  above  described  effects  suggest  some  lytic  action  stemming  from  the  spermatozoon. 

In  Saccoglossus,  electron  microscope  photographs  show  that  acrosome  filaments  penetrating 
the  outer  and  vitelline  membranes  are  spiral  in  shape.  The  outer  egg  membrane  is  pitted 
wherever  spermatozoa  adjoin  it.  These  pits  are  interpreted  as  resulting  from  erosion  of  the 
membrane  by  a  lysin  from  the  spermatozoon,  demonstrating  this  lytic  action  at  the  level  of  the 
individual  spermatozoon.  The  authors  previously  reported  (1954)  such  pits  in  relation  to 
sperm  entry  in  living  material  as  seen  by  light  microscopy. 

Electron  microscope  studies  of  the  egg  surfaces  and  membranes  of  Hydroides  hcx- 
agonus  (Annelida)  and  Saccoglossus  kowalevskii  (Enteropneusta).  LAURA 
HUNTER  COLWIN,  ARTHUR  L.  COLWIN  AND  D.  E.  PHILPOTT. 

\Yith  light  microscopy,  the  vitelline  membrane  of  living  Hydroides  eggs  appears  thick  and 
uniform.  Early  in  fertilization  this  membrane  changes  little  and  fails  to  elevate  rapidly.  The 
perivitelline  space  does  not  enlarge  rapidly.  With  electron  microscopy,  sections  of  unfertilized 
eggs  show  a  wide  felt-like  vitelline  membrane  closely  surrounded  by  a  single  layer  of  very 
evenly  spaced  minute  bodies  linked  in  hexagonal  pattern  by  fine  threads.  Similar  threads  link 
the  layer  to  the  membrane.  Larger,  fewer,  microvilli  project  regularly  from  the  egg  proper, 
deeply  penetrating  the  vitelline  membrane.  In  cross  section,  at  least  the  broader  proximal  part 
of  the  microvillus  shows  a  deeply  staining  periphery  encircling  a  central  unstained  region.  No 
well-defined  layer  of  cortical  granules  is  seen.  No  great  change  is  observed  following  fertili- 
zation. However,  a  narrow  perivitelline  space  does  develop;  across  this  the  microvilli  project 
into  the  membrane  for  some  time. 

Light  microscopy  shows  two  membranes  surrounding  the  living  unfertilized  Saccoglossus 
egg.  Sections  reveal  a  well-defined  cortical  granular  layer.  At  fertilization  both  membranes 
elevate  rapidly.  The  inner  one  (fertilization  membrane)  successively  thickens,  apparently  re- 
ceives additional  material  from  the  cortical  layer,  and  becomes  compressed.  Simultanously, 
the  egg  surface  forms  short-lived  irregular  protuberances,  the  cortical  layer  disintegrates,  hy- 
aline granules  and  threads  erupt  from  the  egg  into  the  rapidly  enlarging  perivitelline  space.  In 
living  eggs  much  of  this  material  becomes  invisible,  though  a  border  of  hyaline  granules  persists 
close  to  the  egg.  Sections  show  a  reticulum  in  the  perivitelline  space.  Electron  microscopy 
verifies  the  above  observations,  shows  release  of  cortical  granular  material,  and  also  reveals 


290         PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

stout  microvilli  between  the  egg  and  the  vitelline  membrane  but  not  penetrating  the  latter. 
Upon  fertilization  these  microvilli  elongate,  apparently  detaching  from  the  membrane ;  their 
ultimate  fate  is  unknown.  Cross  sections  of  elongated  microvilli  show  a  well-defined  circular 
central  structure  apparently  surrounded  by  eight  dot-like  bodies. 

Ionic  regulation  in  the  fiddler  crabs,  Uca  pugna.v  and  U.  pugilator.     JAMES  W. 
GREEN,  MARY  HARSCH,  LLOYD  BARR  AND  C.  L.  PROSSER. 

Three  species  of  Uca  (pugnax,  minax  and  pugilator)  like  Pachygrapsus  but  unlike  Carcinus 
and  Callinectcs  were  found  to  have  lower  concentrations  of  Na  in  urine  when  adapted  to  175% 
than  to  100%  S.W.  In  U.  pugnax  and  pugilator,  the  two  species  selected  for  more  detailed  study, 
the  total  osmotic  concentrations  of  both  urine  and  serum  were  slightly  higher  in  crabs  from  the 
175%  S.W.  than  those  from  the  100%.  Analyses  of  urine  and  serum  from  both  groups  for 
Na,  Mg,  Ca,  K,  Cl  and  SO4  showed  a  cation  deficit  in  the  urine  of  about  20%'  which  was  found 
to  be  largely  NH4.  Although  the  serum  Na  values  of  both  groups  were  approximately  the  same 
there  was  30  to  40%  less  Na  in  the  urine  of  crabs  in  the  175%  S.W.  and  the  cation  deficit  thus 
incurred  was  made  up  by  the  excretion  of  about  3  times  as  much  Mg  as  was  found  in  the  urine 
of  the  100%  crabs.  When  both  groups  were  placed  in  S.W.  containing  Na24  the  isotope  was 
found  to  enter  at  essentially  the  same  rate  in  each  group.  Tissue  analyses  of  Na24-labeled 
crabs  indicated  that  Na  was  not  being  stored.  \Vhen  Na24-labeled  crabs  were  washed  in  S.W. 
without  isotope,  placed  in  dry  finger  bowls  for  short  periods  and  samples  of  gill  fluids  analyzed, 
it  was  found  that  crabs  from  the  175%  S.W.  contained  a  higher  proportion  of  Na24  in  their  gill 
fluids  than  the  100%  group.  This  was  interpreted  to  mean  that  while  both  groups  lost  Na 
through  the  gills  the  crabs  in  175%  S.W.  excreted  more  by  this  route,  thus  accounting  for  the 
lowered  urine  Na  values  found. 

Electron  microscopy  of  the  niitotic  apparatus  in  dividing  Arbacia  eggs.     PAUL  R. 
GROSS,  DELBERT  E.  PHILPOTT  AND  SYLVAN  NASS. 

In  the  course  of  an  investigation  of  the  mechanisms  of  sol-gel  transformations  in  dividing 
cells,  it  became  a  matter  of  interest  to  determine  the  nature  of  the  material  which  constitutes  the 
mitotic  spindle.  Considerable  data  concerning  this  problem  have  been  reported  from  Mazia's 
laboratory,  but  good,  high-resolution  electron  micrographs  of  the  achromatic  figure  are  lacking. 
We  have  devised  a  new  method  for  observing  this  structure  with  the  electron  microscope. 
The  basis  of  the  procedure  is  the  observation  of  Mazia  and  co-workers  that  dividing  cells  can 
be  killed  with  cold  30%  ethanol  without  too  extensive  denaturation  of  cytoplasmic  proteins  and 
without  dissolution  of  the  mitotic  figure. 

The  Cecils  are  fixed  in  the  desired  stage  of  mitosis  with  30%  ethanol  at  --  10°  C.  for  about 
30  minutes,  then  transferred  to  30%  ethanol  containing  1%  osmium  tetroxide  at  --10°  C. 
After  fifteen  minutes  in  the  cold  osmic-ethanol,  the  suspension  is  removed  to  room  temperature 
and  permitted  to  warm  slowly.  After  about  fifteen  minutes  more,  the  eggs  are  quickly  sedi- 
mented  down  and  washed  with  50%  alcohol,  thence  run  through  a  series  of  alcohols  for  dehy- 
dration. The  cells  are  embedded  as  usual  in  a  mixture  of  methyl  and  n-butyl  methacrylates,  and 
sections  are  cut  on  a  microtome  designed  for  ultra-thin  sectioning. 

Using  this  procedure,  we  have  been  able  to  observe  in  detail  the  structure  in  situ  of  the 
metaphase  spindle  and  of  the  chromosomes  at  the  metaphase  plate.  The  spindle  appears  to  be  a 
gel  composed  of  elementary  particles  of  widely  varying  diameter,  with  a  mean  value  near  400  A. 
At  high  magnifications,  these  particles  are  seen  to  be  themselves  composites  of  somewhat  asym- 
metric particles  of  diameter  ca.  30-50  A. 

Participation  by  actin  in  actomyosin  contraction.     TERU  HAYASHI,  RAJA  ROSEN- 
BLUTH,  PETER  SATIR  AND  MICHAEL  VOZICK. 

Any  mechanism  of  muscular  contraction  at  the  molecular  level  must  take  into  considera- 
tion the  behavior  of  the  muscle  proteins,  actin  and  myosin.  The  results  of  recent  in  ritro  ex- 
periments have  been  interpreted  to  indicate  that  only  myosin  is  the  contractile  protein,  with 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY          291 

actin  playing  an  associated  role,  but  not  directly  involved  in  the  configurational  changes  re- 
sulting in  contraction.  Therefore,  a  series  of  experiments  was  done  to  test  whether  myosin 
alone  was  capable  of  contraction,  without  actin. 

Purified  myosin  was  prepared,  and  by  viscosity  tests  and  solubility  tests  shown  to  con- 
tain less  than  0.1%  actin.  Pellicular  fibers  formed  of  this  material  at  pH  7.0,  and  0.05  M  KC1, 
failed  to  develop  tension  upon  addition  of  ATP,  either  at  pH  7.6  or  9.0.  Reconstituted  acto- 
myosin  combining  graded  amounts  of  actin  and  myosin,  developed  graded  tensions  at  pH  7.6, 
whereas  at  pH  9.0,  there  was  no  contraction.  The  graded  tensions  developed  corresponded 
roughly  to  the  "activity"  values  obtained  from  viscosity  tests,  which  are  in  turn  proportional 
to  the  amount  of  actin  present.  Isotonic  contractions  corroborated  the  findings  obtained  from 
experiments  done  under  isometric  conditions.  Myosin  fibers,  formed  at  low  temperatures  with 
ATP  also  applied  at  low  temperature,  failed  to  develop  tension  when  the  temperature  was  raised, 
whereas  actomyosin  fibers  treated  similarly  readily  developed  tension  with  the  increase  in 
temperature. 

Implications  as  to  the  role  of  actin  will  be  discussed.  It  is  concluded  that  the  contraction 
involves  actin  specificially  and  directly,  and  that  myosin  alone  is  not  capable  of  contraction. 

An  interpretation  of  the  action  of  certain  chemical  agents  used  in  cancer  therapy.1 
L.  V.  HEILBRUNN  AND  WALTER  L.  WILSON. 

In  spite  of  the  vast  literature  on  the  chemotherapy  of  cancer,  but  little  is  known  as  to 
why  various  agents  act  as  they  do,  and  there  has  scarcely  been  even  a  hypothesis  proposed  as 
to  why  some  agents  which  have  a  carcinostatic  action  may  also  be  carcinogenic.  We  have  stud- 
ied the  effect  of  nitrogen  mustard,  Nitromin,  6-mercaptopurine  and  urethane  on  eggs  of  the 
marine  worm  Chaetopterus.  All  of  these  substances  have  been  used  in  the  treatment  of  cancer. 
In  dilute  solution,  all  of  them  prevent  cell  division,  and  all  of  them  act  by  suppressing  the  mitotic 
gelation.  In  more  concentrated  solutions,  nitrogen  mustard,  Nitromin  and  urethane  have  the 
opposite  effect,  that  is  to  say  they  tend  to  induce  protoplasmic  clotting.  Thus  they  might  well 
act  to  initiate  mitosis,  for  the  initiation  of  mitosis  is  caused  primarily  by  agents  which  cause 
protoplasmic  clotting.  Indeed  it  can  be  shown  that  solutions  of  urethane  do  actually  induce 
mitosis  in  Chaetopterus  eggs.  These  facts  are  in  line  with  the  colloidal  theories  discussed  in 
some  detail  in  Heilbrunn's  Dynamics  of  Living  Protoplasm,  published  earlier  this  year  by  the 
Academic  Press.  Fractions  of  cow  ovary  extracts  obtained  by  cold  alcohol  fractionation  were 
also  tested  on  Chaetopterus  eggs.  Fractions  which  prevent  mitotic  gelation  act  as  rather  power- 
ful carcinostatic  agents.  This  has  been  established  by  other  members  of  our  group  who  are 
now  working  with  Ehrlich  ascites  tumors  in  mice. 

A  striking  behavioral  change  leading  to  the  formation  of  extensive  aggregations  in  a 
population  of  Nassarius  obsoletus.'2     CHARLES  E.  JENNER. 

The  distribution  of  Nassarius  obsoletus  in  a  given  locality  frequently  presents  problems  of 
considerable  ecological  interest.  Snails  are  sometimes  absent  from  apparently  favorable  areas, 
although  present  in  enormous  numbers  in  adjacent  but  similar  areas.  The  factors  underlying 
this  extreme  patchiness  in  distribution  are  not  readily  apparent. 

The  problem  was  illustrated  by  a  striking  change  in  distribution  pattern  which  occurred 
in  a  population  of  snails  in  Barnstable  Harbor  during  the  summer  of  1956.  The  area  under  ob- 
servation was  an  extensive  mud-sand  flat,  approximately  200  X  800  yards,  exposed  only  at  low 
tide.  In  a  well-populated  area  a  reference  zone  was  staked,  marking  10  adjacent  quadrats,  10  X 
10  yards.  During  visits  on  June  19,  July  1,  7,  15  and  22,  snails  were  present  in  great  numbers 
but  were  widely  distributed  over  most  of  the  flat.  Observations  during  a  brief  visit  on  July 
30  were  hampered  because  the  flat  was  not  exposed  at  the  time.  It  was  apparent,  however, 
that  a  definite  change  in  snail  distribution  had  taken  place  since  the  last  visit ;  snails  could  be 

1  This  investigation  was  supported  by  a  research  grant  from  the  National  Cancer  Insti- 
tute, National  Institutes  of  Health,  Public  Health  Service. 

-  Aided  by  a  grant  from  the  National  Institutes  of  Health,  U.  S.  Public  Health  Service, 
E-356(C4). 


292          PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

found  in  only  one  of  the  10  quadrats.  During  the  next  observation  (August  4),  snails  were 
not  to  be  found  over  most  of  the  flat  and  none  were  present  in  the  marked  area.  The  snails 
occurred  chiefly  in  massive  aggregations ;  in  many  areas  they  literally  covered  the  ground,  in 
some  cases  two  or  three  layers  deep.  No  snails  were  within  180  feet  of  the  marked  area,  in- 
dicating that  most  had  traveled  more  than  this  distance  during  the  13  days  since  the  July  22 
observation. 

The  functional  significance  of  such  mass  migration  and  aggregation  is  not  readily  under- 
standable. In  the  formation  of  the  aggregations  it  seems  clear  that  the  animals  were  not 
responding  independently  to  inanimate  factors.  Some  type  of  interaction  between  snails  must 
have  been  involved. 

The  timing  of  reproductive  cessation  in  geographically  separated  populations  of 
Nassarius  obsoletits.1     CHARLES  E.  JENNER. 

The  termination  of  seasonal  reproductive  activity  was  followed  in  populations  of  Nassarius 
obsoletus  from  Beaufort,  N.  C. ;  Great  Pond,  Mass,  (south  Cape  Cod)  ;  and  Barnstable  Harbor, 
Mass,  (north  Cape  Cod)  during  the  spring  and  summer  of  1956.  In  males  of  N.  obsoletus 
the  copulatory  organ  is  resorbed  at  the  end  of  the  reproductive  season  (Jenner  and  Chamber- 
lain, 1955),  and  the  state  of  reproductive  activity  was  judged  by  recording  the  per  cent  of  un- 
parasitized  males  bearing  this  structure.  The  per  cent  of  unparasitized  females  having  a 
formed  egg  case  in  the  reproductive  duct  was  also  recorded. 

For  a  given  population  cessation  was  found  to  be  abrupt,  with  the  major  part  of  the 
transition  period  being  only  two  to  three  weeks  in  duration.  Curves  for  males  and  females 
were  nearly  superimposed.  Reproductive  activity  was  essentially  completed  for  Beaufort  snails 
by  the  end  of  May,  for  Great  Pond  by  mid-July  and  for  Barnstable  by  the  latter  part  of  August. 
The  displacement  of  the  curves  for  the  different  populations  seems  to  be  related  to  tempera- 
ture, and  agrees  nicely  with  the  known  distribution  of  temperature  along  the  Atlantic  coast 
during  the  spring  and  summer  months  (Parr,  1933).  The  difference  in  timing,  for  example, 
between  the  north  and  south  sides  of  Cape  Cod  (a  latitudinal  difference  of  12  miles)  was  about 
as  great  as  between  south  Cape  Cod  and  Beaufort  (latitudinal  difference  of  over  500  miles). 
A  May  17  collection  from  Mayport,  Florida,  indicated  a  timing  of  reproductive  cessation  simi- 
lar to  that  of  the  Beaufort  snails.  Limited  observations  on  snails  from  Boothbay  Harbor,  Maine, 
showed  these  snails  to  be  two  to  three  weeks  later  in  their  cycle  than  snails  from  Barnstable. 

The  method  employed  allowed  a  high  degree  of  precision  in  the  description  of  this  seasonal 
event. 

Absence  of  membrane  potential  in  presence  of  asymmetrical  ion  distribution  in  the 
Fundulus  egg.     C.  Y.  KAO. 

The  accumulation  of  ions  by  biological  membranes  has  generally  been  assumed  to  be  ac- 
companied by  an  electrical  potential  difference.  In  almost  all  excitable  tissues  at  rest  and  in  the 
egg  of  Asterias  forbesii,  the  membrane  potential  approaches  the  value  obtainable  on  the  basis 
of  the  ratio  of  [K+]  on  the  two  sides  of  the  membrane.  In  the  Fundulus  egg,  no  membrane  po- 
tential can  be  measured,  in  either  the  unactivated  or  activated  state,  across  the  plasma  membrane, 
although  a  high  membrane  resistance  is  present.  By  means  of  flame  spectrophotometry,  po- 
tassium and  sodium  contents  have  been  determined,  using  homogenized  or  aspirated  egg  material. 
K  is  present  in  105  mM /liter  of  egg  material  in  the  unactivated  egg,  and  117  mM  in  the 
activated  egg,  whereas  Na  occurs  in  ca.  50  mJ\l .  The  values  for  the  corresponding  ions  in  sea 
water  are  10  mM  and  452  mM,  respectively.  In  addition  to  a  lack  of  membrane  potential, 
membrane  resistance  of  activated  egg  is  not  materially  affected  by  immersion  in  isotonic  chlo- 
ride solutions  of  Na+,  K+  (540  mM  each),  Ca'1"1',  and  Mg++  (370  mM  each)  for  up  to  10  min. 
The  discordance  between  the  concentrations  of  the  two  major  cations  and  the  electrical  properties 
of  the  egg  plasma  membrane  should  preclude  generalizations  with  respect  to  the  origin  of 
membrane  potentials  in  biological  material. 

1  Aided  by  a  grant  from  the  National  Institutes  of  Health,  U.  S.  Public  Health  Service, 
E-356(C4). 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY          293 

Electron  microscopic  observations  on  the  development  of  the  chorion  of  Fundulus. 
NORMAN  E.  KEMP  AND  MARGARET  D.  ALLEN. 

Pieces  of  ovaries  containing  developing  oocytes  were  fixed  in  buffered  (pH  7.4)  \%  osmic 
acid  in  artificial  sea  water,  embedded  in  methacrylate  and  sectioned  with  a  Porter-Blum  mi- 
crotome set  to  cut  at  0.025  /*.  Sections  reveal  that  cells  of  the  follicular  epithelium  pull  away 
from  the  oocyte  as  deposition  of  the  zona  radiata  begins.  The  zona  is  a  fibrous  membrane  per- 
forated by  so-called  pore  canals  from  the  time  of  its  first  appearance.  It  thickens  chiefly  by 
the  addition  of  new  material  on  its  inner  surface.  The  pore  canals,  which  account  for  the 
striated  appearance  of  the  zona  radiata  with  the  light  microsocpe,  contain  microvilli,  spirally 
coiled  in  older  oocytes,  extending  from  the  surface  of  the  oocyte  outward  to  the  subfollicular 
space.  Branching  protoplasmic  processes  of  the  epithelial  cells  are  in  close  contact  with,  or 
possibly  continuous  with,  the  microvilli  in  the  subfollicular  space.  Among  the  follicular  cell 
processes  are  extracellular  strands  or  filaments.  The  chorion  of  the  ovulated  egg  is  composed 
of  (1)  an  internal  portion,  the  chorion  internum  (Shanklin  and  Armstrong,  1952),  consisting 
of  lamellae  arranged  in  a  herringbone  pattern,  and  (2)  an  outer  portion,  the  chorion  externum, 
consisting  of  tangentially  oriented  filaments  in  an  adhesive  matrix.  The  chorion  internum  is 
the  transformed  zona  radiata  of  the  oocyte ;  and  the  chorion  externum  is  derived  from  the  fila- 
ments and  intercellular  matrix  of  the  follicular  cell  processes.  We  conclude  that  the  chorion  is 
anatomically  a  vitelline  membrane  (lamellated  portion)  combined  with  a  true  chorion  (fila- 
mentous portion). 

The  nature  of  the  metal-to-protein  bond  in  hemerythrin.     IRVING  M.  KLOTZ  AND 
THEMIS  A.  KLOTZ/ 

Hemerythrin,  an  oxygen-carrying  pigment  found  in  sipunculid  worms,  contains  iron  at  its 
active  sites  but  is  devoid  of  porphyrin  groups.  Thus  the  metal  seems  to  be  attached  directly  to 
the  protein.  On  a  stoichiometric  basis  each  active  site,  holding  one  O2  molecule,  corresponds  to 
2.4  Fe  atoms. 

Displacement  experiments  with  other  metal  ions  suggested  that  the  iron  is  attached  to 
sulfide  groups  of  the  protein.  Titrations  for  mercaptan  groups  were  carried  out,  therefore,  with 
silver  ion.  The  color  of  the  oxyhemerythrin  disappeared  as  silver  was  added  and  at  the 
amperometric  end  point  2  Ag  had  been  taken  up  for  each  2.4  Fe  in  the  protein.  It  would 
seem,  therefore,  that  2  of  each  2.4  irons  are  attached  to  mercaptan  side  chains.  Confirmation 
that  the  silver  really  displaces  iron  from  a  sulfide  bond  was  obtained  from  quantitative  studies 
of  the  reaction  of  oxyhemerythrin  with  a  disulfide  dye.  The  dye  should  react  specifically  with 
protein  — SH  groups ;  colorimetric  titration  agreed  well  with  the  silver  amperometric 
titration. 

These  observations  suggest  that  each  active  site  of  hemerythrin  contains  two  iron  atoms 
attached  to  the  protein  through  cysteine  side  chains,  the  iron  atoms  holding  an  O2  molecule 
between  them.  This  configuration  would  also  conform  with  previous  results  on  the  valence 
changes  in  the  metal  during  the  oxygenation  process  as  well  as  with  the  new  observation  that 
approximately  one-half  as  much  mercury-(II)  as  silver-(I)  combines  with  hemerythrin  in 
amperometric  titrations. 

The  molecular  weight  of  hemoglobin  from  Petrornyzon  marinus.     P.  GALEN  LEN- 
HERT,2  WARNER  E.  LOVE  AND  FRANCIS  D.  CARLSON. 

The  present  study  was  undertaken  as  a  preliminary  to  a  proposed  x-ray  diffraction  analy- 
sis of  this  substance.  An  accurate  molecular  weight  of  Petromyzon  hemoglobin  was  desired 
before  attempting  crystallization. 

The  sedimentation  coefficient  corrected  to  water  at  20°  C.  was  found  to  be  1.9  S  units. 
A  molecular  weight  of  23,600  was  calculated  from  data  taken  during  the  approach  to  sedimenta- 

1  This  investigation  was  supported  in  part  by  a  research  grant,  RG-4134,  from  the  National 
Institutes  of  Health,  Public  Health  Service. 

-  National  Science  Foundation  Fellow  1955-56. 


294         PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

tion  equilibrium  by  the  method  of  Archibald  as  modified  by  Klainer  and  Kegeles.  A  value  of 
0.751  given  by  Svedberg  and  Eriksson-Quensel  as  the  partial  specific  volume  of  Petromyzon 
hemoglobin  was  used  in  this  calculation.  This  figure  must  be  considered  approximate  due  to 
our  inability  to  obtain  optimal  photographic  conditions. 

Iron  was  determined  by  Lorber's  sulfosalisylic  acid  method.  The  values  obtained,  using 
an  extinction  coefficient  for  the  iron-sulfosalisylic  acid  complex  of  0.101  per  microgram  of  iron 
per  mililiter,  indicate  one  mole  of  iron  for  every  17,800  grams  of  protein.  Heme  was  measured 
as  the  reduced  pyridine  hemochromogen,  giving  a  value  of  18,600  grams  of  protein  per  mole 
of  heme. 

These  results  in  conjunction  with  the  sedimentation  equilibrium  molecular  weight  given 
above  support  the  conclusion  that  there  is  one  heme  group  per  molecule  of  hemoglobin.  The 
molecular  weight  which  results  from  these  data  is  18,200  ±  400.  This  result  of  approximately 
20,000  is  in  agreement  with  Svedberg's  values  for  other  cyclostome  hemoglobins.  Attempts 
are  now  being  made  to  crystallize  this  protein. 

Hyaline  polymer  of  the  fertilized  egg  of  Arbacia  punctulata.     ARTHUR  K.  PARPART 
AND  JULIEN  CAGLE. 

The  hyaline  layer  which  forms  during  the  first  5  to  10  minutes  after  fertilization  of  the 
egg  of  Arbacia  punctulata  is  the  resultant  of  a  polymerization  of  a  polysaccharide  probably  re- 
leased as  a  monomer  upon  the  explosive  breakdown  of  the  cortical  granules.  The  hyaline 
layer  can  be  depolymerized  completely  and  rapidly,  30  seconds,  in  isosmotic  solutions  of  non- 
penetrating  non-electrolytes  (e.g.,  glucose,  xylose,  erythritol,  glycerol),  and  partly  depoly- 
merized in  the  presence  of  (a)  0.5  M  NaCl,  (b)  0.5  M  NaCl  plus  0.01  M  CaCL,  and  (c)  1.0  M 
glucose  plus  either  0.1  J\I  guanidine  HC1  or  0.001  M  CuCL.  Since  guanidine  and  copper  are 
active  protein  precipitants  it  is  suggested  that  the  hyaline  layer  is  primarily  composed  of  a  poly- 
merized polysaccharide  which  can  be  readily  depolymerized.  Upon  depolymerization  this  poly- 
saccharide exerts  an  osmotic  pressure  equivalent  to  ca.  5%  egg  albumin,  which  draws  water 
into  the  perivitelline  space.  A  depolymerized  hyaline  layer  can  be  repolymerized  by  means  of 
magnesium  ions  (0.005  M  to  0.04  M)  dissolved  in  isosmotic  glucose,  and  an  optically  refractile 
and  dense  hyaline  layer  is  re-formed.  Upon  repolymerization  the  colloid  osmotic  pressure  in  the 
perivitelline  space  decreases. 

Magnifying  the  invisible.     DELBERT  E.  PHILPOTT  AND  GEORGE  G.  LOWER. 

A  16  mm.  color  movie  has  been  made  to  show  the  basic  techniques  used  in  electron  mi- 
croscopy. An  overall  view  of  the  R.C.A.  Model  EMU-2B  and  the  North  American  Phillips 
Company  Model  EM-100  is  shown.  Specimen  grids  are  prepared,  and  the  complete  process  of 
a  preparation  of  liquid  suspension  of  virus  is  demonstrated.  The  shadow  casting  machine  and 
technique  are  illustrated.  To  show  how  living  tissues  are  prepared  for  the  electron  microscope, 
a  frog  heart  is  taken  through  the  steps  of  fixation,  embedding,  and  sectioning.  Actual  opera- 
tion of  the  microscope  is  also  shown.  Various  pictures  of  different  tissues  and  specimens  taken 
with  the  microscope  are  shown  to  demonstrate  the  adaptability  of  the  electron  microscope  to 
a  variety  of  research  problems.  A  wave-length  chart  shows  the  difference  between  light  and 
electrons  as  a  source  for  microscopy,  and  a  comparison  of  magnification  sizes  is  demonstrated. 

This  film  is  meant  to  be  an  introduction  to  electron  microscopy  for  the  layman,  to  clarify 
to  those  who  have  never  had  the  occasion  to  work  with  one,  the  basic  principles  and  techniques 
used. 

Sodium  ion  exchange  in  Ulva  lactitca.     GEORGE  T.  SCOTT,  ROBERT  DEVOE  AND 
GARY  CRAVEN. 

Sodium  ion  was  observed  to  exhibit  an  unexpectedly  high  rate  of  turn-over  between  the 
cells  of  Ulva  lactuca  and  the  surrounding  sea  water.  This  alga  is  a  very  favorable  organism 
for  the  study  of  ion  exchange  since  it  consists  of  but  two  layers  of  cells.  Furthermore,  extra- 
cellular fluid  with  contained  electrolytes  can  be  removed  by  the  combination  of  a  thirty-second 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY          295 

rinse  in  isotonic  sucrose  and  a  triple  blotting  technique  in  absorbent  tissue.  Previously  pub- 
lished data  indicate  that  sodium  and  potassium  ions  diffuse  into  isotonic  sucrose  from  the 
extra-cellular  phases  within  three  seconds. 

The  exchange  experiments  involved  placing  the  alga  for  varying  lengths  of  time  in  sea 
water  containing  0.5  to  1.0  millicurie  of  Na24  per  liter  followed  by  the  thirty-second  sucrose  rinse 
and  the  triple  blotting  procedure.  The  samples  of  algae  consisted  of  small  discs  about  two 
inches  in  diameter  cut  from  a  single  large  frond.  Activities  were  determined,  following  which 
the  samples  were  wet  ashed  and  analyzed  for  sodium  by  flame  photometry ;  specific  activities 
were  then  calculated.  Time  course  curves  reveal  that  at  twenty  degrees  the  specific  activities  of 
the  algae  had  reached  equality  with  that  of  the  sea  water  within  ten  seconds.  At  one  degree 
exchange  was  nearly  complete  in  fifteen  seconds.  Experiments  done  at  twenty  degrees,  in 
which  the  sea  water  contained  1  X  10"3  M  phenylurethane  in  one  case,  and  in  another  5  X  10~* 
uranyl  nitrate  at  pH  4.8,  all  in  addition  to  the  Na24,  showed  a  slight  lessening  of  the  exchange 
rate,  the  significance  of  which  is  in  doubt.  Uranyl  ion  has  been  shown  to  be  preferentially 
surface  adsorbed. 

The  data  suggest,  because  of  the  very  high  rate  of  sodium  turnover,  an  exchange  of  sodium 
ion  between  the  surface  of  peripheral  zone  of  the  cell  and  the  medium  ;  this  exchange  probably 
does  not  directly  involve  an  active  metabolic  pump  per  se. 

Studies  on  parasites  of  the  green  crab,  Carcinides  maenas.     HORACE  W.  STUNKARD. 

Since  Carcinides  maenas  is  a  serious  predator  of  Mya  arenaria.  the  U.  S.  Fish  and  Wildlife 
Service  is  interested  in  determining  whether  or  not  its  parasites  can  serve  as  possible  means 
of  biological  control.  It  has  long  been  known  that  C.  maenas  in  the  Woods  Hole  area  is  in- 
fected by  an  undetermined,  encysted  metacercaria  and  experiments  have  been  conducted  to 
discover  its  identity,  life-history  and  biology.  Metacercariae  were  excysted  to  study  their 
morphology  and  cysts  were  fed  to  recently  hatched,  uninfected  birds,  Sterna  hirundo  and 
Larus  argcntatus.  Large  numbers  of  worms  were  recovered,  including  all  stages  from  juvenile 
to  fully  mature  specimens.  The  structure  of  the  metacercariae  suggested  that  they  may  be 
specifically  identical  with  a  minute,  stylet-bearing  cercaria  which  occurs  in  Littorina  obtusata, 
Littorina  sa.ratilis.  and  rarely  in  Littorina  littorca.  Small  green  crabs  exposed  to  these  cer- 
cariae  became  heavily  infected ;  enormous  numbers  of  worms  entered  the  tissues  and  developed 
to  metacercariae,  identical  with  those  of  the  natural  infections  mentioned  above.  Small  crabs 
exposed  continuously  with  six  to  eight  infected  snails  died  in  ten  to  twenty  days,  and  on  dis- 
section yielded  thousands  of  larvae.  The  stages  in  the  life-cycle  of  the  parasite  agree  with 
descriptions  by  European  investigators  of  corresponding  stages :  the  metacercariae  with  meta- 
cercariae from  C.  maenas,  described  but  not  named  by  MTntosh  (1865)  ;  the  adults  with  Micro- 
phallus  similis  from  Swedish  gulls,  described  and  named  by  JagerskiiJld  (1900)  ;  and  the  cer- 
cariae  with  Cercaria  libiquita  Lebour,  1907.  The  identity  of  these  worms  and  their  relation  as 
stages  in  the  life-cycle  of  a  single  species  is  predicated.  It  is  possible  that  the  parasites  and 
their  intermediate  hosts  are  introduced  species. 

Conduction  velocity  in  the  giant  ax  on  of  the  squid  (Loligo  pcalii)  in  DZ0.     ROGER 
E.  THIES  l  AND  FRANCIS  D.  CARLSON. 

A  partially  cleaned  axon  was  threaded  into  a  glass  chamber  with  a  volume  approximately 
thirty  times  that  of  the  treated  region  of  the  nerve.  A  few  centimeters  of  the  nerve  were 
treated  with  a  D^O  sea  water  (pH  8)  made  by  evaporating  artificial  sea  water  to  dryness  and 
bringing  the  residue  up  to  volume  with  99.5%  D.O.  A  constant  temperature  bath  maintained 
the  preparation  at  17.0  ±0.5°  C.  The  stimulus  was  applied  to  an  untreated  region.  The  di- 
phasic action  potential  was  recorded  within  each  end  of  the  treated  region,  and  both  action  po- 
tentials were  displayed  on  the  same  single  sweep  of  an  oscilloscope.  Measurement  of  the  dis- 
tance between  the  peaks  of  the  action  potentials  gave  the  value  of  the  conduction  time  to  within 
±2%. 

1  National  Science  Foundation  Fellow,  1956-57. 


296         PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

In  eight  experiments  with  five  nerves  the  conduction  velocity  decreased  by  19  ±  3%  after 
treatment  with  the  D.O  solution.  This  agrees  with  the  finding  of  Garby  and  Nordquist  on  simi- 
larly treated  frog  sciatic  nerve.  Recovery  in  ordinary  water  solutions  was  to  within  95  ±  4% 
of  the  original  value.  Both  the  initial  decrease  in  velocity  and  the  recovery  were  complete 
within  three  to  five  minutes.  Lower  concentrations  of  D2O  produced  smaller  decreases  in  the 
conduction  velocity,  the  dependence  being  approximately  linear.  The  effect  is  not  due  to  small 
pH  differences,  since  between  pH  6.1  and  9.7  the  conduction  velocity  remained  unchanged  to 
within  ±  3%.  The  D-O  produced  no  detectable  change  in  the  diameter  of  the  axon. 

The  small  magnitude  of  the  effect  suggests  that  D^O  reduces  conduction  velocity  because 
its  viscosity  is  1.23  times  that  of  ordinary  water,  and  ionic  mobilities  are  correspondingly  re- 
duced. Further  experiments  are  required  to  substantiate  this  hypothesis. 

Change  in  rate  of  release  of  A.'42  upon  fertilization  in  eggs  of  Arbacia  punctulata. 
ALBERT  TYLER  1  AND  ALBERTO  MoNROY.2 

From  previous  experiments  demonstrating  the  existence  of  an  electrical  potential  difference 
across  the  surface  of  echinoderm  eggs,  a  rapid  decrease  followed  by  an  increase  of  this  potential 
upon  fertilization,  reversible  depolarization  by  addition  of  K+  externally,  and  a  much  greater 
concentration  of  K+  inside  the  egg  than  in  the  surrounding  sea  water,  it  appeared  that  the 
process  of  fertilization  has  features  in  common  with  the  stimulation  of  other  excitable  tissue 
such  as  muscle  and  nerve. 

This  leads  to  the  expectation  that  there  should  be  an  increased  rate  of  exchange  of  K+ 
between  the  inside  and  outside  of  the  egg  upon  fertilization. 

By  loading  eggs  of  Arbacia  with  K",  washing  them,  fertilizing  aliquots  and  determining 
radioactivity  of  the  supernatant,  it  has  now  been  found  that  the  rate  of  release  of  the  K42  in- 
creases very  markedly  (1.5  to  3  X)  upon  fertilization.  The  results  of  seven  experiments  were 
consistent  in  showing  that  the  increased  rate  of  release  starts  within  l1/^  to  2  minutes  after 
fertilization.  There  is  also  consistently  a  reduction  of  rate  during  the  5th  to  8th  minute,  fol- 
lowed again  by  a  rise. 

The  reversible  replacement  of  potassium  by  rubidium  ion  in  Ulva  lactuca.     ROBERT 
DEVOE,  GEORGE  T.  SCOTT  AND  GARY  CRAVEN. 

Pieces  cut  from  fronds  of  Ulva  lactuca  were  placed  in  an  artificial  sea  water  containing 
rubidium  instead  of  potassium  ion,  and  samples  harvested  in  triplicate  at  various  times.  After 
ninety-six  hours  the  remaining  pieces  were  placed  in  running  sea  water  and  samples  harvested 
up  to  one  hundred  and  twenty  hours  more.  All  samples  were  rinsed  in  isotonic  sucrose  solution 
and  triple-blotted  with  absorbent  tissue  to  remove  all  extracellular  electrolytes,  then  ashed  and 
analyzed  for  potassium,  rubidium  and  sodium  ion  by  flame  photometry. 

The  uptake  of  rubidium  was  rapid  and  complete  with  four  hours,  the  time  the  first  samples 
were  taken,  and  amounted  to  65%  of  the  original  potassium  present.  The  potassium  ion  con- 
centration, after  an  initial  drop  to  40%  of  its  original  value,  continued  to  decrease  slowly  in  a 
manner  parallel  to  the  potassium  ion  concentration  in  the  control.  After  ninety-six  hours, 
when  the  samples  were  placed  in  running  sea  water,  the  gain  in  potassium  and  the  loss  of  ru- 
bidium were  rapid  for  the  first  ten  hours,  then  more  gradual.  After  one  hundred  and  twenty 
hours  in  running  sea  water,  the  potassium  ion  concentration  had  practically  reached  that  of 
the  control,  but  the  rubidium  ion  concentration  had  dropped  to  only  30%  of  its  highest  value. 
Sodium  ion  concentration  was  relatively  constant  during  the  course  of  the  experiment. 

Ulva  lactuca  does  not  show  a  time  lag  due  to  adaptation  to  rubidium  as  do  some  other  or- 
ganisms. It  does  photosynthesize  when  containing  rubidium,  and  will  form  and  discharge  a 
germinal  ridge. 

1  Supported  by  research  grant  C-2302  from  the  National  Cancer  Institute,  National  Insti- 
tutes of  Health,  Public  Health  Service. 

-  Supported  by  a  grant  from  Consiglio  Nazionale  delle  Ricerche. 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY          297 

PAPERS  READ  BY  TITLE 

Observations   on   autotomy  in   the   starfish,   Asterias  forbcsi.     JOHN    MAXWELL 
ANDERSON. 

Autotomy  of  rays  in  Asterias  has  excited  the  interest  of  many  investigators  but  has  never 
been  adequately  analyzed,  apparently  because  attention  has  been  focussed  chiefly  on  subsequent 
processes  of  regeneration.  Autotomy  normally  follows  severe  injury  to  a  ray  and  can  be  ar- 
tificially induced  by  application  of  electric  current  (8-16  V  DC).  As  King  (1898)  reported, 
separation  always  occurs  at  one  side  or  the  other  of  a  specific  pair  of  ambulacral  ossicles  and 
proceeds  rapidly  at  this  level  around  the  body-wall.  The  logical  supposition  that  separation 
involves  muscular  contraction  is  verified  by  the  fact  that  it  is  prevented  by  treatment  of  the 
animal  with  isotonic  AlgCL  solution.  Under  this  narcosis,  the  animal  can  be  subjected  to  radi- 
cal operations,  such  as  complete  removal  of  its  aboral  body-wall ;  replacement  in  sea  water  and 
recovery  from  narcosis  do  not  then  evoke  spontaneous  autotomy.  Electrically  induced  breakage 
in  the  floor  of  a  ray  thus  exposed  involves  a  sudden  release  of  attachments  binding  the  5th  pair 
of  ambulacral  ossicles  to  its  neighbors ;  these  ossicles,  bound  together  by  transverse  muscular 
and  collagenous  fibers  and  bearing  a  pair  of  tube  feet,  can  be  lifted  out  with  forceps  after  being 
thus  released.  Sections  of  these  elements  reveal  that  transverse  connections  are  intact,  but  that 
the  longitudinal  muscle  bands  and  connective  tissues  have  been  torn  across.  Initiation  of  au- 
totomy does  not  necessarily  depend  upon  this  separation  of  ambulacral  elements ;  it  can  be  in- 
duced by  electrical  stimulation  in  the  isolated  aboral  body-wall,  where  softening  and  tearing  of 
muscular  and  connective  tissues  occur  along  a  predetermined  line.  This  cannot  be  regarded  as 
simply  a  level  of  physical  weakness,  as  attempts  forcibly  to  break  off  a  ray  do  not  result  in 
separation  at  this  point.  Serial  sections  of  rays  have  shown  no  recognizable  structural  pecu- 
liarities of  skeletal,  muscular,  or  nervous  elements  in  the  region  of  the  breaking-joint. 

The  innervation  of  muscle  fibers  in  the  extrinsic  stomach-retractor  strands  of  the 
starfish,  Asterias  forbesi.     JOHN  MAXWELL  ANDERSON. 

The  extrinsic  retractor  strands  are  muscular  bands  extending  in  pairs  from  fibrous  nodules 
on  the  outer  wall  of  the  cardiac  stomach  to  attachments  on  the  ambulacral  ossicles  in  the  floor 
of  each  ray.  Their  position  raises  a  question  as  to  the  source  of  innervation  for  their  muscular 
elements,  whether  by  fiber  tracts  originating  in  the  subepithelial  nerve  plexus  layer  of  the  gut 
wall  or  by  pathways  originating  in  the  radial  nerve  cord  of  the  ray.  Vital  staining  procedures, 
utilizing  leucomethylene  blue,  demonstrate  that  several  groups  of  neurons  traverse  the  mesentery- 
like  sheets  binding  the  strands  to  the  floor  of  the  ray,  penetrate  the  strands,  and  terminate  in 
typical  asteroid  "ribbon  axons"  embracing  the  muscle  fibers.  While  it  has  been  impossible  to 
trace  precisely  the  origin  of  these  neurons,  their  courses  make  it  highly  probable  that  they  arise, 
along  with  nerve  fibers  to  other  sets  of  muscles  in  the  ray,  from  lateral  motor  centers  similar 
to  those  described  for  Astropecten  by  J.  E.  Smith  (1950).  No  fibers  have  been  found  crossing 
the  nodule  from  the  wall  of  the  stomach.  It  may  thus  be  concluded  that  the  coordinated  action 
of  the  retractor  muscles  and  of  the  muscles  in  the  wall  of  the  stomach  does  not  depend  on  com- 
mon pathways  of  innervation. 

The  effects  of  .r-irradiation  on  the  pupae  of  the  yelloiv  mealworm,  Tenebrio  molitor 
Linn.     ALAN  PRIEST  BROCKWAY. 

In  these  experiments  a  genetically  mixed  culture  of  larvae  was  maintained  at  room  tem- 
perature on  a  mixture  of  white  flour  and  dried  brewers  yeast.  Each  morning  the  culture  was 
cleared  of  pupae  and  then  every  four  hours  the  pupae  were  collected  and  irradiated.  Doses 
ranging  from  500  r  to  20,000  r  were  given  at  the  rate  of  2,500  r  per  minute. 

All  pupae  used  as  controls  or  given  500  r  and  1,000  r  hatched  normally.  Of  those  given 
2,000  r  only  33%  hatched  normally.  All  pupae  given  2,500  r  to  20,000  r  hatched  abnormally. 
The  region  between  1,000  r  and  2,500  r  seemed  to  be  quite  critical.  There  was  also  an  effect 
on  the  length  of  the  pupal  stage.  The  average  length  of  pupation  for  the  controls  was  8.15  days 
whereas  that  of  the  pupae  given  500  r  was  9.0  days,  1,000  r  to  4,000  r  was  10.0  days  and  5,000  r 


298          PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

to  20,000  r  was  11  days.  Furthermore  at  doses  from  5,000  r  to  20,000  r  only  50%  of  the  pupae 
hatched.  Work  is  being  carried  on  currently  to  determine  the  radiosensitivity  of  the  pupae  at 
ages  other  than  within  four  hours  of  formation. 

At  2,500  r  the  emerging  adult  was  unable  to  shed  the  pupal  cuticle,  which  remained  in  a 
mass  on  the  tip  of  the  abdomen.  An  increase  of  500  r  (3000  r)  inhibited  the  shedding  of  the 
cuticle  from  most  areas  of  the  body.  This  was  quite  noticeable  on  the  elytra  and  wings  and 
even  if  the  cuticle  was  removed  with  forceps  these  structures  did  not  expand.  The  tanning 
process  of  the  new  cuticle  was  not  complete  in  any  of  the  adults  given  more  than  3,000  r  in 
the  pupal  stage.  Only  some  of  the  cuticle  was  tanned  giving  the  animal  a  mottled  appearance 
and  many  soft  areas.  At  times  there  also  appeared  blisters  on  the  elytra  and  wings.  It  ap- 
peared that  in  the  elytra  the  epidermal  gland  poured  down  an  excess  of  cuticlin.  The  blisters 
on  the  wings  were  filled  with  a  fluid  which  may  be  hemolymph,  though  there  have  been  ob- 
served no  hemocytes  in  the  fluid. 

Chromaio graphic  study  of  crystalline  style  ain\lase.1     ALFRED  B.  CHAET. 

During  the  course  of  an  investigation  it  became  necessary  to  determine  whether  the  amylase 
activity  observed  in  the  crystalline  style  of  Mya  arcnaria  was  due  to  a-amylase,  /3-amylase,  or  a 
mixture  of  the  two.  The  technique  used  in  this  study  was  that  of  paper  chromatography  fol- 
lowed by  incubation  with  the  enzyme's  substrate.  Descending  type  chromatograms  were  run 
in  the  dark  at  18  ±  1°  C.  on  strips  of  Whatman  No.  1  filter  paper.  After  the  front  had  de- 
scended a  certain  distance,  the  paper  was  air  dried  and  placed  face  down  in  close  contact  with 
an  agar-substrate  film.  This  substrate  film  was  prepared  by  allowing  a  4%  solution  of  agar 
(buffered  at  pH  7)  containing  2%  potato  starch  to  gel  on  a  large  glass  plate.  The  chromato- 
gram-substrate  system  was  sealed  and  incubated  for  36  hours  at  30°  C.  The  position  of  a-  and 
j3-amylase  was  observed  after  removing  the  chromatogram  and  spraying  (either  paper  or  agar- 
substrate  sheet)  with  a  dilute  solution  of  iodine-potassium  iodide.  Two  satisfactory  solvents 
were  found  for  distinguishing  between  a-  and  jtf-amylase.  It  was  shown,  using  a  mixture  of 
ether  and  phosphate  buffer  (pH  6.6),  that  a-amylase  has  a  Rf.  of  0.66,  whereas  ^3-amylase  exhibits 
no  activity.  When  using  a  38%  saturated  solution  of  (NH4)2SO4  as  the  solvent,  a-amylase  had 
a  Rf.  value  of  0.02  and  /3-amylase,  0.27.  Although  in  both  solvents  the  spots  are  elongated, 
there  is  no  difficulty  in  distinguishing  the  difference  between  the  two  enzymes.  Experimental 
analysis  of  Mya  crystalline  styles  shows  that  all  of  the  amylolytic  activity  previously  observed 
is  due  only  to  a-amylase. 

Mechanism  of  toxic  factor  release.2     ALFRED  B.  CHAET. 

Additional  experiments  were  carried  out  dealing  with  the  characteristics  of  the  toxic  sub- 
stance obtained  from  scalded  worms  (Phascolosoma  gouldii)  and  an  attempt  made  to  study  the 
mechanism  of  toxin  release.  The  results  indicate  that  in  scalding  experiments  heat  per  sc  does 
not  produce  the  toxin  by  changing  a  non-toxic  molecule  into  a  toxic  one,  for  when  suspensions 
of  washed  cells  taken  from  the  coelomic  fluid  are  ruptured  (by  homogenization)  in  the  absence 
of  heat  a  toxin  is  released  which,  when  injected  into  normal  worms,  causes  death  in  62  hours. 
This  toxin  has  the  same  characteristics  previously  reported  for  the  toxic  factor  extracted  from 
scalded  worms.  It  is  a  heat-stable,  non-dialyzable  molecule  which  precipitates  in  saturated 
(NHi)2SO4.  It  appears  from  these  and  other  experiments  that  the  toxic  factor  is  normally 
present  in  the  form  of  a  toxic  molecule  found  within  the  coelomic  fluid  cells  and  that  rupturing 
the  cell  by  heat,  crushing,  or  hypotonic  solutions  is  sufficient  to  merely  release  this  substance 
which,  when  allowed  to  circulate  through  the  rest  of  the  organism,  results  in  death.  When 
using  Litnulus  as  a  test  object  it  has  been  shown  than  intravenous  injection  of  from  0.002-0.003 
ml.  of  toxin  per  gram  of  Limulus  is  sufficient  to  cause  death  in  less  than  24  hours.  This  test 
object  may  prove  useful  in  studying  the  mode  of  action  of  the  toxic  factor.  The  stability  of 
the  toxin  is  illustrated  in  experiments  whereby  samples  stored  in  a  frozen  state  for  10  months 

1  Supported  by  funds  from  the  State  of  Maine,  Dept.  of  Sea  and  Shore  Fisheries. 
-  Supported  in  part  by  funds  from  Boston  University  School  of  Medicine  and  Coe  Research 
Fund. 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

showed  no  apparent  loss  of  biological  activity.  By  differential  centrifugation  experiments  it 
was  possible  to  separate  the  coelomic  fluid  cells  into  5  distinct  fractions,  but  no  single  fraction 
was  the  storehouse  for  the  toxic  factor  since  at  least  4  out  of  5  released  the  toxic  material  when 
heated  in  vitro. 

A  combined  effect  of  urea  and  borate  buffer  on  uricase  activity.1     AURIN  M.  CHASE. 

The  effect  of  urea  and  borate  buffer  concentrations  on  the  oxidation  of  uric  acid  by  uricase 
was  studied  by  measuring  the  change  in  extinction  at  300  m/i  using  the  Beckman  DU  spectro- 
photometer,  maintained  at  26°  C.  by  water  cooling.  Reaction  mixtures  contained  0.33  nig. /ml. 
of  uricase  (Worthington  crude  preparation)  and  8  Mg/ml.  of  uric  acid,  plus  the  desired  urea 
concentration ;  all  dissolved  in  pH  9  borate  buffer  of  the  required  molarity.  These  molarities 
were  0.10,  0.23,  0.37,  and  0.50;  each  used  in  conjunction  with  urea  concentrations  up  to  9.0  M. 
After  adding  uricase  the  extinction  was  measured  for  10  minutes,  during  which  time  the  reaction 
was  zero  order.  The  slope  of  the  resulting  line  was  taken  as  the  measure  of  enzyme  activity. 

Borate  buffer  by  itself  had  an  inhibitory  effect  on  uricase  activity,  the  initial  rate  in  0.5  M 
being  about  75%  that  in  0.1  M  concentration.  With  4.8  M  urea  present,  activity  in  0.5  M 
borate  was  about  half  that  in  0.1  M.  In  0.5  M  buffer  regular,  increasing  inhibition  occurred 
with  increasing  urea  concentrations  whereas,  in  0.1  M  buffer,  there  was  no  inhibition  until 
nearly  4.0  M  urea  concentration  was  reached.  Then  activity  dropped  to  zero  as  8.0  M  urea  con- 
centration was  approached.  The  intermediate  borate  concentrations  gave  intermediate  results. 

Uricase  inactivation  at  the  lower  urea  concentrations  was  essentially  instantaneous.  Above 
6.4  M,  however,  a  slower  inactivation  occurred  in  addition  to  the  rapid  effect.  The  pH  of  all 
four  buffers  was  increased  from  9.0  to  9.7  by  added  urea  up  to  9.0  M,  and  this  may  well  be  a 
complicating  factor. 

It  is  known  (Canellakis  and  Cohen,  1955)  that  intermediates  and  end  products  of  this  re- 
action may  differ  in  different  buffers.  The  present  results  show,  in  addition,  that  the  kinetics 
observed  for  urea  inactivation  can  be  considerably  influenced  by  the  concentration  of  the 
buffer  used. 

Dimcthylatcd  dioxypurmes  and/or  .r-ray  inhibition  of  Arbacia  egg  development. 
RALPH  HOLT  CHENEY. 

Current  dimethylxanthine  and  x-ray  inter-relationship  studies  regarding  their  inhibitory 
action,  separately  and  combined,  on  growth  phenomena  were  done  using  the  Arbacia  punctnlata 
egg  as  the  test  material.  Af/400  theophylline  (1:3  CH3  2:6  dioxypurine)  and  M/400  theo- 
bromine  (3:7  CH3  2:6  dioxypurine)  and  30,000  r  x-ray  dosage  were  employed  for  comparison 
with  reports  (Cheney  1948,  1949)  on  inhibition  by  Tp,  Tb,  and  caffeine  (1:3:7  CH3  2:  6  dioxy- 
purine) ;  upon  caffeine  with  x-ray  (Cheney  and  Rugh  1954)  ;  and  the  report  by  Cheney  (1955) 
on  purine  (C5H4N4).  Tb  is  somewhat  more  inhibitory  than  Tp  at  JW/400.  30,000  r  delays  but 
does  not  arrest  egg  development  prior  to  the  stage  at  which  M/400  of  either  drug  separately 
stops  growth  without  irradiation.  30,000  r  does  not  affect  the  fertilizing  power  of  the  sperm. 
Eggs  were  x-rayed  before  fertilization. 

Development  of  non-irradiated  eggs,  x-irradiated  with  or  without  drug ;  drug-treated  30 
minutes  with  or  without  x-rays ;  also,  immediate  mixing  for  insemination  in  drug  without  pre- 
treatment,  was  observed  until  controls  reached  pluteus.  Subsequent  to  experimental  conditions 
cited  as  prior  to  mixing  with  normal  sperm,  equivalent  egg  numbers  were  transferred  to  stender 
dishes,  inseminated  by  normal  sperm,  and  maintained  in  running  sea  water  (SW).  Develop- 
mental time  and  form  were  recorded  in  SW,  SW  with  drug,  irradiated  SW  without  drug,  and 
irradiated  SW  with  drug. 

The  over-all  effect  of  x-irradiation  alone,  M/400  Tb  and  Tp  with  and  without  30,000  r  was 
delay  in  development :  first,  at  prophase  amphiaster ;  second,  at  blastula-gastrula  sequence ;  and 
third,  to  eventually  arrest  development.  No  evidence  was  found  to  indicate  any  significant  syner- 
gistic,  summation,  nor  antagonistic  action  between  either  Tb  or  Tp  and  30,000  r  irradiation 
although  each  of  the  three  factors  is  separately  inhibitory.  This  suggests  that  their  individual 
effects  are  via  different  mechanisms. 

1  Supported  in  part  by  a  National  Science  Foundation  grant. 


300          PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

The  distribution  of  mitochondria  and  tipid  droplets  during  early  cleavage  in  Ilya- 
nassa  obsoleta.     A.  C.  CLEMENT  AND  F.  E.  LEHMANN. 

Mitochondria  were  identified  by  their  size  and  affinity  for  Janus  green.  In  centrifuged 
eggs  they  form  a  band  between  two  layers  of  clear  C}  toplasm.  The  Janus  green  stain  of  the 
mitochondria!  band  may  be  fixed  with  OsO4.  The  normal  localization  of  mitochondria  and  lipid 
droplets  has  been  followed  from  the  one-cell  stage  through  the  time  of  formation  of  the  fourth 
quartet  of  micromeres.  The  position  of  mitochondria  was  revealed  by  vital  staining  with  Janus 
green  (1:  100,000  solution),  and  that  of  lipid  droplets  by  staining  with  Sudan  III  or  OsO4. 

Before  cleavage  the  mitochondria  are  concentrated  mainly  in  a  broad  cytoplasmic  cap 
around  the  animal  pole.  Each  of  the  first  4  blastomeres  shows  abundant  mitochondria.  All  of 
the  cells  of  the  first  three  quartets  of  micromeres,  and  their  derivatives,  also  receive  abundant 
mitochondria.  After  the  formation  of  the  first  and  second  micromere  quartets,  the  mitochondria 
in  the  macromeres  are  massed  in  a  broad  crescentic  zone  where  the  cytoplasmic  cap  meets  the 
yolk  zone.  The  mesentoblast  cell  4d  receives  numerous  mitochondria ;  the  fourth  quarter  ento- 
blasts  (4a,  b,  c)  also  contain  numerous  mitochondria,  whereas  the  4A,  B,  C  and  D  macromeres 
show  relatively  few. 

Lipid  droplets  are  concentrated  near  the  animal  pole  of  the  uncleaved  egg  and  are  appor- 
tioned rather  evenly  to  the  first  4  blastomeres.  In  the  early  macromeres  the  lipids  form  a 
crescentic  band  between  the  cytoplasmic  and  yolk  zones.  In  all  of  these  respects  the  general 
pattern  of  lipid  distribution  is  rather  similar  to  that  of  the  mitochondria.  However,  a  gradient 
of  lipid  distribution  appears  during  the  formation  of  the  micromere  quartets.  The  first  quartet 
micromeres  receive  very  few  lipid  droplets,  the  second  somewhat  more  and  the  third  still  more. 
The  4d  cell  receives  nearly  all  of  the  lipids  from  the  3D  macromere ;  there  is  a  more  even 
distribution  of  lipids  at  the  subdivision  of  3A-3C. 

The  uptake  and  distribution  of  radioactive  allo.ran  in  islet  and  other  tissues  of  the 
toad  fish.     S.  J.  COOPERSTEIN,  ARNOLD  LAZAROW  AND  WILMA  LAUFER. 

As  part  of  a  broad  research  program  on  the  problem  of  diabetes  mellitus  we  have  been  in- 
terested in  determining  the  mechanism  by  which  alloxan  selectively  kills  the  insulin-producing 
cells  of  the  islets  of  Langerhans  and  thereby  produces  diabetes.  One  of  the  immediate  prob- 
lems in  elucidating  the  mechanism  of  alloxan  action  is  to  determine  whether  this  chemical  agent 
is  selectively  concentrated  by  the  insulin-producing  beta  cells  or  whether  the  selectivity  of  al- 
loxan for  the  beta  cells  is  due  to  their  specialization  for  insulin  synthesis.  The  toadfish  is 
ideally  suited  for  these  studies.  Whereas  in  mammals  the  islet  tissue  is  distributed  throughout 
the  pancreas  and  constitutes  only  1%  of  its  total  weight,  in  the  toadfish  the  islet  tissue  is  seg- 
regated into  a  discrete  mass  which  is  separated  from  the  pancreatic  acinar  tissue.  Therefore 
we  have  studied  the  uptake  of  radioactive  alloxan  by  the  principal  islet  and  other  tissues  of  the 
toadfish,  Opsanus  tan, 

We  have  developed  suitable  techniques  for  injecting  alloxan  directly  into  the  circulation 
through  a  gill  arch  vessel.  In  this  manner  we  have  determined  the  distribution  of  radioactive 
alloxan  in  the  various  tissues  as  early  as  90  seconds  after  injection  and  at  various  later  times 
(2,  2  and  */£,  5,  15  and  30  minutes).  The  absolute  amount  of  radioactivity  found  in  the  various 
tissues  increased  rapidly  and  then  gradually  decreased  with  time.  Its  relative  distribution  among 
the  different  tissues,  however,  did  not  change  significantly.  Blood  had  the  highest  activity  at 
all  times.  The  activity  in  the  other  organs  decreased  in  the  following  order :  gill,  heart,  kidney, 
islet,  brain,  liver  and  skeletal  muscle.  The  islet  tissue  had  only  %  to  ^5  of  the  radioactivity 
found  in  blood,  and  at  no  time  did  its  radioactivity  exceed  that  which  would  have  resulted  from 
uniform  distribution  of  the  radioactive  alloxan  throughout  the  body. 

Phosphorylase  system  in  the  lobster}-     ROBERT  W.  COWGILL. 

Phosphorylase  catalyzes  the  reversible  formation  of  glycogen  and  inorganic  phosphate  from 
glucose- 1 -phosphate.  This  enzyme  exists  in  tail  muscle  of  the  lobster  in  three  forms:  phos- 
phorylase  a  is  active  enzymatically,  phosphorylase  b  requires  muscle  adenylic  acid  for  activity, 

1  This  work  was  supported  by  a  grant  from  the  National  Institutes  of  Health. 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY          301 

and  phosphorylase  c  is  completely  inactive.  Tfie  relative  levels  of  these  forms  of  the  enzyme 
change  with  the  physiological  condition  of  the-  animal  (Cowgill  and  Cori,  1955)  but  the  prin- 
cipal form  extracted  from  the  normal  lobster  is  phosphorylase  c. 

Phosphorylase  c  has  been  separated  from  phosphorylase  a  and  phosphorylase  b  by  am- 
monium sulfate  fractionation  of  crude. muscle  extracts.  It  has  been  tested  in  the  presence  of  a 
wide  variety  of  metal  ions  and  nucleotides  and  it  has  been  found  completely  inactive.  The 
ammonium  sulfate  fractions  also  contain  enzymes  for  the  interconversion  of  the  various  forms 
of  phosphorylase.  Two  of  these  are  activating  enzymes.  Activating  enzyme  No.  1  converts 
phosphorylase  c  to  phosphorylase  b ;  this  enzyme  requires  Fe+++,  Cd++,  Pb++,  or  VO++  for  activity. 
Activating  enzyme  No.  2  converts  phosphorylase  b  to  phosphorylase  a,  and  requires  Mn++  and 
adenosine  triphosphate  for  activity.  Both  the  metal  ion  and  nucleotide  requirement  are  highly 
specific  for  the  latter  enzyme.  In  addition  to  these  enzymes,  there  is  at  least  one  inactivating 
enzyme  that  converts  phosphorylase  a  to  phosphorylase  c.  This  enzyme  requires  Mn++  or  cer- 
tain other  divalent  cations  for  activity,  but  unlike  the  second  activating  enzyme  it  is  completely 
inhibited  by  fluoride  ions.  All  of  the  activating  and  inactivating  enzymes  were  inhibited  by 
ethylenediaminetetraacetic  acid. 

It  is  interesting  that  these  enzymes  that  interconvert  the  various  forms  of  lobster  phos- 
phorylase also  are  capable  of  interconverting  phosphorylase  a  and  phosphorylase  b  from  rabbit 
muscle.  That  is,  the  inactivating  enzyme  of  lobster  will  convert  rabbit  phosphorylase  a  to 
phosphorylase  b  (but  not  to  a  phosphorylase  c  form)  ;  and  activating  enzyme  No.  2  of  lobster 
will  convert  rabbit  phosphorylase  b  to  phosphorylase  a. 

The  action  of  Nessler's  reagent  and  ATP  on  extracted  and  denatured  muscle* 

DAVID  DlBBELL  AND  HOWARD  HOLTZER.2 

The  following  experiments  suggest  that  Nessler's  reagent  has  more  than  one  mode  of  action 
on  the  muscle  model ;  one  simulates  the  action  of  ATP,  the  other  does  not. 

Muscle  fibers  contracted  maximally  in  either  ATP  or  Nessler's  and  inspected  under  phase, 
exhibited  extreme  contraction  bands.  Such  contracted  fibers  may  be  stretched  to,  and  main- 
tained at,  300%  of  rest  length  by  treatment  in  Nessler's  followed  by  versine  (Lacki).  Stretched 
fibers  can  be  recontracted  by  Nessler's  but  not  by  ATP.  If  a  contracted  fiber  is  stretched  an 
expanded  contraction  band  pattern  is  observed.  Subsequent  changes  in  length  do  not  change 
this  contraction  band  pattern.  Fibers  lightly  prefixed  in  formalin  to  inhibit  contraction  and  ex- 
tended in  Nessler's,  yield  the  stretched  A,  I,  Z  and  H  band  pattern.  Thus  ATP  or  Nessler's 
induced  maximal  contractions  produce  the  same  irreversible  submicroscopic  reorganization  which 
is  expressed  cytologically  as  contraction  bands. 

Differences  between  ATP  and  Nessler's  were  demonstrated  by  treating  muscle  models  with 
heat,  formalin,  acetone,  absolute  alcohol,  low  pH,  and  extracting  in  0.5  M  KI,  0.9  M  LiCL  or 
urea.  In  all  these  instances,  Nessler's  induced  contractions  after  the  muscle  proved  refractory  to 
ATP.  For  example,  0.5  M  KI  extracted  fibers  do  not  contract  to  ATP  after  one  hour,  but 
react  to  Nessler's  after  6  hours.  Extension  in  Nessler's  followed  by  contraction  can  be  observed 
in  treated  fibers  that  will  not  contract  from  rest  length.  For  example,  fibers  extracted  in  0.5  M 
KI  for  two  weeks  though  not  contracting  when  initially  placed  in  Nessler's,  when  stretched, 
contract  vigorously.  Nessler's  will  contract  living  muscle,  whereas  ATP  will  not.  These  ex- 
periments indicate  that  Nessler's,  in  addition  to  its  effect  on  the  ATP-sensitive  actomyosin 
complex,  may  act  on  the  muscle  fiber  skeleton. 

Fermentation  studies  in  nine  varieties  of  Tetrahytnena  pyrijormis?     ALFRED  M. 
ELLIOTT  AND  DARRYLL  E.  OUTKA. 

In  a  search  for  biochemical  differences  in  T.  pyriformis,  clones  representing  each  of  the  45 
mating  types  of  the  nine  known  varieties  were  tested  for  their  capacity  to  ferment  the  following 

1  Supported  by  Multiple  Sclerosis  Society  and  National  Foundation  for  Infantile  Paralysis 
grants. 

2  American  Cancer  Society  Scholar. 

3  This  investigation  was  supported  by  grants  from  the  National  Institutes  of  Health  (PHS 
G3588C3)   and  the  Horace  H.  Rackham  School  of  Graduate  Studies,  University  of  Michigan. 


302          PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

sugars :  dextrose,  levulose,  galactose,  mannose,  sucrose,  maltose,  and  lactose.  To  the  basic 
medium,  containing  0.5%  proteose  peptone,  0.5%  tryptone  and  phenol  red  as  the  indicator,  fil- 
tered sugars  were  added  aseptically  to  make  up  final  concentrations  of  0.5%.  The  pH  was 
initially  set  at  7.2.  Light  inoculations  (0.2  ml.)  of  the  organisms  in  log  growth  were  dispensed 
into  the  test  and  control  media  which  were  then  incubated  at  27°  C.  Records  of  color  changes 
were  made  at  one,  two,  three  and  5  days ;  in  doubtful  cases  the  tubes  were  maintained  for  as 
long  as  12  days. 

All  clones  fermented  dextrose,  mannose,  and  maltose.  Levulose  was  degraded  by  all  ex- 
cept two  clones  in  variety  1.  None  fermented  sucrose  and  only  four  of  the  five  clones  tested 
in  variety  9  attacked  lactose.  The  widest  variation  occurred  with  galactose  which  was  fer- 
mented by  all  clones  in  varieties  1,  3,  7  and  9  but  was  unaffected  by  clones  from  varieties  2,  4, 
5,  6  and  8. 

These  results  indicate  considerable  variation  in  fermenting  capacity  among  the  clones 
tested,  and  although  varietal  differences  are  striking,  the  number  of  clones  tested  is  insufficient 
for  any  final  generalizations.  Only  after  a  large  number  of  clones  from  each  variety  are  ex- 
amined can  general  conclusions  be  drawn. 

Electron  microscope  studies  of  conjugating  Tetrahymcna  pyriformis.1     ALFRED  M. 
ELLIOTT  AND  JOHN  W.  TREMOR. 

The  sequence  of  events  occurring  during  conjugation  in  T.  pyriformis  (strains  WH6  and 
WH14,  variety  1,  mating  types  I  and  II)  was  observed  under  the  electron  microscope.  Con- 
centrated suspensions  of  washed  cells  in  various  stages  of  conjugation  were  prepared  from  both 
homogenized  and  sectioned  material.  The  former  proved  unsatisfactory  owing  to  destruction 
of  nuclear  elements.  Sectioned  material  revealed  certain  details  of  conjugation  not  observed 
with  the  light  microscope.  The  organisms  were  fixed  in  1%  veronal-acetate  buffered  formalin 
and  treated  with  2%  osmic  acid.  They  were  then  concentrated,  embedded  in  methacrylate,  and 
sectioned. 

Sections  through  the  region  of  contact  between  the  two  mates  showed  regularly  spaced 
protoplasmic  bridges  of  approximately  0.2  micron  in  diameter  which  could  conceivably  serve 
for  the  exchange  of  cytoplasmic  materials.  These  were  well  established  at  the  time  of  the  third 
prezygotic  division  and  possibly  earlier.  Since  they  possess  about  the  same  diameter  and  spac- 
ing as  cilia,  it  is  possible  that  they  were  derived  from  them.  The  morphology  of  the  membranes 
in  the  region  of  nuclear  exchange  was  clearly  seen.  Most  of  the  nuclear  stages  occurring  dur- 
ing conjugation  were  observed.  Examination  of  early  and  late  macronuclear  anlagen  stages 
revealed  structure  which  could  not  be  clearly  interpreted  from  the  preparations.  Chromosomes 
were  readily  observed  but  showed  nothing  that  had  not  already  been  seen  under  the  light  micro- 
scope. It  is  hoped  that  with  better  fixation  other  details  may  be  seen  that  will  supplement  our 
knowledge  of  the  morphology  of  this  and  other  ciliates. 

Influence  of  hematoporphyrin  and  phenol  on  .v-radiation  sensitivity  of  Paramecium. 
FRANK  H.  J.  FIGGE  ~  AND  RALPH  WICHTERMAN.S 

In  previous  experiments,  hematoporphyrin  solutions  containing  phenol  as  a  preservative  had 
been  employed  to  alter  the  radiation  sensitivity  of  Paramecium  caudatum.  Paramecia  placed  in 
the  solutions  of  hematoporphyrin  prior  to  irradiation  exhibited  an  LD  50  (24  hours)  of  18  kr 
while  the  control  groups  required  a  dose  of  340  kr.  Experiments  reported  here  were  designed 
to  ascertain  the  influence  of  phenol  and  hematoporphryin  on  radiation  sensitivity  when  used  in- 

1  This   investigation   was   supported   by   research    grants    from    the    National    Institutes    of 
Health  (PHS  G3588C3)  and  the  Horace  H.  Rackham  School  of  Graduate  Studies,  University 
of  Michigan. 

2  Supported  by  grants  from  the  Anna  Fuller   Fund  and   the   American   Cancer   Society, 
Maryland  division. 

3  Part  of  a  project  aided  by  a  contract  between  the  Office  of  Naval  Research,  Department 
of  the  Navy,  and  Temple  University   (NR  135-263)   and  the  Committee  on  Research,  Temple 
University. 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY          303 

dependency  and  combined.  The  conditions  of  the  radiation  experiments  were  essentially  the 
same  as  those  described  in  our  earlier  publications.  Paramecium  bursaria  and  P.  multiniicro- 
nucleatum  were  irradiated  in  Nylon  syringes  so  that  samples  could  be  taken  at  various  dose 
intervals  up  to  100,000  r.  The  highest  dose  killed  none  of  the  controls.  All  of  the  animals 
placed  in  porphyrin  and  phenol  combined  were  killed  by  50  to  100  kr  doses.  The  LD  50  for 
P.  bursaria  was  16  kr,  the  same  as  observed  previously.  The  1 :  20,000  hematoporphyrin  solu- 
tions without  phenol  had  relatively  little  observable  effect  since  nearly  all  animals  of  both  spe- 
cies survived  the  maximum  dose  given.  Animals  placed  in  1  :  10,000  phenol  solutions  corre- 
sponding to  the  concentration  of  phenol  in  the  porphyrin  and  phenol  mixtures  were  sensitized 
to  x-radiation.  None  of  the  animals  survived  a  dose  of  33  kr.  The  LD  50  for  the  phenol- 
treated  group  was  even  lower  (14  kr,  P.  bursaria)  than  the  solutions  containing  hematopor- 
phyrin and  phenol. 

It  was  also  observed  that  the  irradiated  animals  in  the  phenol  solution  survived  much 
longer  (12  hours)  when  kept  in  Nylon  syringes  and  deprived  of  air  while  specimens  that  were 
expressed  from  syringes  into  spot  plates  died  within  30  minutes. 

Thus  the  increased  sensitivity  of  paramecia  is  due  mainly  to  the  phenol  which  is  used  as 
a  preservative  for  the  porphyrin  solutions.  Attempts  will  be  made  to  utilize  phenol  in  com- 
bination with  porphyrin  and  phenol  alone  in  cancer  therapy. 

The  effect  of  argon  at  high  pressures  on  the  cleavage  time  of  the  sea  urchin,  Arbacia 
punctulata.     CHARLOTTE  HAYWOOD. 

Several  investigators  have  shown  that  the  inert  gases,  nitrogen  and  argon,  at  sufficiently 
high  atmospheric  pressures  can  exert  a  narcotic  effect  on  certain  tissues  and  on  animals,  in- 
cluding man.  A  previous  study  of  nitrogen  at  pressures  up  to  61  atmospheres  upon  fertilized 
Arbacia  eggs  failed  to  demonstrate  a  delay  in  the  cleavage  rate.  A  similar  study  has  now  been 
made  with  argon  since  it  might  be  expected  to  be  more  effective  than  nitrogen  because  of  its 
greater  lipid  solubility. 

The  method  is  essentially  the  same  as  that  used  with  nitrogen  at  high  pressures  (already 
published).  A  pressure  chamber  which  permitted  microscopic  observation  was  employed.  Ade- 
quate oxygen  was  available  in  the  air  initially  present  in  the  chamber.  Controls  in  air  at  at- 
mospheric pressure  were  run  simultaneously.  Temperatures  were  21.5°  to  22.0°  C. 

The  time  required  for  the  first  cleavage  to  appear  in  50%  of  the  eggs  was  regarded  as  the 
cleavage  time.  In  ten  experiments  with  argon  at  41  atmospheres  the  cleavage  rates  were  re- 
tarded 4  to  8  minutes  (average  =  5.9  minutes).  These  values  represent  delays  of  8  to  15  per 
cent  (average  =  11%)  beyond  the  control  cleavage  times.  In  six  experiments  at  55  atmospheres 
of  argon  the  delays  were  9%  to  18  minutes  (average  =  14.2  minutes),  or  cleavage  times  16.4  to 
34%  (average  =  26.2%)  longer  than  the  controls.  At  still  higher  pressures  the  delays  were 
greater,  although  pressures  up  to  68  atmospheres  failed  to  give  complete  suppression  of  cleavage. 
Abnormalities  were  frequent  at  the  pressures  above  55  atmospheres. 

Hydrostatic  pressure  per  se  is  not  involved  with  41  and  55  atmospheres  at  least,  as  shown 
by  earlier  negative  results  with  nitrogen  and  with  helium  at  61  atmospheres. 

The  enhancement  of  somitic  muscle  maturation  by  the  embryonic  spinal  cord.1 
HOWARD  HoLTZER,2  JAMES  LASH  AND  SYBIL  HOLTZER. 

Recent  experiments  in  this  laboratory  indicated  that  in  the  chick  maturation  of  somitic 
muscles  was  enhanced  by  the  spinal  cord  but  unaffected  or  inhibited  by  the  notochord.  This 
relationship  prompted  a  re-examination  of  the  situation  in  amphibian  embryos,  where  it  has  been 
claimed  that  the  notochord  is  essential  to  somitic  myogenesis.  The  following  experiments  will 
demonstrate  that  in  salamander,  as  well  as  chick  embryos,  the  spinal  cord  stimulates  the  growth 
of  somitic  muscle,  whereas  the  notochord  is  inert. 

Clusters  of  6  to  10  isolated  somites  from  tail-bud  embryos  \vere  implanted  into  the  dorsal 
fin  of  young  larval  hosts.  At  time  of  sacrifice  three  to  5  weeks  later,  small  strands  of  muscle 
and  pronephric  tissue  were  found  in  the  quasi-culture  chamber  of  the  dorsal  fin  of  the  host. 

1  Supported  by  U.S.P.H.  Service  and  Multiple  Sclerosis  Society  grants. 

2  American  Cancer  Society  Scholar. 


304          PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

Implanted  clusters  of  somites  plus  notochord  differentiated  into  a  similar  small  mass  of  muscle. 
Irregular  and  inconstant  mounds  of  cartilage  adhering  to  the  notochord  and  scattered  kidney 
tubules  were  also  present.  In  contrast,  the  same  mass  of  somites  plus  a  piece  of  spinal  cord 
cultured  in  the  dorsal  fin  formed  a  large  mass  of  well  differentiated  muscle,  similar  to  what 
the  somites  would  have  formed  if  left  in  situ.  Cartilaginous  vertebral  elements,  including  pre- 
cociously formed  centra,  were  also  present.  The  enhancement  of  muscle  growth  is  not  a  gen- 
eralized property  of  neural  tissue.  Implants  of  forebrain  and  somites  yielded  the  small  numbers 
of  muscle  strands  found  in  cases  of  somites  alone  or  somites  plus  notochord.  That  this  action 
of  the  embryonic  spinal  cord  on  muscle  growth  need  not  be  mediated  by  motor  nerves  is  indi- 
cated by  implants  of  somites  from  stage  33  embryos.  Sizeable  muscle  masses  developed  in  this 
series  though  motor  nerves  were  not  present. 

The  relation  of  the  cortex  to  the  formation  of  a  perivitelline  space  in  the  eggs  of 
Funduhts  heteroclitus.     CHARLES  W.  HUVER. 

Unfertilized  Fundulus  eggs  were  centrifuged  in  an  effort  to  determine  if  the  cortically 
located  cytoplasm  is  necessary  for  the  formation  of  a  perivitelline  space.  An  International  clini- 
cal centrifuge  was  used.  Temperatures  ranged  from  22°  C.  to  26°  C.  during  the  experiments. 
All  eggs  were  in  a  medium  of  0.95  M  sucrose  solution.  The  control  group  consisted  of  41  un- 
fertilized eggs  which  were  neither  centrifuged  nor  pricked. 

Preliminary  centrifugation  experiments  at  846  X  g.  and  at  3,390  X  g.  for  10  minutes  caused 
the  cytoplasm  to  concentrate  at  the  centrifugal  end  of  the  egg.  Cytoplasm  could  be  seen  because 
of  its  grey  or  brown  color  when  concentrated.  In  all  28  eggs  centrifuged  in  these  early  ex- 
periments the  blastodisc  formed  at  the  concentration  of  cytoplasm.  Cortical  alveoli  were  densely 
packed  in  the  blastodisc  and  a  few  were  scattered  on  the  egg  surface.  The  size  of  the  blastodisc 
was  inversely  correlated  with  the  number  of  alveoli  left  in  the  egg  cortex.  Therefore,  cortical 
alveoli  may  be  regarded  as  indicators  for  the  presence  of  cortical  cytoplasm. 

When  40  eggs  were  centrifuged  at  4,320  X  g.  for  5  minutes,  the  cortex  appeared  to  have 
been  moved  inside  the  egg  in  15  cases.  Since  no  cortical  alveoli  were  seen  on  the  egg  surface, 
it  is  inferred  that  little  or  no  cortical  cytoplasm  was  present.  The  former  cortex  was  clearly 
visible  as  a  grey  area  filled  with  alveoli.  This  internally  displaced  cortex  rounded  up  to  form 
a  blastodisc  surrounded  by  yolk.  Seven  of  the  15  eggs  with  internal  blastodiscs  were  pricked 
with  a  glass  needle  25  M  in  diameter.  In  none  of  these  eggs  was  there  any  formation  of  a 
perivitelline  space.  While  in  the  controls  and  in  the  25  other  experimentals  which  had  some 
cytoplasm  remaining  in  a  cortical  position,  a  perivitelline  space  was  formed  regardless  of 
whether  they  were  pricked  or  not.  These  results  suggest  that  cortically  located  cytoplasm  is 
necessary  for  the  formation  of  a  perivitelline  space. 

The  occurrence  of  a  crystalline  style  in  the  marine  snail,  Nassarius  obsolctns.^ 
CHARLES  E.  JENNER. 

The  crystalline  style  is  a  specialized  digestive  apparatus  found  in  all  bivalve  mollusks,  but 
in  gastropods  its  occurrence  is  believed  to  be  restricted.  The  present  report  of  its  occurrence 
in  Nassarius  obsoletus  is  of  special  interest  since :  ( 1 )  this  snail  belongs  to  the  order  Steno- 
glossa,  a  group  in  which  the  style  is  believed  not  to  occur;  (2)  this,  snail  is  frequently  de- 
scribed as  carnivorous,  but  the  style  is  said  not  to  occur  in  carnivorous  snails;  (3)  this  snail 
passes  large  quantities  of  sand  and  other  particles  of  similar  size  through  its  digestive  tract 
by  peristalsis,  a  procedure  not  followed  by  any  other  snail  reported  to  have  a  crystalline  style. 
The  characterization  of  this  snail  as  primarily  carnivorous  or  as  essentially  a  scavenger  on 
dead  animals  is  clearly  in  error  for  the  following  reasons:  (1)  the  concentration  of  snails  fre- 
quently encountered  must  require  a  food  source  far  greater  than  provided  by  available  animal 
substance;  (2)  the  gut  is  frequently  filled  with  bottom  materials,  mud  and  sand;  (3)  the  pos- 
session of  a  crystalline  style  is  primarily  an  adaptation  for  the  digestion  of  starch,  a  plant 
product. 

1  Aided  by  a  grant  from  the  National  Institutes  of  Health,  U.  S.  Public  Health  Service, 
E-356(C4). 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY          305 

Seasonal  resorption  of  the  copulatory  organ  in  Nassarius  trivittatus  and  Littorina 
littorea.'1     CHARLES  E.  JENNER. 

In  Nassarius  obsolctus  the  termination  of  seasonal  reproductive  activity  is  marked  by  the 
resorption  of  the  copulatory  organ  in  males  (Jenner  and  Chamberlain,  1955).  This  has  been 
found  to  be  true  also  for  Nassarius  trivittatus  and  Littorina  littorea,  the  phenomenon  for  the 
latter  having  previously  been  recorded  for  European  waters  (Tattersall,  1920,  and  others).  As 
judged  by  copulatory  organ  resorption,  Nassarius  trivittatus  from  the  shallow  water  of  Eel 
Pond,  Woods  Hole,  terminated  seasonal  reproduction  by  early  July  1956.  By  the  same  criterion, 
Littorina  littorea  from  mid-tide  level  at  Nobska  Point,  Woods  Hole,  showed  evidence  of  repro- 
ductive decline  by  late  July ;  approximately  25  per  cent  reduction  in  males  had  occurred  by 
August  25. 

Electron  microscopic  observations  on  changes  in  the  cortical  cytoplasm  after  fertili- 
sation of  Fundulus  eggs.     NORMAN  E.  KEMP  AND  MARGARET  D.  ALLEN. 

Eggs  were  fixed  in  buffered  (pH  7.4)  \%  osmic  acid  in  artificial  sea  water  at  various 
times:  (1)  before  fertilization;  (2)  during  breakdown  of  cortical  alveoli,  a  process  which 
starts  at  the  animal  pole  one-two  min.  after  insemination  and  is  completed  at  the  vegetative  pole 
about  one  min.  later;  (3)  after  breakdown  of  cortical  alveoli  was  complete;  and  (4)  after  the 
blastodisc  was  well-formed.  In  order  to  eliminate  most  of  the  yolk,  eggs  were  fixed  lightly 
(three-five  min.)  in  osmic  acid,  then  transferred  to  50%  sea  water  for  removal  of  the  chorion 
and  bisection  of  the  egg  with  iridectomy  scissors.  Unfixed  yolk  was  washed  out  with  a  pipette 
and  the  partially  fixed  half-shells  of  cytoplasm  and  adherent  yolk  returned  to  osmic  acid  for 
about  15  min.  to  complete  osmication,  following  which  they  were  processed  for  methacrylate 
embedding  and  thin  sectioning.  Unfertilized  eggs  have  short  microvilli  scattered  over  the  sur- 
face. Some  cortical  alveoli  are  covered  only  by  a  thin  layer  of  cytoplasm ;  others  are  embedded 
more  deeply,  some  even  resting  against  the  interior  yolk  mass.  After  fertilization,  alveoli  come 
to  the  surface,  burst  and  liberate  their  contents  into  the  perivitelline  space.  Many  alveoli  form 
crater-like  depressions  in  the  surface  cytoplasm  during  the  process  of  extrusion.  Cytoplasm 
may  heap  up  to  form  a  prominent  elevated  rim  around  an  alveolar  crater.  Sides  and  floor  of 
a  crater  come  to  be  part  of  the  surface  of  the  egg  as  it  smooths  out  after  the  cortical  reaction 
is  completed.  The  surface  of  the  blastodisc  possesses  long  pseudopodial  processes  indicative 
of  great  activity  of  the  cortical  cytoplasm.  By  contrast,  the  thin  yolk  gel  layer  peripheral  to 
the  blastodisc  has  a  perfectly  smooth  surface. 

Dchydrogenase  activity  in  developmental  stages  of  Asterias  as  measured  with  tetra- 
soliurn  salts.     EVELYN  KIVY-ROSENBERG  AND  BENJAMIN  W.  ZWEIFACH. 

A  quantitative  estimate  of  endogenous  dehydrogenous  activity  during  developmental  stages 
of  Asterias  was  sought  using  tetrazolium  salts  as  indicators.  The  toxicity  of  the  tetrazoles 
made  such  studies  not  feasible.  As  a  result,  an  attempt  was  made  to  investigate  specific  sub- 
strate-dependent dehydrogenases  at  given  stages  (uninseminated  ova,  inseminated  ova,  blastulae, 
gastrulae).  A  method  similar  to  the  frozen  tissue-slice  technique  was  employed  in  which  most 
endogenous  activity  is  eliminated  by  freezing,  and  circumvents  the  problem  of  toxicity. 

Having  been  removed  from  deep  freeze,  samplings  of  chosen  developmental  stages  were 
thawed  at  room  temperature.  These  were  incubated  anaerobically  at  37-38°  C.  in  a  medium 
containing  sea  water,  DPN,  nicotinamide  and  one  of  a  series  of  substrates  (which  had  been 
used  previously  in  tissue  work)  :  succinate,  alpha-glycerophosphate,  glucose,  glutamate,  malate, 
lactate,  beta-hydroxybutyrate.  No  cofactors  were  used  with  succinate.  Two  tetrazolium  salts 
were  employed:  2,  3,  5  triphenyltetrazolium  chloride  (TTC)  and  2-(p-iodophenyl)-3-(p-nitro- 
phenyl)-5-phenyl  tetrazolium  chloride  (INT).  Final  concentration  of  tetrazolium  in  the  me- 
dium was  under  0.5%.  The  amount  of  formazan  was  determined  colorimetrically  by  a  spectro- 

1  Aided  by  a  grant  from  the  National  Institutes  of  Health,  U.  S.  Public  Health  Service, 
E-356(C4). 


306         PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

photometer.     The  substrate-dependent  dehydrogenase  activity  was  expressed  in  terms  of  micro- 
grams  per  cubic  milliliter  of  ova  (or  embryos). 

It  is  evident  that  particular  substrate-dependent  dehydrogenases  are  more  metabolically  ac- 
tive than  others  and  that  there  is  a  quantitative  variation  during  development.  Although  the 
absolute  quantity  of  reduced  tetrazolium  varied  somewhat  from  one  batch  of  eggs  (or  embryos) 
to  another,  it  appears  when  TTC  was  used  as  the  acceptor,  that  malate  dehydrogenase  activity 
is  highest,  with  beta-hydroxybutyrate  and  alpha-glycerophosphate  next  in  order  though  defi- 
nitely lower.  Other  substrates  yielded  negligible  values  or  none  at  all.  With  INT,  malate, 
alpha-glycerophosphate  and  beta-hydroxybutyrate  were  highly  active.  Other  substrates  gave 
positive  reactions  but  of  much  lower  intensity.  Limited  observations  indicate  that  alpha- 
glycerophosphate,  malate,  and  beta-hydroxybutyrate-dependent  dehydrogenases  become  less  ac- 
tive soon  after  insemination  and  then  increase  again  at  gastrulation. 

The  chemical  nature  of  bound  oxygen  in  hemerythrin  and  in  hemocyanin.1     IRVING 
M.  KLOTZ  AND  THEMIS  A.  KLOTZ. 

Since  ferrous  ion  can  be  released  from  deoxygenated  hemerythrin  and  ferric  ion  from 
oxyhemerythrin,  it  has  been  suggested  that  the  bound  oxygen  in  the  oxygenated  protein  is  in 
the  form  of  peroxide  ion.  Several  tests  for  peroxide  ion  have  been  carried  out,  therefore. 
The  most  delicate  test  for  HL,O2  is  the  formation  of  a  yellow  color  with  a  solution  of  TiO2  in 
dilute  sulfuric  acid.  To  five  drops  of  Ti(IV)  test  reagent  in  a  spot  plate  was  added,  therefore, 
some  oxyhemerythrin  crystals.  An  orange  color  formed  immediately  around  the  protein  ;  on 
further  mixing  the  color  was  diluted  to  a  strong  yellow.  Corroboration  of  the  presence  of 
peroxide  ion  was  also  obtained  with  the  benzidine  test.  A  few  drops  of  a  4%  solution  of  ben- 
zidine  in  glacial  acetic  acid  were  placed  in  a  spot  plate.  To  this  solution  were  added  a  few 
milligrams  of  horse-radish  peroxidase  and  then  some  crystals  of  oxyhemerythrin.  A  blue 
color  developed  immediately  in  the  vicinity  of  the  hemerythrin.  Concentrated  solutions  of 
crystalline  oxyhemerythrin,  as  well  as  laked  blood,  also  gave  positive  benzidine  tests.  In  con- 
trast deoxygenated  hemerythrin  did  not  give  the  blue  color.  Both  tests  thus  show  that  peroxide 
ion  is  released  from  oxyhemerythrin  in  acidified  solutions. 

In  hemocyanin  valence  changes  of  the  copper  on  oxygenation  suggest  that  the  bound 
oxygen  is  in  the  form  of  perhydroxyl  free-radical  ion.  This  species  is  a  reactive  intermedi- 
ate known  to  appear  in  the  metal-catalyzed  decomposition  of  peroxide.  Oxyhemocyanin  was 
added,  therefore,  without  peroxidase,  to  a  solution  of  benzidine  in  glacial  acetic  acid.  At  0°  C., 
a  stable  blue  color,  characteristic  of  oxidized  benzidine,  was  obtained,  as  would  be  expected  if 
perhydroxyl  radical  were  released  from  hemocyanin. 

Crystallisation  of  Busycon  hemocyanin.1     IRVING  M.  KLOTZ,  THEMIS  A.  KLOTZ 
AND  GEORG  H.  CZERLINSKI. 

Attempts  to  prepare  crystalline  hemocyanin  from  the  blood  of  Busycon  canaliculatum  by 
standard  procedures  used  for  other  species  have  not  proved  successful.  It  has  been  found 
now,  however,  that  an  adaptation  of  the  heparin  method  described  by  S.  Cohen  (1942)  for  other 
macromolecules  does  produce  crystals. 

Blood  drained  from  the  tissues  of  the  conch  is  filtered  through  glass  wool  and  then  centri- 
fuged  at  4°  C.  at  2000  r.p.m.  in  the  International  Refrigerated  Centrifuge.  The  solution  is 
then  decanted  and  dialyzed  against  distilled  water  at  4°  C.  for  two  days,  with  frequent  re- 
placement of  the  water  outside  the  dialysis  bags.  The  hemocyanin  is  then  separated  from  the 
blood  by  ultracentrifugation  at  35,000  r.p.m.  in  the  Spinco  Model  L.  The  colorless  super- 
natant is  discarded,  the  concentrated  hemocyanin  at  the  bottom  of  the  tube  is  redissolved  in 
distilled  water  and  the  ultracentrifugation  repeated.  After  the  third  ultracentrifugation,  the 

1  This  investigation  was  supported  in  part  by  a  research  grant,  RG-4134,  from  the  National 
Institutes  of  Health,  Public  Health  Service. 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY          307 

concentrated  hemocyanin  is  removed  and  to  it  is  added  solid  sodium  heparin,  or  a  10%  aqueous 
solution,  to  a  final  concentration  of  3%.  Turbidity  appears  immediately  but  the  mixture  is 
permitted  to  stand  overnight.  The  solid  is  then  separated  by  centrifugation  at  1500  r.p.m. 
Examination  under  the  microscope  reveals  crystals,  perfect  ones  being  in  the  shape  of  hexagons. 
Even  this  crystalline  hemocyanin,  in  aqueous  solution,  shows  two  widely  separated  com- 
ponents in  the  Spinco  analytical  ultracentrifuge  Model  E.  It  is  likely  that  the  faster  moving 
one  is  a  dimer  or  higher  aggregate  of  the  monomeric  form. 

Pathways  of  glucose-C1*  utilization  in  eggs  of  Arbacia,  Mactra,  and  Chaetopterus. 
M.  E.  KRAHL,  A.  K.  KELTCH,  C.  P.  WALTERS  AND  G.  H.  A.  CLOWES. 

From  experiments  with  glucose-1-C14  (Gl),  glucose-2-C14  (G2),  and  glucose-6-C14  (G6), 
the  authors  reported  here  (1955)  that:  (a)  glucose  is  oxidized  in  Arbacia  eggs  principally  via 
the  TPN  shunt,  the  ratio  of  C14O2  from  G6  to  that  from  Gl  being  0.07  for  unfertilized  and  0.12 
for  fertilized  eggs ;  the  glycolytic  pathway  is  more  important  in  24  hour-old  swimming  em- 
bryos where  the  ratio  is  0.28;  (b)  C14  from  glucose  appears  in  nucleoprotein ;  (c)  dinitrocresol 
(DNC)  inhibits  glucose  oxidation  by  the  shunt, ^tending  to  divert  glucose-6-phosphate  into  the 
glycolytic  pathway;  DNC  also  inhibits  glucose-C14  conversion  to  nucleoprotein.  These  findings 
have  been  extended  for  Arbacia  and  found  to  apply  in  general  to  eggs  and  embryos  of  Mactra 
and  to  embryos  of  Chaetopterus.  Representative  total  c.p.m.  collected  after  2  hr.  incubation  of 
80  mg.  eggs  at  20°  in  4  ml.  sea  water  containing  glucose-C14  (0.0006  M ;  600,000  c.p.m.)  are  as 
follows.  For  Arbacia,  just  fertilized,  in  DNA  from  Gl,  200,  from  G6,  700;  in  RNA  from  Gl, 
200,  from  G6,  400.  For  24  hr.  embryos,  in  DNA,  from  Gl,  20,000,  from  G6,  18,000;  in  RNA, 
from  Gl,  12,000,  from  G6,  12,000.  For  Mactra  the  total  c.p.m.  into  respiratory  CO2  with  0 
and  0.0001  M  DNC  present  were,  respectively:  unfertilized,  from  Gl,  200,  and  300;  G2,  100 
and  500;  G6,  25  and  200;  just  fertilized,  Gl,  300  and  400;  G2,  200  and  1600;  G6,  200  and 
700;  24  hr.  embryos,  Gl,  16,000  and  22,000;  G2,  7000  and  13,000;  G6,  4000  and  11,000.  The 
total  c.p.m.  into  nucleoprotein  of  24  hr.  embryos  with  0  and  0.0001  M  DNC  were,  respectively: 
from  Gl,  27,000  and  4900;  G2,  33,000  and  5900,  G6,  33,000  and  6600.  For  Chaetopterus  the 
total  c.p.m.  into  respiratory  CO..  with  0  and  0.0001  M  DNC  were,  respectively:  for  24  hr.  em- 
bryos, from  Gl,  5500  and  5000;  G2,  1500  and  6500;  G6,  800  and  3600.  The  total  c.p.m.  into 
nucleoprotein  of  24  hr.  embryos  with  0  and  0.0001  M  DNC  were,  respectively :  from  Gl,  13,000 
and  1400;  G2,  16,000  and  2100;  G6,  18,000  and  2000. 

Improved  fixing  and  staining  methods  for  cellular  structures  in  Ilyanassa  eggs. 
F.  E.  LEHMANN. 

1.  Fixation.     Previous  studies  had  shown  that  in  Amoeba  fibrous  structures  in  cytoplasm 
are  not  sufficiently  preserved  by  osmic  or  strongly  acidic  fixatives.     A  suitable  rapidly  pene- 
trating and  partly  dehydrating  fixative  was  found  in  a  mixture  of  acetone  and  formalin ;  good 
preservation  of  cytoplasmic  fibers  was  shown  by  the  electron  microscope.     For  Ilyanassa  two 
mixtures  were  tried.     A  mixture  of  80  ml.   of  20%   formaldehyde  and   16  ml.   of  acetone  is 
sufficient  to  fix   fibrillar   structures  in  the  cytoplasm    (cytoplasmic   reticulum   and  asters).     A 
slightly  acidified  fixative  (80  ml.  of  20%  formaldehyde,  16  ml.  of  acetone,  and  0.5  ml.  of  glacial 
acetic  acid)   gave  good  preservation  also  of  interphase  nuclei,  spindles  with  chromosomes,  and 
asters.     In  contrast  to  this,  OsO4  fixation  of  Ilyanassa  eggs  destroys  astral  structures,  which 
are  visible  in  living  eggs.     In  order  to  stabilize  structures  containing  nucleic  acids  and  to  pre- 
vent shrinkage  by  increasing  rigidity  of  cell  parts,  chromic  acid  was  added  to  the  fixative    (2 
ml.  of  10%  chromic  acid  for  every  5  ml.  of  fixative)   20  minutes  after  eggs  had  been  placed 
into  the  fixative.     After  5-20  minutes  of  exposure  to  this  combination,  eggs  were  rinsed  for 
1-2  minutes  in  distilled  water ;  they  were  then  ready  for  staining  and  mounting. 

2.  Staining  by  bromphenol  blue-sublimate   (after  Mazia,   Brewer  and  Alfert)    showed  cell 
boundaries,   cytoplasmic   reticulum,    spindles   and   astral   fibers   clearly.      For   staining   of   total 
mounts  of  Ilyanassa  eggs  the  original  mixture  (0.05  mg.  bromphenol  blue,  5  g.  sublimate  and 
50  ml.  tLO)  was  diluted  20  times.     Eggs  were  stained  10-20  minutes,  transferred  to  0.5%  acetic 
acid  for  20  minutes,  and  then  to  tap  water  for  5-20  minutes.     They  were  then  passed  through 
alcohol,    cleared   in   xylene    and   mounted    in    Canada    balsam.     Besides    the    blue    cytoplasmic 


308          PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

structures  the  yolk  granules  stained  partly  purple,  violet  and  green.     The  shape  of  the  egg  is 
very  little  distorted  as  compared  with  the  living  stages. 

Factors  inhibiting  metamorphosis  in  tadpoles  of  the  tunicate  Amaroecium  constel- 
latum.     WILLIAM  F.  LYNCH. 

Tadpoles  were  placed  in  sea  water  solutions  of  0.001  M  potassium  cyanide,  0.001  M  sodium 
azide,  1.5%  urethane  (all  at  a  pH  of  7.8-8.0)  and  in  sea  water  acidified  to  pH  values  of  4.8, 
5.2  and  5.7.  The  number  undergoing  metamorphosis  was  determined:  (1)  after  the  last  con- 
trol had  begun  metamorphosis  (30-70  min.)  in  eight  observations,  (2)  at  7  hours,  and  (3)  at  21 
hrs.  in  three  experiments.  (1)  After  the  last  larva  in  the  controls  had  begun  metamorphosis 
the  inhibition  was:  urethane,  100%;  azide,  98%;  cyanide,  97%;  acidified  sea  water  (pH  = 
4.8-5.2),  94%.  Inhibition  was  completely  reversible  on  removal  of  the  tadpoles  to  sea  water. 
(2)  By  7  hours  none  of  the  tadpoles  in  urethane  or  azide  (other  experiments)  and  only  2.8% 
of  those  in  cyanide  and  8.3%  of  those  in  acidified  sea  water  (pH  — 5.7)  had  begun  metamor- 
phosis. Inhibition  was  reversible.  (3)  In  another  set  of  experiments  inhibition  was  100%  in 
urethane  and  in  acidified  sea  water  (pH  =  5.2)  at  21  hrs.  Larvae  removed  from  either  of  these 
solutions  showed  a  tendency  for  persistent  swimming.  Those  removed  from  urethane  changed 
their  axes  from  45  to  90°  by  24  hrs.  Those  removed  from  acidified  sea  water  only  partially 
metamorphosed  and  then  died.  Inhibition  was  incomplete  at  21  hrs.  in  azide  and  in  cyanide. 
LTnmetamorphosed  tadpoles  removed  at  21  hrs.  from  azide  developed  normally  and  those  from 
cyanide  at  a  somewhat  retarded  rate. 

Tadpoles  placed  in  sea  water  solutions  of  0.01%  2,4-dinitrophenol  and  1.5%  thiourea 
(both  at  a  pH  of  7.8-8.0)  began  metamorphosis  and  tail  resorption  as  quickly  as  the  controls, 
but  subsequent  stages  were  inhibited.  The  larvae  contracted,  became  ovoid  and  the  tunic  im- 
bibed much  sea  water ;  none  of  them  changed  their  axes  nor  elongated  by  24  hrs.  Inhibition 
was  completely  reversible  when  tadpoles  were  removed  from  dinitrophenol  at  six  hrs.  or  from 
thiourea  at  1.5  hrs.  Tadpoles  in  0.15%  chloral  hydrate  (pH  =  8.0)  showed  17%  inhibition 
when  the  last  larva  of  the  controls  had  begun  metamorphosis  but  they  changed  their  axes  from 
10  to  90°  by  24  hours ;  subsequent  development  was  somewhat  retarded. 

A  North  American  record  of  Rhopalura  sp.  (Orthonectida:  Mesosoa),  a  parasite  of 
the  nemertean  Amphiporus  ochraccns  (Vcrrill).     NORMAN  A.  MEINKOTH. 

On  July  3,  1956,  a  specimen  of  the  armed  nemertean  Amphiporus  ochraccus  (Verrill),  taken 
from  among  growths  of  the  compound  ascidian  Amaroucium  pellucldum  dredged  off  Nobska 
Point  near  Woods  Hole,  Mass.,  was  found  harboring  a  strange  parasite.  These  minute,  cili- 
ated, cylindrical,  annulated  organisms  were  noted  both  within  the  body  of  the  host  and  swim- 
ming beside  it  on  the  slide. 

The  parasite  measured  126  ^  by  18  M.  Its  body  consisted  of  an  outer  layer  surrounding  an 
inner  core  of  large  cells,  19  to  23  in  number,  disposed  in  a  single  row  anteriorly  and  double 
posteriorly,  extending  the  length  of  the  organism.  Constrictions  of  the  outer  layer  divided 
the  body  into  a  series  of  annuli,  all  of  which  with  certain  exceptions  contained  numerous  re- 
fringent  granules.  Three  annuli  constituting  an  anterior  cone  were  followed  by  two  prominent 
annuli,  each  delimited  by  deep  furrows.  Behind  these  followed  a  series  of  six  large  annuli,  each 
succeeded  by  a  smaller  granule-free  annulus.  The  remaining  posterior  part  was  rounded,  and 
somewhat  longer  than  wide.  All  annuli  bore  cilia.  Those  on  the  first  annulus  were  held 
rather  rigidly  anteriad  as  a  tuft,  while  those  on  the  posterior  part,  longer  than  the  other  body 
cilia,  did  little  vibrating  and  trailed  behind.  The  remainder  of  the  body  cilia,  of  about  equal 
length  and  distribution,  beat  actively.  All  individuals  encountered  were  essentially  identical 
in  the  above  characteristics.  To  date,  of  171  more  A.  ocliracciis  examined,  none  has  been  found 
harboring  the  parasite. 

Consultation  with  the  literature  indicates  that  these  animals  are  females  of  a  species  of 
the  genus  Rhopalnra,  order  Orthonectida,  phylum  Mesozoa.  The  species  is  similar  to  if  not 
identical  with  R.  metchnikovi  Caullery  and  Mesnil  1901  or  R.  linci  (Giard)  1879.  This  is  be- 
lieved to  be  the  first  reported  occurrence  of  an  orthonectid  from  North  America. 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY          309 

Studies  on  accelerator  and  retarding  factors  of  one  species  on  the  developing  ova 
of  an  unrelated  form.1  VALY  MENKIN,  GABRIEL  MENKIN  AND  LOUISE 
MENKIN. 

Recent  observations  have  indicated  the  presence  of  two  distinct  factors  in  the  aqueous 
homogenate  of  the  ovaries  of  Arbacia  punctulata.  These  two  factors  conceivably  play  a  sig- 
nificant role  in  the  regulation  of  cell  division.  One  of  them  markedly  accelerates  the  rate  of 
cleavage  of  sea  urchin  ova ;  the  other  displays  considerable  retarding  activity  of  cleavage  de- 
velopment. Since  from  a  single  homogenate  two  opposed  factors  are  obtained  by  methods 
previously  described,  it  is  conceivable  that  the  rate  of  cleavage  of  the  fertilized  ova  is  a  re- 
sultant of  these  two  opposed  effects. 

In  a  new  series,  it  has  now  been  shown  in  10  distinct  experiments  that  the  accelerator  factor 
derived  from  the  homogenate  of  sea  urchin  ovaries  speeds  up  the  cleavage  of  the  ova  of  a 
mollusk,  Spisula  solidissima.  The  accelerator  factor  is  obtained  by  centrifuging  the  aqueous 
homogenate  at  about  10,000  r.p.m.  in  a  Servall  angle  centrifuge  for  about  one  hour.  The  su- 
pernatant is  then  dialyzed  against  distilled  water  in  a  refrigerator  for  several  days.  The 
diffusate  contains  the  accelerating  factor.  Its  activity  on  the  ova  of  Spisula  solidissima  has 
yielded  the  following  results :  the  average  number  of  ova  is  about  150  per  cent  greater  ir 
the  experimental  than  that  of  the  controls  in  the  two-blastomeric  stage.  In  the  succeeding 
segmentation  the  incidence  averages  100  per  cent  in  the  experimental  over  that  of  the  controls. 
In  a  series  of  13  experiments  with  the  retarding  factor  of  Arbacia  ovaries  on  Spisula  ova,  the 
average  retarding  effect  on  the  two-cell  stage  is  60.9%  and  38.7%  in  the  succeeding  cleavage 
stage.  Thus  the  factors  obtained  from  an  echinoderm  are  equally  effective  on  the  developing 
eggs  of  a  mollusk,  indicating  that  basic  substances  are  evidently  involved  in  the  mechanism. 

Further  studies  on  some  factors  concerned  in  the  regulation  of  cell  division.1  VALY 
MENKIN,  GABRIEL  MENKIN  AND  WILLIAM  ROGERS. 

Since  there  is  a  definite  suggestion  that  the  accelerator  factor  seems  to  be  a  nucleotide,  some 
studies  were  undertaken  to  determine  the  effect  of  a  few  derivatives  of  nucleic  acid  on  the 
cleavage  rate  of  Arbacia  ova.  The  pyrimidine  base  uracil  appears  in  preliminary  experiments 
to  cause  an  initial  acceleration  in  the  incidence  of  the  first  segmentation,  the  figures  being  63% 
in  the  experimental  and  26%  in  the  controls.  In  the  succeeding  cleavage  the  experimentals  re- 
veal an  incidence  of  75%  as  against  58%  in  the  controls.  This  base  is  to  be  studied  further. 
Cytosine  seems  to  be  incapable  of  altering  the  cleavage  rate.  For  the  pyrimidine  bases  doses  of 
757  per  ml.  were  employed.  Adenosine  triphosphate  (ATP),  757  per  ml.  on  Arbacia  punctu- 
lata ova  yielded,  in  a  series  of  six  experiments,  the  following  results :  either  a  retardation  or  an 
acceleration  in  the  cleavage  rate.  In  the  first  segmentation  ATP  induced  an  average  retardation 
of  13.1%,  whereas  in  the  succeeding  cleavages  an  accelerating  effect  was  noted  amounting  to 
84.4%.  The  effects  with  ATP  are  therefore  of  a  different  order  than  those  obtained  with 
the  accelerator  cleavage  factor  fractionated  from  sea  urchin  ovaries. 

The  effects  of  the  accelerator  and  the  retarding  factors  derived  from  Arbacia  ovaries,  on  the 
sperms  of  the  clam,  Spisula  solidissima,  were  studied.  Sperms  were  exposed  for  varying 
intervals  (33  minutes  to  4  hours)  to  the  two  factors,  and  to  sea  water  as  control.  Ova  of 
Spisula  were  then  added,  and  the  incidence  of  cleavage  subsequently  determined.  The  results 
of  9  experiments  indicated  44.5%  cleavage  in  the  controls;  34.7%  in  the  dish  containing  the 
accelerator  cleavage  factor ;  and  9.8%  in  the  one  with  the  retarding  factor.  Prolonged  ex- 
posure to  the  accelerator  factor  seems  to  injure  the  sperms.  This  detrimental  effect  is  very 
pronounced  with  the  retarding  cleavage  factor.  However,  addition  of  the  retarding  factor  fol- 
lowing fertilization  induced  likewise  retarding  cleavage  effects,  indicating  also  a  probable  effect 
on  the  eggs  themselves. 

1  Aided  by  grants  from  the  U.  S.  Public  Health  Service,  Sigma  Xi,  and  Dr.  A.  Wander, 
S.A.,  Berne,  Switzerland. 


310          PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

Effect   of   electric   current   on   the   contraction   of   the   chloroplasts   of  Spirogyra. 

W.  J.  V.  OSTERHOUT. 

Experiments  were  made  on  Spirogyra  cells  which  contained  single  spiral  chloroplasts. 
These  cells  were  placed  in  0.001  M  NaCl  solution  which  brought  about  no  visible  change  in 
the  chloroplasts.  When  a  small  amount  of  direct  or  alternating  current  (less  than  0.2  milli- 
ampere )  was  passed  through  the  solution  each  spiral  chloroplast  in  less  than  ten  minutes  became 
detached,  straightened,  and  contracted  until  it  formed  a  small  rounded  mass.  This  contraction 
continued  even  after  the  current  was  turned  off.  A  cell  whose  chloroplast  was  not  visibly  af- 
fected by  the  current  sometimes  showed  signs  of  contraction  after  the  current  was  turned  off. 
This  was  not  reversible.  If  the  direct  current  was  reversed  there  was  no  additional  effect. 

In  order  to  make  certain  that  these  effects  were  not  due  to  the  heat  produced  in  the  circuit 
the  following  was  done.  A  drop  of  gelatin  whose  melting  point  was  35°  C.  was  put  in  the  path 
of  the  current  along  with  Spirogyra  cells.  Since  the  gelatin  did  not  melt  the  temperature  was 
less  than  35°  C.  If  a  drop  of  gelatin  was  placed  in  a  stender  dish  containing  some  water  at 
35°  C.  it  melted.  Cells  of  Spirogyra  exposed  to  a  temperature  at  40°  C.  for  one  half  hour  in 
a  stender  dish  of  water  showed  no  signs  of  contraction. 

When  the  normal  cells  of  Spirogyra  were  centrifuged  the  spiral  chloroplasts  became  de- 
tached, straightened,  and  contracted.  If  the  cells  were  centrifuged  until  only  slight  shortening 
of  the  chloroplasts  was  evident  and  removed  to  a  stender  dish  of  water  no  further  contraction 
occurred. 

Retinal  action  potentials  in  the  e\e  of  the  scallop.     FLOYD  RATLIFF. 

The  scallop,  Pcctcn  irradians,  has  approximately  one  hundred  eyes,  each  of  which  contains 
two  retinae.  Hartline  found  that  nerve  fibers  from  the  proximal  retina  of  an  excised  eye  re- 
spond to  the  onset  and  continuance  of  illumination,  while  those  from  the  distal  retina  respond 
only  to  the  cessation  of  illumination.  In  the  present  study  retinal  action  potentials  were  meas- 
ured by  placing  one  electrode  on  the  posterior  pole  of  an  eye  and  inserting  another  into  the  eye 
near  the  margin  of  the  lens.  Upon  stimulation  by  light  the  anterior  portion  of  the  eye  be- 
comes negative  with  respect  to  the  posterior  pole.  This  retinal  action  potential  passes  quickly 
through  a  maximum  (approximately  one  millivolt  when  the  eye  is  fully  dark-adapted)  and 
then  subsides  toward  the  resting  level.  Upon  cessation  of  illumination  the  potential  drops  more 
rapidly  toward  the  resting  level.  An  "off"  component,  such  as  that  found  in  many  vertebrate 
and  invertebrate  eyes  which  respond  to  the  cessation  of  illumination,  is  lacking.  The  absence 
of  an  "off"  component  is  not  due  to  inactivity  of  the  distal  retina,  however.  Simultaneous 
observations  of  retinal  potentials  in  the  eye  and  action  potentials  in  the  optic  nerve  showed  that, 
at  intensities  of  illumination  great  enough  to  produce  a  measurable  retinal  response,  there  was 
always  a  vigorous  "off"  discharge  in  the  optic  nerve  when  the  light  was  turned  off. 

The  course  of  the  recovery  of  maximal  sensitivity  of  the  eye  in  the  dark,  following  pro- 
longed exposure  to  light,  was  determined  by  measuring  the  amplitudes  of  retinal  potentials 
produced  by  stimuli  of  fixed  intensity  and  one  second  duration  spaced  five  minutes  apart.  Re- 
covery is  complete  within  about  forty  minutes. 

Strain  differences  in  inability  following  conjugation  ivitliin  variety  9   of   Tctra- 
hymcna  pyrifonnis.1     CHARLES  RAY,  JR.  AND  ALFRED  M.  ELLIOTT. 

Per  cent  of  viable  clones  following  conjugation  of  strains  of  variety  9  of  T.  pyriformis  was 
studied  as  part  of  a  program  concerning  strain  relationships  and  genetics  of  biochemical  differ- 
ences between  strains.  Strains  of  variety  9  are  known  only  from  collections  made  in  Panama 
or  Colombia.  Seven  strains  (TC.  105,  147,  156,  160,  258,  267  and  84)  representing  all  collection 
sites  and  all  five  mating  types  were  mixed  in  all  possible  combinations.  Tests  for  selfing  and 
for  intervarietal  mating  were  negative.  Two  hundred-twenty  pairs  were  isolated  singly  into 
proteose-peptone  from  conjugating  mixtures.  Length  of  refractory  period,  amount  of  pairing, 

1  This  investigation  was  supported  in  part  by  research  grants  from  the  National  Science 
Foundation  and  from  the  National  Institutes  of  Health  (PHS  G3588). 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY          311 

per  cent  of  pairs  giving  viable  clones,  and  stage  at  which  death  occurred  for  inviable  pairs,  were 
recorded. 

All  combinations  of  opposite  mating  types  showed  pairing.  The  refractory  period  of  most 
combinations  was  about  thirty-six  hours  (25°  C.),  although  one  paired  at  24  hours  and  three  at 
60  hours.  Per  cent  of  pairing  in  various  mixtures  was  poor  to  good.  No  relation  was  observed 
between  length  of  refractory  period,  amount  of  pairing,  and  per  cent  of  exconjugant  clones.  Of 
all  crosses  made,  about  70%  of  the  pairs  died  before  fission  of  exconjugants,  about  20%  died 
after  several  fissions,  and  about  10%  gave  viable  clones.  Yield  of  viable  exconjugant  clones 
varied  for  different  strains:  e.g.,  crosses  involving  TC267  gave  2%  viable  exconjugants;  those 
with  TC160  gave  20%.  Three  mating  type  IV  strains  gave  20,  13  and  2  per  cent  viable  ex- 
conjugants. TC156  (III)  and  TC160  (IV)  were  isolated  from  the  same  original  collection; 
they  produced  from  all  crosses  5%  and  20%  viable  exconjugants,  respectively,  but  when  crossed 
with  each  other  they  gave  no  viable  exconjugants. 

Observation  of  cytological  events  associated  with  this  widespread  lethality  and  behavior 
of  viable  clones  with  continued  cross  and  inbreeding  is  in  progress. 

Carbohydrates  metabolised  b\>  ccstodc  parasites  of  dogfish^     CLARK  P.  READ. 

CalUobothriuin  verticillatum  (Tetraphyllidea)  and  Lacistorhynchus  tennis  ( Trypano- 
rhyncha)  were  removed  from  naturally-infected  Mustelus  canis.  The  worms  were  washed  for 
2  hours  in  several  changes  of  filtered  40%  sea  water  at  room  temperature  (20-22°  C.).  Groups 
of  ten  worms  (70  to  90  mg.  of  wet  tissue)  were  transferred  to  Warburg  flasks  containing  40% 
sea  water-bicarbonate  (pH  7.2)  and  equilibrated  for  15  minutes  under  95%  N,-5%  CO2  in  the 
20°  C.  bath.  Flasks  were  incubated  for  60  minutes  to  determine  the  endogenous  rate  of  acid 
production.  Substrates  were  than  added  to  make  a  final  concentration  of  0.01  M  and  the  flasks 
incubated  for  an  additional  60  minutes.  Throughout  each  experiment  readings  were  made  at 
20-minute  intervals.  Addition  of  acid  to  initial  control  flasks  and  to  the  experimental  flasks 
showed  that  anaerobic  metabolic  gas  is  not  produced  by  either  of  these  cestode  species.  As 
indicated  by  an  increase  in  acid  production  glucose  and  galactose  are  utilized  by  both  tape- 
worms. Fructose,  mannose,  xylose,  maltose,  trehalose,  sucrose,  lactose,  and  raffinose  are  not 
metabolized.  Non-utilization  of  the  latter  substrates  was  confirmed  by  analyses  of  the  media 
before  and  after  a  three-hour  incubation,  using  Roe's  anthrone  procedure  for  the  sugar  deter- 
minations. The  rate  of  utilization  of  glucose  and  galactose  is  independent  of  the  concentration 
in  the  range  0.001  to  0.02  M. 

The  extremely  limited  spectrum  of  carbohydrates  metabolized  by  CaUiobothrinm  and 
Lacistorhynchus  resembles  that  of  the  cyclophyllidean  cestodes,  Hymenolepis  diininiita,  Oocho- 
ristica  syminetrica,  Raillictina  cesticillus,  and  Moniczia  e.rpansa.  Additional  experiments  are 
in  progress  to  determine  whether  the  dogfish  cestodes  resemble  the  cyclophyllideans  in  re- 
quiring the  inclusion  of  carbohydrate  in  the  host  diet  for  normal  growth  and  reproduction. 

Recovery  from  .r-irradiation  effects  at  the  cellular  level.     ROBERTS  RUGH   AND 
JOAN  WOLFF. 

It  is  more  difficult  to  conceive  of  repair  of  structural  damage  consequent  to  x-irradiation 
than  to  a  re-synthesis  by  the  living  system  of  molecules  rendered  unusable.  Recovery  of  func- 
tion at  the  cellular  level  is  conceivable  to  a  degree  inversely  related  to  time. 

Henshaw  originally  described  the  recovery  of  Arbacia  gametes  following  x-irradiation. 
The  present  experiments  extend  his  work  to  determine  the  degree  of  recovery  following  different 
levels  of  exposure  and  different  time  intervals  between  x-irradiation  and  the  fertilization  of 
the  Arbacia  egg. 

Eggs  of  Arbacia  were  washed  and  concentrated  into  a  40-cc.  volume  in  filtered  sea  water, 
placed  in  a  covered  plastic  dish,  and  x-irradiated  at  2160  r/min.  to  from  36,000  r  to  86,000  r. 
At  intervals  up  to  three  hours  after  irradiation  eggs  were  fertilized  and  the  degree  of  cleavage 
determined  at  1.5  and  2.5  hours  after  fertilization.  To  do  this,  eggs  were  fixed  in  10%  formalin 
with  a  trace  of  acetic  acid,  in  sea  water.  Counts  were  made  of  200  eggs  from  each  sample. 

1  These  studies  were  aided  by  a  contract  between  the  Office  of  Naval  Research,  Department 
of  the  Navy,  and  Johns  Hopkins  University,  NR  119-353. 


312          PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

Cleavage  was  delayed  in  all  eggs  x-irradiated,  the  greater  the  exposure  the  greater  the  de- 
lay. However,  eggs  which  were  irradiated  but  not  fertilized  for  one,  two  or  three  hours 
showed  increasing  recovery  value  (cleavage  percentage)  with  increasing  time.  At  the  higher 
levels  of  exposure,  which  allowed  no  eggs  to  cleave,  some  eggs  developed  when  the  time  be- 
tween irradiation  and  fertilization  was  extended. 

The  above  facts  indicate  that  the  type  of  damage  inflicted  upon  the  single-celled  Arbacia  egg 
by  x-irradiation  is  reparable  to  a  degree. 

The  effect  of  substrate  on  the  length  of  planktonic  existence  in  Nassarins  obsoleta. 
RUDOLF  S.  ScHELTEMA.1 

Veliger  larvae  of  the  mud  snail  Nassarins  obsoleta  (Say)  were  successfully  reared  through 
metamorphosis.  In  culture  the  larvae  grew  rapidly  in  15-liter  crocks  with  Nitzschia  clostcrinm 
(200,000/ml.)  as  a  source  of  food.  The  length  of  pelagic  existence  extended  from  20  to  approxi- 
mately 30  days.  Planktonic  life  ended  with  the  loss  of  the  velum.  Contrary  to  the  accounts 
generally  given  for  gastropod  larvae,  the  velum  of  Nassarins  obsoleta  was  cast  off  rather 
than  resorbed.  Shell  height  of  the  larvae  at  the  time  of  setting  varied  between  0.5  and  1.1  mm. ; 
the  average  was  between  0.7  to  0.8  mm.  Within  a  few  days  after  metamorphosis,  the  shell 
darkened  and  become  opaque.  In  a  series  of  experiments,  40  cultures,  each  containing  20 
Nassarius  veligers,  were  maintained  through  metamorphosis  in  the  laboratory.  A  substrate 
of  sand  and  organic  material  (<140M,  sieved  from  natural  bottom  sediment  of  Barnstable 
Harbor)  was  added  to  one-half  of  the  cultures.  To  the  remainder  of  the  cultures  no  sub- 
strate was  added.  In  cultures  of  20-day  old  larvae,  17%  of  the  organisms  with  a  substrate 
metamorphosed  within  24  hours,  while  in  those  without  substrate  only  3%  set  during  this  pe- 
riod. Similarly  in  cultures  containing  31 -day  old  larvae,  90%  of  the  larvae  with  a  natural 
substrate  metamorphosed,  while  only  19%  of  the  larvae  without  substrate  completed  meta- 
morphosis. Considering  the  average  of  all  cultures  between  20  and  31  days,  in  those  with  sub- 
strate added,  76%  of  the  veligers  set  within  24  hours  while  in  cultures  without  substrate  only 
24%  completed  metamorphosis  within  this  time.  These  laboratory  observations  have  con- 
siderable ecological  significance  since  they  may  help  to  explain  the  distribution  of  newly 
metamorphosed  Nassarius  in  the  natural  environment. 

Electrophoretic  separation  of  chroniatophorotropic  principles  of  the  fiddler  crab, 
Uca.     G.  C.  STEPHENS,  F.  FRIEDL  AND  B.  GUTTMAN. 

The  following  observations  were  carried  out  in  order  to  attempt  to  isolate  and  characterize 
the  chromatophorotropic  principles  present  in  the  sinus  gland  of  the  fiddler  crab,  Uca  puc/ilator. 
Sinus  glands  were  dissected  and  isolated  in  sea  water  as  rapidly  as  possible  until  twelve  glands 
were  obtained.  These  were  then  placed  on  a  strip  of  filter  paper  one-half  inch  wide  and  eight- 
een inches  long  which  had  been  moistened  with  M/15  phosphate  buffer  at  pH  7.0.  The  glands 
were  then  crushed  and  exposed  to  450  volts  DC  at  5  to  8  ma.  for  12  to  18  hours.  On  a  separate 
strip  in  the  same  apparatus  a  sample  of  serum  albumen  was  run  to  serve  as  a  marker. 

The  results  of  this  procedure  were  observed  as  follows.  The  strip  of  filter  paper  on  which 
the  glands  had  been  placed  was  divided  into  ten  equal  units,  five  on  each  side  of  the  point  at 
which  the  glands  had  been  crushed.  Each  unit  was  extracted  with  0.5  cc.  of  sea  water  and  0.05 
cc.  of  the  extract  was  injected  into  each  of  five  male  fiddler  crabs  whose  eyestalks  had  been  re- 
moved at  least  twelve  hours  before  use.  The  stages  of  the  black,  white,  red,  and  yellow  chro- 
matophores  of  these  assay  animals  were  estimated  before  injection  and  observed  again  20,  40, 
60,  and  120  minutes  after  injection  of  the  test  extract.  A  quite  comparable  assay  procedure 
was  used  to  study  chromatophore  concentrating  activity  of  regions  of  the  strip.  In  this  case, 
the  chromatophores  (black,  red,  and  yellow)  of  the  assay  animals  to  be  used  were  dispersed  by 
injection  of  a  sinus  gland  extract  one  to  two  hours  before  injection  of  the  filter  paper  extract. 

By  this  procedure  we  were  able  to  distinguish  three  distinct  peaks  of  black  dispersing  ac- 
tivity, one  dubious  area  of  black  concentrating  activity,  and  at  least  one  peak  of  dispersing  ac- 

1  This  work  was  done  with  the  assistance  of  a  summer  fellowship  from  the  Woods  Hole 
Oceanographic  Institution. 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY          313 

tivity  and  one  peak  of  concentrating  activity  for  the  red  and  yellow  chromatophores.  At  least 
two  peaks  of  white  concentrating  activity  were  discernible.  Each  of  these  peaks  mentioned  can 
be  stated  to  be  distinct  from  any  of  the  others  by  comparing  the  variation  obtained  in  our  several 
observations. 

The  rate  of  disappearance  of  the  melanophore-dispersing  hormone  from  the  blood  of 
the  fiddler  crab,  Uca.     G.  C.  STEPHENS,  A.  STRICKHOLM  AND  F.  FRIEDL. 

The  rate  of  disappearance  of  the  melanophore  hormone  in  Uca  pugilator  was  studied  by 
the  following  technique.  Assay  animals  were  prepared  by  removing  the  eyestalks  of  male 
fiddler  crabs  at  least  twelve  hours  before  they  were  to  be  used.  Within  this  time  the  melano- 
phores  had  become  punctate  so  that  the  dispersing  activity  of  an  extract  could  be  readily 
ascertained  by  observing  the  response  induced  on  injection  of  the  material  concerned.  The 
melanophore-dispersing  hormone  to  be  assayed  was  prepared  by  dissecting  sinus  glands  from 
normal  donor  animals  and  grinding  them  to  prepare  a  sea  water  extract. 

An  initial  group  of  twenty-five  to  thirty  animals  was  injected  with  0.05  cc.  of  sea  water 
containing  the  extract  from  one-half  a  sinus  gland.  One  group  of  five  of  these  animals  was 
followed  to  ascertain  the  effect  of  this  full  strength  injection.  The  remaining  animals  were  di- 
vided into  groups  of  five  each.  At  suitable  intervals  after  the  initial  injection  (5  minutes,  15 
minutes,  30  minutes,  60  minutes)  0.05  cc.  of  blood  was  withdrawn  from  each  member  of  a  group 
and  injected  into  another  group  of  five  assay  animals.  The  dispersing  effect  of  the  blood  could 
then  lie  followed  in  these  secondary  assay  animals.  As  a  control,  blood  from  uninjected  assay 
animals  was  withdrawn  and  injected  into  a  second  group  of  assay  animals;  no  dispersion  was 
obtained. 

These  experiments  indicated  that  the  dispersing  hormone  was  present  in  the  circulating 
blood  of  destalked  assay  animals  in  discernible  amounts  for  approximately  three  hours  after 
injection.  In  order  to  get  a  quantitative  estimate  of  the  amount  present,  readings  of  each  assay 
group  were  made  at  fifteen-minute  intervals  after  injection.  The  first  seven  estimates  of 
melanophore  dispersion  were  summed  to  give  a  measure  of  the  effect  of  the  extract  or  the 
blood  which  the  animals  had  received.  This  information  has  been  supplemented  by  a  dilution 
curve  measuring  the  effect  of  various  concentrations  of  sinus  gland  extract,  and  by  a  measure- 
ment of  typical  blood  volumes  of  the  crabs  (average  26.4%  of  body  weight).  This  additional 
information  permits  calculation  of  the  circulating  hormone  in  sinus  gland  units. 

Studies  on  activation  in  eggs  of  Urechis  caupo,  Nereis  limbata  and  Asterias  jorbesi. 
HOWARD  M.  TEMiN.1 

Activation  in  eggs  of  Urechis  caupo,  Nereis  limbata  and  Asterias  jorbesi  involves  a  series 
of  linked  depolymerizations  of  preformed  layers  or  membranes :  breaking  of  secondary  valence 
and  salt  bonds  in  the  cortex  and  disulfide  bonds  in  the  germinal  vesicle.  The  process  was 
studied  by  use  of  various  reagents  of  known  chemical  action. 

In  eggs  of  Urechis  the  vitelline-fertilization  membrane  is  an  outer  protein  layer,  soluble  in 
non-electrolytes,  and  an  inner  calcium-proteinate  layer.  Activation  involves  breaking  of  sec- 
ondary valence  bonds  in  the  egg  cortex  with  a  decrease  in  surface  area ;  breaking  of  secondary 
electrostatic  and  then  salt  bonds  in  the  sub-vitelline  membrane  layer  causing  membrane  eleva- 
tion and  release  of  a  compound  which  breaks  disulfide  bonds  in  the  germinal  vesicle. 

In  eggs  of  Nereis  the  vitelline-fertilization  membrane  is  an  outer  layer  soluble  in  alkaline 
thioglycolate  and  an  inner  layer  soluble  in  citrate.  The  un-ionized  jelly  is  secondarily  bonded 
to  the  cortical  gel.  In  solutions  containing  only  monovalent  ions  (NaCl)  or  of  agents  which 
disrupt  the  cortex  (sea  water,  pH  2.5  ;  urea;  sodium  lauryl  sulfate)  the  jelly  is  set  free  and  the 
egg  loses  its  depression.  At  a  pH  less  than  9  the  jelly  passes  through  the  outer  membrane 
and  then  releases  protons.  At  a  pH  of  10.5  the  jelly  does  not  pass  through  the  membrane 
(Costello).  Precipitation  of  the  jelly  to  give  a  viscous  mass  depends  on  complexing  with 
divalent  ions.  The  germinal  vesicle  is  soluble  in  alkaline  thioglycolate,  but  not  in  urea,  citrate, 
alkaline  NaCl  or  solutions  of  ions. 

1  National  Science  Foundation  Pre-doctoral  Fellow. 


314          PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

In  eggs  of  Asterias  the  easily  separable  vitelline  membrane  is  soluble  in  alkaline  thiogly- 
colate.  On  activation  granules  are  released  by  breaking  first  secondary  valence  and  then 
hydrogen  bonds.  These  granules  in  the  presence  of  ions  precipitate  on  the  vitelline  membrane 
to  give  the  fertilization  membrane,  which  is  also  soluble  in  alkaline  thioglycolate.  The 
fertilization  membrane  expands  in  a  divalent  ion-free  medium. 

Spermatozoa  in  the  oviducal  gland  of  the  smooth  dogfish,  Mustelus  canis.     Lois  E. 

TEWlNKEL. 

Fertilization  of  the  elasmobranch  egg,  known  to  be  internal,  was  thought  to  occur  in  the 
oviduct  anterior  to  the  oviducal  gland  until  Metten  reported  (1939)  that  in  Scyliorhinus 
canicula  the  oviducal  gland,  itself,  serves  as  a  seminal  receptacle.  Metten  demonstrates  the 
presence  of  spermatozoa  exclusively  within  the  shell-secreting  tubules  of  the  gland  and  con- 
siders that  sperm  are  swept  out,  together  with  shell  material,  as  the  egg  passes  through  this 
region. 

Oviducal  glands  of  Mustelus  canis  also  contain  sperm.  Six  mature  females  collected  be- 
tween June  28  and  July  11  fall  into  the  following  three  groups:  a)  two  were  post-partum  and 
pre-ovulatory ;  b)  one  had  ovulated  two  eggs;  c)  three  had  recently  completed  ovulation. 
Living  sperm  were  found  in  washings  of  the  oviducal  gland  in  all  specimens,  but  were  not 
present  in  washings  of  more  anterior  or  more  posterior  portions  of  the  oviduct.  Longitudinal 
sections  of  glands  from  each  of  the  three  groups  of  females  show  spermatozoa  singly,  in  small 
groups,  or  in  dense  clusters,  in  the  mouths  or  deep  within  the  lumina  of  tubules,  not  only  of  the 
shell-secreting  type,  but  also  in  more  caudal  "mucous"  tubules.  Occasional  clusters  or  single 
sperm  lie  near  the  lamellar  lining  of  the  oviducal  gland,  but  no  sperm  have  been  seen  in  al- 
bumen-secreting tubules  or  in  more  anterior  mucous  tubules. 

Unlike  the  oviparous  Scyliorhinus,  which  breeds  throughout  the  year,  the  viviparous 
Mustelus  presumably  mates  only  between  the  birth  of  pups  (late  April  to  early  June)  and  the 
onset  of  ovulation  (June  to  early  July).  One  would  expect,  therefore,  to  find  few  or  no  sperm 
in  oviducal  glands  after  gestation  is  advanced.  In  the  glands  studied,  even  after  ovulation  has 
ceased,  sperm  in  large  numbers  are  present,  especially  in  the  caudal  "mucous"  tubules,  but 
whether  this  is  the  case  in  later  pregnancy  cannot  be  answered  until  glands  from  such  females 
are  examined. 

Acetylcholine  and  frog  brain  oxygen  consumption.     ELBERT  TOKAY. 1 

Since  there  seems  relatively  little  known  about  the  influence  of  acetylcholine  (ACh)  on 
brain  metabolism,  a  study  of  the  effect  of  the  drug  on  frog  brain  oxygen  consumption  (standard 
Warburg  technique)  was  undertaken  for  the  purpose  of  correlating  the  findings  with  electro- 
encephalographic  results. 

Optic  lobes  and  cerebral  hemispheres  were  separated  (razor  blade  slicing)  from  freshly 
isolated  frog  (Rana  pipicns]  brains.  Each  Warburg  flask  contained  four  optic  lobes  or  cerebral 
hemispheres  (from  different  brains)  in  Ringer's.  Flasks  were  gassed  with  pure  oxygen  and 
equilibrated  at  30°  ±  0.5°  C.  At  10-minute  intervals,  readings  of  "normal"  oxygen  consump- 
tion were  taken  for  one  hour  and  then  for  two  additional  hours  after  tipping  in  side-arm  con- 
tents (acetylcholine  chloride  Merck  or  Ringer  solutions).  Each  ACh  flask  was  duplicated  by 
a  Ringer  flask,  both  containing  similar  parts  of  the  same  brains.  Oxygen  consumption  (mm.8 
per  g.  of  wet  tissue  weight)  was  calculated  and  the  Qo-  (mm.3  OL.  consumed  per  g.  per  hour) 
derived  from  the  slope  of  the  plotted  curve. 

The  "normal"  Qo-'s  are  in  agreement  with  those  of  previous  workers.  ACh  was  used  in 
concentrations  ranging  from  1CT3  to  10~9  g.  per  ml.,  in  steps  of  tenfold  dilution.  Current  re- 
sults indicate  that  high  concentrations  of  ACh  depress  oxygen  consumption,  1CT3  markedly  and 
1CT4  somewhat  less  markedly.  Lower  concentrations  (1CT5  and  below)  tend  to  increase  oxygen 
consumption  more  often  than  decrease  it.  More  definitive  conclusions  and  any  differential 
effects  on  parts  of  the  brain  must  await  further  experimentation  and  statistical  evaluation. 

1  This  investigation  was  supported  in  part  by  research  grant  B-918  from  the  National  In- 
stitute of  Neurological  Diseases  and  Blindness,  of  the  National  Institutes  of  Health,  Public 
Health  Service. 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY          315 

A  comparison  of  two  inhibiting  agents  in  Tubularia.     KEN  YON  S.  TWEEDELL. 

Regeneration  in  Tubularia  can  be  inhibited  with  either  a  culture  medium  nitrate  or  with 
adult  tissue  extracts.  Suspected  differences  in  the  active  components  were  investigated. 

An  effective  concentration  of  inhibitor  water  is  dependent  upon  the  tissue  source  and  the 
concentration.  Pieces  of  stems  only  showed  no  inhibition  while  amputated  hydranths  gave 
initial  inhibition  at  a  concentration  of  125.  Stems  plus  intact  hydranths  were  the  best  source ; 
the  minimum  effective  number  necessary  is  about  200  to  250  per  200  ml.  collected  for  18  to  24 
hours.  During  this  time  an  average  drop  in  pH  of  the  collecting  medium  of  0.5  was  noted. 
The  inhibitor  strength  is  reduced  when  it  is  collected  in  boiled  sea  water.  Refrigeration  for  24 
hours  at  7°  C.  or  boiling  will  destroy  it.  Adsorption  with  Norite  A  or  synthetic  resins  will 
completely  remove  its  activity  but  centrifugation  at  21,000  G  has  no  effect.  The  fresh  filtrate 
gives  a  positive  test  with  ninhydrin  and  the  Feulgen-Schiff  reagents.  The  regenerating  stems 
are  most  susceptible  to  inhibition  during  the  first  21  hours  (proximal  ridge  stage).  Beyond 
this,  little  effect  is  noted.  Aeration  of  the  regenerates  during  the  first  20  hours  improves  re- 
generation in  inhibitor  water. 

The  supernatant  from  tissue  breis  of  mature  hydranths  was  collected  by  centrifugation 
(21,000  G).  The  minimum  inhibitory  dose  was  between  20  and  25  hydranth  equivalents  wherein 
stems  required  longer  to  regenerate  (96  hours)  and  were  reduced  in  size.  Complete  inhibition 
occurred  between  hydranth  equivalents  of  35  to  50  (a  concentration  of  ^5  to  y\$  in  the  culture 
medium).  The  tissue  extract  is  highly  resistant  to  sterilization,  centrifugation  and  can  be  re- 
frigerated indefinitely.  The  supernatant  gives  positive  protein  tests  but  freshly  collected  or 
boiled  extract  will  readily  dialyze  and  completely  inhibit  regeneration.  Dialysis  of  the  extract 
against  running  sea  water  for  24  hours  with  subsequent  application  to  regeneration  stems 
show  a  total  loss  of  activity. 

Attempts  to  breed  an  x-ray  resistant  clone  of  Paramecium.     RALPH  WiCHTERMAN.1 

In  an  attempt  to  breed  radio-resistant  paramecia  by  selection  and  cultivation  of  survivors 
of  x-radiation,  clonal  cultures  of  Paramecium  multimicronucleatnm  were  irradiated  repeatedly 
at  intervals  extending  over  a  period  of  13  months.  For  irradiation,  200  specimens  were  placed 
in  each  of  4  Nylon  syringes  (2  cc.)  and,  in  one  irradiation  operation,  given  different  dosages 
by  the  removal  of  a  syringe  at  intervals  from  the  x-ray  generator.  Most  of  the  paramecia  were 
irradiated  with  100  to  250  kr  in  steps  of  50  kr  but  occasionally  higher  dosages  were  used. 
Immediately  after  all  irradiation  exposures,  survivors  of  the  different  dosages  were  placed  in 
test-tubes  of  lettuce  medium  containing  Acrobactcr  aerogenes  as  the  food  source.  Upon  re- 
gaining reproductive  ability,  progeny  of  survivors  were  then  harvested,  placed  again  in  the 
syringes  and  irradiated  as  before.  In  some  cases  the  next  dosage  was  increased.  As  an  ex- 
ample a  clonal  sample  which  received  an  earlier  dosage  of  100  kr  later  received  150  kr. 
After  13  successive  and  varied  irradiation  exposures  of  4  sets  of  syringes,  the  clonal  cultures 
of  paramecia  have  received  in  this  manner  a  cumulative  dosage  amounting  to  1800  kr.  Speci- 
mens are  presently  reproducing  although  at  a  slower  rate  of  division  than  the  unirradiated 
controls  even  after  one  year  following  the  last  irradiation.  In  addition  to  the  reduced  fission 
rate,  the  heavily  irradiated  clones  after  one  year  Contain  specimens  which  reveal  the  following: 
reduced  swimming  activity  and  altered  behavior,  reduced  size,  altered  body  shape  and  complete 
loss  of  all  micronuclei.  Unirradiated  controls  have  three  micronuclei. 

Instead  of  being  stream-line  as  in  the  controls,  the  paramecia  from  cumulatively  irradiated 
clones  are  more  ellipsoidal.  Fission  rate  of  well-fed  controls  occasionally  reaches  three  divisions 
per  day,  generally  not  less  than  two.  The  successively  irradiated  clones  that  have  received 
1800  kr  contain  specimens  which  rarely  reach  two  divisions  per  day  even  when  last  x-rayed  one 
year  earlier. 

Using  the  same  dosages,  results  indicate  that  specimens  from  the  successively  irradiated 
clones  are  more  radio-sensitive  than  those  irradiated  for  the  first  time. 

1  Part  of  a  project  aided  by  a  contract  between  the  Office  of  Naval  Research,  Department 
of  the  Navy,  and  Temple  University  (NR  135-263)  and  the  Committee  on  Research,  Temple 
University. 


316          PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

The  micromanipulation  of  Arbacia  eggs.     F.  J.  WIERCINSKI. 

Preliminary  to  a  series  of  experiments  with  Arbacia  punctulata  egg  cells  it  was  necessary 
to  develop  a  suitable  microinjection  technique.  Egg  and  sperm  suspensions  were  obtained  from 
Arbacia  by  the  electric  method  of  stimulation.  Fertilized  and  unfertilized  eggs  were  pricked 
with  a  fine  micropipette  in  a  thin  film  of  sea  water  suspended  over  a  moist  chamber.  The 
micropipette  was  mounted  in  the  upward  position.  With  this  technique  many  of  the  fertilized 
eggs  did  not  develop  to  the  two-cell  stage  because  of  injury  from  high  surface  tension  phenome- 
non along  the  edge  of  the  hanging  droplet.  A  depression  slide  marked  into  two-millimeter 
squares  containing  150  X  of  sea  water  for  50  to  80  eggs  was  found  to  be  adequate  for  normal 
development.  After  ten  to  fifteen  minutes  following  fertilization,  eggs  were  successfully  mi- 
croinjected  with  approximately  3  cubic  micra  per  egg  of  0.5%  gelatin  solution  and  showed 
normal  development.  The  micropipette  with  a  fine  shaflet  and  a  one-micron  opening  at  the  tip 
was  mounted  downward  so  that  the  injection  was  made  into  the  cell  as  it  rested  on  the  surface 
of  the  slide.  Dark  field  illumination  with  a  magnification  of  100  X  was  used  to  advantage  to  ob- 
serve the  position  of  the  tip  of  the  micropipette.  The  location  of  the  eggs  for  control  and 
experiment  was  noted  by  the  squares  on  the  slide.  In  late  June,  1956,  the  fertilization  percentage 
was  80.  The  jelly  coat  did  not  cling  to  the  micropipette.  In  the  middle  of  July,  the  jelly  coat 
of  the  fertilized  eggs  was  very  tacky  and  the  cells  clung  to  the  micropipette.  The  fertilization 
percentage  was  95. 

The  physiology  of  flic  heart  in  marine  fish.     CHARLES  G.  WILBER. 

For  several  years  a  variety  of  physiological  studies  have  been  made  on  the  heart  of  marine 
fish  available  in  the  Woods  Hole  area.  There  have  been  suggestions  in  the  literature  that 
cardiac  rate  in  fish  may  vary  with  size  as  in  mammals.  In  order  to  test  this,  data  from  numerous 
species  have  been  assembled.  There  is  strong  evidence  that  at  a  given  temperature  large  fish 
have  appreciably  slower  hearts  than  do  smaller.  For  example  here  are  a  few  average  values  in 
beats  per  minute  for  fish  arranged  in  order  of  decreasing  size :  Roccus,  20 ;  Opsanus,  40 : 
Prionotus,  50 ;  Tautogolabrus,  60 ;  Fundulus,  100.  These  are  values  taken  at  an  ambient  tem- 
perature of  22°  C.  The  toadfish,  Opsanus,  has  a  heart  which  is  very  refractory  to  many  drugs : 
fairly  large  doses  of  decamethonium,  atropine,  and  darstine  cause  no  changes  in  the  electrocardio- 
gram of  this  species.  Massive  doses  of  darstine  (80  mg.  intravenously)  cause  an  A-V  dis- 
sociation and  an  eventual  doubling  of  conduction  time  from  pacemaker  to  ventricle.  The 
latter  chamber  seems  to  be  more  sensitive  to  the  drug  than  is  the  pacemaker.  Decamethonium 
in  the  tautog  results  in  impure  auricular  flutter ;  conduction  time  thru  the  ventricular  muscle 
is  unchanged  but  there  is  a  significant  prolongation  of  the  refractory  period.  These  studies 
are  continuing. 

A   rapid  method  for  recognition  of  specimens  of  Littorina  littorea  injected  with 
treniatode  larvae.     CHARLES  H.  WILLEY. 

In  response  to  the  need  for  snails  infected  with  Cryptocotylc  lingua  for  experimental  pur- 
poses an  attempt  was  made  to  discover  some  external  feature  of  the  snail  which  would  indicate 
that  it  was  infected.  The  isolation  method  with  examination  of  the  water  for  emerged  cercariae 
is  too  time-consuming  and  the  snails  show  a  high  mortality  rate  following  long  isolation. 
Some  snails  even  though  infected  will  not  shed  cercariae  during  isolation  for  several  days  and 
the  isolation  method  fails  to  identify  immature  stages  of  infection. 

Observation  indicated  that  the  foot  of  an  infected  snail  (Littorina  littorea}  becomes  a  dark 
yellow  to  brown  in  color  in  contrast  to  the  whitish  foot  of  uninfected  specimens.  To  detect 
infected  individuals,  collections  of  snails  are  placed  in  sea  water  in  tall  glass  containers  such 
as  battery  jars  and  allowed  to  crawl  up  the  sides.  On  the  basis  of  the  color  of  the  foot  seen 
from  outside  the  glass,  specimens  can  readily  be  sorted  into  infected  and  uninfected  categories. 
In  five  collections  of  snails,  the  results  have  consistently  been  positive,  checked  by  crushing  and 
examining  all  the  snails,  both  brown-  and  white-footed.  Unless  the  specimens  had  a  brownish 
foot,  they  were  not  infected.  In  one  experiment  82  snails  were  assorted  into  two  groups,  60 
white  foot  and  22  with  brown  foot.  Crushing  and  examination  showed  no  infections  among 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY          317 

those  with  white  foot  and  21  of  the  brown-footed  specimens  were  infected.  Similar  results  were 
obtained  with  other  collections.  Most  of  the  infected  snails  harbored  Cryptocotyle  lingua  but 
two  other  species  were  encountered  in  the  infected  group.  Some  were  in  early  stages  of  in- 
festation which  would  not  have  been  detected  by  isolation  techniques. 

Induction    of   premature   cleavage   furrows   in    the    eggs    of   Arbacia    punctulata. 
ARTHUR  M.  ZIMMERMAN  x  AND  DOUGLAS  MARSLAND.2 

These  experiments  indicate  that  the  furrowing  reaction,  which  normally  is  not  scheduled 
to  occur  until  telophase,  can  be  induced  to  occur  much  earlier,  starting,  in  fact,  at  about  the  12th 
minute  (at  20°  C.)  following  insemination. 

The  induction  treatment  consists  of  pressure-centrifuging  the  fertilized  eggs  at  high  (8000- 
12,000  lbs./in.2)  pressure  and  at  high  (41,000  X  G)  force,  for  periods  ranging  up  to  5  minutes. 
The  temperature  in  all  the  experiments  was  kept  constant  at  20  ±  0.3°  C. 

The  premature  furrows  appear  2-4  minutes  subsequent  to  centrifugation,  always  at  right 
angles  to  the  centrifugal  axis.  Usually  the  furrows  impinge  from  the  equator  of  the  cell,  al- 
though sometimes  they  are  displaced  toward  the  centripetal  end.  Frequently  they  cut  completely 
through  the  cell  and  do  not  recede.  However,  premature  furrows,  induced  not  more  than  10 
minutes  prior  to  the  normal  time  of  furrowing,  usually  recede  as  soon  as  the  normal  furrows 
appear ;  and  the  normal  furrows  almost  always  come  in  at  right  angles  to  the  premature  ones. 
The  eggs  do  not  appear  to  be  damaged  appreciably,  since  the  treated  specimens  gave  rise  to 
apparently  normal  plutei. 

The  greatest  frequency  of  premature  furrowing,  which  in  many  experiments  involved 
virtually  100%  of  the  eggs,  was  observed  at  30-35  minutes  after  insemination.  At  12,000  Ibs./ 
in.2,  a  maximum  frequency  was  obtained  with  300  seconds  of  centrifugation,  and  there  was  a 
gradual  decline  in  frequency  with  lesser  durations  of  treatment.  At  lower  pressures  (8000 
and  10,000  lbs./in.2)  the  centrifugation  times  were  longer. 

Preliminary  observations  indicate  that  the  induction  of  premature  furrowing  may  be  re- 
lated to  the  rupturing  of  the  nuclear  membrane.  No  intact  nuclei  can  be  seen,  either  by  phase 
or  ordinary  microscopy,  in  the  pressure-centrifuged  eggs,  whereas  nuclei  can  be  seen  in  com- 
panion eggs  centrifuged  at  the  same  force  but  not  under  pressure.  Moreover,  pressure-centri- 
fuged premature  furrowing  eggs,  stained  by  Feulgen  (or  the  acetocarmine)  technique,  do  not 
show  intact  nuclei,  whereas  the  control  cells  do.  The  experimental  eggs  show  just  a  small 
clump  (sometimes  two  clumps)  of  densely  packed  Feulgen-positive  material,  lying  in  the  vicinity 
of  the  furrow  and  displaying  a  diameter  about  %  that  of  an  intact  nucleus.  It  is  suggested, 
therefore,  that  the  furrowing  may  be  induced  by  a  substance  or  substances  released  by  the 
breaking  of  the  nuclear  membrane. 

Pressure -centrifuge  studies  on  mast  cells.     ARTHUR  M.  ZIMMERMAN/  JACQUES 
PADAWER  3  AND  DOUGLAS  MARSLAND.2 

The  pressure-centrifuge  has  been  used  extensively  for  studying  sol-gel  equilibria  in  Amoeba, 
Elodea  and  various  marine  eggs,  but  not  in  somatic  mammalian  cells.  In  this  study,  mast  cells 
from  rat  peritoneal  fluid  were  centrifuged  under  varying  hydrostatic  pressures  at  41,000  X  G 
and  20°  C.  It  was  found  that  the  relative  gel  strength  of  these  cells  (expressed  as  the  logarithm 
of  the  centrifugation  time  required  to  effect  a  distinct  deformation  in  25%  of  the  cell  popula- 
tion) is  inversely  proportional  to  the  pressure.  This  relationship  is  similar  to  that  found  in 
all  other  cellular  types  that  have  been  studied.  Control  mast  cells,  centrifuged  at  41,000  X  G 
at  atmospheric  pressure  for  periods  up  to  8  minutes  (the  longest  centrifugation  used  in  this 

1  Fellow  of  the  Lalor  Foundation,  1956. 

2  Work  supported  by  the  National  Cancer  Institute,  Grant  C-807   (cont.) 

3  Post-doctoral    Research   Fellow,    American    Heart    Association.      Supported    in    part    by 
grants  from  the  Damon  Runyon  Memorial  Fund  for  Cancer  Research    (DRG  360)    and  from 
the  American  Heart  Association. 


318          PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

study),  were  not  measurably  deformed.  The  deformation  effected  by  the  centrifugation  was 
found  to  be  spontaneously  reversible  at  room  temperature  within  approximately  30  minutes 
following  return  to  atmospheric  pressure. 

Preliminary  experiments  show  that  mast  cells  obtained  from  22-week-old  rats  may  be 
appreciably  more  gelated  than  cells  similarly  obtained  from  5-week-old  animals.  This  result 
suggests  that  the  aberrant  morphology  of  mast  cells  encountered  in  old  rats  may  be  related 
to  a  progressively  increasing  gelational  state  of  these  cells  during  the  aging  process  of  the  ani- 
mal. Other  cellular  types  of  the  peritoneal  fluid  (macrophagic  elements  and  eosinophils)  were 
not  studied  quantitatively.  However,  these  cells  were  also  deformed  by  the  pressure-centrifu- 
gation.  In  fact,  they  seem  to  display  a  weaker  gel  structure  than  the  mast  elements. 

LALOR  FELLOWSHIP  REPORTS 

Sensory  and  motor  relationships  of  a  crustacean  central  ganglion.     MELVIN   J. 
COHEN. 

Input-output  relationships  of  the  supraoesophageal  ganglion  in  the  lobster  Homarus 
americanus  were  studied  by  stimulating  statocyst  afferents  and  recording  the  response  evoked 
in  occulomotor  nerve  fibers  leaving  the  ganglion.  Circulation  to  the  exposed  ganglion  must  be 
intact  and  the  exposed  portion  of  the  central  nervous  system  bathed  in  a  balanced  physiological 
solution  in  order  for  transmission  through  the  ganglion  to  occur. 

Movement  of  the  statocyst  sensory  hairs  evoked  a  response  in  occulomotor  fibers  which 
usually  did  not  participate  in  the  "spontaneous"  activity  of  this  nerve.  Cutting  both  cir- 
cumoesophageal  connectives  caused  a  burst  of  activity  in  the  motor  nerve  followed  by  a  gradual 
decrease  in  the  number  of  spontaneously  active  units  until  only  1-3  fibers  remained  firing  15 
minutes  after  cutting  the  connectives.  The  spontaneous  activity  in  these  remaining  units  was 
very  rhythmic  in  contrast  to  the  irregular  spacing  of  impulses  in  the  statocyst  afferent  neurons. 
Some  occulomotor  fibers  increased  in  frequency  up  to  40/sec.  when  ipsilateral  statocyst  hairs 
were  moved  laterally  toward  the  vertical,  and  decreased  to  1-2  impulses/sec,  when  the  same 
hairs  were  moved  medially  toward  the  horizontal.  Here  the  frequency  changes  in  the  motor 
fibers  seem  to  parallel  those  of  the  sensory  neurons.  Other  occulomotor  fibers  increased  in 
frequency  only  when  statocyst  hairs  were  moved  medially  toward  the  horizontal.  This  direction 
of  hair  movement  is  usually  associated  with  a  decrease  in  the  frequency  of  firing  in  statocyst 
neurons.  It  appears,  therefore,  that  a  decrease  in  the  level  of  spontaneous  firing  in  certain 
statocyst  afferents  can  serve  as  an  adequate  signal  to  the  central  nervous  system  and  evoke  a  rise 
in  frequency  in  specific  fibers  of  the  occulomotor  nerve. 

Invertebrate  metabolism  in  ritro  not  affected  by  estradiol.     DWAIN  D.  HAGERMAN. 

Estradiol-17 13  stimulates  the  oxidative  metabolism  of  human  endometrium  and  placenta 
in  -vitro.  The  stimulation  can  be  demonstrated  in  tissue  slices  or  cell-free  soluble  enzyme  prepa- 
rations, and  is  the  result  of  a  specific  activation  of  a  DPN-linked  isocitric  dehydrogenase.  In 
a  search  for  metabolic  effects  of  estrogens  in  other  species,  a  variety  of  invertebrate  tissues 
were  incubated  in  Warburg  vessels  in  the  presence  or  absence  of  estradiol  (4  X  10"°  moles  per 
liter).  The  incubation  medium  was  filtered  sea  water,  to  which  was  added  glucose  (11.1  milli- 
moles  per  liter)  or  potassium  pyruvate  (10  millimoles  per  liter)  in  some  experiments.  The  gas 
phase  was  air.  Tissues  were  incubated  at  24—25°  C.  for  four  hours.  Oxygen  consumption  was 
measured  manometrically  and  conventional  chemical  techniques  were  used  for  the  analysis  of 
glucose,  pyruvic  acid,  glycogen,  and  lactic  acid. 

No  effect  of  estradiol  on  the  rates  of  oxygen  consumption,  glycogen  utilization,  or  lactate 
production  was  found  in  the  ovaries  and  contained  eggs  of  Arbacia  punctulata,  Astcrias  forbesi, 
Mactra  solidissima,  Venus  mercenaria,  Busycon  canaliculatiim,  Carcinidcs  macnas,  Homarus 
americanus,  or  Liimilus  Polyphemus.  No  effect  of  estradiol  on  glucose  utilization  or  pyruvate 
utilization  was  found  in  the  ovaries  and  contained  eggs  of  Arbacia  punctulata,  Astcrias  forbesi, 
Mactra  solidissima,  or  Busycon  canaliculatum.  Moreover,  no  effect  of  estradiol  on  these  meta- 
bolic functions  was  found  in  the  whole  body  of  Microciona  prolifera,  the  ctenidia  or  testes  of 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY          319 

Loligo  pcalci,  the  gills  or  testes  of  Callincctcs  sapidus,  the  testes  of  Homarus  amcricamis,  or  the 
liver  of  Limulus  polyphcmns. 

Estrogens  have  been  reported  to  be  present  in  some  of  these  tissues.  Our  metabolic  experi- 
ments do  not  reveal  any  invertebrate  estradiol-enzyme  relationship  similar  to  that  found  in  man, 
and  it  is  concluded  that  the  estradiol-sensitive,  DPN-linked  isocitric  dehydrogenase  is  not  pres- 
ent in  invertebrate  tissues. 

Methods  for  investigating  the  location  of  the  photoperiodic  receptors  in  insects. 
A.  D.  LEES. 

Although  it  is  well  known  that  the  induction  of  diapause  in  many  insects  and  mites  is  con- 
trolled by  the  length  of  day,  the  site  of  absorption  of  the  photoperiodic  light  energy  has  not 
yet  been  identified.  In  this  connection  the  observation  by  Tanaka  that  the  larvae  of  the  oak 
silkworm  Antheraea  pernyi  still  respond  to  photoperiod  after  extirpation  of  the  ocelli  is  of 
considerable  interest.  Two  techniques  which  may  prove  of  use  in  the  identification  of  the  recep- 
tors are  being  tested  currently,  using  the  larvae  of  A.  pernyi  as  material.  (I)  Localized  il- 
lumination can  be  achieved  by  the  topical  application  of  a  transparent  cellulose  paint  containing 
a  blue-fluorescing  substance,  such  as  anthracene.  The  insects  are  then  exposed  for  part  of  the 
photoperiod  to  a  U.V.  source  with  maximum  emission  at  365  m/i.  The  feasibility  of  this 
method  rests  upon  the  fact  that  insects  in  general  exhibit  greatest  sensitivity  to  the  blue  region 
of  the  spectrum.  (II)  Using  low  incident  light  intensities  to  minimize  light  scattering,  it  may  be 
possible  to  "silhouette"  the  sensitive  areas  by  covering  them  with  an  opaque  black  paint.  Since 
all  five  larval  instars  of  A.  pernyi  are  light-sensitive,  the  treated  areas  must  be  re-covered 
after  each  moult. 

Contractility  of  glycerinated  Vorticellae.     LAURENCE  LEVINE. 

The  technique  of  glycerination  was  applied  to  various  species  of  Vorticellae  in  an  attempt 
to  elucidate  some  features  of  intracellular  environment  necessary  for  coiling  of  the  spasmoneme 
(condensed  myonemes). 

Three  species  of  Vorticellae,  campanula,  ncbulifcra  and  convallaria,  maintained  on  an  egg 
yolk  infusion  were  used.  Each  species  was  very  sensitive  to  glycerol  because  they  invariably 
coiled  tightly  when  immersed  at  0°  C.  even  in  concentrations  as  low  as  0.05  per  cent.  However, 
if  the  glycerol  was  4  mM  in  EDTA  (pH  7)  such  coiling  was  prevented.  The  glycerination 
procedure  adopted,  therefore,  was  immersion  of  animals  previously  washed  in  de-ionized  water 
in  20  or  50%  0°  C.  glycerol  and  4  mM  in  EDTA.  Vorticellae  glycerinated  according  to  this 
recipe  for  as  long  as  one  month  showed  excellent  preservation  of  the  spasmoneme  and  other 
cellular  features. 

Physiological  integrity  of  the  spasmoneme  was  also  retained.  The  coiling  so  character- 
istic of  the  living  spasmoneme  was  produced  by  the  addition  of  CaCL  in  concentrations  as  low 
as  0.3  mM  in  0.5  M  KC1.  Coiling  was  reversed  through  application  of  4  mM  EDTA  and 
preparations  were  cycled  repeatedly  before  contractility  was  lost.  Contractility  could  not  be 
restored  through  application  of  ATP  and  ions  known  to  contract  glycerinated  rabbit  psoas. 
Divalent  ions  such  as  magnesium  and  manganese  also  produced  coils  but  were  not  as  effective 
as  calcium. 

The  retention  of  contractility  was  dependent  upon  species,  concentration  of  glycerol  and 
extraction  time.  Stalks  of  Vorticella  convallaria  lost  contractility  after  short  extraction  in  20% 
glycerol,  whereas  coiling  ability  was  maintained  for  longer  periods  in  50%. 

These  observations  suggest  that  an  extractable  calcium-activated  factor  is  responsible  for 
spasmoneme  coiling. 

Regulation  of  arginine  biosynthesis  in  Escherichia  coli.     W.  K.  MAAS. 

Although  E.  coli  cells  are  able  to  synthesize  their  amino  acids  from  simple  nitrogen  and 
carbon  sources  such  as  ammonia  and  glucose,  when  an  amino  acid  is  supplied  in  the  culture 
medium,  the  bacteria  will  utilize  it  in  preference  to  synthesizing  their  own.  In  order  to  eluci- 
date this  regulatory  mechanism,  the  effect  of  externally  supplied  arginine  on  its  own  biosynthesis 


320         PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

was  studied.  Arginine  was  chosen  because  most  of  the  enzymes  involved  in  its  biosynthetic 
pathway  have  been  extracted  and  the  reactions  catalyzed  by  them  characterized.  It  was  found 
that  arginine  inhibits  the  synthesis  of  transcarbamylase,  the  enzyme  which  couples  ornithine  with 
carbamyl  phosphate  to  form  citrulline.  In  growing  cultures,  in  the  presence  of  10  micrograms/ 
ml.  of  arginine,  no  transcarbamylase  is  formed.  After  removal  of  arginine  the  enzyme  is  re- 
synthesized  rapidly.  Citrulline,  on  the  other  hand,  does  not  inhibit  synthesis  of  transcarbamyl- 
ase. Studies  are  in  progress  on  the  effect  of  arginine  on  other  enzymes  involved  in  arginine 
biosynthesis. 

The  transcarbamylase  system  also  offers  an  opportunity  for  studying  the  conditions  re- 
quired for  the  synthesis  of  a  constitutive  enzyme.  Experiments  have  been  carried  out  to  see 
whether  or  not  the  presence  of  ornithine  is  necessary  for  enzyme  synthesis.  For  these  studies, 
a  mutant  unable  to  synthesize  ornithine  was  used.  The  cells  were  first  grown  on  arginine  to 
exhaust  transcarbamylase  and  then  transferred  to  either  citrulline  or  ornithine.  It  has  found 
that  transcarbamylase  was  resynthesized  as  rapidly  in  the  presence  of  citrulline  as  in  the  pres- 
ence of  ornithine.  These  results  indicate  that,  in  contrast  to  the  synthesis  of  adaptive  enzymes, 
here  the  substrate  may  not  function  as  an  inducer  for  enzyme  formation. 

The  ATPase  activity  of  frog  myosin.     G.  W.  DE  VILLAFRANCA. 

In  studying  the  ATPase  activity  of  frog  muscle  it  was  found  that  methods  normally  used 
for  preparation  of  myosin  from  rabbit  muscle  always  resulted  in  actomyosin.  Even  centrifuga- 
tion  of  purified  actomyosin  at  110,000  g  in  the  presence  of  ATP  failed  to  yield  actin-free  myosin. 
Myosin,  however,  was  obtained  by  centrifuging  twice  precipitated  actomyosin  in  0.6  M  KC1  and 
0.1  M  MgCln  at  110,000  g  for  30  minutes  after  the  actomyosin  had  been  previously  cleared  by 
centrifugation  in  0.6  M  KC1  at  that  force  for  20  minutes.  The  supernatant  after  Mg++  treat- 
ment was  then  treated  with  an  equivalent  amount  of  Versene  to  remove  the  ATPase  inhibiting 
Mg(++.  It  was  then  precipitated  twice  by  dilution  with  10  volumes  of  cold,  ion-free  water  and, 
between  precipitations,  dialyzed  overnight  against  3  changes  of  0.6  7l/  KC1.  There  was  no 
change  in  viscosity  of  the  myosin  upon  addition  of  ATP,  it  had  an  intrinsic  viscosity  approxi- 
mately that  of  rabbit  myosin,  and  it  had  pronounced  ATPase  activity  (Qp  about  1200). 

Three  types  of  preparations  (short  extraction  with  Hasselbach-Schneider  solution,  24- 
hour  extraction  with  Weber-Edsall  solution,  and  myosin  prepared  as  described )  showed  the 
same  ATPase  characteristics  and  yielded  Qp-s  ranging  from  500-1400  at  24°  C.  The  pH 
optimum  was  found  to  lie  between  pH  9  and  9.5  with  occasional  smaller  peaks  in  the  pH  6-7 
region.  Calcium  activated  strongly  with  an  optimal  concentration  of  5  X  10"3  M  while  mag- 
nesium either  inhibited  or  gave  the  same  activity  as  the  absence  of  divalent  ions.  Increasing 
the  KC1  concentration  from  0.03  to  0.24  M  progressively  decreased  activity.  The  enzyme  split 
only  40-50%  of  the  10  min.  P.  Optimal  activity  was  obtained  at  24°  C. ;  pre-incubation  for  15 
minutes  resulted  in  slight  inactivation  (10%)  at  37°  C.  and  almost  complete  inactivation 
(94%-)  at  45°  C. 


Vol.  Ill,  No.  3  December,  1956 

THE 

BIOLOGICAL  BULLETIN 

PUBLISHED  BY  THE  MARINE  BIOLOGICAL  LABORATORY 


A  NEW  PHYCOERYTHRIN  FROM  PORPHYRA  NAIADUM  1 

R.  L.  AIRTH  2  AND  L.  R.  BLINKS 

Hopkins  Marine  Station  of  Stanford  University,  Pacific  Grove,  California 

Svedberg  and  Katsurai  (1929)  proposed  a  phylogenetic  nomenclatural  system 
for  the  classification  of  the  phycobilin  pigments  of  the  algae.  They  designated  the 
phycoerythrin  and  phycocyanin  from  the  red  algae  as  R-phycoerythrin  and  R- 
phycocyanin,  respectively,  and  the  corresponding  pigments  from  the  blue-green 
algae  as  C-phycoerythrin  and  C-phycocyanin.  In  general,  these  pigments  exhibit 
the  following  absorption  maxima  : 

Approximate  absorption  maxima 
Pigment  m/< 

R-phycoerythrin  495           540           560 

C-phycoerythrin  550 

R-phycocyanin  550                            615 

C-phycocyanin  615 

This  system  has  proved  inadequate  in  several  instances  (Kylin,  1912;  Lemberg, 
1930;  Kylin,  1940;  Haxo,  ct  a!.,  1955)  in  that  phycobilin  pigments  other  than  the 
above  types,  as  judged  from  their  absorption  spectra,  have  been  isolated.  In  spite 
of  the  apparent  shortcomings  of  the  Svedberg  and  Katsurai  system  of  classification, 
no  new  system  has  been  proposed. 

A  new  phycoerythrin  has  now  been  isolated  from  Porphyra  naiadum  and  it  is 
proposed  that  this  pigment  be  called  B-phycoerythrin  (tentatively  so  designated  by 
Blinks,  1954).  This  differs  from  known  phycoerythrins  in  having  two  absorption 
peaks,  at  545  and  565  nip..  The  isolation,  purification  and  some  properties  of  this 
pigment  will  be  discussed. 

SOURCE 

Porphyra  naiadum  Anderson  is  a  member  of  the  most  primitive  red  algal  order, 
the  Bangiales.  There  is  now  some  question  as  to  whether  it  belongs  in  the  genus 
Porphyra,  since  its  life  cycle  is  different.  This  is  currently  under  study  by  Prof. 
G.  J.  Hollenberg ;  pending  his  description  of  a  new  genus,  we  must  use  the  current 
name.  The  thallus  is  one  cell  thick,  extremely  delicate,  and  yields  its  pigment 
readily  into  fresh  water  in  a  few  hours.  It  is  found  growing  only  upon  a  marine 
flowering  plant,  Phyllospadi.v. 

1  Research  supported  under  contract  with  the  Office  of  Naval  Research  (No.  NR  120-050). 

2  Present  address :  McCollum-Pratt  Institute,  The  Johns  Hopkins  University,  Baltimore  18, 
Maryland. 

321 


322  R.  L.  AIRTH  AND  L.  R.  BLINKS 

MATERIALS  AND  METHODS 

Masses  of  thalli  were  stripped  from  the  host  plant  and  washed  with  distilled 
water.  The  washed  algal  mass  was  then  just  covered  with  distilled  water  and 
kept  at  5°  C.  for  about  15  hours.  The  supernatant,  which  contains  the  water- 
soluble  phycobilin  pigments,  was  separated  from  the  algae  by  centrifugation  at 
15.000  times  gravity  for  one  hour.  By  this  procedure  approximately  75  per  cent 
of  the  total  phycobilins  present  can  be  extracted.  The  supernatant  was  filtered 
twice  through  Whatman  No.  1  filter  paper  and  the  filtrate  centrifuged  at  20.000  g 
for  twenty  minutes.  The  pH  of  the  pigment  solution  at  this  stage  was  6.8-7.0. 
The  pigment  solution  was  then  dialyzed  in  "Visking"  tubing  for  12  hours  at  1°  C. 
against  0.1  M  acetate  buffer,  pH  5.0.  The  dialyzed  pH  5.0  pigment  solution  con- 
stituted the  stock  solution  and  will,  in  the  future,  be  referred  to  as  such. 

RESULTS  AND  DISCUSSION 
Purification  and  crystallization 

The  general  method  of  purification  and  crystallization  of  phycobilin  pigments, 
which  has  varied  slightly  from  investigator  to  investigator,  involved  precipitation 
with  ammonium  sulfate  (Kylin,  1912;  Kitasato,  1925;  Lemberg,  1928).  This  pre- 
cipitation is  carried  out  after  the  algae  have  been  extracted  for  several  days  at  room 
temperature  under  slightly  basic  conditions. 

This  procedure  was  attempted  on  freshly  extracted  stock  pigment  solution  from 
P.  naiad  it  in:  it  consistently  failed  to  crystallize  phycoerythrin  and  phycocyanin  al- 
though concentrated  ammonium  sulfate  precipitated  an  amorphous  mass.  If  the 
stock  pigment  solution  was  allowed  to  stand  at  room  temperature  for  several  days, 
then  phycoerythrin  (but  not  phycocyanin)  could  be  crystallized  by  this  method. 
Bannister  (1954)  working  with  the  blue-green  alga  Synechocystis  apparently  en- 
countered similar  difficulties  in  trying  to  crystallize  with  a  freshly  extracted  pig- 
ment solution.  It  seems  very  likely  that  the  phycoerythrin  obtained  by  the  classical 
procedure  may  be  a  modified  pigment. 

In  contrast  to  ammonium  sulfate  treatment  a  freshly  prepared  stock  pigment  so- 
lution yielded  well-formed  phycoerythrin  crystals  on  simply  standing,  in  the  cold,  for 
about  24  hours  at  pH  4.5  These  crystals  were  separated  by  centrifugation,  washed 
in  acetate  buffer  at  pH  4.5,  and  redissolved  in  water  adjusted  to  pH  7.5.  Recrys- 
tallization  could  be  carried  out  by  reacidification  to  pH  4.5.  The  first  crystalliza- 
tion gave  an  estimated  20  per  cent  yield  of  the  total  phycoerythrin  present,  while 
the  second  and  subsequent  recrystallizations  were  quantitative.  The  reason  for 
this  low  yield  in  the  original  crystallization  will  be  discussed  in  a  subsequent  com- 
munication. (Suffice  it  to  say  here  that  the  non-crystallizable  fraction  appears  to 
have  an  iso-electric  point  (if  at  all)  in  very  acid  ranges.) 

The  absorption  spectra  of  the  original  crystals  and  three-times  recrystallized 
B-phycoerythrin  are  presented  in  Figure  1.  The  main  absorption  maximum  is  at 
545  mp.;  this  value  did  not  vary  between  pH  5.0  to  7.0.  Phycoerythrin  that  is 
kept  at  pH  9.0  for  6  hours  has  the  same  absorption  maximum  but  the  blue  (400- 
450  m/j.)  and  red  (600-700  m^)  absorption  is  increased  by  about  300  per  cent.  Re- 
cently Haxo  ct  al.  (1955)  have  found  that  the  phycoerythrin  from  Porphyndinm 
was  modified  above  pH  7.3.  Our  findings  are  also  consistent  with  theirs  in  that 


PHYCOERYTHRIN  FROM  PORPHYRA  NAIADl'M 


323 


we  find  that  phycocyanin  from  P.  naiaduin  is  irreversibly  bleached  at  pH  9.0.  In 
contrast  to  the  phycoerythrin  from  Porpliyridinni,  however,  neither  the  native  nor 
the  crystalline  B-phycoerythrin  from  Porphyra  naiad  inn  tended  to  form  additional 
"shoulders"  or  absorption  maxima  under  alkaline  conditions. 

In  addition,  it  is  apparent  that  the  original  B-phycoerythrin  crystals  exhibit  a 
"shoulder"  in  the  565  m^  spectral  region  which  is  lacking  in  the  three-times  re- 
crystallized  preparation.  Whether  this  absorption  represents  an  impurity  in  the 
original  crystallization  or  a  modification  of  phycoerythrin  on  repeated  recrystalliza- 


UJ 
Q 


U 

t- 
Q_ 

o 


/     /ORIGINAL 
/      '    CRYSTALS 


\3x  RECRYSTALLIZED 
V 


400 


450 


500     550     600 
WAVE  LENGTH  (mp) 


650 


700 


FIGURE  1.     Absorption  curves  of  once-  and  three-times  recrystallized   B-phycoerythrin.     Solid 
line,  original  crystals ;  dashes,  three-times  recrystallized. 

tion  is  unknown  at  the  present  time.  There  are,  however,  reasons  for  believing  that 
the  latter  may  be  the  case.  On  repeated  recrystallization  it  was  found  that  the  B- 
phycoerythrin  solubility  at  pH  7.5  decreased  with  the  number  of  times  the  pigment 
was  recrystallized.  Also  the  stability  of  the  native  and  crystalline  phycoerythrin  to 
hydrogen  peroxide  is  different.  Table  I  gives  the  reduction  in  optical  density  at 
540  m/A  of  the  two  pigment  solutions  when  treated  with  varying  hydrogen  peroxide 
concentrations.  The  pH  of  both  solutions  was  5.8. 

B-phycoerythrin  was  also  isolated  by  the  chromatographic  method  of  Swingle 
and  Tiselius  (1951).  Haxo  ct  al.  (1955)  have  successfully  separated  the  phyco- 
bilin  pigments,  including  allophycocyanin,  from  several  algal  species  by  this  method. 
The  latter  pigment  is  also  present  in  P.  naiadum  and  this  was  the  only  method  we 
found  to  isolate  allophycocyanin  and  phycocyanin  in  relatively  pure  form. 


324  R.  L.  AIRTH  AND  L.  R.  BLINKS 

The  order  of  pigment  elution  from  the  column  depends  upon  the  pH  of  the  elut- 
ant.  Using  1  to  2  M  acetate  buffer  at  pH  5.0  as  an  elutant,  the  pigments  come 
off  the  column  in  the  following  order  :  phycocyanin,  B-phycoerythrin,  a  mixture  of 
phycoerythrin  and  phycocyanin.  Allophycocyanin  could  not  be  eluted  from  the 
column  at  pH  5.0;  however,  it  was  eluted  with  0.1  M  phosphate  buffer,  pH  7.0. 
When  phosphate  buffer  (0.05  to  0.1  M),  pH  7.0,  was  used  the  elution  order  of  the 
pigment  was  :  B-phycoerythrin,  a  mixture  of  phycoerythrin,  phycocyanin  and  allo- 
phycocyanin,  and  finally  allophycocyanin. 

The  phycoerythrin  isolated  by  this  method  was,  however,  never  completely  free 
of  phycocyanin  and  repeated  chromatography  did  not  remove  this  impurity. 

Ultracentrifugation 

Svedberg  and  his  collaborators  determined  the  molecular  weight  of  crystalline 
phycoerythrin  from  several  species  of  red  algae  (Svedberg  and  Lewis,  1928;  Sved- 
berg and  Katsurai,  1929;  Svedberg  and  Eriksson,  1932).  In  a  final  paper  of  this 
series  Eriksson-Quensel  (1938)  found  the  molecular  weight  of  R-phycoerythrin 

TABLE  I 

The  reduction  in  optical  density  (at  540  MH)  of  stock  pigment  solution  and  crystalline 
B-phycoerythrin,  treated  with  H^Oz  for  four  hours 

Stock  pigment  Crystalline 

Percent  solution  B-phycoerythrin 

H2Os  -.iE540 


0  0.040  0.000 

1  0.874  +0.204 
3                                                 1.146  0.000 
5                                                 1.060  0.000 

from  Ccramhim  rubnim  to  be  290,000  between  pH  3.0  to  10.0.  At  other  pH  values 
the  R-phycoerythrin  molecule  breaks  down  into  units  with  a  molecular  weight  of 
34,600  or  a  multiple  thereof. 

In  view  of  the  fact  that  we  were  dealing  with  a  new  type  of  phycoerythrin  it  was 
considered  desirable  to  determine  its  molecular  weight  and  compare  it  with  that  of 
R-phycoerythrin.  The  ultracentrifugation  experiments  were  carried  out  for  us 
through  the  very  kind  courtesy  of  Drs.  H.  Cook  and  J.  M.  Luck  of  the  Department 
of  Chemistry,  Stanford  University. 

B-phycoerythrin  that  had  been  recrystallized  several  times  was  dissolved  at 
pH  7.0,  centrifuged,  and  dialyzed  at  1°  C.  in  0.05  M  acetate  buffer,  pH  5.0.  The 
sedimentation  constant  (s20  X  1013)  was  determined  on  the  dialyzed  preparation  in 
a  Spinco  ultracentrifuge  and  found  to  be  12.0.  This  value  is  comparable  to  that 
found  by  Eriksson-Quensel  (1938).  Consequently  it  seems  very  probable  that  the 
molecular  weights  of  R-  and  B-phycoerythrin  are  the  same.  The  crystalline  B- 
phycoery  thrin  was  judged  to  be  homogeneous  from  the  fact  that  only  one  schlieren 
peak  was  observed  and  the  absorption  boundary  of  the  pigment  corresponds  almost 
exactly  with  it.  It  may  also  be  noted  that  Svedberg  and  Eriksson  (1932)  deter- 
mined the  molecular  weight  of  what  they  designated  native  R-phycoerythrin  from 
Ceramiwn  and  found  it  to  be  similar  to  the  crystalline  phycoerythrin  from  this 
species. 


PHYCOERYTHRIN  FROM  PORPHYRA  NAIADUM 


325 


Electrophoresis 

The  mobility  of  crystalline  B-phycoerythrin  was  tested  by  dissolving  it  at  pH  7.5, 
centrifuging  and  then  dialyzing  at  1°  C.  in  acetate  buffer  of  0.1  ionic  strength, 
pH  5.0.  The  movement  is  toward  the  anode  at  the  rate  of  about  2.0  X  10~5  cm.2/ 
sec./volt. 

The  above  value  was  determined  by  visually  measuring  the  movement  of  the 
ascending  and  descending  boundary  in  a  Tiselius  apparatus  (no  schlieren  optics 
were  available).  Fairly  reproducible  results  could  be  obtained,  because  of  the  in- 
tense color  of  the  pigment. 

Crystalline  B-phycoerythrin  had  charge  characteristics  different  from  those  of 
the  remainder  of  the  pigments  in  the  stock  solution  and  it  was  possible  to  determine 


+  20.0 


+  I  6.0 


4-12.0 

LJ 
CO 


CVJ 


O 

If) 

I 

O 


8.0 


4.0 


8 


,H 


FIGURE  2.     pH-mobility  curve  of  crystalline  B-phycoerythrin.     Mobility  in   10""  cm.2/sec./volt. 
Determinations  by  visual  measurement  in  Tiselius  apparatus. 

the  pH-mobility  characteristics  of  this  pigment.  In  this  case  the  mobility  values 
were  determined  from  either  the  ascending  or  descending  boundary,  depending  upon 
the  pH  of  the  determination.  The  pH-mobility  curve  of  B-phycoerythrin  is  pre- 
sented in  Figure  2.  No  values  were  determined  below  pH  5.0  as  the  pigment 
tended  to  precipitate  below  this  pH  value.  The  isoelectric  point  as  extrapolated  by 
this  method  would  be  approximately  at  pH  4.5  which  corresponds  wyell  with  that 
found  for  crystallization. 

Other  optical  properties 

The  fluorescence  spectrum  of  B-phycoerythrin  has  been  determined  by  French, 
Smith,  Virgin  and  Airth  (unpublished  data)  ;  at  pH  7.0  the  fluorescence  maximum 
is  at  578 


326  R.  L.  AIRTH  AND  L.  R.  BLINKS 

The  phycobilin  constituents  of  P.  naiadnm  are  B-phycoerythrin,  phycocyanin 
and  allophycocyanin.  At  least  the  two  former  pigments  are  photosynthetically  ac- 
tive in  that  they  pass  absorbed  light  energy  on  to  chlorophyll.  In  such  studies  it  is 
often  essential  to  know  the  percentage  of  the  total  light  absorbed  by  each  pigment 
at  various  wave-lengths.  As  purified  pigments  were  available  a  curve  analysis  of 
the  absorption  of  the  stock  pigment  solution  was  carried  out.  The  percentage  of 
the  total  light  absorbed  by  each  of  the  phycobilin  pigments  at  their  respective  ab- 
sorption maxima  is  presented  in  Table  II.  These  values  are  fairly  consistent  with 
those  presented  by  Yocum  and  Blinks  (1954). 

TABLE  II 

Percentage  absorption  of  the  total  light  absorbed  by  the  various  phycobilin  pigments  at  different 
wave-lengths  of  an  extracted  pigment  solution  of  Porphyra  naiadnm 

Per  cent 

Wave-length  total  light 

m/j  Pigment  absorbed 

545                                              B-phycoerythrin  88 

phycocyanin  9 

allophycocyanin  3 

615  B-phycoerythrin 

phycocyanin  77 

allophycocyanin  21 

655                                            B-phycoerythrin  0 

phycocyanin  18 

allophycocyanin  82 

The  authors  gratefully  acknowledge  the  many  helpful  suggestions  of  Dr.  C.  B. 
van  Niel  during  the  course  of  this  work. 

SUMMARY 

A  new  phycoerythrin,  B-phycoerythrin,  isolated  from  PorpJiyni  naiadnm,  is  de- 
scribed. The  purification  and  crystallization,  ultracentrifugation,  electrophoretic 
and  optical  properties  of  this  pigment  are  discussed.  It  has  a  major  absorption 
peak  at  545  nip,  with  a  minor,  and  transient,  one  at  565  m/x,  which  tends  to  dis- 
appear on  repeated  crystallization.  The  molecular  weight  is  apparently  the  same 
as  that  of  R-phycoerythrin  (ca.  290,000).  Its  iso-electric  point  is  close  to  pH  4.5, 
and  its  mobility  (toward  the  anode)  at  pH  5.0  is  about  2  X  10'5  cm. -/sec. /volt. 

LITERATURE  CITED 

BANNISTER,  T.  L.,  1954.  Energy  transfer  between  chromophore  and  protein  in  phycocyanin. 
Arch.  Biochcm.  Biophys.,49:  222-233. 

BLINKS,  L.  R.,  1954.  The  role  of  accessory  pigments  in  photosynthesis.  In :  Symposium  on 
Autotrophic  Micro-organisms.  Cambridge,  England ;  Cambridge  University  Press. 

ERIKSSON-QUEXSEL,  L,  1938.  The  molecular  \veights  of  phycoerythrin  and  phycocyanin.  Bio- 
chcm. /.,  32:  585-589. 

HAXO,  F.,  C.  O'nEocHA  AND  P.  NORRIS,  1955.  Comparative  studies  of  chromatographically 
separated  phycoerythrins  and  phycocyanins.  Arch.  Biochcm.  Biophys.,  54:  162-173. 

KITASATO,  Z..  1925.  Biochemische  Studien  iiber  Phykoerythrin  und  Phykocyan.  A  eta  Phyto- 
cliima  (Japan),  11  :  75-97. 

KYLIN,  H.,  1912.  Uber  die  roten  und  blauen  Farbstoffe  der  Al^en.  Zcitschr.  f.  Physiol.  Chan., 
76 :  397-425. 


PHYCOERYTHRIN  FROM  PORPHYRA  NAIADUM 

KYLIN,   H.,   1940.     Uber    Phykoerythrin   und   Phykocyan   bci   Ccraniiiini    nihntin.     Zcitschr.   f. 

Phys.  Chcm,,  69:  169-239. 

LEMBERG,   R.,   1928.     Die  Chromoproteide  der   Rotalgen.     Ann.   Chcmic    (Licbi<i),  461:   46-89. 
LEMBERG,  R.,  1930.     Chromoproteide  der  Rotalgen  II.     Spaltung  mit  Pepsin  und  Saiiren.     Iso- 

lierung  eines  Pyrrolfarbstoffs.     Ann.  Chcmic   (Licln/i),  477:   195-245. 
SVEDBERG,  T.,  AND  I.  B.  ERIKSSON,  1932.     The  molecular  weights  of  phycocyanin  and  phyco- 

erythrin.     /.  Amcr.  Chcm,  Soc.,  54:  3998-4010. 
SVEDBERG,  T.,  AND  I.  T.  KATSURAI,  1929.     The  molecular  weights  of  phycocyanin  and  of  phyco- 

erythrin  from  Porphyra  tcncra  and  of  phycocyanin  from  Aphanizomenon  flos  aquae. 

J.  Amcr.  Chcm.  Soc.,  51 :  3573-3583. 

SVEDBERG,  T.,  AND  N.  B.  LEWIS,  1928.     The  molecular  weights  of  phycoerythrin  and  of  phyco- 
cyanin.    /.  Amer.  Chcm.  Soc.,  50:  525-536. 
SWINGLE,  S.  M.,  and  A.  TISELIUS,  1951.     Tricalcium  phosphate  as  an  adsorbent  in  the  chro- 

matography  of  proteins.     Biochcm.  J.,  48:  171-174. 
YOCUM,  C.  S.,  AND  L.  R.  BLINKS,   1954.     Photosynthetic  efficiency  of  marine  plants.     /.   Gen. 

PhysioL,  38 :  1-16. 


HERMAPHRODITISM  IN  ECHINOIDS  * 

R.  A.  BOOLOOTIAN  AND  A.  R.  MOORE 

Hopkins  Marine  Station  of  Stanford  University,  Pacific  Grove,  California 

In  his  chapter  on  "Hermaphroditism"  Goldschmidt  (1923,  p.  165)  speaks  of 
this  as  "the  most  unsatisfactory  chapter  in  the  whole  sex  problem,  and  up  to  date 
our  material  is  insufficient  to  permit  of  a  correct  genetic  or  physiological  under- 
standing." The  problem  is  still  further  rendered  difficult  by  the  fact  that  it  cannot 
be  dealt  with  experimentally.  In  the  echinoids  hermaphroditism,  as  a  rule,  is  shown 
by  entire  gonads  of  an  individual  being  of  one  sex  or  the  other,  i.e.,  testes  and 
ovaries  in  the  same  animal.  More  rarely  ovarian  and  testicular  tissues  develop  side 
by  side  in  the  same  gonad — an  ovotestis.  In  the  five  gonads  radially  symmetrically 
disposed,  all  possible  combinations  have  been  found,  the  rarest  being  the  ovotestis. 

Such  a  condition  is  of  sufficiently  infrequent  occurrence  to  be  noted  in  the  litera- 
ture in  a  number  of  cases  in  which  it  has  been  observed.  The  paucity  of  known 
cases  is  well  expressed  by  E.  B.  Harvey  (1939,  p.  74)  :  "Among  the  many  thou- 
sands of  Arbacia  punctulata  opened  in  the  course  of  ten  summers  at  Woods  Hole, 
and  many  hundreds  of  Arbacia  pustitlosa,  Sphaer echinus  granularis,  Paraccntrotus 
lividns,  and  ParccJiiniis  microtuberculatus  opened  during  several  springs  at  Naples, 
and  many  hundreds  of  Strongylocentrotus  droebacJiiensis  from  Maine,  I  observed 
last  summer  for  the  first  time  an  hermaphroditic  sea  urchin,  an  Arbacia  punctulata 
opened  on  July  4,  1938." 

This  situation  renders  an  analysis  of  the  phenomenon  difficult,  and  the  impos- 
sibility of  attacking  the  problem  experimentally  prevents  a  precise  causative  analy- 
sis. Nevertheless,  an  examination  of  the  cases  described,  the  frequency  of  their 
incidence  geographically  and  in  the  classification  may  give  some  clue  to  the  phe- 
nomenon of  what  may  be  called  "accidental  hermaphroditism."  It  is  hoped  that 
this  collection  of  records  may  serve  to  stimulate  an  interest  in  the  problem  and  re- 
sult in  further  information  being  published. 

At  the  present  time  very  few  statistical  records  of  bisexual  echinoids  are  avail- 
able. H.  B.  Moore  (1932)  reported  one  hermaphrodite  in  3000  Echinus  csculoitiis 
opened  during  the  season  1931-32  at  Port  Erin.  Shapiro  (1935)  kept  an  exact 
account  of  the  Arbacia  punctulata  which  he  opened  during  the  summer  of  1935. 
He  found  one  hermaphrodite  in  2350  animals  opened.  Albert  Tyler  (personal 
communication)  at  Corona  del  Mar  has  kept  the  most  extensive  records  and  reports 
that  10,000  Strongylocentrotus  purpiiratits  opened  over  a  period  of  several  years 
yielded  approximately  20  hermaphrodites,  or  1  in  500.  This  is  the  highest  inci- 
dence so  far  reported.  Tyler  found  that,  in  addition  to  normal  development  as  the 
result  of  selfing,  agglutination  of  the  sperm  by  autologous  sea  water  was  positive. 
Edward  Chambers,  working  at  Berkeley  with  5".  purpuratus.  in  the  course  of  two 
seasons  observed  three  hermaphrodites. 

1  This  work  was  supported  in  part  by  a  grant  from  the  National  Science  Foundation  and 
Grant  B-160  (C)  from  National  Institutes  of  Health,  Public  Health  Service. 

328 


HERMAPHRODITISM  IN  ECHINOIDS 


329 


At  Pacific  Grove,  although  the  use  of  sea  urchins  has  gone  on  for  many  years, 
only  one  bisexual  individual  each  of  .S".  pnrpitnitus  and  .S".  franciscanus  has  heen 
detected.  In  all  these  cases  the  selfed  eggs  gave  normal  plutei. 

In  the  specimen  of  .V.  pnrpnratns  opened  at  Pacific  Grove  in  December,  1950 
(A.R.M.),  the  gonads  were  swollen  with  ripe  products  and  easily  broken.  There 
were  three  ovaries,  one  testis  and  one  ovotestis.  None  of  the  eggs  could  have  been 
fertilized  in  corf>orc,  since  no  fertilized  or  segmenting  eggs  or  embryos  were  seen 
at  the  time  the  animal  was  opened.  However,  as  soon  as  the  eggs  and  sperm  were 
free  in  the  sea  water,  fertilization  took  place.  Development  of  the  selfed  eggs  was 
entirely  normal,  the  plutei  differing  in  no  way  from  normals. 


Fn;ri<K   1.     Topographical  scheme  showing  details  of  female  and  male  gonad  elements  of 
S.  franciscanus,  viewed  from  the  aboral  surface. 

In  March,  1955,  a  bisexual  individual  .S.  franciscanus  was  found  (R.  A.  B.)  at 
Pacific  Grove.  This  was  the  first  such  individual  of  this  species  to  be  discovered. 
The  gonads  were  separate  as  to  sex,  four  ovaries,  one  testis,  and  no  ovotestis.  The 
distribution  of  the  gonads  is  shown  in  Figure  1.  The  selfed  eggs  produced  normal 
plutei  as  did  the  outcrossing  of  both  eggs  and  sperm.  It  is  to  be  noted  that  this  is 
the  first  case  of  bisexuality  found  in  this  species.  The  very  short  breeding  season 
may  be  a  factor. 

In  addition  to  these  examples  of  sea  urchins,  several  hermaphroditic  individuals 
of  Dendrastcr  have  been  found  at  Pacific  Grove.  The  first  was  in  19.29  (Needham 
and  Moore,  192()),  when  hundreds  of  the  animals  were  being  used  to  obtain  ma- 
terial for  chemical  work.  In  every  case  the  entire  gonadal  disk  was  removed  and 
any  disk  containing  white  sperm  and  red  eggs  would  have  been  detected  at  once. 
Since  only  one  was  found,  it  may  be  assumed  that  the  incidence  was  of  the  order 
of  one  in  1000.  In  this  one  case  the  eggs  and  sperm  were  not  fertile  inter  sc.  The 


330  R.  A.  BOOLOOTIAN  AND  A.  R.  MOORE 

second  specimen  of  D  end  raster  was  taken  in  1943,  and  contained  both  ripe  sperm 
and  eggs.  The  spermaries  occupied  a  little  more  than  half  the  gonadal  disk  and 
yielded  abundant  sperm.  The  ovarian  half  contained  a  few  ripe  eggs.  Less  than 
half  of  these  were  fertilizable  with  the  sperm  of  the  same  individual.  Such  eggs 
segmented  at  the  normal  cleavage  rate  and  gave  rise  to  swimming  blastulae  in  nor- 
mal time,  but  the  plutei  were  not  vigorous,  some  larvae  remaining  blastulae.  At 
55  hours  and  20°  C.  these  selfed  larvae  were  in  all  stages  of  juvenility.  It  is  evi- 
dent that  the  eggs  were  defective  in  their  potential,  but  the  sperm  was  normal  since 
it  brought  about  normal  development  in  normal  eggs. 

THE  OVOTESTIS 

The  occurrence  of  eggs  and  sperm  in  the  same  gonad  has  been  observed  in  sev- 
eral widely  different  groups  of  animals  and  is  a  type  of  accidental  hermaphroditism 
according  to  Goldschmidt.  Ishikawa  (1891)  described  a  case  in  Gcbia  major  in 
which  the  anterior  part  of  the  gonad  was  testis,  the  posterior  part  ovary.  The 
latter  was  not  functional  since  the  eggs  were  unable  to  pass  out  through  the  vas 
deferens  and  consequently  atrophied  in  situ.  Paul  Buchner  (1911)  described  in 
careful  detail  the  gonad s  of  a  bisexual  starfish.  He  observed  eggs  about  the  sperm 
in  the  testicular  vesicles,  and  ripe  sperm  infiltrated  into  the  ovary.  Harvey  (1939) 
has  shown  similar  conditions  in  a  hermaphrodite  Arhacia.  All  of  the  gonads  con- 
tained both  types  of  cells,  four  being  mainly  female,  one  predominantly  male. 
Therefore  all  five  gonads  were  ovotestes.  Normal  fertilization  took  place  inter  se 
and  development  proceeded  to  normal  plutei.  Similar  instances  have  been  de- 
scribed by  Neefs  (1953)  and  by  Reverberi  (1947).  An  account  of  a  hermaphro- 
ditic sea  urchin  ^.  pulcherrimus  has  been  published  by  Okada  and  Shimoizumi 
(1952),  in  which  they  give  a  very  complete  analysis.  One  gonad  was  an  ovary, 
the  others  were  ovotestes.  The  eggs  and  sperm  did  not  yield  normal  larvae  when 
used  inter  se,  but  gave  normal  larvae  on  out-crossing. 

As  stated  above,  the  hermaphroditic  S.  f>urf>nnitns  found  at  Pacific  Grove  con- 
tained one  ovotestis.  This  gonad  was  preserved  and  imbedded  by  Dr.  D.  P.  Abbott 
at  this  station.  The  specimen  remained  in  block  until  the  present  season  when  it 
was  sectioned  and  mounted  by  Mr.  W.  K.  Bowen  of  the  Biology  Department  of 
Stanford  University.  The  gonad  appeared  to  be  divided  into  an  upper  and  lower 
half  which  were,  respectively,  ovary  and  testis.  Sections  were  made  of  parts  that 
were  clearly  unisexual  and  of  the  mixed  median  zone.  Sections  of  the  ovarian  half 
show  the  ovarian  lobes  well  filled  with  eggs  both  ripe  and  immature  (Fig.  2),  while 
sections  of  the  testicular  half  show  normal  testicular  structure  and  dense  collections 
of  sperm  in  the  vesicles  and  ducts  (Fig.  3).  In  the  median  section  ovarian  and 
testicular  tissue  lie  side  by  side,  the  acini  intermingled  in  the  same  section  (Fig.  4). 
Ripe  ova  occur  among  the  sperm  (Fig.  5),  but  no  eggs  were  found  fertilized,  a  fact 
which  presumably  was  due  to  the  immobility  of  the  sperm.  This  duplicates  the 
situation  in  the  case  described  by  Harvey. 

Two  clear  exceptions  to  the  general  rule  that  fertilization  does  not  occur  in 
corpore  before  the  extrusion  of  the  sex  cells  from  an  ovotestis  have  been  noted. 
H.  B.  Moore  (1935)  describes  one  such  case  in  Echinocardiutn  cordatuni.  In  his 
sketches  he  figures  apparently  normal  early  segmentation  stages,  morulae  and  blas- 
tulae present  in  the  gonadal  ducts  of  the  ovotestis.  Reverberi  (1947)  has  described 


HERMAPHRODITISM  IN  ECHINOIDS 


331 


'' '          ; 


FIGURE  2.     Ovarian  fraction  of  ovotestis. 
FIGURE  3.     Testicular  fraction  of  ovotestis. 

FIGURE  4.     Boundary  zone  showing  ovarian  tissue  above,  testicular  below. 
FIGURE  S.     From  boundary  zone  showing  vesicle  with  eggs  in  mass  of  sperm. 


R.  A.  BOOLOOTIAN  AXD  A.  R.  MOORE 

a  similar  case  in  Arbacia  pnstitlusa  at  Naples.  However.  XeetY  (1953)  figure  of 
a  cell-mass  in  the  lobe  of  an  ovary  of  a  bisexual  Arbacia  li.ritla  is  of  doubtful  sig- 
nificance, for  the  reason  that  the  structure  appears  to  be  a  relatively  unorganized 
mass  of  cells  without  recognizable  embryonic  form,  and  is  apparently  a  solitary 
instance  in  the  specimen. 

DISCUSSION 

Various  causative  factors  have  been  proposed  to  account  for  hermaphroditism 
in  echinoids.  The  oldest  is  the  suggestion  of  seasonal  dimorphism  by  Giard  (  1900) 
who,  working  at  Wimereux.  found  evidence  which  he  thought  sufficient  for  con- 
cluding that  Echinocardium  cordatnin  is  normally  a  protandrous  hermaphrodite, 
for  the  reason  that  in  July  ova  begin  to  appear  in  individuals  which  up  to  that  time 
he  believed  had  been  male.  However,  Giard's  conclusions  were  later  emphatically 
denied  by  Caullery  (  1(>25  )  who  states  that  the  gonads  of  this  form  at  Wimereux  are 
entirely  quiescent  during  autumn,  the  gametes  developing  during  the  winter,  the 
phase  of  maturity  beginning  in  April  and  ending  in  August,  with  a  maximum  in 
May.  As  a  result  of  numerous  observations,  Caullery  says  (p.  29)  :  "I  have  never 
found  a  single  case  of  hermaphroditism  (in  this  form)  and  1  cannot  explain  how 
Giard  could  think  that  at  Wimereux.  Echinocardium  shows  successive  sexuality 
with  protandry,  the  eggs  beginning  to  appear  toward  mid-July  in  the  gonads  which 
up  to  that  time  were  apparently  male  and  full  of  sperm."  Since  Caullery.  the  emi- 
nent zoologist  and  director  of  the  Wimereux  Station,  has  written  from  the  vantage 
point  of  25  years  after  Giard's  paper,  it  must  be  considered  established  that  Giard 
observed  a  rare  case  of  hermaphroditism  in  Echinocardium  (two  others  have  been 
recorded  at  RoscofT  and  one  at  Port  Erin  ),  and  that  he  was  clearly  in  error  in  pos- 
tulating seasonal  sexual  dimorphism  for  the  whole  population  of  this  species.  Re- 
cently Reverberi  (1940.  1947)  in  Italy  and  Neefs  (1937,  1938,  1<)52,  1953)  in 
France  have  sought  to  revive  Giard's  hypothesis  and  give  it  substantial  support. 
Reverberi  considers  the  fact  of  an  ovotestis  in  itself  to  be  a  significant  indication 
of  sexual  metamorphosis.  But  to  the  unbiased  worker  it  is  not  clear  why  the  fact 
that  normal  eggs  and  sperm  occur  side  by  side  in  an  ovotestis,  with  no  evidence 
whatever  of  either  type  of  gland  degenerating,  should  indicate  a  process  of  sexual 
metamorphosis.  It  must  be  confessed  that  Reverberi's  observations  do  not  give 
convincing  basis  for  his  hypothesis.  Xeefs  has  used  two  lines  of  argument  for  her 
belief  in  the  seasonal  change  of  sex  as  the  basis  of  bisexuality.  (  )ne  is  the  presence 
of  degenerate  gonads  of  two  colors  in  an  animal.  Such  a  case  was  described  by 
Gray  (1921).  Wre  occasionally  find  them  among  the  -V.  pitrpitratns.  Since  these 
pathological  individuals  often  do  not  have  either  eggs  or  sperm,  the  more  conserva- 
tive view  should  be  taken,  namely,  that  the  appearance  is  an  indication  of  disease. 
The  other  attempt  of  Neefs  to  show  seasonal  sexual  dimorphism  has  been  made  by 
means  of  statistical  counts  of  sex.  It  must  be  said  that  her  tables  are  not  con- 
vincing, for  the  reason  that  at  each  station  a  relatively  limited  number  of  individuals 
was  examined,  and  animals  of  both  sexes  appeared  in  each  month  of  the  year- 
a  very  different  picture  from  that  given  by  Giard.  It  should  also  be  noted  that  no 
one  except  Giard  has  reported  seasonal  incidence  of  bisexuality. 

Recently   Kgami    (1955)    has   proposed   a   nutritional    basis   for   bisexuality    in 
fishes.      He  has  shown  that  periods  of  starvation,   succeeded  by  food   in   plentiful 


HERMAPHRODITISM  IN  ECHINOIDS 


333 


Author 


TABLK  I 
Table  of  reported  cases 

Species  and 
Normal  Eggs  and  Sperm 


Boolootian  (this  paper) 

Chambers  (personal  communication) 

Cornman  (Harvey,  1956) 

Fisher  (Harvey,  1956) 

Fox  (Gray,  1921) 

Giard  (1900) 

Harvey  (1939) 

Herbst  (1925) 

Heilbrunn  (1929.) 

Herlant  (1918) 

Moore,  A.  R.  (this  paper) 

Moore,  H.  B.  (1932) 

Moore,  H.  B.  (1935) 

Neefs  (1938) 

Neefs  (1952) 

Neefs  (1937  and  1953) 

Reverberi  (1940) 

Tyler  (personal  communication) 


5'.  franc/scan u.', 
S.  f>nrf>iiratus 
Arbacia  punctulafa 
Arbacia  punctulata 
Pa  racentrotiis  1  ivid  u  s 
Echinocardiuiu  cordatum 
Arbacia  punctulata  (2) 
Psammechinus  tubcrculatns 
Arbacia  punctulata  (2) 
Paracentrotus  lividus  (12) 
S.  purpuratiis 
Echinus  esculentus 
Echinocardium  cordatum 
Paracentrotus  lividus  (7) 
Spha  erech  in  us  gra  H  n!n  r  is 
Arbacia  lixula  (2) 
.4  rbacia  pustulosa 
S.  purpuratiis  (20) 


Drzewina  and  Bohn  (1924) 
Moore,  A.  R.  (this  paper) 


Defective  Eggs — Normal  Sperm 

Paracentrotus  lividiis 
Echinocardium  cordatum  (2) 
Dend raster  excentricus 

Normal  Eggs — Defective  Sperm 


Okacla  and  Shimoizumi  (1952) 

Yignier  (1900) 

Needham  and  Moore  (1929) 

Shapiro  (1935) 

Reverberi  (1947) 


Chambers  (personal  communication) 

Gadd  (1907) 

Ruloii  (personal  communication) 


5.  pulcherrimus 
Sphaerechin us  gra  >i uluris 
Dend  raster  excentricus 
Arbacia  punctulata 
Arbacia  pustulosa  (5) 
Psa  in ii/ech  inns  m ic ro- 
tuberciilatus 

No  Test 

S.  purpuratiis  (2) 
S.  droebachiensis 
Dendraster  excentricus 


Arbacia  15 

Paracentrotus  21 

Strongylocentrolus      27 


Genus  Incidence — Summary  of  Cases 

Dendraster  3 

Echinocardium         4 
Echinus  1 


Locality 


Pacific  Grove 
Berkeley 

Woods  Hole 

Woods  Hole 

Naples 

\Yimereiix 

Woods  Hole 

Naples 

Woods  Hole 

Yillefranche 

Pacific  Grove 

Port  Erin 

Port  Erin 

Roscoff 

Roscoff 

Banyuls 

Naples 

Corona  del  Mar 


Roscoff 
Roscoff 
Pacific  Grove 


Japan 
Algiers 
Pacihc  Grove 
Woods  Hole 
Naples 
Naples 


Berkeley 
Murmansk 
Pacific  Grove 


Psam m ech  in  u s  2 

Sphaer  echinus  2 


supply,  in  rare  cases  result  in  hermaphroditism.  It  is  possible  that  other  causative 
factors  will  have  to  be  taken  into  account  in  any  final  analysis.  Two  of  these  are 
the  incidence  of  hermaphroditism  in  the  classification,  and  the  distribution  of  the 
phenomenon  geographically.  On  the  first  point,  it  is  a  striking  fact  that  instances 
of  hermaphroditism  so  far  described  are  from  relatively  few  genera  and  species, 
and  these  belong  almost  entirely  to  the  order  of  the  true  sea  urchins  (Table  I). 
Here  we  find  five  genera  and  ten  species  represented,  while  the  sand  dollars  and 


334  R.  A.  BOOLOOTIAN  AND  A.  R.  MOORE 

heart  urchins  furnish  examples  in  one  species  each.  One  factor  which  may  in  part 
account  for  this  preponderance  of  the  true  sea  urchins  is  the  accessibility  and  ex- 
tensive use  of  these  animals  in  scientific  work.  Most  of  them  are  littoral  dwellers 
and  are  easily  obtained,  while  members  of  the  other  two  orders  as  a  rule  are  dwellers 
of  deeper  waters  and  do  not  yield  the  quantity  of  eggs  to  be  found  in  most  of  the  sea 
urchins  during  the  breeding  season,  and  hence  are  little  used.  In  view  of  the  rarity 
of  the  phenomenon,  the  total  number  of  individuals  of  a  species  examined  is  an 
important  factor. 

Despite  the  fact  that  ten  or  more  species  of  sea  urchins  have  been  extensively 
used  in  experimental  work,  most  of  the  cases  of  hermaphroditism  have  come  from 
three  genera,  namely,  Arbacia,  Paraccntrotits  and  Strongyloccntrotus.  It  is  notable 
that  Lyt echinus,  which  has  been  extensively  used  in  southern  stations,  has  not 
yielded  a  single  case.  Nor  have  examples  of  hermaphroditic  sea  urchins  been  re- 
ported from  northern  waters,  except  for  the  observation  of  a  specimen  of  6".  droe- 
bachiensis  found  fifty  years  ago  at  Murmansk  by  Gadd  ( 1907) .  No  cases  have  been 
reported  from  Scandinavia,  none  from  Maine,  none  from  the  Oregon  and  Wash- 
ington coasts.  The  distribution  of  hermaphroditic  echinoids  so  far  reported  is  in 
a  band  between  N  35°  and  55°  in  Europe,  and  between  N  32°  and  45°  in  North 
America. 

More  information  and  precise  records  with  publication  are  needed  to  give  a 
basis  for  assessing  possible  causative  factors.  It  is  important  that  those  engaged 
in  work  with  echinoid  material  examine  animals  opened  for  possible  bisexuality.  and 
where  this  condition  is  found,  a  sketch  record  be  made  of  the  positions  of  ovaries, 
testes,  and  ovotestes.  Harvey's  suggestion  that  cases  of  ovotestis  may  have  been 
passed  over  as  due  to  contamination  when  a  worker  has  found  occasional  fertilized 
eggs  among  those  freshly  shed,  is  worth  bearing  in  mind.  Experiments  to  deter- 
mine developmental  potentialities  both  inter  sc  and  in  outcrossing  are  of  great  in- 
terest and  importance. 

SUMMARY 

Two  new  cases  of  hermaphroditism  in  sea  urchins  are  described.  In  a  search 
for  causative  factors  of  bisexuality,  a  survey  of  incidence  of  the  phenomenon  in 
echinoids.  both  as  to  genera  and  geographical  distribution,  has  been  made.  The 
suggestion  of  seasonal  dimorphism  is  rejected. 

LITERATURE  CITED 

BUCHNER,  PAUL,  1911.     Ueber  hermaphroditische  Seesterne.     Zool.  Anz.,  38:  315-319. 

CAULLERY,  M.,  1925.  Sur  la  structure  et  le  fonctionnement  des  gonades  chez  les  echinides. 
Traraux  Stat.  Zool.  de  Wimcreux,  9 :  21-35. 

DRZEWINA,  A.,  AND  G.  BOHN,  1924.  Un  nouveau  cas  d'hermaphroditisme  chez  1'oursin  Stron- 
gyloccntrotus liridtis.  C.  R.  Acad.  Sci.,  178:  662-663. 

EGAMI,  N.,  1955.  Production  of  testis  ova  in  adult  males  of  Oryzias  latipcs.  III.  Testis  ovum 
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421-428. 

GADD,  G.,  1907.  Ein  Fall  von  Hermaphroditismus  bei  dem  Strongyloccntrotus  drocbachicnsis. 
Zool.  Anz..  31:  365. 

GIARD,  A.,  1900.  A  propos  de  la  parthenogenese  artificielle  des  oeufs  d'echinodermes.  C.  R. 
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H.  Doran  Co.,  New  York.     P.  165. 
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2233-2235. 
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la  France  et  de  la  Belgique,  87  :  461-468. 
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THE  METABOLISM  OF  RADIONUCLIDES  BY  MARIXE  ORGANISMS. 

I.  THE  UPTAKE,  ACCUMULATION,  AND  LOSS  OF 

STRONTIUM  89  BY  FISHES 

HOWARD  BOROUGHS,  SIDNEY  J.  TOWNSLEY  AND 
ROBERT  W.  HIATT  L 2 

Hawaii  Ularinc  Laboratory,   University  of  Haivaii,  Honolulu    14.  Hawaii 

The  revolution  in  biology  which  occurred  about  twenty  years  ago  as  a  result  of 
the  utilization  of  isotopes  has  led  to  some  problems  which  could  hardly  have  been 
foreseen  at  the  time.  Biologists  can  no  longer  use  radioactive  material  simply  as 
a  tool  with  which  to  solve  specific  problems  ;  they  are  now  forced  to  consider  the 
effects  of  radioactivity  which  is  introduced  beyond  their  own  control  into  an  en- 
vironment which  they  are  studying.  This  becomes  particularly  important  to  marine 
and  fresh  water  biologists  who  are  interested  in  physiological  or  ecological  prob- 
lems which  may  be  influenced  in  certain  regions  by  an  increase  in  radioactivity  above 
the  background  which  existed  before  the  first  atomic  detonation  at  Alamagordo  in 
1945. 

The  weapons  testing  program  of  several  nations,  regardless  of  the  type  of  blast, 
has  increased  the  radioactivity  of  the  seas.  Underwater  detonations,  of  course, 
contribute  the  largest  percentage  of  their  radioactivity  directly  to  the  water,  but 
most  of  the  fall-out  from  aerial  bursts  can  be  expected  to  appear  ultimately  in  the 
ocean,  since  the  land  area  of  the  earth  is  only  about  30  per  cent  of  the  total.  Run- 
off from  the  land  will  also  increase  the  radioactivity  of  the  seas.  Far  more  impor- 
tant than  the  radioactivity  which  appears  as  a  result  of  weapons  testing,  however, 
is  the  radioactivity  which  inevitably  will  be  introduced  into  the  oceans  from  nuclear 
power  plant  wastes  and  atomic-powered  ships. 

There  are  several  important  reasons  for  studying  the  metabolism  of  fission  prod- 
ucts and  other  radionuclides  in  marine  organisms.  First,  several  fission  products 
are  known  to  be  potential  hazards  from  a  public  health  standpoint  (  N.B.S.  Hand- 
book 52).  Second,  almost  nothing  is  known  about  the  metabolism  of  these  radio- 
elements  by  marine  species.  Third,  we  cannot  tell  at  present  what  potential  eco- 
logical effects  may  be  brought  about  through  the  deleterious  action  of  radiation 
on  the  marine  biota,  but  the  possibility  exists  that  some  adverse  changes,  such  as 
those  which  apparently  occurred  in  White  Oak  Lake  ( Krumholz,  1956),  might 
occur  in  estuaries  and  other  inshore  regions.  It  is  therefore  important  to  study 
these  problems  now,  before  the  oceans  become  polluted  with  radioactivity,  because 
the  changes  which  may  occur  will  be  irreversible  at  least  for  several  centuries. 

The  problems  raised  by  the  above  considerations  can  best  be  solved  by  studying 
the  metabolism  of  these  radionuclides  not  only  in  individual  organisms,  but  also  in 
relation  to  the  various  trophic  levels  by  way  of  the  food  chains.  As  desirable  as 
such  studies  may  be,  it  is  impossible  to  undertake  investigations  of  this  magnitude 

1  This  work  was  carried  out  with  the  aid  of  Contract  No.  AT  ( 04-3 )  -50  between  the  U.  S. 
Atomic  Energy  Commission  and  the  University  of  Hawaii. 

-  Contribution  No.  82,  Hawaii  Marine  Laboratory,  University  of  Hawaii. 

336 


METABOLISM  OF  RADIOSTRONTIUM  BY  FISH  337 

for  the  entire  marine  biota.  Thus,  because  of  our  special  facilities  at  the  Hawaii 
Marine  Laboratory,  we  have  confined  our  research  to  fishes  which  are  representative 
of  three  distinct  marine  habitats,  and  include  herbivores  belonging  to  the  second 
trophic  level,  and  carnivores  from  the  third  and  fourth  trophic  levels. 

Strontium  was  selected  for  our  initial  studies  for  two  reasons.  First,  it  is  chemi- 
cally similar  to  calcium,  and  is  therefore  a  "bone  seeker."  As  such,  if  radioactive, 
it  may  interfere  with  the  blood  cell  formation  of  many  animals.  Second,  Sr90  has 
a  half-life  of  about  28  years,  so  that  the  deposition  of  an  atom  of  Sr90  into  a  tissue 
which  has  a  slow  rate  of  turnover  may  result  in  radiation  exposure  for  the  entire 
life  of  the  animal.  These  characteristics  make  radiostrontium  a  particularly  haz- 
ardous fission  product.  In  these  experiments  we  have  used  Sr8'1  because  of  its 
much  shorter  half-life  (—'53  days)  which  decreases  the  danger  of  contamination 
in  the  laboratory. 

The  particular  objective  of  the  present  study  was  to  measure  the  uptake,  accu- 
mulation, and  loss  of  radiostrontium  by  the  various  tissues  and  organs  of  selected 
species  of  fish  when  the  isotope  was  given  orally,  by  intramuscular  injection,  and 
by  the  immersion  of  the  fish  in  sea  water  enriched  with  Sr89. 

MATERIALS  AND  METHODS 

The  large  pelagic  fishes  used  in  these  experiments  consisted  of  the  black  skipjack 
(Euthynnus  yaito),  the  yellowfin  tuna  (Ncotlmnnus  macropterus},  and  the  so- 
called  dolphin  (Coryphaena  Jiif>f>itrus).  These  species  are  fast-swimming,  wide- 
ranging  carnivores  which  occupy  the  fourth  trophic  level.  Among  the  small  fish 
used,  the  papio  (Carangoides  a/a.r),  and  the  aholehole  (Kuhlia  sandvicensis) ,  are 
small  carnivores  common  along  the  reefs  and  shores  in  the  Hawaiian  Islands  and 
occupy  the  third  trophic  level  primarily.  The  aholehole  is  also  able  to  adapt  itself 
to  brackish  water  environments,  and  has  even  been  found  well  into  fresh  water 
streams  (Tester  and  Takata,  1953;  Tester  and  Trefz,  1954).  The  third  small 
species  used,  Tilapia  niossauibica,  is  a  sluggish  fish,  predominantly  herbivorous, 
but  facultatively  omnivorous,  and  may  be  placed  in  the  second  trophic  level.  It 
prefers  brackish  water,  but  is  well  adapted  to  either  fresh  water  or  sea  water. 

Carrier-free  strontium89  was  obtained  from  Oak  Ridge  and  fed  to  the  large  fishes 
by  filling  gelatine  capsules  with  cracker  crumbs  and  a  measured  quantity  of  the 
isotope  solution.  The  capsule  was  sewn  into  a  small  piece  of  fish  muscle  which 
was  held  just  under  the  surface  of  the  water  by  a  weak  thread.  As  the  fish  swal- 
lowed the  bait,  the  thread  was  broken  off.  In  this  way  the  capsule  could  be  given 
to  a  particular  fish.  In  some  instances  small  fishes  were  force-fed  a  gelatine  cap- 
sule prepared  in  the  same  way.  Others  were  fed  by  incorporating  a  measured 
amount  of  Sr89  into  a  gelatine  solution  which  was  allowed  to  solidify  in  a  small 
plastic  tube.  The  tube  was  put  into  the  fish's  stomach  and  the  gelatine  was  ex- 
truded with  the  aid  of  a  syringe.  A  dose  solution  was  prepared  by  extruding  the 
same  quantity  of  radioactive  gelatine  into  a  volumetric  flask. 

The  large  fish  were  perfused  with  a  mixture  of  two  parts  sea  water  and  three 
parts  distilled  water.  The  brain,  eyes,  spinal  cord  and  integument  were  removed, 
and  the  excised  internal  organs  were  further  soaked  in  distilled  water  until  no  blood 
was  apparent  in  the  water.  All  the  rinsings  were  added  to  the  blood.  The  gut 
was  opened,  and  any  material  remaining  in  it  was  flushed  out.  Only  the  eyes  and 


338 


BOROUGHS,  TOWNSLEY  AND  HIATT 


the  visceral  organs  were  removed  from  the  small  fish  which  were  not  perfused. 

The  remainder  of  the  fish,  consisting  of  muscle  and  skeleton  for  the  large  fish, 
or  muscle,  skeleton  and  integument  for  the  small  fish,  was  then  put  in  a  pressure 
cooker,  brought  to  20  pounds  pressure  and  allowed  to  cool.  After  this  treatment, 
the  muscle  was  easily  removed  from  the  bones,  and  any  flesh  remaining  on  the  gill 
arches  was  removed  with  warm  formamide.  Control  experiments  indicated  that 
no  leaching  or  loss  of  strontium  occurred  as  a  result  of  the  pressure  cooker  treatment. 

Wet  weight  of  organs  was  obtained  without  blotting,  and  the  tissues  were  dried 
at  about  110°  C.  for  48  hours.  The  dried  tissues  were  put  in  a  muffle  furnace  which 
was  brought  to  about  550°  C.  The  furnace  was  then  shut  off  and  the  samples  left 
overnight.  A  slow  stream  of  air  was  introduced  in  the  oven  to  aid  combustion. 

The  ash  was  ground  and  spread  evenly  on  aluminum  planchettes  with  the  aid 
of  water  and  a  detergent,  and  dried  under  infra-red  lamps.  The  samples  were 


100 


10 


1000 


24  100 

Hours    after    dose 

FIGURE  1.     The  decrease  of  ingested   Srs<1  in  pelagic  fishes  as  a  function  of  time. 

counted  in  triplicate  when  possible,  using  commercial  counters  and  sealers.  A 
minimum  of  2560  counts  was  taken  on  each  sample,  including  background.  No 
corrections  were  made  for  back-scatter,  self-scatter,  or  self-absorption.  The  latter 
is  very  small  at  the  ash  densities  which  were  used  «  5  mg./cm.2).  The  sealers 
were  calibrated  daily  with  Bureau  of  Standards  nuclides,  and  the  only  correction 
applied  was  for  radioactive  decay.  The  counts/minute  of  the  samples  were  com- 
pared with  aliquots  of  the  actual  close  given  in  each  instance.  Specific  details  for 
each  experiment  will  be  described  at  the  appropriate  place. 

RESULTS  AND  DISCUSSION 
A.  Ingcstion  of  5VS"  by  large  pelagic  fishes 

Figure  1  shows  that  the  excretion  of  a  single  dose  is  very  rapid  :  about  50  per 
cent  disappeared  within  a  few  hours,  and  only  1-2  per  cent  was  left  after  24 
hours.  This  latter  value  persisted  for  the  remainder  of  the  experiment  which 
lasted  27  days. 


METABOLISM  OF  RADIOSTRONTIUM  BY  FISH 


339 


Table  I  shows  the  distribution  of  the  Sr89  in  the  various  organs  and  tissues  of 
these  fishes,  and  Figure  2  is  a  graph  showing  some  of  these  data.  This  graph  is 
presented  as  the  percentage  of  radioactivity  of  the  different  organs  and  tissues  in 
terms  of  the  total  radioactivity  found  in  the  entire  fish  when  it  was  killed.  It  is 


100 


Head  operc. 
Gills 


Integument 
Muscle 


10  100 

Hours    after  dose 


*"A  kidney,  Gonad 
_^<  Y^'sC&ecum 
\X  \Livcr 

Fore  gut 
Heart 

1000 


FIGURE  2. 


The  distribution  of  a  single  ingestion  of  Sr89  in  organs  and  tissues  of  pelagic  fishes 

as  a  function  of  time. 


apparent  that  the  tissues  are  segregated  into  two  groups  with  regard  to  strontium 
retention :  the  visceral,  and  the  structural.  The  visceral  organs  and  tissues,  in- 
cluding the  blood,  kidney,  foregut,  midgut,  hindgut,  spleen,  liver,  caecum  and  heart, 
show  a  continuing  decrease  in  radioactivity  beginning  one  hour  after  the  adminis- 
tration of  the  dose.  The  structural  tissues,  including  the  skeleton,  head  and 


340 


BOROUGHS,  TOWNSLEY  AND  HIATT 


opercular  bones,  gill  arches,  integument  and  muscle  appear  to  concentrate  strontium 
rapidly  to  a  level  which  is  maintained  more  or  less  constant  for  a  relatively  long 
period  of  time.  The  turnover  or  excretion  of  strontium  in  these  structures  is 
therefore  slow. 

TABLE  I 

Accumulation  of  ingested  Sr89  in  the  various  organs  and  tissues  of  pelagic 
carnivorous  fishes  expressed  as  percentage  of  total  activity 


Tissue 

Species 

EYi 

EY 

EY 

CH? 

EY 

EY 

EY 

NM3 

EY 

EY 

Dose  in 

MC 

5.55 

480 

240 

51.0 

240 

464 

464 

464 

371 

371 

Duration 
in  hours 

1 

21 

6 

7 

11* 

24 

96 

264 

480 

648 

Heart 

0.04 

0.01 

0.03 

0.049 

0.11 

0.05 

0.028 

0.01 

0.014 

0.007 

Gall  bladder 

0.05 

0.04 

0.07 

0  ID 

0.08 

0.03 

0.0001 

0.01 

0.004 

0.002 

Blood 

4.21 

6.68 

15.00 

0.85 

8.07 

2.73 

1.14 

0.35 

0.51 

0.12 

Gill  flesh 

12.44 

0.18 

0.91 

8.56 

5.06 

30.61 

25.72 

2.42 

1.42 

2.21 

Gill  bone 

1.34 

6.47 

26.39 

16.80 

19.48 

22.76 

Caecum 

37.01 

7.67 

7.84 

2.70 

2.64 

0.34 

0.15 

0.05 

0.04 

0.029 

Foregut 

0.89 

9.32 

1.12 

0.74 

1.03 

0.20 

0.24 

0.04 

0.01 

0.018 

Midgut 

2.28 

14.16 

16.50 

1.08 

1.48 

0.65 

0.25 

0.05 

0.036 

0.003 

Hindgut 

11.78 

3.98 

21.26 

2.26 

0.11 

0.15 

0.024 

0.03 

0.015 

0.016 

Gut  contents 

— 

48.32 

12.73 

0.056 

19.65 

— 

0.10 

0.013 

— 

0.0008 

Head,  operculum 

— 

0.41 

1.09 

24.99 

6.28 

18.33 

24.58 

28.18 

29.91 

24.58 

Appendicular  skeleton 

3.60 

0.40 

1.19 

36.21 

8.45 

23.69 

30.32 

29.15 

30.47 

31.43 

Liver 

3.34 

1.48 

3.04 

0.39 

2.46 

0.15 

0.04 

0.03 

0.04 

0.027 

Spleen 

0.20 

0.32 

1.39 

0.08 

(l.(,o 

0.03 

0.008 

0.03 

0.010 

0.003 

Tail 

— 

0.42 

0.15 

— 

0.00 

— 

— 

— 

— 

— 

Brain,  spinal  cord 

0.23 

0.00 

0.01 

1.24 

0.05 
0.60 

1.66 

1.70 

1.33 

0.030 

0.004 

Eyes 

0.04 

0.06 

2.02 

1.34 

1.34 

Integument 

5.  28 

1.69 

0.86 

10.20 

5.89 

7.69 

11.37 

13.73 

10.25 

10.51 

Integument  flesh 

(aliquot) 

— 

0.01 

0.01 

— 

0.05 

— 

— 

— 

— 

0.065 

Integument  scales  (aliquot) 

— 

0.02 

0.02 

— 

0.11 

— 

— 

— 

— 

0.091 

Gonad 

2.40 

0.09 

0.47 

0.22 

0.08 

0.06 

0.004 

0.03 

0.023 

0.020 

Kidney 

O.OS 

O.K. 

0.09 

0.027 

0.07 

0.035 

0.022 

Light  muscle 

16.23 

3.23 

8.74 

4.19 

10.01 

12.84 

3.94 

5.26 

5.69 

5.79 

Dark  muscle 

0.10 

0.86 

5.25 

0.70 

0.72 

0.48 

0.47 

0.63 

0.95 

1  EY    =  Euthynnus  yaitn. 

-  CH    =  Coryphaena  hippitnis. 

3  NM   =  Neothunnus  macropterus. 


We  do  not  interpret  the  departures  from  a  smooth  curve  for  any  one  organ  to 
indicate  a  sequential  pattern.  In  other  words,  a  rise  in  the  radioactivity  of  one 
organ  and  the  fall  of  radioactivity  in  another  are  not  necessarily  linked  by  way  of 
precursor  relations.  Each  point  on  the  graph  represents  a  single  fish,  and  indi- 
vidual differences  can  most  likely  account  for  the  small  deviations  of  the  curves. 


METABOLISM  OF  RADIOSTRONTIUM  BY  FISH 


341 


In  order  to  study  the  sequential  pattern  of  strontium  metabolism,  a  much  larger 
group  of  fish  would  have  to  be  used,  particularly  since  it  is  known  that  there  is  a 
very  large  difference  in  the  time  a  food  bolus  remains  in  a  fish  as  compared  with 
another  fish  of  the  same  species  living  in  the  same  tank. 

The  rank  order  of  radioactivity  in  the  organ  systems  of  these  fishes  is  :  skeleton, 
gills,  integument,  muscle  and  viscera.  It  is  interesting  to  note  that  the  dark  muscle, 
which  has  a  better  supply  of  blood,  has  less  radioactivity/gram  ash  than  has  the 
light  muscle.  Similarly,  the  "specific  activity"  of  the  gills,  that  is,  the  counts/ 
minute/mg.  ash,  was  considerably  higher  than  that  of  the  axial  skeleton.  Goldberg 
(personal  communication)  has  analyzed  yellowfin  tuna  for  various  metals,  and  found 
that  the  gill  arches  and  filaments  had  considerably  more  strontium  in  them  than  had 


80r 


70 


60 


I   50 
o 

E 


o> 
to 
o 


30 


CD 
O 


10 


_L 


-o- 
_JL 


_L 


_L 


6789 
Days    after    dose 


10 


12 


13 


-o 

_J 

14 


FIGURE  3.     The  decrease  of  ingested  Sr89  in  Tilapia  mossambica  as  a  function  of  time. 

the  bones.  His  values  for  strontium  in  the  gills  would  therefore  be  a  minimum 
value,  since  any  flesh  adhering  to  the  sample  would  tend  to  dilute  the  strontium 
concentration.  The  main  chemical  difference  between  the  gill  arches  and  the  re- 
maining skeletal  tissue  is  the  presence  of  cartilage  in  the  gill  rakers.  In  our  own 
work,  and  in  the  work  of  others  (Jones  and  Copp,  1951),  there  is  a  suggestion 
that  cartilage  may  have  a  greater  capacity  to  exchange  ionic  calcium  for  strontium 
than  has  bone.  For  example,  we  have  found  a  higher  "specific  activity"  in  the 
eye,  which  has  cartilaginous  ossicles,  than  we  have  found  in  skeletal  bone.  More- 
over, Jones  and  Copp  found  that  the  uptake  of  strontium  by  the  skeleton  is  more 
rapid  in  young  rats  than  it  is  in  adults.  It  is  possible  that  the  explanation  of  these 
differences  might  lie  with  an  increased  amount  of  Sr++  binding  by  the  protein  of 
the  cartilage  as  compared  to  that  bound  by  the  protein  of  calcified  bones.  Perhaps 
differences  in  the  amount  of  blood  supplied  to  ossified  and  cartilaginous  tissue,  or 
some  other  properties  of  cartilage  may  also  be  involved. 


342 


BOROUGHS,  TOWNSLEY  AND  HIATT 


TABLE  II 

Percentage  of  total  radioactivity  recovered  in  various  organs  of  Tilapia  mossambica. 
(Each  horizontal  column  represents  an  individual  fish  given  a  dose  of  20  ^c) 


Duration 

Tissue 

Integument 

Eyes 

Visceral 
organs 

Gills 

Muscle 

Skeleton 

2hr. 

10.58 

0.26 

31.88 

28.02 

9.44 

19.81 

1.73 

0.08 

88.01 

1.43 

0.83 

7.92 

4hr. 

12.54 

0.36 

28.64 

23.50 

6.45 

28.52 

0.32 

0.07 

65.81 

11.34 

10.88 

11.58 

1.70 

0.29 

24.41 

36.18 

6.09 

31.34 

8hr. 

10.64 

1.38 

44.50 

6.90 

3.72 

32.85 

8.01 

0.24 

44.40 

15.83 

5.46 

26.06 

19.66 

1.62 

11.99 

17.10 

1.74 

47.88 

12  hr. 

23.07 

0.24 

42.34 

3.65 

7.61 

23.08 

4.63 

1.26 

78.36 

1.95 

1.61 

12.19 

7.86 

0.67 

40.92 

8.94 

4.26 

37.36 

24  hr. 

25.67 

0.32 

1.45 

9.43 

8.96 

54.18 

24.93 

0.36 

1.60 

8.60 

3.52 

60.99 

28.31 

0.53 

3.57 

8.74 

4.82 

54.03 

48  hr. 

22.67 

0.31 

0.62 

4.81 

1.62 

69.97 

5.79 

0.83 

3.32 

14.35 

7.39 

68.32 

26.48 

0.29 

1.76 

8.40 

2.30 

60.77 

4  days 

23.10 

0.16 

0.40 

5.99 

2.17 

68.17 

26.71 

0.27 

0.64 

8.08 

2.38 

61.92 

23.63 

0.17 

0.93 

8.20 

3.35 

63.72 

7  days 

21.88 

0.22 

0.97 

8.02 

2.26 

66.65 

20.84 

0.21 

0.57 

8.28 

1.92 

68.18 

14  days 

31.42 

0.24 

0.17 

8.92 

1.97 

56.73 

29.63 

0.41 

1.10 

10.06 

2.10 

56.69 

20.39 

0.22 

0.51 

8.07 

1.24 

69.57 

B.  Ingcstion  of  5YS9  by  Tilapia 

Figure  3  shows  that  the  rate  of  excretion  of  Srso  by  Tilapia  is  much  slower  than 
that  by  the  pelagic  fishes.  About  50  per  cent  is  still  present  after  one  day,  and  the 
time  required  to  reach  a  more  or  less  constant  level  is  about  four  days.  The  amount 


METABOLISM  OF  RADIOSTRONTIUM  BY  FISH 


343 


which  persists  is  also  larger  than  that  observed  with  the  pelagic  fishes,  although 
the  variability  among  the  Tilapia  was  fairly  large.  The  Tilapia  used  in  these  ex- 
periments were  fed  the  Sr89  in  gelatine  capsules  containing  cracker  crumbs.  Occa- 
sionally crumbs  were  observed  in  the  carboys  used  to  hold  three  of  the  experimental 
fish,  and  therefore  the  true  dose  could  not  be  ascertained.  The  incorporation  of 
the  isotope  in  gelatine  for  the  later  experiments  has  apparently  obviated  this 
difficulty. 

Table  II  presents  the  data  concerning  the  percentage  of  the  total  radioactivity 
recovered  in  the  various  organs  and  tissues.  Figure  4  is  a  graph  of  this  informa- 
tion except  that  the  ordinate  is  given  in  microcuries/gram  fresh  weight  of  fish. 


10000 


1000 


0010 


0001 


678 
Doys     offer      dose 


10 


12 


13 


14 


FIGURE  4.     The  distribution  of  a  single  ingestion  of  Sr89  in  organs  and  tissues  of 
Tilapia  mossambica  as  a  function  of  time. 


The  structural  tissues  account  for  the  bulk  of  the  radioactivity  where  approximately 
90-95  per  cent  of  the  activity  is  present  in  both  Tilapia  and  the  pelagic  fishes. 
Roughly,  about  60  per  cent  is  accounted  for  by  the  skeleton,  30  per  cent  by  the 
integument,  10  per  cent  by  the  gills,  2  per  cent  by  the  muscle,  and  1  per  cent  by  the 
viscera.  A  larger  percentage  of  radioactivity  is  found  in  the  integument  of  Tilapia 
than  in  the  integument  of  the  other  fishes,  probably  because  a  larger  percentage  of 
the  body  weight  of  this  species  is  due  to  the  large  scales.  The  percentage  of  the 
total  radioactivity  in  Tilapia  was  found  to  decrease  in  the  following  order :  skeleton, 
integument,  gills,  muscle  and  viscera.  The  order  in  the  pelagic  fishes  studied  was 
skeleton,  gills,  integument,  muscle  and  viscera. 


344  BOROUGHS,  TOWNSLEY  AND  HIATT 

C.  The  ingcstion  of  Srso  by  aholehole 

Five  aholehole  were  each  fed  48  /xc  of  Srs9  in  gelatine  capsules  and  kept  in  run- 
ning sea  water  24  hours  before  killing.  The  entire  fish  was  then  dried  and  ashed, 
and  Table  III  shows  that  the  results  are  much  more  reproducible  than  they  were 
using  Tilapia.  After  24  hours  the  latter  fish  retained  approximately  the  same  per- 
centage of  the  dose  as  did  the  aholehole,  but  the  range  was  between  2  and  20.  No 
further  experiments  were  conducted  with  aholehole  at  this  time,  but  because  of  their 
apparent  superiority  as  a  laboratory  animal  we  plan  to  use  them  in  experiments 
which  will  be  reported  at  a  later  date. 

Although  we  have  completed  some  experiments  on  the  repetitive  feeding  of  Sr89, 
the  uncertainty  of  the  exact  dose  in  some  instances,  and  the  excessive  range  of  re- 
tention by  Tilapia  during  short  periods  have  caused  us  to  omit  these  data  here. 
The  results  of  such  experiments  on  other  fish  will  be  reported  at  a  later  date. 

TABLE  III 

Percentage  of  Srw  retained  by  aholehole  24  hours  after  ingest  ion 

Per  cent 
Fish  of  dose 

1  10.43 

2  7.42 

3  9.16 

4  5.70 

5  7.97 
Aver.  8.13 

D.  The  uptake  and  accumulation  of  5Y89  injected  intramuscularly  into  tuna  and 
Tilapia 

Because  our  results  showed  that  the  biological  half-life  3  of  strontium  in  fish 
muscle  fell  in  the  same  range  as  that  found  for  the  bones  and  integument,  rather 
than  with  the  soft  tissues  as  was  expected,  additional  experiments  were  devised  to 
study  the  retention  of  Sr  by  muscle.  These  experiments  also  enabled  us  to  study 
the  metabolism  of  radiostrontium  which  \vas  introduced  by  a  method  other  than 
feeding. 

A  yellowfin  tuna  was  injected  with  288  ^c  of  Sr89  at  the  base  of  the  pectoral  fin. 
The  fish  was  killed  and  analyzed  after  it  had  been  in  running  sea  water  for  19  hours. 
A  comparison  of  the  percentage  of  Sr8!)  recovered  in  the  organs  and  tissues  of  a 
tuna  which  received  the  isotope  by  injection  with  that  of  a  tuna  receiving  an  oral 
dose  (Table  IV)  shows  that  the  digestive  organs  of  the  latter  were  relatively  more 
radioactive  than  were  the  corresponding  organs  of  the  injected  fish.  This  result  is 
perhaps  to  be  expected,  but  there  are  several  other  outstanding  differences  in  the 
distribution  of  the  isotope  within  this  time  period.  The  muscle  tissue  of  the  fish 
receiving  the  injection  had  almost  three  times  the  percentage  of  Sr89  as  had  the 
muscle  of  the  fish  receiving  the  radioisotope  orally.  The  gills  of  the  former,  on 
the  other  hand,  had  only  about  half  the  percentage  of  Srs9  as  had  the  orally  dosed 

3  We  define  the  term  "biological  half-life"  in  this  paper  to  mean  the  time  required  for  half 
the  labelled  strontium  to  be  removed  from  the  tissue  or  animal  in  question,  exclusive  of  radio- 
active decay. 


METABOLISM  OF  RADIOSTRONTIUM  BY  FISH  345 

fish.  The  percentage  of  radioactivity  in  the  integument  and  the  skeleton  of  both 
fish  was  about  the  same,  and  it  is  not  possible  to  state  whether  or  not  the  differences 
found  in  the  remaining  organs  are  significant. 

The  fact  that  such  a  large  percentage  of  the  Sr"'1  was  retained  by  the  muscle 
suggested  that  it  might  be  informative  to  study  this  process  over  a  longer  period 
of  time.  Thus,  Tilapia  were  injected  intramuscularly  between  the  caudal  and  anal 
fin  with  Sr89  C12  neutralized  to  about  pH  6.  The  fish  were  kept  in  aerated,  but 
not  circulating,  sea  water  for  the  first  24  hours,  during  which  time  samples  of  the 
water  in  the  aquaria  were  removed.  After  24  hours  the  fish  were  put  in  running 
sea  water  and  removed  at  intervals.  Figure  5  shows  that  the  radiostrontium  was 
rapidly  excreted  for  about  the  first  twelve  hours,  and  that  the  rate  decreased 
thereafter. 

TABLE  IV 

A  comparison  of  the  percentage  of  Srw  recovered  in  the  organs  and 
tissues  of  tuna  related  to  the  route  of  administration 

Tissue  Oral1  Injected2 

Integument  7.69  7.15 

Gill  bones  Ln  ,,  1  15.25 

/-•it  a      u  1  3U.61  I  1    01 

Gill  flesh  l.ol 

Head,  operculum  18.33  14.41 

Appendicular  skeleton  23.69  23.25 

Light  muscle  12.84  33.24 

Dark  muscle  0.72  1.51 

Foregut  0.20  0.11 

Midgut  0.65  0.36 

Hindgut  0.15  0.07 

Kidney/gonad  0.06  0.20 

Heart  0.05  0.04 

Caecum  0.34  0.16 

Liver  0.15  0.13 

Spleen  0.03  0.02 

Gall  bladder  0.03  0.37 

Blood  2.73  1.79 

1  Skipjack,  duration  24  hours. 

2  Yellowfin,  duration  19  hours. 

An  analysis  of  the  percentage  of  the  dose  retained  by  the  fish  confirmed  the 
fact  that  after  24  hours  comparatively  little  strontium  was  excreted,  and  that  most 
of  the  strontium  remaining  was  held  by  the  fish  for  the  duration  of  the  experiment. 
These  results  are  shown  graphically  in  Figure  6.  The  points  on  the  graph  repre- 
sent the  average  of  three  fish.  The  greater  retention  of  the  five-day  fish  as  com- 
pared to  the  one-day  fish  can  be  ascribed  to  individual  variation.  The  range  of 
retention  between  the  one-  and  the  21 -day  fish  is  of  the  same  order  of  magnitude 
as  the  range  of  retention  at  any  single  time  interval.  In  other  words,  the  curve, 
neglecting  individual  differences,  is  very  likely  parallel  to  the  abscissa. 

The  internal  distribution  of  the  injected  Sr89  requires  longer  to  reach  a 
"levelling-off"  than  does  Sr89  given  orally  (Fig.  7).  In  the  former  instance,  the 
time  required  is  about  one  week,  whereas  in  the  latter  instance  the  "levelling" 
occurs  within  two  days.  In  both  situations,  however,  the  percentage  of  the  total 
radioactivity  retained  by  each  of  the  organ  systems  is  the  same. 


346  BOROUGHS,  TOWNSLEY  AND  HIATT 

2400 
2200  - 
2000- 

£  1800  - 

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1400- 


1200- 


1000- 


800- 


600- 


400 


2  34 


8  12  16 

Hours    offer  injection 


24 


FIGURE  5.     The  rate  of  excretion  of  Srs9  injected  intramuscularly  into  Tilapia  iiwssaiubica. 

The  aquaria  used  in  these  experiments  were  inverted  five-gallon  carboys  with 
the  bottoms  removed.  Feces  and  other  solid  material  thus  settled  to  the  neck  of 
the  carboy  and  could  be  removed  through  glass  tubing  which  just  penetrated  the 
rubber  stopper.  In  this  way  the  feces  were  removed  from  the  tanks  six  times 
during  the  first  24  hours  at  each  sampling  of  the  tank  water.  The  average  total 


METABOLISM  OF  RADIOSTRONTIUM  BY  FISH 


347 


100 


80 


TJ 

O> 


2!  60 
8 

k_ 

to 

•5  40 


01 

o 


g.   20 


16 


18 


20 


2  4  6  8  10  12  14 

Days     after     injection 

FIGURE  6.     The  continuing  retention  of  SrS!l  injected  intramuscularly  into  Tilapia  mossambica. 


70 


60 


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isceral  organs 


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Skeleton 


Integument 


GiliS 


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Days     after    injection 


16 


18 


FIGURE  7.     The  internal  distribution  of  Srs9  injected  intramuscularly  into   Tilapla  mossambica. 


348  BOROUGHS,  TOWNSLEY  AND  HIATT 

Sr89  recovered  in  the  feces  was  0.35  per  cent  of  the  dose  at  24  hours,  but  the  amount 
of  leaching  is  unfortunately  unknown. 

The  injected  fish  retained  a  much  greater  percentage  of  the  dose  than  did  the 
fish  which  received  the  strontium  orally.  These  results  suggest  that  the  injected 
Sr89  was  actually  absorbed  to  a  greater  extent,  and  that  the  absorption  of  strontium 
through  the  gut  is  not  efficient.  Slow,  continuous  diffusion  from  the  site  of  injec- 
tion may  also  allow  a  larger  amount  to  become  incorporated  into  the  various  tissues. 
If  the  rate  of  blood  supply  to  the  muscles  is  less  than  that  to  the  visceral  organs, 
one  might  expect  the  muscle  to  retain  the  strontium  for  a  comparatively  longer 
period.  The  fact  that  the  dark  muscle  of  tuna  retained  much  less  strontium  than 
did  the  light  muscle  (Table  I),  and  the  fact  that  the  dark  muscle  is  better  supplied 
with  blood  than  is  the  light  muscle,  suggest  that  the  degree  of  vascularization  is  of 
some  importance.  One  might  therefore  reasonably  expect  the  cartilaginous  tissues, 
such  as  the  gill  rakers  and  gill  rays,  to  retain  the  strontium  for  longer  periods  than 
does  the  bone  which  is  better  supplied  with  capillaries. 

Although  the  amount  of  strontium  in  tuna  muscles  and  visceral  organs  is  of  the 
same  order  of  magnitude  (Goldberg,  personal  communication),  some  specific  bind- 
ing of  strontium  may  occur  with  muscle  protein  which  does  not  occur  with  the 
proteins  of  the  visceral  organs.  Further,  the  very  long  biological  half-life  of 
strontium  in  the  muscle  suggests  that  fish  muscle  may  not  be  in  such  a  "dynamic 
state"  as  one  ordinarily  assumes  according  to  the  researches  of  Schoenheimer  and 
his  associates  (Schoenheimer,  1942).  Moreover,  there  is  evidence  that  mammalian 
muscle  protein  has  a  very  much  slower  turnover  rate  than  has  the  proteins  of  the 
visceral  organs  ( Tarver  and  Schmidt,  1942).  Therefore,  the  slow  turnover  of 
strontium  in  the  fish  muscle  may  be  only  a  reflection  of  the  slow  turnover  of  muscle 
in  general. 

E.  The  uptake  and  accumulation  of  SYs9  in  solution  bv  Tilapia 

Because  the  pattern  of  distribution  of  Sr89  in  the  tissues  and  organs  of  several 
species  of  fish  appears  to  be  similar,  both  when  the  isotope  was  given  orally  and  by 
injection,  one  might  extrapolate  and  conclude  that  regardless  of  the  mode  of  entry, 
the  internal  distribution  of  Sr89  ultimately  would  be  the  same.  However,  to  secure 
more  information  on  this  point,  and  to  ascertain  whether  or  not  fish  could  take  up 
strontium  directly  from  the  sea  water,  a  situation  which  is  possible  in  nature,  the 
experiments  described  below  were  carried  out. 

Six  Tilapia  were  put  into  each  of  four  tanks  containing  20  liters  of  filtered  sea 
water  and  1744  ^c  of  Sr89.  The  water  was  aerated  during  the  experiment,  but  the 
fish  were  not  fed.  The  total  amount  of  Srs9  available  during  the  experiment  can  lie 
considered  constant,  since  even  at  21  days,  the  total  Sr89  removed  by  six  fish  in  a 
tank  was  less  than  one  per  cent  of  the  available  dose. 

Figure  8  shows  the  rate  of  uptake  from  solution  in  terms  of  microcuries  of 
Sr89/gram  fresh  weight  of  fish.  The  rate  slows  down  considerably  after  about  a 
week,  but  uptake  is  apparently  still  continuing.  The  ordinate  on  the  right  indicates 
that  within  21  days,  the  ratio  of  internal  Sr89  to  external  Sr*9  is  still  less  than  one. 

The  internal  distribution  of  the  Sr89  taken  up  from  solution  is  shown  in  Figure 
9.  The  Sr89  found  in  the  skeleton  is  about  40  per  cent  as  compared  with  a  value 
close  to  60  per  cent  when  the  strontium  is  fed  or  injected.  The  amount  found  in 


METABOLISM  OF  RADIOSTRONTIUM  BY  FISH 


349 


the  integument  in  all  cases  is  about  the  same,  and  the  gills  and  muscles  show  little 
variation.  The  amount  found  in  the  visceral  organs,  however,  is  markedly  differ- 
ent. The  individual  organs  were  not  ashed  and  counted  separately  because  of  their 
very  small  size,  so  it  is  not  possible  to  state  in  what  organ  or  organs  the  very  large 
percentage  of  Srso  was  located. 

Since  marine  fish  in  general  swallow  more  water  than  do  fresh  water  fish  to 
maintain  proper  osmotic  balance,  it  is  possible  that  the  principal  route  of  entry  of 
the  isotope  in  solution  is  by  way  of  the  gut.  However,  our  direct  feeding  experi- 


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FIGURE  8.     The  uptake  of  Sr89  in  solution  by  Tilapia  mossambica,  expressed  as  the 

concentration  ratio. 

nients  with  a  variety  of  fishes  indicate  that  strontium  is  rapidly  eliminated  from 
all  the  visceral  organs.  How  is  it,  then,  that  so  much  strontium  remains  in  the 
visceral  organs  when  the  fish  is  immersed  in  the  isotope?  A  probable  explanation 
is  that  the  fish  is  being  fed  the  isotope  continually,  in  effect,  every  time  it  swallows. 
Since  the  concentration  of  Sr89  was  found  to  be  higher  in  the  sea  water  than  in  the 
fish,  only  a  small  amount  of  sea  water  present  in  the  gut  would  account  for  the  large 
percentage  of  total  Sr89  which  was  found  in  the  visceral  organs.  The  concentra- 
tion of  Sr89  in  the  sea  water  was  8.7  X  10"2  /xc/ml.  Assuming  arbitrarily  that  50 
per  cent  of  the  total  Sr89  of  the  fish  was  in  the  visceral  organs  (Fig.  9),  this  amount 
equals  about  12  X  10~3  /xc/gram  fresh  weight  of  the  organs.  If  only  from  0.1-0.2 
ml.  of  sea  water  was  present  in  the  gut,  this  would  account  for  the  radioactivity 


350 


BOROUGHS,  TOWNSLEY  AND  HIATT 


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o  Muscles 
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Days    in    Sr89  sea    water 

FIGURE  9.     The  internal  distribution  of  Srs9  taken  up  by  Tilapia  iiwssambica  from   sea  water. 


found.  What  is  important,  then,  is  the  rank  order  of  radioactivity  in  the  various 
organs  and  systems.  Excluding  the  visceral  organs  for  the  reasons  given  above, 
it  is  seen  that  the  other  organ  systems  studied  fall  in  the  same  rank  order  regardless 
of  the  mode  of  entry  of  the  radiostrontium. 


SUMMARY 

1.  The  ingestion  of  Srsfl  by  large  pelagic  fishes  results  in  the  excretion  of  most 
of  the  isotope  in  a  few  hours.     The  small  percentage  remaining  after  one  day  per- 
sisted for  the  27  days  of  the  experiment.     The  strontium  is  rapidly  eliminated  from 
the  visceral  organs  and  tissues,  but  the  structural  tissues,  including  the  bones,  gills, 
integument  and  muscle,  maintain  their  strontium  level  more  or  less  constant.     The 

o 

turnover  of  strontium  in  these  latter  tissues  is  therefore  slow. 

2.  Dark  muscle,  which  has  a  better  blood  supply  than  light  muscle,  retains  less 
Sr89.     Similarly,  bone,  which  is  better  supplied  with  blood  than  is  cartilage,  retains 
less  Sr89  than  the  gill  arches  or  the  cartilaginous  eye  ossicles. 

3.  The  excretion  of  Sr*"  by  Tilapia  uiossauibica  is  much  slower  than  it  is  by  the 
pelagic  fishes.     The  percentage  of  the  dose  retained  is  somewhat  larger,  and  most 
of  the  radioactivitv  is  found  in  the  structural  tissues. 


METABOLISM  OF  RADIOSTRONTIUM  BY  FISH  351 

4.  About  three  times  as  much  Sr89  was  found  in  the  muscle  of  an  injected  tuna 
as  compared  with  another  fish  receiving  the  isotope  orally.     The  gills  of  the  former 
fish  had  only  about  half  the  activity  found  in  the  latter.     From  60-70  per  cent  of 
the  dose  injected  into  Tilapia  muscle  was  retained  by  these  fish  for  14  days.     The 
long  biological  half-life  of  Sr*9  in  fish  muscle  is  contributing  evidence  for  the  slow 
turnover  of  muscle  tissue  in  comparison  with  such  tissues  as  liver  or  kidney. 

5.  Tilapia  were  able  to  concentrate  Sr89  directly  from  the  sea  water,  although 
the  ratio  of  Srso  in  the  fish  to  the  Sr89  in  an  equal  weight  of  sea  water  was  only 
about  0.3  after  three  weeks.     Except  for  the  visceral  organs,  the  rank  order  of  the 
retention  of  radioactivity  in  the  various  tissues  is  skeleton,  integument,  gills  and 
muscle.     This  is  the  same  distribution  as  was  observed  after  oral  administration  of 
Sr89.     Because  marine  fish  swallow  water  continually,  a  small  amount  of  water  in 
the  gut  might  account  for  the  relatively  large  percentage  of  radioactivity  found  in 
the  viscera. 

LITERATURE  CITED 

JOXES,  D.  C.,  AND  D.  H.  COPP,  1951.     The  metabolism  of  radioactive  strontium  in  adult,  young, 

and  rachitic  rats.     Biol.  Chan.,  189:  509. 
KRUMHOLZ,  Louis  A.,   1956.     Observations  on  the  fish  population  of  a  lake  contaminated  by 

radioactive  wastes.     Bull.  Aincr.  Mus.  Nat.  Hist.,  110(4)  :  281-367. 

NATIONAL  BUREAU  OF  STANDARDS  HANDBOOK  52,  1953.     Maximum  permissible  amounts  of  radio- 
isotopes  in  the  human  body  and  maximum  permissible  concentrations  in  air  and  water. 
SCHOENHEIMER,    R.,    1942.     The   dynamic   state   of   body   constituents.     Harvard    Univ.    Press, 

Cambridge,  Mass. 

TARVER,  H.,  AND  C.  L.  A.  SCHMIDT,  1942.     Radioactive  sulfur  studies.     Biol.  Chein.,  146:  69. 
TESTER,  ALBERT  L.,  AND  MICHIO  TAKATA,  1953.     Contribution  to  the  biology  of  the  aholehole, 

a  potential  baitfish.     hid.  Res.  Adris.  Council,  Terr,  of  Haivaii,  Grant  No.  29,  Final 

Report. 
TESTER,  ALBERT  L.,  AND  S.  M.  TREFZ,   1954.     The  food  of  the  aholehole,   Kulilia  sand-cicensis 

(Steindachner),  in  Hawaiian  waters.     Pacific  Science,  8(1)  :  3-10. 


THE  METABOLISM  OF  RADIONUCLIDES  BY  MARINE  ORGANISMS. 
II.  THE  UPTAKE,  ACCUMULATION,  AND  LOSS  OF  YTTRIUM91 
BY  MARINE  FISH,  AND  THE  IMPORTANCE  OF  SHORT- 
LIVED RADIONUCLIDES  IN  THE  SEA  ^  - 

HOWARD  BOROUGHS,  SIDNEY  J.  TOWNSLEY  AND  ROBERT  W.  HIATT 
Hawaii  Marine  Laboratory,  University  of  Hazvaii,  Honolulu  14,  Hazvaii 

A  study  of  the  metabolism  of  radioyttrium  is  important  for  two  reasons :  first, 
yttrium90  occurs  as  the  daughter  of  long-lived  strontium90,  an  element  which  has 
considerable  interest  from  the  standpoint  of  public  health,  and  second,  yttrium91 
occurs  as  a  direct  fission  product  to  the  extent  of  about  four  per  cent  of  the  total 
radioactivity  present  in  a  fission  product  mixture  one  year  old.  Fission  products 
are  being  introduced  into  the  seas  to  a  small  extent  as  a  result  of  fall-out,  and  also 
from  nuclear  reactor  plants  located  near  the  seaboard.  The  latter  situation  now 
occurs  in  the  Irish  Sea  near  Harwell,  and  more  activity  of  this  tvpe  may  occur  as 

J  J   J7  J 

reactor  plants  increase  in  number.  Spooner  (1949),  in  a  noteworthy  study  on  the 
metabolism  of  radioyttrium  by  marine  algae,  showed  that  this  element  was  capable 
of  being  concentrated  by  certain  algae,  and  thus  stimulated  the  interest  of  marine 
biologists  to  learn  more  about  the  metabolism  of  the  nuclide  of  mass90. 

Because  we  had  begun  experiments  on  the  metabolism  of  strontium89  and 
strontium90,  both  of  which  contain  a  certain  amount  of  yttrium90,  we  were  interested 
in  comparing  the  metabolism  of  these  two  elements  in  marine  fish.  Although  the 
ratio  of  Sr90 :  Y90  in  an  equilibrium  mixture  is  about  3000:  1,  it  was  conceivable 
that  some  of  the  results  we  obtained  might  have  come  from  the  small  amount  of 
yttrium  present. 

In  a  number  of  instances  the  concentration  of  certain  elements  is  sometimes 
greater  within  a  marine  organism  than  it  is  in  the  surrounding  sea  water  (Noddack 
and  Noddack,  1939;  Vinogradov,  1953).  For  example,  one  cannot  predict,  let 
alone  explain,  why  one  species  of  green  algae  will  accumulate  yttrium  almost  ex- 
clusively from  a  mixture  of  strontium  and  yttrium,  \vhile  another  accumulates  stn5n- 
tium  exclusively.  Thus,  Rice  (1956)  reports  that  Carteria  sp.  takes  up  100  per 
cent  of  its  radioactivity  from  the  strontium  in  a  Sr90-Y90  mixture,  but  Chlorella  sp., 
which  is  also  a  member  of  the  Chlorophyceae,  has  95  per  cent  of  its  radioactivity  in 
the  form  of  yttrium.  Such  tremendous  differences  in  accumulation  cannot  be  ex- 
plained on  the  basis  of  size,  that  is,  surface  per  gram  protoplasm.  Rather,  the  ex- 
planation is  more  likely  to  be  one  involving  the  chemical  nature  of  the  surfaces 
among  the  different  algae.  Because  closely  related  algae  are  able  to  concentrate 
one  element  more  than  another,  it  is  not  unreasonable  to  suspect  that  one  organ  of 
a  fish  might  have  a  greater  avidity  for  yttrium  than  it  has  for  strontium.  Unfor- 

1  This  work  was  carried  out  with  the  aid  of  Contract  No.  AT  (04-3) -56  between  the  Atomic 
Energy  Commission  and  the  University  of  Hawaii. 

2  Contribution  No.  83,  Hawaii  Marine  Laboratory,  University  of  Hawaii. 

352 


METABOLISM  OF  RADIOYTTRIUM  BY  FISH 

Innately ,  the  small  size  of  the  fish  used  in  these  experiments  did  not  permit  us  to 
separate  the  visceral  organs  in  detail,  so  that  we  can  only  report  the  amount  of  radio- 
activity found  in  the  integument,  skeleton,  gills,  muscle,  and  in  the  combined  visceral 


organs. 


MATERIALS  AND  METHODS 

Yttrium01,  obtained  from  Oak  Ridge,  was  incorporated  into  a  two  per  cent 
gelatine  solution,  and  0.5  ml.  was  drawn  up  into  a  piece  of  Tygon  tubing  of  small 
diameter.  When  the  solution  solidified,  it  was  extruded  directly  into  the  fish's 
stomach  with  the  aid  of  a  syringe.  The  dose  was  5.5  microcuries.  The  fish  used 
were  Tilapia  inossainbica,  each  of  which  weighed  about  100  grams.  After  the 
radioyttrium  was  administered,  three  fish  were  put  into  a  single  carboy  with  20  liters 
of  sea  water  which  had  been  filtered  through  a  No.  4  Mandler  filter.  Four  carboys 
were  used  for  the  four  time  intervals  of  1,  2,  4  and  14  days.  The  water  was  aerated, 
but  the  fish  were  not  fed.  Twenty-four  hours  after  the  administration  of  the 
yttrium,  the  fish  were  put  in  tanks  with  running  sea  water  where  they  were  kept 
until  killed  rapidly  by  flooding  the  gill  chamber  with  ether.  After  removing  the 
eyes  and  the  visceral  organs,  the  remainder  of  the  fish  was  put  in  a  pressure  cooker 
which  was  brought  to  20  pounds  pressure  and  then  allowed  to  cool.  This  process 
allows  the  skeleton  and  integument  to  be  separated  easily  from  the  muscle  with  no 
leaching  of  the  radioisotope.  The  separated  organs  and  tissues  were  dried  and 
ashed  at  about  550°  C.  The  ash  was  spread  on  aluminum  planchettes  with  the  aid 
of  a  wetting  agent,  dried  under  infra-red  lamps,  and  counted  with  a  G-M  tube  and 
a  conventional  sealer  for  a  minimum  of  2560  counts.  Yttrium91  has  a  maximum 
energy  of  1.5  Mev.  No  corrections  were  made  for  the  self-absorption  which  was 
very  small  at  the  densities  employed  «  6  mg./cm.2).  An  aliquot  of  the  dose  was 
counted  for  reference  and  to  correct  for  decay. 

RESULTS 

Figure  1  shows  that  radioyttrium  is  very  rapidly  excreted  by  Tilapia.  After  two 
days,  the  fishes  retained  only  about  two  per  cent  of  the  ingested  dose  (1.6  ±  0.5). 
In  a  similar  experiment,  the  average  amount  of  Sr89  retained  by  Tilapia  after  two 
days  wTas  20  per  cent,  and  even  after  14  days,  the  average  retention  was  about  six 
per  cent  (Boroughs,  Townsley  and  Hiatt,  1956).  The  actual  absorbed  dose  may 
be  something  less  than  these  values,  because  marine  fishes  swallow  water  in  order 
to  maintain  their  osmotic  balance.  A  small  amount  of  recently  swallowed  water 
would  therefore  be  trapped  in  the  gut  unabsorbed,  but  contributing  to  the  radio- 
activity in  the  visceral  organs.  Any  feces  remaining  in  the  gut  would  also  add  to 
the  radioactivity  of  the  visceral  organs.  In  both  instances,  however,  the  amount 
would  be  small. 

Figure  2  indicates  that  most  of  the  yttrium  is  retained  in  the  viscera.  In  rats, 
the  liver,  kidney  and  spleen  accumulate  yttrium  (Hamilton,  1948),  but  our  methods 
did  not  permit  us  to  localize  the  accumulation  of  yttrium  in  the  visceral  organs. 
Future  experiments  to  disclose  this  are  underway.  Assuming  that  all  the  radio- 
activity recovered  in  the  14-day  fishes  was  absorbed  (1.3  per  cent  of  the  dose),  the 


354 


BOROUGHS,  TOWNSLEY  AND  HIATT 


viscera  retained  43  per  cent,  the  muscle  29  per  cent,  the  skeleton  16  per  cent,  the 
integument  8  per  cent,  and  the  gills  4  per  cent.  The  large  percentage  of  yttrium 
accumulated  in  the  fish  muscle  was  wholly  unexpected,  for  in  rats  65  per  cent  of 
the  absorbed  dose  was  found  in  the  bones  (Hamilton,  1948). 


100 


J L 


J L 


24  6  8  10  12 

DAYS     AFTER     INGESTION 

FIGURE  1.     The  loss  of  yttrium91  after  ingestion  by  Tilapia  mossambica. 


14 


METABOLISM  OF  RADIOYTTRIUM  BY  FISH 


355 


10000 


1000 


.  0 100 


0010  • 


000 


5678 
Days     after      ingeslion 


10 


12 


13 


14 


FIGURE  2.     The  internal  distribution  of  yttrium91  fed  to  Tilapia  mossambica  via  stomach  tube. 

DISCUSSION 

Similar  experiments  with  radiostrontium  (Boroughs,  Townsley  and  Hiatt, 
1956)  have  indicated  that  in  14  days,  Tilapia  have  about  60  per  cent  of  the  absorbed 
dose  in  the  skeleton,  28  per  cent  in  the  integument,  9  per  cent  in  the  gills,  2  per  cent 
in  the  muscle,  and  1  per  cent  in  the  viscera.  The  rapid  excretion  of  radioyttrium 
by  Tilapia,  coupled  with  the  different  patterns  of  internal  distribution,  indicate  that 
the  metabolism  of  strontium  and  yttrium  in  this  species  is  markedly  different. 
However,  this  is  not  too  surprising  in  view  of  the  difference  in  chemical  behavior 
between  these  two  elements.  Studies  on  the  accumulation  of  strontium  by  pelagic 
fish  (Boroughs,  Townsley  and  Hiatt,  1956)  showed  that  this  element  is  rapidly 
absorbed  by  all  the  tissues,  but  is  also  rapidly  lost  from  the  visceral  organs  and  the 
blood.  Similar  experiments  with  Tilapia,  a  small  sluggish  fish,  were  unsatisfactory 
because  of  the  large  variability  in  the  amount  of  Sr89  absorbed  over  periods  up  to 
24  hours.  We  therefore  made  no  attempt  to  follow  the  pathway  of  yttrium  imme- 
diately after  ingestion,  but  very  likely  yttrium  is  not  actually  absorbed  to  the  extent 
that  is  strontium.  At  24  hours,  about  60  per  cent  of  the  dose  was  still  in  the  entire 
fish,  but  most  of  this  was  in  the  gut.  However,  at  least  5  per  cent  of  the  dose  was 
absorbed  by  the  muscle  alone.  This  is  at  least  100  times  more  than  the  amount 
absorbed  by  rats  from  an  oral  dose  (Hamilton,  1948),  and,  moreover,  represents  a 
minimum  value  for  Tilapia. 

The  maximum  percentage  of  ingested  strontium  which  was  recovered  in  the  feces 
of  Tilapia  during  24  hours  was  less  than  1  per  cent.  The  feces  were  removed  six 
or  seven  times  during  this  interval,  but  some  leaching  of  Sr89  may  have  occurred. 


356  BOROUGHS,  TOWNSLEY  AND  HIATT 

The  percentage  of  Y91  recovered  in  parallel  experiments  has  been  as  much  as  20 
per  cent  of  the  dose. 

If  the  length  of  time  required  for  an  organ  to  excrete  one-half  of  its  concentra- 
tion of  a  particular  element  is  very  long  ( biological  half-life  ) ,  then  the  effective 
biological  half-life  approaches  the  half-life  of  the  radioisotope  involved  (radioactive 
decay).  Elements  which  lodge  in  mammalian  bones  appear  to  have  a  very  long 
biological  half-life.  According  to  Figure  2,  however,  the  biological  half-life  of 
yttrium  in  all  the  tissues  of  Tilapia  is  of  the  order  of  one  month.  This  value  is  of 
course  only  a  guess,  and  long  term  experiments  will  have  to  be  carried  out  to  verify 
this  estimate.  In  man,  the  body  burden  tolerance  for  Y91  is  given  as  about  15  times 
that  of  Sr90  (N.B.S.  Handbook  52),  and  in  both  instances  the  bulk  of  the  radio- 
activity appears  in  the  skeleton.  In  one  year,  the  radioactivity  owing  to  Y91  would 
be  reduced  to  a  negligible  amount  because  of  both  excretion  and  decay,  but  very 
little  Sr90  would  be  lost  in  this  time.  However,  in  organisms  other  than  man, 
particularly  marine  organisms,  one  year  may  be  a  substantial  part  of  their  life  span, 
and  it  is  for  this  reason  that  we  urge  that  attention  be  paid  to  the  metabolism  of 
radioactive  fission  products  other  than  strontium.  While  strontium  may  constitute 
the  most  serious  direct  health  hazard  to  man,  long  term  effects  of  other  shorter- 
lived  fission  products  may  have  significant  effects  on  the  shorter-lived  biota,  and 
thus  ultimately  may  also  prove  of  importance  to  man. 

There  is  no  evidence  that  the  slight  increase  in  the  radioactivity  of  the  oceans 
has  as  yet  caused  any  adverse  ecological  changes.  Moreover,  there  is  no  evidence 
that  adverse  ecological  changes  will  occur  even  as  a  result  of  the  introduction  of 
much  larger  amounts  of  radioactivity  from  nuclear  reactor  plants  which  are  certain 
to  be  established  within  the  next  decade  or  so.  If  the  present  power  requirements 
of  the  world  are  to  be  met  with  the  aid  of  atomic  energy,  it  is  likely  that  a  sort  of 
steady-state  condition  will  occur  with  regard  to  the  added  radioactivity  in  the 
oceans — the  result  of  a  balance  between  the  rate  of  introduction  of  radioactive 
wastes,  the  rate  of  physical  decay,  and  the  rate  of  biological  turnover.  It  is  there- 
fore imperative  that  marine  biologists  study  in  great  detail  the  problems  of  the  up- 
take and  accumulation  of  fission  products,  the  transfer  of  these  nuclides  back  and 
forth  among  the  trophic  levels,  and  the  direct,  long-term  effects  of  the  nuclides  in 
specific  regions.  Estuaries  and  the  littoral  zone  will  most  likely  have  a  higher 
concentration  of  radioactivity  than  the  open  sea,  and  it  is  in  these  regions  that  the 
bulk  of  the  world's  marine  resources  is  produced. 

SUMMARY 

Only  about  2  per  cent  of  an  ingested  dose  of  yttrium91  was  left  in  Tilapia  inos- 
sainbica  after  two  days.  This  is  much  less  than  the  amount  of  strontium  retained 
by  Tilapia  in  similar  experiments.  About  40  per  cent  of  the  radioisotope  remaining 
is  found  in  the  visceral  organs,  but  the  muscles  retain  about  30  per  cent  after  14 
days.  The  skeleton  retained  less  than  20  per  cent,  the  integument  about  10  per 
cent,  and  the  gills  5  per  cent.  These  findings  are  in  marked  contrast  with  those 
obtained  with  strontium80  in  similar  experiments.  Attention  is  focused  on  the  fact 
that  yttrium91  may  have  little  direct  effect  on  man  compared  with  the  possible  ef- 
fects of  Sr90,  but  the  retention  of  this  and  other  short-lived  fission  products  in 


METABOLISM  OF  RADIOYTTRIUM  BY  FISH 

marine  organisms  having  a  brief  life  span  may  possibly  affect  the  biota,  and  thus 
affect  man  indirectly. 

LITERATURE  CITED 

BOROUGHS,  HOWARD,  SIDNEY  J.  TOWNSLEY  AND  ROBERT  W.  HIATT,  1956.  The  metabolism  of 
radionuclides  by  marine  organisms.  I.  The  uptake,  accumulation,  and  loss  of  stron- 
tium89 by  fishes.  Biol.  Bull,  111:  336-351. 

HAMILTON,  JOSEPH  G.,  1948.  The  metabolic  properties  of  the  fission  products  and  actinide  ele- 
ments. Rev.  Mod.  Physics,  20:  718-728. 

NATIONAL  BUREAU  OF  STANDARDS  HANDBOOK  52,  1953.  Maximum  permissible  amounts  of 
radioisotopes  in  the  human  body  and  maximum  permissible  concentrations  in  air  and 
water. 

NODDACK,  IDA,  AND  WALTER  NODDACK,  1939.  Die  Haufigkeiten  der  Schwermetalle  in  Meere- 
stieren.  Arkiv.  Zool,  32(4)  :  1-35. 

RICE,  THEODORE  R.,  1956.  The  accumulation  and  exchange  of  strontium  by  marine  planktonic 
algae.  Limnol.  and  Occanogr.,  1  :  123-128. 

SPOONER,  G.  M.,  1949.  Observations  on  the  absorption  of  radioactive  strontium  and  yttrium  by 
marine  algae.  /.  Alar.  Biol.  Assoc.,  U.K.,  28 :  587-625. 

VINOGRADOV,  A.  P.,  1953.  The  elementary  chemical  composition  of  marine  organisms.  Scars 
Found.  Mar.  Res.  Mem.  No.  II. 


IMMOBILIZING  AND  PRECIPITATING  ANTIGENS  OF 

PARAMECIUM 

IRVING  FINGER  1,  = 
Zoological  Laboratory,  University  of  Pennsylvania,  Philadelphia,  Pennsylvania 

The  pattern  of  inheritance  of  the  immobilization  antigens  of  Paramecium  aurelia 
and  the  serological  basis  for  the  specific  immobilization  reactions  have  been  studied 
by  Sonneborn  (1951),  Beale  (1952),  van  Wagtendonk  and  van  Tijn  (1953)  and 
F*inger  (1955).  The  systems  of  transformation  from  one  serotype  to  another  have 
also  been  investigated  (Sonneborn,  1950a;  Beale,  1948)  and  possible  mechanisms 
suggested  (Kimball,  1947;  Sonneborn,  1950b ;  Delbruck,  1949).  Further  work  on 
the  unique  systems  of  inheritance  and  stability  exhibited  by  the  immobilization  an- 
tigens has  been  hampered  by  the  lack  of  a  rapid,  reproducible  method  for  assaying 
them.  We  have  now  been  able  to  detect  these  antigens  through  the  development 
of  a  method  which  involves  a  modification  of  the  techniques  of  Oudin  (1952)  and 
Oakley  and  Fulthorpe  (1953).  Specifically,  a  particular  band  formed  by  the  dif- 
fusion of  two  reactants  (antigen  solution  and  antiserum )  from  opposite  ends  of  an 
agar  column  has  been  identified  as  a  complex,  in  part,  of  precipitated  immobilization 
antigen  and  its  homologous  antibody.  The  present  paper  deals  with  the  immobili- 
zation antigens  and  this  in  vitro  method  for  detecting  them. 

MATERIALS  AND  METHODS 

Sera  against  whole  animals  prepared  according  to  the  methods  described  by 
Sonneborn  (1950c)  against  several  stocks  of  varieties  2,  4  and  8  were  employed. 
These  were  tested  with  variety  2  antigen  extracts  of  stock  3  and  extracts  of  animals 
that  were  derived  from  the  variety  2  stocks  7,  30  and  35.  The  survey  of  antigenic 
types  found  in  variety  2,  upon  which  much  of  the  work  reported  here  is  based,  has 
been  submitted  for  publication.  The  antigen  solutions  were  prepared  in  several 
ways :  ( 1 )  Cilia  were  obtained  from  paramecia  and  extracted  as  follows  ( Preer  and 
Finger,  unpublished).  Six  million  animals  were  concentrated  by  centrifugation  and 
one  volume  placed  in  twenty  volumes  of  0.22  %  sodium  chloride  buffered  at  pH  7.8 
with  0.01  M  sodium  phosphate.  At  the  end  of  ten  minutes  the  animals  were  cen- 
trifuged  lightly  at  890  g  for  two  minutes ;  the  supernatant  (containing  mainly  cilia) 
was  retained  and  again  centrifuged  lightly.  Any  sediment  of  animals  and  tricho- 
cysts  formed  was  then  removed  and  the  process  repeated  until  the  supernatant  was 
free  of  animals.  The  supernatant  was  then  centrifuged  for  two  minutes  at  24,000  g 
in  order  to  sediment  the  cilia.  Then  one  ml.  of  0.9 %  sodium  chloride,  buffered  to 
pH  7.0  with  0.01  M  sodium  phosphate,  was  added  to  the  centrifugate.  After  24 

1  Philadelphia  Brewers  Association  Fellow,  1954-55.     This  work  was  also  supported  by  a 
grant  from  the  Phi  Beta  Psi  Sorority  administered  by  Dr.  John  R.  Preer. 

2  Present  address  :  Departments  of  Neurology  and  Microbiology,  College  of  Physicians  and 
Surgeons,   Columbia  University   and  the   Neurological   Institute,    Presbyterian   Hospital,    New 
York. 

358 


ANTIGENS  OF  PARAMECIUM  359 

hours  at  room  temperature  this  suspension  was  centrifuged  at  24,000  g  for  two 
minutes  and  the  clear  supernatant  used  as  antigen.  (2)  Lyophilized  animals  were 
placed  in  a  0.9%  sodium  chloride  solution  overnight  in  a  refrigerator,  centrifuged 
at  24,000  g  for  two  minutes  and  the  supernatant  used  as  antigen.  (3)  Animals 
were  concentrated  by  centrifugation,  placed  in  a  deep  freeze,  and,  after  thawing  at 
room  temperature,  the  entire  brei  was  used  as  antigen.  (4)  A  brei  was  made  by 
repeatedly  forcing  living  animals  from  a  syringe  against  the  walls  of  a  glass  cylinder 
at  room  temperature.  The  resulting  homogenate  was  then  centrifuged  for  ten 
minutes  at  24,000  g  and  the  supernatant  used  as  antigen.  These  four  preparations 
were  not  equally  effective  in  yielding  the  immobilization  antigen  and  are  listed  in 
increasing  order  of  efficiency.  When  quantitatively  comparable  data  were  desired 
the  antigen  solutions  used  were,  of  course,  obtained  by  the  same  method. 

The  method  of  gel  diffusion  has  been  given  in  detail  by  Freer  (1956).  It  is  as 
follows :  Pyrex  tubes,  approximately  2-mm.  inside  diameter,  were  coated  with  a 
0.1%  agar  solution,  evacuated,  cut  into  4-cm.  lengths  and  flame-sealed  at  one  end. 
To  set  up  a  double-diffusion  test  0.01  ml.  of  antiserum  was  added  with  a  syringe 
to  a  tube  held  upright  in  a  Cartesian  diver  loader.  Then  0.6%  washed,  merthio- 
lated,  and  buffered  (pH  7.0)  agar  maintained  at  60°  C.  was  carefully  layered  with  a 
warm  syringe  until  an  agar  column  of  6-8  mm.  height  was  reached.  A  third  layer 
of  0.01  ml.  antigen  solution  was  placed  on  the  agar  after  it  had  solidified.  The  tube 
was  then  sealed  with  Picene  cement  and  placed  in  a  horizontal  position  at  24°  C. 
When  serum  dilutions  were  used,  normal  serum  was  used  as  the  diluent.  This  was 
to  prevent  mixing  of  antiserum  with  the  warm  agar  at  the  time  of  layering.  The 
diluent  for  the  antigen  was  usually  0.9%  sodium  chloride  or,  less  frequently,  10% 
Ringer's  solution. 

The  location  of  a  zone  of  precipitation  within  the  agar  was  used  as  a  measure  of 
the  concentration  of  one  of  the  reactants  (antigen  or  antibody)  contributing  to  the 
band  when  the  concentration  of  the  other  reactant  was  kept  constant  (Freer,  1956). 
The  position  of  a  band  was  measured  after  24-72  hours  with  a  binocular  dissecting 
microscope  provided  with  an  eyepiece  micrometer. 

Variations  on  these  general  techniques,  e.g.,  mutual  dilution,  absorption  experi- 
ments, etc.  are  described  below. 

RESULTS 

When  a  solution  of  several  antigens  is  diffused  against  homologous  antiserum, 
bands  of  precipitate  will  be  formed  as  a  result  of  the  specific  complexing  of  antigens 
with  antibody.  Because  the  position  of  a  band  is  dependent  on  several  factors 
(concentration  of  reactants,  diffusion  coefficients,  etc.),  each  serologically  distinct 
precipitating  system  usually  appears  as  a  separate  zone  of  precipitation.  Oc- 
casionally, however,  two  or  more  precipitating  systems  may,  through  a  fortuitous 
combination  of  these  factors,  appear  as  a  single  band.  Therefore  the  number  of 
bands  will  represent  the  minimum  number  of  antigen-antibody  systems  present. 

Many  workers  have  demonstrated  that  P.  anrelia  may  manifest  a  number  of  dif- 
ferent antigenic  types,  called  "serotypes,"  and  designated  by  the  letters  A,  B,  C, 
etc.  When  animals  of  a  given  serotype  are  placed  into  a  suitable  dilution  of  anti- 
serum  prepared  against  that  type,  their  locomotion  slows,  and  they  become  im- 
mobilized. Each  serotype  is  serologically  distinct  from  any  of  the  others,  an  animal 


360  IRVING  FINGER 

of  a  particular  serotype  generally  becoming  immobilized  only  when  placed  in  ho- 
mologous antiserum.  If  a  number  of  antisera  are  allowed  to  diffuse  against  ex- 
tracts of  homologous  serotypes  and  against  extracts  of  heterologous  serotypes,  then 
it  should  be  possible  to  correlate  the  presence  of  certain  bands  with  these  specific 
immobilization  antigen-antibody  systems. 

When  animals  of  the  G  serotype  were  used  as  the  source  of  antigens  and  G 
antiserum  diffused  against  the  antigen  solution,  a  band  formed  that  was  not  present 
when  an  antigen  solution  prepared  from  non-G  animals  reacted  with  a  G  antiserum. 
Six  different  stocks  of  P.  aiirelia,  all  of  serotype  G,  possessed  a  similar  antigen,  an 
antigen  absent  in  extracts  of  two  stocks  of  a  serotype  other  than  G  and  in  non-G 
animals  of  two  other  species,  P.  caudatinn  and  P.  polycaryuui. 

Analogous  results  were  obtained  with  C  antigen-antiserum  systems.  A  band 
appeared  in  these  homologous  systems  that  was  missing  when  the  antigen  solution 
was  obtained  from  non-C  animals. 

To  provide  further  evidence  that  this  antigen  was  found  only  in  homologous 
serotypes,  absorption  experiments  were  performed.  It  was  found  that  extracts  of 
several  stocks  manifesting  the  G  serotype  were  able  to  remove  the  specific  precipi- 
tin  from  G  antisera  while  several  stocks  of  a  different  serotype  and  non-G  P.  cau- 
datiiin  and  P.  polycarymn  preparations  were  ineffective. 

The  precipitating  antigen  associated  with  a  particular  serotype  was  further 
homologized  between  stocks  through  the  use  of  a  mutual  dilution  technique  ( Oudin, 
1952;  Telfer  and  Williams,  1953).  Although  all  antigen  preparations  were  able 
to  form  a  distinctive  band  when  diffused  against  homologous  antisera,  it  was  pos- 
sible that  the  bands  formed  with  different  antigen  preparations  did  not  represent 
the  same  antigen-antibody  systems,  the  correspondence  being  coincidental.  An 
antigen  forming  a  band  against  a  particular  serum  may  be  identified  with  an  antigen 
from  a  second  extract  by  mutually  diluting  the  two  extracts,  one  with  the  other, 
and  noting  whether  the  bands  formed  behave  independently,  as  though  diluted  with 
a  neutral  reagent,  or  act  to  reinforce  each  other.  In  order  to  employ  the  mutual 
dilution  technique  with  maximum  effectiveness,  the  concentrations  of  serum  and 
antigen  solution  were  chosen  so  as  to  eliminate  most  or  all  of  the  bands  in  the  sys- 
tem, aside  from  the  ones  being  compared,  and  still  leave  a  band  intense  enough  to 
withstand  a  two-fold  dilution  of  antigen.  For  optimum  resolution  in  a  mutual  di- 
lution series  the  concentrations  were  adjusted  so  that  the  bands  in  the  two  prepa- 
rations being  compared  formed  at  widely  separated  positions  in  the  original  un- 
diluted systems.  As  a  result  of  the  mutual  dilution  studies,  the  bands  formed  with 
different  preparations  of  the  same  serotype  were  shown  to  represent  the  same 
antigen-antibody  system. 

In  summary,  then,  a  study  of  serotypes  G  and  C  has  shown  that  animals  of  each 
serotype  have  a  specific  precipitating  antigen  which  is  lacking  in  animals  of  other 
serotypes.  It  seems  likely,  then,  that  these  antigens  are  the  immobilizing  antigens. 
Such  an  immobilization  antigen  can  be  identified  in  gel-diffusion  systems  in  several 
ways :  ( 1 )  a  comparison  of  bands  formed  with  homologous  and  heterologous  anti- 
gens;  (2)  specific  absorption  by  heterologous  extracts  of  all  antibody  from  a  ho- 
mologous antiserum,  but  the  one  that  forms  a  zone  of  precipitation  with  homologous 
extracts;  and  (3)  the  mutual  dilution  of  an  extract  with  an  antigen  solution  which 
forms  several  bands,  one  of  which  is  known  to  be  formed  by  the  specific 
precipitinogen. 


ANTIGENS  OF  PARAMECIUM 


361 


Although  the  immobilization  antigen  and  the  specific  precipitating  antigen  are 
probably  the  same,  there  is  evidence  that  the  precipitated  band  may  not  be  composed 
solely  of  immobilizing  antigen  and  immobilizing  antibody.  The  evidence  has  been 
obtained  from  a  comparison  of  immobilization  titers  of  antisera  and  precipitin  titers 
as  measured  by  the  specific  band  position. 

This  specific  band  was  identified  as  above  by  diffusing  antisera  against  antigens 
extracted  from  two  clones  with  the  same  genotype  but  of  contrasting  serotypes. 
The  eleven  sera  titered  for  antibody  forming  this  band  were  prepared  against  sev- 
eral different  stocks  and  were  known  to  immobilize  animals  of  the  G  serotype  in 
concentrations  of  1 :  12.5  or  less  after  two  hours  at  room  temperature.  Eight  of 
these  anti-G  sera,  all  having  an  immobilization  titer  of  1  :  50  or  greater,  precipitated 

TABLE  I 

Comparison  of  immobilization  titers  and  gel  diffusion  band  titers  of  antisera  against  P.  aurelia 


Serum 

Anti-G 
titer 

G-  pre- 
cipitin 
titer 

Anti-C 
titer 

C-pre- 
cipitin 
titer 

Seru  in 

Anti-G 
titer 

G-  pre- 
cipitin 
titer 

Anti-C 
titer 

C-pre- 
cipitin 
titer 

P#23 

100 

40 

0 

0 

F#10 

0 

0 

100 

100 

P#16 

100 

9 

0 

0 

P#8 

0 

0 

100 

65 

P#2 

75 

100 

0 

0 

F#17 

0 

0 

50 

30 

P#12 

75 

100 

0 

0 

F#ll 

0 

0 

40 

90 

P#7 

50 

15 

0 

0 

F#4 

0 

0 

25 

25 

P#14 

50 

30 

0 

0 

F#5 

0 

0 

25 

45 

P#17 

50 

15 

0 

0 

F#12 

0 

0 

20 

9 

F#15 

20 

45 

15 

4 

P#18 

0 

0 

15 

4 

F#18 

15 

3 

0 

0 

F#3 

0 

0 

15 

7 

F#14B 

5 

5 

20 

11 

P#19 

0 

0 

5 

7 

F#13B 

3 

1 

0 

1 

F#l 

0 

0 

3 

0 

P#4 

0 

2 

0 

0 

P#21 

0 

3 

0 

0 

The  immobilization  antibody  titers  (anti-G  and  anti-C  titers)  and  the  gel  diffusion  precipitin 
titers  are  presented  on  a  scale  with  sera  having  the  greatest  concentration  of  antibody  listed  as 
100,  and  the  antibody  concentration  of  all  other  sera,  as  compared  with  these  sera,  being  denoted 
by  numbers  from  0-100.  In  this  way,  a  serum  with  half  the  antibody  content  of  the  strongest 
sera  would  be  given  the  number  50,  etc.  The  titers  presented  above  represent  the  means  of 
several  series  of  titrations. 

a  single  band  against  the  G  antigen  solution  that  was  absent  when  preparations  of 
the  C  serotype  \vere  used  as  reactants.  Of  the  three  anti-G  sera  that  showed  the 
same  number  and  type  of  bands  with  both  G  and  C  antigen  solutions,  two  had  im- 
mobilization titers  against  both  serotypes  (F^tlS  and  F#14B)  and  one  (F#13B) 
had  negligible  anti-C  precipitin  titer.  These  data  are  presented  in  Table  I  where 
the  immobilization  titers  of  24  antisera  against  the  G  and  C  serotypes  are  compared 
with  the  precipitating  antibody  concentration  as  determined  by  band  position. 

Similar  results  were  obtained  with  anti-C  sera  when  the  sera  were  titered 
against  homologous  and  heterologous  solutions.  Ten  of  thirteen  antisera  gave  a 
band  with  C  antigen  solutions  not  present  when  G  antigen  solutions  were  used. 
Of  the  three  homologous  antisera  that  did  not  precipitate  this  band,  one  had  a  very 
low  immobilization  titer  (F#l),  and  two  also  had  anti-G  immobilization  titer. 
Thus,  any  differences  that  may  have  existed  due  to  the  C  immobilizing  antibodies 


362  IRVING  FINGER 

would  be  obscured  (F#15  and  F#14B).  Seventeen  sera  prepared  against  sev- 
eral serotypes  (omitted  from  Table  I)  and  with  neither  G  nor  C  immobilization 
antibody  presented  essentially  identical  band  series  with  G  and  C  antigen  solutions. 
Following  the  procedure  used  in  the  studies  on  antigens,  absorption  experiments 
were  employed  to  confirm  the  identification  of  the  immobilization  antibody.  The 
standard  G  antiserum  was  diffused  against  a  G  antigen  extract  that  had  previously 
been  incubated  with  antiserum  at  12°  C.  for  24  hours.  Nine  sera  which  immo- 
bilized G  animals  in  two  hours  when  used  in  dilutions  of  1  :  200  or  greater  success- 
fully absorbed  the  antigen  responsible  for  the  band  found  in  G  antigen-antibody 
systems.  Three  with  poor  G  titers  and  26  without  any  G  titer  when  used  as  ab- 
sorbents had  no  effect  on  the  appearance  or  position  of  the  band. 

A  final  corroborative  group  of  experiments  was  carried  out  using  the  mutual 
dilution  method.  A  single  serum  having  a  high  immobilization  titer  against  G 
animals  and  precipitating  antibody  capable  of  withstanding  several-fold  dilution  was 
chosen  as  a  standard  serum.  When  eight  anti-G  sera  were  mutually  diluted  with 
the  standard  serum,  it  \vas  found  that  the  antibody  restricted  to  the  G  antigen- 
antibody  reaction  and  present  in  all  systems  involving  these  sera  was  either  related 
to  or  identical  with  the  antibody  found  in  the  standard  G  antiserum,  the  band  formed 
being  reinforced  upon  the  addition  of  the  sera  being  tested.  Additional  mutual  di- 
lutions among  the  eight  sera  confirmed  this  finding. 

Although  the  evidence  for  the  identity  of  the  band  characteristic  of  homologous 
systems  with  the  immobilization  antigen  and  antibody  is  convincing,  there  appears 
to  be  a  rather  poor  correlation  between  the  titer  of  a  serum  as  determined  by  im- 
mobilization tests  and  the  concentration  of  precipitating  antibody.  Thus,  serum 
P#16  with  about  the  same  G  immobilization  titer  as  sera  P#23,  P#2,  and 
P^12  has  less  than  25%  of  the  precipitating  antibody  of  these  antisera  (Table  I). 
Other  "exceptional"  sera  are  P#4  and  P#21  which  possess  precipitating  antibody 
and  yet  do  not  immobilize.  It  is  apparent,  then,  that  immobilization  antibody  and 
precipitating  antibody,  although  closely  correlated,  are  not  identical.  The  serologi- 
cal  nature  of  the  relationship  of  the  two  kinds  of  antibodies  is  being  studied.  As 
would  be  expected,  preliminary  absorption  experiments  have  demonstrated  that  it 
is  possible  to  remove  all  precipitating  antibody  from  certain  antisera  without  abol- 
ishing all  immobilizing  activity. 

As  for  the  immobilization  antigens,  although  these  studies  have  demonstrated 
that  in  animals  of  serotype  G  one  particular  precipitating  antigen  is  found  and  in 
animals  of  serotype  C  it  is  not  found,  but  a  second  precipitating  antigen  is,  it  is  not 
known  whether  immobilization  antigens  and  precipitating  antigens  are  identical. 
It  is  possible  that  only  a  portion  of  the  immobilization  antigen  is  capable  of  pre- 
cipitation or  that  there  may  be  precipitating  antigen  that  does  not  take  part  in 
immobilization. 

SUMMARY 

1.  A  study  of  serotypes  G  and  C,  of  variety  2,  Paramecium  aurelia,  has  been 
made,  using  diffusion  in  agar.     It  has  been  shown  that  animals  of  each  serotype 
have  a  specific  precipitating  antigen  which  is  lacking  in  animals  of  other  serotypes. 
Consequently,  it  seems  probable  that  these  antigens  are  the  immobilizing  antigens. 

2.  Comparisons   of  antibody   concentration,   as   determined   by   immobilization 
titers  and  by  band  position,  show  that  the  precipitated  band  may  not  be  composed 


ANTIGENS  OF  PARAMECIUM  363 

solely  of  immobilizing  antigen  and  antibody,  and  that  there  is  precipitating  antibody 
that  is  not  capable  of  immobilizing. 

LITERATURE  CITED 

BEALE,  G.  H.,  1948.  The  process  of  transformation  of  antigenic  type  in  P.  aurclia,  variety  4. 
Proc.  Nat.  Acad.  Sci.,  34:  418-423. 

BEALE,  G.  H.,  1952.     Antigenic  variation  in  P.  aurelia,  variety  1.     Genetics,  37:  62-74. 

DELBRUCK,  M.,  1949.  See  discussion  of  Sonneborn  and  Beale,  Influence  des  genes,  des  plasma- 
genes  et  du  milieu  dans  le  determinisme  des  caracteres  antigenique  chez  Paramecium 
aurclia  (variete  4).  Collogues  internationaux  du  Centre  National  de  le  Recherche 
scicntifiqne,  8 :  33. 

FINGER,  I.,  1955.  The  inheritance  of  ciliary  antigens  in  Paramecium  aurelia,  variety  2.  Ph.D. 
thesis,  University  of  Pennsylvania. 

KIMBALL,  R.  F.,  1947.  The  induction  of  inheritable  modification  in  reaction  to  antiserum  in 
P.  aurelia.  Genetics,  32  :  486-499. 

OAKLEY,  C.  L.,  AND  A.  J.  FULTHORPE,  1953.  Antigenic  analysis  by  diffusion.  /.  Path.  Bact., 
65 :  49-60. 

OUDIN,  J.,  1952.  Specific  precipitation  in  gels  and  its  application  to  immunochemical  analysis. 
Methods  in  Medical  Research,  5 :  335-378. 

FREER,  J.  R.,  1956.  A  quantitative  study  of  a  technique  of  double  diffusion  in  agar.  /.  Im- 
iininol.,  77:  52-60. 

SONNEBORN,  T.  M.,  1950a.     Cellular  transformations.     Harvey  Lectures,  44:  145-164. 

SONNEBORN,  T.  M.,  1950b.     The  cytoplasm  in  heredity.     Heredity,  4:  11-36. 

SONNEBORN,  T.  M.,  1950c.  Methods  in  the  general  biology  and  genetics  of  P.  aurelia.  J.  Exp. 
Zool,  113:  87-143. 

SONNEBORN,  T.  M.,  1951.  The  role  of  the  genes  in  cytoplasmic  inheritance.  In:  Genetics  in  the 
twentieth  century,  edited  by  L.  C.  Dunn.  Macmillan  Co.,  New  York. 

TELFER,  W.  H.,  AND  C.  M.  WILLIAMS,  1953.  Immunological  studies  of  insect  metamorphosis. 
I.  Qualitative  and  quantitative  description  of  the  blood  antigens  of  the  Cecropia  silk- 
worm. /.  Gen.  Physiol,  36 :  389-413. 

VAN  WAGTENDONK,  W.  J.,  AND  B.  VAN  TIJN,  1953.  Cross  reaction  of  serotypes  51A,  51B  and 
51D  of  P.  aurelia,  variety  4.  Exp.  Cell  Res.,  5:  1-9. 


OOGENESIS  IN  HABROTROCHA  TRIDENS   (MILNE) 

W.  SIANG  HSU 
Zoology  Department,   University  of  Washington,  Seattle,   Washington 

The  bdelloid  rotifers  of  about  200  species,  classified  into  19  genera  and  4 
families,  reproduce  exclusively  by  parthenogenesis,  males  being  unknown  in  this 
group.  It  is  therefore  interesting  to  study  the  behavior  of  their  chromosomes 
during  oogenesis.  I  have  reported  such  a  study  on  one  of  them,  Philodina  roseola 
(1956).  Some  of  the  findings  reported  in  that  paper  are  as  follows:  1.  In  Philo- 
dina roseola,  there  are  two  maturation  divisions,  both  equational.  2.  No  indica- 
tion of  synapsis  has  been  observed  between  any  two  of  the  chromosomes.  Indi- 
vidual chromosomes  even  in  the  earliest  oocytes  were  observed  to  be  in  a  con- 
densed state.  The  anaphase  chromosomes  of  the  oogonial  division  do  not  de- 
spiralize  in  forming  the  nuclei  found  in  the  syncytial  ovary.  The  chromosomes, 
after  the  last  oogonial  division,  remain  condensed,  and,  by  progressive  packing 
together,  they  form  first  a  ring  and  then  a  homogeneous  and  spherical  mass  of 
chromatin  occupying  the  center  of  the  nucleus.  When  one  of  these  nuclei  is  iso- 
lated by  the  ovary  to  form  an  oocyte,  its  condensed  chromosomes  do  not  despira- 
lize  into  leptotene  threads,  but  persist  in  a  condensed  state.  As  the  germinal 
vesicle  increases  in  size  they  separate  from  each  other  until  finally  13  condensed 
chromosomes  can  be  easily  counted.  3.  The  zygoid  chromosome  number  in  this 
rotifer  is  13.  Three  of  the  13  chromosomes,  two  dot-shaped  ones  and  one  that  is 
appreciably  longer  than  the  rest,  are  morphologically  distinguishable  from  one 
another  and  from  any  one  of  the  other  ten  (Fig.  37).  It  was  suggested  that  the 
chromosomes  in  this  group  of  animals  may  have  lost  their  homology. 

The  present  paper  reports  observations  made  on  Habrotrocha  tridcns,  which 
belongs  to  the  family  of  Habrotrochidae.  For  methods  employed  in  this  study 
reference  may  be  made  to  my  paper  dealing  with  Philodina  roseola. 

OBSERVATIONS 

As  in  Philodina  roseola,  the  ovary  and  its  accessory  structure,  the  vitellarium, 
in  Habrotrocha  tridcns  are  syncytial.  In  mature  animals,  the  ovary  consists  of 
about  30  nuclei,  with  the  chromosomes  in  each  nucleus  grouped  so  tightly  together 
that  they  form  a  single  spherical  body  of  smooth  outline.  When  one  of  the  nuclei 
is  isolated  by  the  ovary  to  form  an  oocyte,  the  individual  chromosomes  do  not  go 
through  the  meiotic  changes  characteristic  of  oocytes  in  other  animals.  They  sim- 
ply remove  themselves  from  each  other  as  contracted  bodies  while  the  nucleus  in- 
creases in  size. 

Figure  1  illustrates  the  condition  of  the  chromosomes  in  the  nucleus  of  a  young 
oocyte.  At  this  stage,  it  is  still  difficult  to  differentiate  the  individual  chromosomes. 
But  as  the  nucleus  enlarges,  the  chromosomes  begin  to  stand  out  clearly  as  con- 
densed bodies.  If  the  whole  history  of  the  chromosomes  in  the  developing  egg  is 

364 


OOGENESIS  IN  HABROTROCHA  TRIDENS  365 

not  studied  carefully,  the  thread-like  structures  of  a  less  basophilic  character  in  the 
nuclei  illustrated  in  Figures  2  and  3,  for  instance,  may  be  erroneously  interpreted 
as  leptotene  threads,  and  the  intensely  stained  true  chromosomes  in  a  condensed 
state  regarded  as  heterochromatic  sections  of  thread-like  chromosomes.  But  as  the 
oocyte  and  the  germinal  vesicle  increase  in  size,  the  true  chromosomes  become  more 
and  more  separated  from  each  other  and  more  easily  differentiated  tinctorially  from 
the  thread-like  material.  The  true  situation  can  be  clearly  seen  in  Figures  2-9. 
The  stage  of  maximum  growth  of  a  germinal  vesicle  is  seen  in  Figure  10.  At  this 
stage  the  nuclear  sap  appears  in  fixed  material  as  a  fine-meshed  net.  Thirteen 
chromosomes  are  spread  out  widely  apart  from  each  other  and  can  be  most  easily 
counted  at  this  stage.  Two  of  them  are  appreciably  smaller  than  the  others  and 
are  dot-shaped.  These  two,  however,  are  not  of  equal  size.  It  will  be  recalled 
that  in  Philodina  roseola  there  are  also  two  dot-shaped  chromosomes  of  unequal 
size.  But  unlike  Philodina  roseola,  this  form  does  not  possess  among  the  remain- 
ing 1 1  chromosomes  one  that  is  conspicuously  longer  than  the  rest. 

Further  development  from  this  stage  is  indicated  by  a  shrinking  in  mass  on  the 
part  of  the  germinal  vesicle ;  the  nucleus  thus  becomes  reduced  in  size  and  irregular 
in  shape  (Fig.  11).  But  as  this  takes  place,  the  nuclear  sap  seems  to  react  differ- 
ently to  the  fixative.  The  fine-meshed  appearance  no  longer  prevails,  and  threads 
begin  to  make  their  appearance  within  the  nucleus  (Figs.  12-16).  At  this  stage 
there  is  a  difference  between  the  present  form  and  Philodina  roseola.  In  the  latter, 
the  nucleus  keeps  on  decreasing  in  size  to  a  much  more  extreme  degree,  and  finally 
becomes  again  rounded  in  outline ;  whereas  in  Habrotrocha  tridcns,  the  nucleus 
stops  shrinking  much  earlier,  and  I  have  not  observed  any  well-rounded  germinal 
vesicle  of  reduced  size  (Figs.  15-17).  On  the  contrary,  when  the  nucleus  has  de- 
creased in  size  to  the  extent  shown  in  Figure  17,  it  begins  to  break  up.  Figure  17a 
gives  the  condition  of  the  same  nucleus  at  a  lower  level  of  focus  than  the  one  at 
which  Figure  17  was  drawn.  At  this  level  of  focus,  the  nuclear  membrane  shows 
unmistakable  signs  of  disintegration.  After  the  membrane  is  broken,  the  chromo- 
somes are  set  free  in  the  cytoplasm  (Figs.  18  and  19).  In  Philodina  roseola  the 
chromosomes  next  spread  out  into  a  more  or  less  irregular  line  pressed  close  to  the 
wall  of  the  oocyte.  This  line  formation  has  not  been  observed  in  the  present  form, 
and  Figure  19  represents  the  distribution  of  the  chromosomes  most  frequently  ob- 
served at  this  time  of  development. 

At  first  the  cytoplasm  immediately  surrounding  the  free  chromosomes  does  not 
appear  any  different  from  that  seen  in  any  other  area  within  the  egg.  But  when 
the  chromosomes  have  pulled  away  from  the  periphery  of  the  oocyte  and  have  be- 
come more  separated  from  each  other,  they  are  seen  to  be  embedded  in  an  area  of 
cytoplasm  which  appears  to  be  more  vacuolated  than  the  rest  of  the  cytoplasm  in 
the  developing  egg  (Fig.  20).  This  was  also  observed  in  Philodina  roseola.  Then, 
also  as  in  Philodina  roseola,  it  seems  that  under  the  influence  of  the  chromosomes, 
a  homogeneous  and  light-staining  material  is  developed  in  which  the  chromosomes 
are  embedded  (Fig.  21).  It  is  within  the  area  occupied  by  this  material  that  the 
spindle  is  developed  later.  There  seems  to  be  a  change  going  on  in  this  material, 
as  a  result  of  which  the  area  formerly  occupied  by  the  homogeneous  material  now 
appears  to  be  traversed  by  threads.  These  threads  are  not  as  taut  and  trim  in  out- 
line as  the  regular  spindle  fibers  (Fig.  22).  Such  stages  have  been  frequently  ob- 
served, and  in  some  cases  the  fibers  do  give  a  rather  close  resemblance  to  the  regular 


366 


W.  SIANG  HSU 


PLATE  I 


OOGENESIS  IN  HABROTROCHA  TRIDENS  367 

spindle  fibers.  In  Philodina  roseola,  the  next  stage  has  been  found  to  be  a  mono- 
polar  spindle  which  eventually  develops  into  an  orthrodox  bipolar  one  (Hsu,  1956). 
But  in  Habrotrocha  tridcns,  I  have  seen  a  single  tripolar  spindle  which  may  be 
described  as  a  compound  structure  formed  by  three  different  bipolar  spindles,  the 
long  axes  of  which  all  lie  on  the  same  plane  and  so  arranged  with  regard  to  each 
other  that  the  structure  forms  a  somewhat  triangular  configuration.  The  chromo- 
somes form  three  separate  equatorial  plates,  one  on  each  component  spindle  (Fig. 
23).  Unfortunately,  this  is  the  only  one  I  have  observed  in  my  slides. 

Another  spindle,  also  the  only  one  I  have  found  in  my  material,  is  represented 
in  Figure  24.  There  are  two  cones  placed  at  an  angle  as  shown.  The  chromo- 
somes are  gathered  loosely  at  the  general  area  toward  which  the  truncate  ends  of 
the  two  cones  converge.  The  chromosomes  are  not  all  in  one  level  of  focus.  But 
due  to  the  rarity  of  these  spindles,  it  is  simply  unsafe  to  consider  them  definitely 
as  structures  normal  in  Habrotrocha  tridcns.  They  should  be  merely  recorded  and 
left  for  future  discussion  when  more  evidence  becomes  available.  However,  in  view 
of  the  peculiar  spindle  and  its  manner  of  development  observed  in  Philodina  roseola 
where  evidence  was  more  abundant,  the  possibility  that  the  two  peculiar  spindles 
observed  in  Habrotrocha  tridens  may  express  normal  stages  of  development  in  this 
rotifer  cannot  be  entirely  excluded.  If  these  spindles  be  considered  as  normal  struc- 
tures, I  should  then  think  that  they  represent  stages  following  those  depicted  in 
Figures  21  and  22  and  preceding  that  represented  by  Figure  25.  I  would  assume 
that  Figure  24  shows  a  stage  in  which  the  compound  spindle  is  breaking  up  and  a 
bipolar  structure  is  in  the  process  of  forming.  This  process  would  consist  of  a 
disintegration  of  the  base  spindle  in  Figure  23  and  a  movement  on  the  part  of  the 
chromosomes.  Then  a  proper  rotation  of  the  two  remaining  cones  shown  in  Figure 
24  would  produce  an  orthodox  bipolar  spindle  such  as  that  shown  in  Figure  25. 
Of  course,  this  is  largely  a  conjecture. 

Whatever  may  be  the  true  significance  of  these  two  peculiar  spindles,  there  is  no 
question  that  a  bipolar  spindle  does  finally  form  to  effect  the  first  maturation  divi- 
sion in  Habrotrocha  tridcns.  Figures  25,  26,  27  and  28  represent  lateral  and  polar 
views  of  the  first  maturation  division.  In  Figure  29,  we  see  two  anaphase  groups 
of  chromosomes  which  demonstrate  beautifully  that  this  division  involves  no  reduc- 
tion in  chromosomes.  Figure  30  shows  a  polar-body  and  the  chromosomes  within 
the  secondary  oocyte  being  regrouped  to  form  the  metaphase  plate  of  the  second 
division.  Figures  31  and  32  both  show  the  metaphase  spindle  of  the  second  divi- 
sion, each  with  a  polar-body  directly  over  it  at  a  higher  level  of  focus  (Figs.  3 la 
and  32a),  which  fact  indicates  that  the  long  axes  of  the  spindles  of  the  two  divisions 
are  perpendicular  to  each  other.  The  number  of  chromosomes  which  could  be 
made  out  in  each  one  of  these  two  metaphase  spindles,  counting  each  dumb-bell 
shaped  granule  as  a  unit,  indicates  that  the  second  division  is  also  equational  in 
Habrotrocha  tridens.  Usually  in  such  cases  something  like  10  to  11  units  could  be 

All  figures  are  camera  lucida  drawings  made  at  1500  X 

PLATE  I 

FIGURES  1-9.  Oocytes  showing  the  condensed  chromosomes  in  their  germinal  vesicles, 
and  becoming  progressively  more  easily  distinguishable  from  the  less  intensely  stained  thread- 
like structures  as  the  oocytes  mature.  In  Figures  2,  3  and  4,  an  idiozome  is  shown  in  contact 
with  the  germinal  vesicle. 


368 


W.  SIANG  HSU 


13 


17. 


14 


20 


15 


16 


17 


19 


21 


PLATE  II 


OOGENESIS  IN  HABROTROCHA  TRIDENS  369 

counted.  Having  seen  many  eggs  of  about  this  stage,  I  cannot  help  feeling  that 
the  second  division  takes  place  immediately  after  the  first  without  giving  the  chro- 
mosomes enough  time  to  be  included  within  a  nucleus.  In  Philodina  roseola,  how- 
ever, a  metabolic  nucleus  is  achieved  between  the  two  divisions. 

After  the  two  maturation  divisions,  the  comparatively  large  nucleus  of  the  ma- 
ture egg  goes  into  a  resting  stage  in  which  the  chromosomes  lose  their  staining  in- 
tensity (Fig.  33).  Figure  34  is  a  polar  view  of  the  metaphase  plate  of  the  first 
cleavage  division  in  which  13  chromosomes  with  two  relatively  smaller  ones  are 
clearly  visible.  As  in  Philodina  roseola,  during  anaphase  of  the  first  few  cleavage 
divisions  of  the  embryo,  "elimination  bodies"  are  visible  (Fig.  35). 

DISCUSSION 

In  both  Philodina  roseola  and  Habrotrocha  tridens,  the  oocyte  undergoes  two 
maturation  divisions,  and  the  zygotic  chromosome  number,  13,  is  maintained  in  the 
mature  egg  because  both  these  divisions  are  equational.  No  sign  of  chromosome 
pairing  has  been  observed.  I  have  examined  as  yet  too  few  species  to  venture  an 
opinion  on  the  question  as  to  whether  or  not  all  the  species  of  Bdelloidea  follow  this 
pattern  of  oogenesis.  I  can  only  point  out  the  fact  that  the  two  species  examined 
belong  to  two  different  families  of  Bdelloidea. 

In  view  of  the  genetic  principles  which  should  apply  to  ameiotic  parthenogenetic 
animals,  we  should  not  be  surprised  to  find  in  their  chromosomes  evidence  of 
aneuploidy,  polyploidy,  structural  rearrangement  and  the  loss  of  diploid  character 
in  both  the  genetic  and  the  cytological  sense.  In  this  connection  I  cannot  do 
better  than  to  quote  White  (1954)  (p.  341)  :  "In  ameiotic  parthenogenesis  genetic 
segregation  wall  not  occur.  Recessive  mutations  and  structural  rearrangements  will 
tend  to  accumulate  indefinitely  in  such  organisms,  only  the  ones  which  are  imme- 
diately deleterious  being  eliminated  by  natural  selection.  Such  forms  must  conse- 
quently be  expected  to  become  gradually  more  and  more  heterozygous,  but  all  the 
offspring  of  a  single  female  will  resemble  their  mother  exactly,  except  for  newly 
arisen  dominant  mutations  and  differences  due  to  the  action  of  the  environment. 
An  ameiotic  form  evolving  for  a  long  period  of  time  might  be  expected  eventually 
to  lose  its  diploid  character  in  both  the  genetic  and  the  cytological  sense,  its  two 
chromosome  sets  having  become  almost  completely  unlike.  Moreover,  since  no 

PLATE  II 

FIGURE  10.  A  germinal  vesicle  of  full  growth  in  which  the  thread-like  structures  have  dis- 
appeared and  a  fine-meshed  net  has  made  its  appearance.  Note  the  13  chromosomes  :  two  dot- 
shaped,  the  rest  all  dumb-bell  shaped  indicating  doubleness. 

FIGURE  11.     A  germinal  vesicle  beginning  to  shrink,  exhibiting  an  irregular  outline. 

FIGURES  12-17.  Germinal  vesicles  of  increasingly  reduced  size  in  which  the  fine-meshed  net 
is  in  turn  replaced  by  fibers. 

FIGURE  17a.  The  same  germinal  vesicle  as  depicted  in  Figure  17  but  at  a  lower  level  of 
focus,  showing  signs  of  disintegration  of  its  membrane. 

FIGURES  18  AND  19.  Chromosomes  lying  free  in  the  cytoplasm  close  to  the  wall  of  the 
oocyte. 

FIGURE  20.  Chromosomes  have  pulled  away  from  the  cell  wall  and  become  more  scattered 
in  an  area  of  material  which  clearly  appears  to  be  more  vacuolated  than  the  cytoplasm  elsewhere 
in  the  egg. 

FIGURE  21.  Chromosomes  lying  in  an  area  of  light-staining  material  in  which  short  sec- 
tions of  fiber  can  be  vaguely  seen. 


370 


W.  SIANG  HSU 


32 


OOGENESIS  IN  HABROTROCHA  TRIDENS  371 

pairing  of  chromosomes  takes  place  during  the  maturation  of  the  eggs,  there  is  no 
'mechanical'  barrier  to  the  establishment  of  any  type  of  polyploidy  in  such  forms 
and  various  forms  of  aneuploidy,  due  to  irregular  reduplication  of  some  chromo- 
some elements,  must  be  expected  to  occur." 

It  seems  to  me  that  at  least  three  chromosomes  in  Philodina  roseola  may  very 
well  have  been  involved  in  structural  rearrangement  of  some  kind,  though  not  nec- 
essarily just  among  themselves  (Fig.  37).  This  conclusion  should  hold  unless  we 
assume  that  the  bisexual  ancestor  of  this  form  had  one  pair  of  dimorphic  chromo- 
somes and  another  one  without  a  mate.  But  this  seems  to  me  unlikely.  Besides, 
although  reports  on  chromosome  number  in  rotifers  are  very  confusing,  none  of 
them  besides  the  two  forms  under  discussion  has  been  reported  to  possess  an  odd 
number  of  chromosomes  (Makino,  1951)  (p.  11).  It  would  seem,  then,  that  the 
odd  number  of  chromosomes  seen  in  the  two  species  of  Bdelloidea  under  discussion 
may  indicate  aneuploidy  due  either  to  irregular  reduplication  of  some  chromosome 
elements  or  some  such  structural  rearrangements  as  centric  fusion  accompanied  by 
the  loss  of  one  chromosome. 

It  is  difficult  to  say  whether  or  not  the  two  dot-shaped  chromosomes  were  origi- 
nally members  of  the  same  homologous  pair.  But  since  they  are  present  in  both 
Philodina  roseola  and  Habrotrocha  tridcns,  and  since  there  is  no  other  chromosome 
that  is  comparable  to  them  in  morphology,  it  may  be  safe  to  look  upon  them  as 
originally  forming  a  pair.  In  that  event,  their  disparity  in  size  and  the  fact  that 
the  smaller  one  of  the  two,  especially  in  Philodina  roseola,  often  stains  less  intensely 
than  the  bigger  one  could  perhaps  be  regarded  as  indications  of  loss  of  homology 
between  them. 

Turning  next  to  the  chromosome  which  in  Philodina  roseola  is  conspicuously 
longer  than  the  rest,  I  must  say  I  cannot  confidently  identify  it  in  Habrotrocha 
tridens.  This  is  the  only  morphological  difference  I  can  point  out  with  confidence 
between  the  chromosome  complexes  of  these  two  forms. 

It  should  perhaps  be  stressed  here  that  the  absence  of  pairing  of  chromosomes 
in  these  two  forms  should  not  be  interpreted  necessarily  as  evidence  of  loss  of 
homology  on  the  part  of  their  chromosomes,  since  according  to  the  genetic  principle 
applying  to  ameiotic  parthenogenetic  organisms  the  loss  of  the  diploid  character  be- 
tween homologous  member  chromosomes  is  possible  precisely  because  of  asynapsis. 

PLATE  III 

FIGURE  22.  Chromosomes  in  an  area  in  which  the  light-staining  material  is  replaced  by 
coarse  fibers  reaching  between  the  chromosomes  and  connecting  them  to  the  boundary  of  this 
area. 

FIGURE  23.  A  compound  spindle  consisting  of  three  bipolar  spindles,  each  with  its  own 
equatorial  chromosome  plate. 

FIGURE  24.     A  compound  spindle  disintegrating. 

FIGURE  25.     Lateral  view  of  a  first  polar  spindle. 

FIGURES  26-28.     Polar  view  of  three  equatorial  plates  of  the  first  polar  division. 

FIGURE  29.     A  mitotic  figure  at  anaphase  of  the  first  maturation  division. 

FIGURE  30.  The  first  polar-body  and  the  chromosomes  within  the  secondary  oocyte  re- 
grouping to  undergo  the  second  maturation  division. 

FIGURE  31.     A  metaphase  spindle  of  the  second  maturation  division. 

FIGURE  3 la.  Chromosomes  belonging  to  the  first  polar-body  seen  at  a  higher  level  of  focus 
directly  above  the  metaphase  spindle  represented  in  Figure  31. 

FIGURES  32-32a.  The  same  as  Figures  31  and  31a  except  in  this  case  the  first  polar-body 
nucleus  is  already  formed. 


372 


W.  SIANG  HSU 


34 


36 


37 


PLATE  IV 


OOGENESIS  IN  HABROTROCHA  TRIDENS  373 

In  other  words,  asynapsis  is  here  supposed  to  be  antecedent  to  the  loss  of  homology. 
Moreover,  the  persistent  condensed  state  of  the  chromosomes  in  my  material  com- 
plicates the  situation,  since  our  current  theory  explaining  pairing  of  chromosomes 
takes  into  account,  besides  the  singleness  of  the  threads,  also  the  degree  of  their 
uncoiling.  In  this  connection  we  should,  of  course,  recall  that  in  Neurospora, 
MacClintock  (1945)  has  reported  pairing  of  relatively  contracted  chromosomes. 
Incidentally,  it  may  be  mentioned  that  since  daughter  chromosomes  in  these  forms 
can  separate  without  difficulty,  the  coiling  which  their  chromonemata  assume  must 
be  of  the  paranemic  type,  using  the  term  which  Sparrow,  Huskins  and  Wilson 
(1941)  have  proposed. 

With  regard  to  the  first  polar  spindle,  it  must  be  said  that  due  to  the  paucity 
of  observations  the  situation  in  this  rotifer  is  not  clear.  It  is  difficult  to  venture 
an  opinion  as  to  whether  or  not  a  tripolar  spindle  represents  a  normal  stage  of  de- 
velopment. More  observations  are  needed  before  a  reliable  answer  can  be  given. 
At  present,  I  can  only  say  with  confidence  that  the  two  spindles  shown  in  Figures 
23  and  24  are  very  distinct  and  unmistakable  structures.  Although  I  have  not  made 
an  attempt  to  study  the  mitochondria  condition  in  Habrotrocha  tridcns,  I  feel  quite 
certain  that  mitochondria  are  not  involved  in  this  case,  Devise  (1922)  and  Junger 
(1931,  1934)  notwithstanding.  The  three  separate  equatorial  plates  of  chromo- 
somes, one  on  each  spindle,  ought  to  settle  the  question. 

This  research  was  supported  by  the  National  Science  Foundation  and  the 
Washington  State  Initiative  171  Research  Fund  for  Biology  and  Medicine.  I  am 
indebted  to  my  colleague.  Dr.  W.  Thomas  Edmondson,  for  identification  of  the 
rotifer  used  in  this  investigation,  and  to  Miss  Lorraine  Pilon  for  efficient  technical 
assistance. 

SUMMARY 

1.  The  pattern  of  chromosome  behavior  during  egg  formation  in  Habrotrocha 
tridens  is  the  same  as  that  found  in  Philodina  roseola.     The  oocytes  undergo  two 
maturation  divisions,  both  equational. 

2.  The  zygoid  chromosome  number  is  13,  the  same  as  that  of  PJiilodina  roseola. 

3.  The  pair  of  dot-shaped  chromosomes  of  unequal  size  is  found  in  each  of 
these  forms,  though  the  conspicuously  longer  one  seen  in  Philodina  roseola  (Fig. 
37)  cannot  be  identified  in  the  present  form  (Fig.  36). 

PLATE  IV 

FIGURE  33.  A  portion  of  a  mature  egg  with  a  comparatively  large  nucleus  about  ready  to 
undergo  the  first  cleavage  division.  Note  the  two  polar-bodies. 

FIGURE  34.  Polar  view  of  the  equatorial  plate  of  the  first  cleavage  division.  Note  the  13 
chromosomes,  two  of  which  are  appreciably  smaller  than  the  rest. 

FIGURE  35.  An  anaphase  figure  of  the  first  cleavage  division.  Note  the  polar-body  and 
the  "elimination  bodies." 

FIGURE  36.  One  late  prophase  and  four  metaphase  chromosome  plates  seen  in  the  em- 
bryonic cells  of  Habrotrocha  tridcns.  Note  the  two  dot-shaped  chromosomes. 

FIGURE  37.  Two  metaphase  chromosome  plates  seen  in  the  embryonic  cells  of  Philodina 
roseola.  Note  the  two  dot-shaped  chromosomes  and  the  one  that  is  conspicuously  longer  than 
the  rest. 


374  W.  SIANG  HSU 

4.  No  sign  of  synapsis  has  been  observed  in  either  form. 

5.  The  chromosomes  exist  in  a  condensed  condition  in  the  nuclei  of  the  ovary 
after  the  last  oogonial  division,  and  remain  condensed  throughout  at  least  the  first 
maturation  division. 

LITERATURE  CITED 

DEVISE,  R.,  1922.     La  figure  achromatique  et  la  plaque  cellulaire  dans  les  microsporocytes  du 

Lari.v  europaea.     La  Cellule,  32:  249-312. 
Hsu,  W.   S.,   1956.     Oogenesis   in  the   Bdelloidea   rotifer,   Philodina   roseola.     La   Cellule,  57 : 

283-296. 
JUNGERS,  V.,  1931.     Figures  caryocinetiques  et  cloisonnement  du  protoplasme  dans  1'endosperm 

d'Iris  pseudo-acorns.    La  Cellule,  40 :  5-82. 
JUNGERS,   V.,   1934.     Mitochondries,   chromosomes   et   fuseau   dans    les    sporocytes   d'Equisetuiu 

linwsum.    La  Cellule,  43  :  321-340. 
MACCLINTOCK,   B.,    1945.      Neurospora.      I.    Preliminary   observations   of   the   chromosomes   of 

Ncurospora  crassa.    Amcr.  J.  Bot.,  32:  671-675. 
MAKING,   S.,   1951.     Chromosome  number  in  animals.     The  Iowa   State   College   Press,   Ames, 

Iowa. 
SPARROW,  A.  H.,  C.  L.  HUSKINS  AND  G.  B.  WILSON,  1941.     Studies  on  the  chromosome  spirali- 

zation  cycle  in  Trillium.     Canad.  J.  Res.,  19  :  323-336. 
WHITE,  M.  J.  D.,  1954.     Animal  cytology  and  evolution.     The  Cambridge  University  Press. 


SELECTIVE  LIGHT  ABSORPTION  BY  THE  LENSES  OF  LOWER 

VERTEBRATES,  AND  ITS  INFLUENCE  ON 

SPECTRAL  SENSITIVITY 

DONALD  KENNEDY1  AND  ROGER  D.  MILKMAN2 

The  Biological  Laboratories,  Harvard  University;  Marine  Biological  Laboratory;  and 
U.  S.  Fish  and  Wildlife  Service  Laboratory,  Woods  Hole,  Mass. 

Visual  processes  in  all  vertebrates  apparently  depend  upon  a  group  of  closely 
similar  carotenoid-proteins.  Since  the  spectral  distribution  of  sensitivity  is  deter- 
mined by  the  absorption  spectra  of  these  pigments,  it  is  no  accident  that  most  ver- 
tebrates are  sensitive  to  approximately  the  same  band  of  wave-lengths.  In  man, 
this  range  lies  between  the  rough  limits  of  400  m/A  and  700  m/A.  As  an  expression 
of  these  limitations,  we  have  come  to  call  wave-lengths  longer  than  700  m/A  "infra- 
red" and  those  shorter  than  400  m/A  "ultra-violet." 

The  long-wave-length  limit  of  sensitivity  is  relatively  inflexible  among  verte- 
brates, because  the  visual  pigments  so  far  isolated  from  retinas  do  not  absorb  sig- 
nificantly above  700  m/A.  At  the  other  end  of  the  spectrum,  however,  the  limit  im- 
posed is  of  quite  a  different  sort.  The  visual  pigments  rhodopsin,  porphyropsin  and 
iodopsin  (Wald,  1955;  Wald,  1939;  Wald,  Brown  and  Smith,  1955)  all  show  con- 
siderable absorption  between  300  and  400  m/A,  with  a  secondary  maximum  present 
in  this  region.  In  the  case  of  rhodopsin,  the  absorption  at  360  m/A  is  nearly  30% 
of  that  at  the  500  m/i,  maximum ;  the  absorption  at  600  m/A,  by  contrast,  is  less  than 
10%.  Light  of  these  "ultra-violet"  wave-lengths  is  thus  potentially  available  for 
utilization  in  the  sensory  process. 

In  the  case  of  human  vision,  Wald  (1952)  has  shown  that  the  short-wave- 
length limit  at  approximately  400  HI/A,  is  imposed  by  selective  absorption  in  ocular 
tissues.  Belo\v  310  m/A,  in  the  region  of  general  protein  absorption,  almost  all 
light  is  absorbed  by  the  cornea,  since  it  is  the  first  tissue  encountered.  The  lens, 
which  appears  yellow  in  color  (especially  in  aged  persons),  is  an  effective  cut-off 
filter  for  radiations  below  400  m/A.  Aphakics  (persons  with  lenses  excised  for 
cataracts)  tested  by  Wald  could  read  an  optometrist's  chart  by  the  isolated  365  m/A 
line  from  a  mercury  arc,  under  which  conditions  a  normal  person  could  see  nothing 
at  all.  Although  the  pigment  responsible  for  the  coloration  of  the  human  lens— 
and  for  its  properties  as  a  filter — has  not  been  definitely  identified,  the  indications 
are  that  it  is  a  melanin  (Gourevitch,  1949). 

Walls  and  Judd  (1933)  and  Walls  (1940)  attempted  a  comparative  survey  of 
the  occurrence  of  such  filtering  lenses  in  other  vertebrates.  They  found  yellow 
lenses  in  the  eyes  of  some  diurnal  reptiles  (snakes  and  certain  geckoes),  lampreys, 
squirrels,  tree  shrews  and  ground  squirrels  (Citellus).  None  were  seen  in  fishes 

1  Predoctoral  research  fellow  of  The  National  Science  Foundation.  Present  address : 
Department  of  Zoology,  Syracuse  University,  Syracuse  10,  New  York. 

2Predoctoral  research  fellow  of  the  National  Science  Foundation.  Present  address : 
Laboratoire  de  Genetique,  13  rue  Pierre  Curie,  Paris  IV,  France. 

375 


376  DONALD  KENNEDY  AND  ROGER  D.  MILKMAN 

or  amphibians;  but  the  yellow  perch  and  the  bowfin  (Auiia)  both  had  yellow  cor- 
neas. Walls  believes  that  the  functional  advantage  in  the  selective  removal  of 
short-wave-length  radiations  lies  in  promoting  visual  acuity.  Chromatic  aberra- 
tion, since  it  increases  exponentially  as  the  wave-length  decreases,  can  produce  se- 
rious errors  in  the  violet  and  ultra-violet  (Wald  and  Griffin,  1947).  It  is  there- 
fore adaptive  for  diurnal  animals — whose  requirement  is  for  acuity  and  not  sensi- 
tivity— to  remove  this  region  of  the  spectrum  before  the  light  reaches  the  retina. 
This  idea  is  supported  by  Walls'  finding  that  yellow  lenses  occur  only  in  diurnal 
forms. 

The  mere  presence  of  a  yellow  coloration,  however,  does  not  mean  that  the  lens 
is  a  successful  ultra-violet  filter.  Many  yellow  pigments  (for  example,  xantho- 
phyll  and  carotene)  absorb  in  the  blue  region  of  the  spectrum  and  not  in  the  near 
ultra-violet.  Conversely,  lack  of  a  visible  yellow  color  does  not  mean  that  the  lens 
is  not  an  effective  filter  for  the  near  ultra-violet.  A  filter  absorbing  nothing  above 
400  m/A  but  cutting  off  sharply  at  390  m/j,,  for  example,  would  appear  colorless  to 
the  human  eye,  but  it  would  be  a  powerful  aid  to  visual  acuity  for  the  animal  pos- 
sessing it. 

The  fact  that  the  human  eye  is  a  poor  instrument  with  which  to  assess  these 
properties  prompted  the  present  investigations.  These  experiments  are  an  at- 
tempt to  measure  quantitatively  the  selective  absorption  of  lenses,  comparing  those 
of  a  wide  variety  of  lower  vertebrates.  Such  measurements  may  clarify  the  func- 
tional significance  of  this  interesting  visual  adaptation ;  in  addition,  they  provide 
concrete  information  about  an  important  dimension  of  sensory  capacity  in  these 
animals.  Some  studies  on  the  chemical  basis  of  this  selective  absorption  are  also 
reported.  Finally,  the  influence  of  these  filtering  lenses  upon  spectral  sensitivity 
is  assessed  by  electrophysiological  measurements  comparing  ultra-violet  sensitivity 
in  animals  with  and  without  their  lenses.  A  preliminary  report  of  some  of  these 
experiments  has  appeared  elsewhere  (Milkman  and  Kennedy,  1955). 

METHODS 

For  measurements  of  intact  lens  absorption,  fresh  lenses  were  dissected  from  the 
experimental  animals  and  placed  in  a  holder  designed  to  fit  a  spectrophotometer 
cuvette.  The  holder  was  constructed  in  such  a  way  that  light  passed  through  the 
lens  and  out  an  exit  pupil  of  approximately  one  mm.  diameter,  corresponding  in 
position  to  the  central  axis  of  the  lens,  and  thence  to  the  photocell  of  the  spectro- 
photometer. Thus  the  absorption  of  only  a  small  axial  section  of  lens  tissue  was 
measured.  The  lens  and  holder  were  immersed  in  a  cuvette  filled  with  mineral 
oil,  which  was  chosen  to  match  as  closely  as  possible  the  refractive  index  of 
the  lens  and  thereby  eliminate  errors  due  to  refraction.  The  mineral  oil  also  pre- 
vented the  lens  from  growing  opaque  during  the  period  of  measurement. 

A  Beckman  DU  quartz  spectrophotometer  was  used  to  measure  the  transmission 
of  the  lenses  to  light  of  different  wave-lengths.  Measurements  were  checked  re- 
peatedly, and  it  was  found  that  the  transmission  at  a  particular  wave-length  did 
not  change  significantly  during  the  course  of  an  experiment.  A  "blank"  was  used 
which  consisted  of  an  adapter  without  the  lens,  suspended  in  a  similar  medium. 
In  certain  species,  the  transmission  of  the  cornea  was  measured  using  the  same  ap- 
paratus and  a  similar  technique. 


VERTEBRATE  LENSES  AS  FILTERS  377 

In  attempts  to  discover  the  chemical  basis  of  the  selective  absorption  found  in 
the  lenses  of  certain  of  the  species  tested,  aqueous  extracts  were  made  from  large 
amounts  of  homogenized  lens  tissue.  Lenses  were  ground  with  distilled  water  in 
a  glass  mortar ;  protein  material  was  removed,  either  by  dialysis,  precipitation  by 
heating,  or  making  up  the  solutions  to  50%  ethanol. 

Extracts  were  chromatographed  on  Whatman  No.  1  filter  paper  in  butanol-acetic 
acid- water  mixtures  (5  :  1:4),  using  ascending  strips  or  cylinders  of  paper.  They 
were  viewed  under  ultra-violet  light  from  a  mercury  arc  lamp,  equipped  with  a  filter 
which  removed  almost  all  visible  wave-lengths.  The  chromatograms  were  treated 
in  various  ways.  Some  were  sprayed  with  ninhydrin ;  in  others,  the  spots  were  cut 
out  and  eluted  with  a  small  volume  of  water.  All  absorption  spectra  were  measured 
in  a  Beckman  spectrophotometer.  In  some  cases,  it  was  desirable  to  obtain  ab- 
sorption spectra  directly  from  spots  on  the  paper ;  these  were  measured  directly  in 
the  spectrophotometer,  using  a  "blank"  of  dry  butanol-saturated  paper  and  employ- 
ing a  photo-multiplier  attachment  for  extra  sensitivity.  Satisfactory  spectra  could 
be  obtained  in  this  way  under  conditions  when  eluted  samples  might  have  been  too 
dilute  to  yield  satisfactory  measurements. 

In  order  to  measure  directly  the  effect  of  selective  lens  absorption  on  spectral 
sensitivity,  the  spectral  sensitivity  function  of  intact  frogs  was  compared  with  that 
of  animals  deprived  of  their  lenses.  Briefly,  the  technique  involved  recording  the 
electroretinogram  ( the  slow  action  potential  of  the  retina )  from  either  the  cornea  or, 
in  the  case  of  excised  eyes,  from  the  vitreous  body.  Moist  cotton  wick  electrodes 
were  used  with  a  capacity-coupled  pre-amplifier  and  oscilloscope.  Monochromatic 
light  produced  through  interference  filters  (or,  in  the  case  of  365  m/j.,  by  isolation 
of  that  mercury  arc  line)  was  directed  onto  the  eye  of  the  preparation  through  a 
pair  of  opposed  annular  wedges  which  regulated  the  intensity.  The  optical  system 
was  calibrated  with  a  thermopile  and  sensitive  galvanometer.  In  each  experiment, 
a  certain  amplitude  of  b-wave  (the  large  positive  potential  of  the  electroretinogram) 
was  chosen  as  the  "criterion  response."  The  intensity  of  constant-duration  flash 
necessary  to  produce  a  response  of  this  amplitude  was  then  found  for  each  wave- 
length, and  the  reciprocals  of  these  "threshold"  intensities  wrere  plotted  as  a  spectral 
sensitivity  curve.  Frogs  were  curarized  and  dark-adapted  before  each  experiment. 
Each  wave-length  was  then  tested  in  turn,  with  control  flashes  frequently  inter- 
spersed to  assure  that  a  constant  level  of  sensitivity  was  maintained.  Then  the  lens 
was  removed  and  the  experiment  repeated.  In  a  number  of  experiments,  excised 
eyes  were  used  instead  of  intact  animals ;  this  procedure  proved  equally  satisfactory. 

RESULTS 

The  lenses  of  many  of  the  fish  and  amphibians  studied  showed  marked  filtering 
properties.  Representative  absorption  curves  of  intact  lenses  are  shown  in  Figure 
2,  with  Wald's  data  on  the  Rhesus  monkey  lens  included  for  comparison.  The 
species  studied  are  divided  into  roughly  three  groups.  Members  of  the  first  of 
these  possess  lenses  which,  like  the  human  lens,  show  a  cut-off  at  about  400  m/j., 
but  they  are  clearly  better  filters  in  that  their  rise  in  extinction  is  sharper.  As  a 
result  of  their  lack  of  absorption  above  400  niju,.  these  lenses  do  not  appear  yellow. 
The  group  includes  the  yellow  perch  (Perca  flavesccns),  the  calico  bass  (Pomo.ris 
sparoidcs],  and  the  blue-gill  sunfish  (Lcpoinis  pallidus),  all  common  fresh-water 


378 


DONALD  KENNEDY  AND  ROGER  D.  MILKMAN 


species;  the  scup  (Stenotomus  versicolor),  the  summer  flounder  (Pardichthys 
dentatus},  the  rudderfish  (Serolia  sonata],  and  the  sea  robin  (Prionotus  evolans), 
all  marine  species;  and  the  grass  frog  (Rana  pipiens).  The  absorption  spectra  of 
lenses  in  this  group  are  roughly  similar  (Fig.  1),  except  for  that  of  the  frog,  in 
which  the  cut-off  occurs  at  a  definitely  shorter  wave-length.  This  will  be  discussed 
below. 


2.0 


OPTICAL 
DENSITY 


1.0 


.8 


.6 


.4 


.2 


YELLOW     PERCH 


SPECTRAL    TRANSMISSION     OF 


VARIOUS      LENSES 


RHESUS    MONKEY 


1  X~H 

1        ,        1        ,2     f 

_A-  -                     -f 

n 

1 

T            T 

320 


360 


400  440  480 

WAVELENGTH-  MJJL 


520 


560 


FIGURE  1.     Measurements  of  the  spectral  transmission  of  lenses  from  various  lower  vertebrates. 
Data  on  the  lens  of  the  Rhesus  monkey  from  Wald  (1949)  are  included  for  comparison. 

The  butterfish  (Poronotus  triacanthus]  shows  instead  a  steep  rise  in  lens  ex- 
tinction near  350  m^,  and  thus  is  in  a  group  by  itself. 

A  third  group,  represented  by  the  tautog  (Tantoga  onitis),  the  smooth  dogfish 
(Mustelus  canis)  and  the  toadfish  (Opsanus  tail),  all  marine  bottom  species,  and 
the  catfish  (Amieurus  nebulosus),  a  fresh-water  bottom  scavenger,  appear  to  have 
only  a  gradual,  slight  rise  in  lens  extinction  down  to  320  m/*..  The  brook  trout 
(Salvelinus  fontinalis)  is  also  in  this  category,  but  possesses  a  cornea  which  shows 
strong  absorption  beginning  at  400 


VERTEBRATE  LENSES  AS  FILTERS 


379 


Aqueous  extracts  of  all  lenses  tested  belonging  to  the  first  group  showed  ab- 
sorption spectra  similar  to  that  given  in  Figure  2,  a  preparation  from  lenses  of  the 
cod  (Gadus  callarias).  Flounder,  rudderfish  perch,  calico  bass  and  sea  robin  pre- 
parations were  also  tested  and  found  to  be  spectrally  identical ;  in  most  further 
studies,  cod  lenses  were  used  because  they  could  be  obtained  in  large  quantities, 
fresh,  through  the  courtesy  of  the  Booth  Fisheries  Corporation  of  Boston. 


1.0 


OPTICAL 
DENSITY 


.5 


ABSORPTION     SPECTRA        OF      CRUDE 

LENS       EXTRACTS 


COD 


220 


260  300  340 

WAVELENGTH—  MU 


380 


420 


FIGURE  2.     Absorption  spectra  of  aqueous  extracts  from  lenses  of  the  butterfish   (dotted  line) 
and  the  cod  (solid  line).     Maxima  have  been  adjusted  to  same  height. 


The  absorption  spectra  of  these  extracts,  as  shown  in  Figure  2,  show  a  maxi- 
mum at  360  m/A,  and  this  absorption  band  is  responsible  for  the  action  of  the  lens 
as  a  cut-off  filter.  In  the  case  of  the  frog,  similar  extracts  had  their  maxima  at 
345  m/A,  consistent  with  the  slight  displacement  of  the  extinction  of  intact  frog  lenses. 

Extracts  of  butterfish  lenses  (Fig.  2)  had  absorption  maxima  at  320  m/u,  which 
explains  the  fact  that  intact  lenses  in  this  species  have  their  steep  rise  in  extinction 
at  350  m/A  instead  of  near  400  m/x.  No  absorption  bands  between  300  m/x.  and 


380 


DONALD  KENNEDY  AND  ROGER  D.  MILKMAN 


400  mp.  were  found  in  extracts  from  lenses  which  lacked  the  steep  rise  in  extinction 
(third  group). 

The  term  "pigment"  is  usually  restricted  to  those  substances  which  absorb  in  the 
human  visible  range.  This  range,  however,  is  restricted  by  the  presence  of  an 
ultra-violet-absorbing  lens ;  it  is  appropriate  in  this  context  to  refer  to  the  visual 


O.6.- 


0.5 


OPTICAL 
DENSITY 


0.4 


0.3 


O.2 


O.I 


360  M)X    ABSORPTION 

PAPER    CHROMATOGRAM      OF 
COO      LENS      EXTRACT 


320    MJJ  ABSORPTION 


CO 

%        @0 

0 

0 

0               .1 
ORIGIN 

.2               .3                .4               .5 
R.F. 

.6 

.7              .8 

.9              I.C 
FRONT 

FIGURE  3.  Chromatography  of  cod  lens  extract.  The  shaded  spots  at  R.  f.  0.19  and  0.43 
are,  respectively,  the  presumed  oxidation  product  of  360-pigment  and  360-pigment  itself  :  the 
first  spot  corresponds  with  a  peak  of  absorption  measured  at  320  rru*,  and  the  second  with  a  peak 
of  absorption  measured  at  360 


range  of  vertebrates  in  general  as  extending  from  310  m/z,  below  which  all  ocular 
tissues  absorb  strongly,  to  700  m/t,  the  upper  limit  of  visual  pigment  absorption, 
provided  no  special  intra-ocular  filters  intervene.  In  this  sense,  then,  the  filtering 
substances  of  the  fish  lens  qualify  as  pigments,  since  they  absorb  light  which  is 
visible-  —  though  not  to  humans.  We  therefore  will  refer  to  these  substances  as  pig- 
ments, labeling  them  specifically  by  their  absorption  maxima:  for  example,  360- 


VERTEBRATE  LENSES  AS  FILTERS 


381 


pigment  for  the  substance  extracted  from  lenses  in  the  first  group,  and  320-pigment 
for  the  material  isolated  from  butterfish  lenses. 

After  it  was  found  that  selective  absorption  by  these  lenses  had  a  specific  chemi- 
cal basis,  some  attempts  were  made  to  characterize  the  substances  responsible. 
Both  360-pigment  and  320-pigment  are  water-soluble,  somewhat  soluble  in  methanol 
and  ethanol,  and  insoluble  in  all  organic  solvents  tried.  They  are  stable  in  acid 
(pH  1),  but  in  alkali  (pH  12)  they  break  down  slowly  and  their  characteristic  ab- 
sorption bands  disappear. 

CHROMATOGRAPHED         COD 


1-0 


OPTICAL 
DENSITY 


.5 


.2 


LENS       EXTRACT 


OXIDATION    PRODUCT  -    RF    0.19 
\ 


LENS    PIGMENT  -  R.F.   0.43 


X 


220 


260 


380 


FIGURE  4. 


300  340 

WAVELENGTH  -  MJ_l 

Absorption  spectra  of  eluates  from  the  two  spots  shown  in  Figure  3. 
been  adjusted  to  the  same  height. 


420 


Maxima  have 


360-pigment  is  apparently  readily  oxidized  on  standing,  or  by  bubbling  oxygen 
into  the  solution.  The  absorption  maximum  at  first  shifts  from  360  m/x  to  320  m//, ; 
this  latter  band  later  disappears,  and  the  final  product  shows  only  a  rising  general 
absorption  into  the  ultra-violet,  often  developing  a  tan  color  suggesting  the  forma- 
tion of  a  melanin-like  polymer. 

Paper  chromatography  of  lens  extracts  in  butanol-acetic  acid-water  mixtures 
(5 :  1:4)  reveals  a  series  of  fluorescent  and  ninhydrin-positive  spots.  Presumably, 
a  variety  of  amino  acids  and  polypeptides  is  present,  together  with  other  sub- 


382 


DONALD  KENNEDY  AND  ROGER  D.  MILKMAN 


stances  such  as  riboflavin.  Figure  3  shows  the  presence  of  360-pigment  as  a 
ninhydrin-positive,  non-fluorescent  spot  at  R.  f.  0.43 ;  another  spot,  yellow- 
fluorescent  and  ninhydrin-positive,  is  present  at  R.  f.  0.19. 

Concentrated  lens  extracts  were  then  chromatographed  in  streaks,  and  the  bands 
at  R.  f.  0.43  were  cut  out  and  eluted  with  distilled  water  to  yield  a  quantity  of 
purified  360-pigment.  When  such  eluates  were  allowed  to  stand  overnight  at 
room  temperature  and  rechromatographed,  two  spots  were  seen :  one  was  identical 


ioor 


RELATIVE 
SENSITIVITY 

(%) 

50 


--o--8  APHAKIC  FROGS 
—  x  —  6  NORMAL  FROGS 
RHODOPSIN 


340 


400 


460 


520 


560 


WAVELENGTH  -   MJJ. 

FIGURE  5.     Average  spectral  sensitivity  curves  at  short  wave-lengths  for  normal  frogs  and  frogs 
with  lenses  excised,  compared  with  Wald's  absorption  spectrum  for  frog  rhodopsin. 

in  position  and  absorption  spectrum  to  the  original  eluted  spot,  and  a  second  was 
present  at  R.  f.  0.19.  This  spot  was  ninhydrin-positive  and  yellow-fluorescent,  and 
had  an  absorption  maximum  at  320  m/*.  This  is  the  presumed  oxidation  product 
of  360-pigment ;  as  can  be  seen  from  Figure  4,  it  is  spectrally  similar  to  320-pigment 
from  the  butterfish. 

The  ninhydrin-positive  nature  of  both  substances  in  these  experiments  indicates 
that  an  amino  group  is  found  in  360-pigment  and  its  derivative.  The  absorption 
spectra  of  chromatographically  purified  360-pigment  and  of  its  derivative  indicate 
that  a  second  absorption  band  at  225  m^  is  characteristic  of  both,  though  in  the 
latter  it  is  present  as  a  shoulder. 


VERTEBRATE  LENSES  AS  FILTERS  383 

When  iodine  is  added  to  360-pigment  in  solution,  a  quantitative  shift  of  the 
absorption  maximum  to  320  m/j.  is  produced.  The  conversion  apparently  produces 
a  single  product,  since  there  is  a  clear  isosbestic  point.  It  is  not  certain  whether 
the  observed  shift  is  due  to  saturation  of  a  conjugated  double-bond  system  or  an- 
other sort  of  oxidation.  The  product  is  spectrally  identical  with  320-pigment  from 
the  butterfish  and  with  the  previously-described  derivative  of  360-pigment;  320- 
pigment  from  the  butterfish  will  not  react  with  iodine. 

The  pigment  from  the  frog  lens  has  an  absorption  maximum  at  345  m^.  It 
differs  from  the  360-pigment  not  only  spectrally,  but  in  that  it  will  not  add  iodine 
and  is  acid-unstable  and  alkali-stable. 

The  effect  of  selective  lens  absorption  upon  the  spectral  sensitivity  of  the  frog 
is  shown  in  Figure  5.  The  spectral  sensitivity  function  of  animals  with  their  lenses 
removed  is  in  satisfactory  agreement  with  the  absorption  spectrum  of  rhodopsin 
down  to  365  m/j.  in  the  ultra-violet.  Intact  frogs,  however,  begin  to  show  low  sen- 
sitivity at  425  m/x,  and  at  365  in/A,  sensitivity  is  only  approximately  1%  of  that  at 
the  500  nifi  maximum. 

DISCUSSION 

It  appears  from  these  results  that  a  great  many  lower  vertebrates,  as  well  as 
mammals,  possess  an  intra-ocular  system  for  filtering  out  ultra-violet  radiations 
which  might  otherwise  impair  visual  acuity.  A  rough  correlation  is  observed  here, 
too,  between  the  existence  of  such  filters  and  an  apparent  ecological  requirement 
for  acute  vision  on  the  part  of  their  possessors.  The  species  found  to  lack  such 
filters  are  bottom-feeders  like  the  dogfish  which  rely  primarily  on  other  sensory 
systems  in  their  feeding.  Active,  surface-living  forms  all  seem  to  have  filtering 
lenses ;  in  the  butterfish.  however,  the  lens  transmits  a  considerable  band  of  ultra- 
violet. 

The  correlation  is  rather  better  among  higher  vertebrates.  Squirrels,  tree- 
shrews  and  primates,  among  the  mammals,  are  largely  diurnal,  and  have  yellow 
lenses;  no  nocturnal  animal  has  been  found  to  possess  one,  and  Weale  (1953)  has 
shown  that  the  cat  lens  has  a  high  transmission  down  to  400  m/j,. 

In  gauging  the  adaptive  value  of  an  intra-ocular  filtering  mechanism  in  aquatic 
animals,  a  number  of  complications  must  be  considered.  First,  though  it  is  gen- 
erally believed  that  ultra-violet  light  penetrates  water  poorly,  the  transmission  of 
water  for  near  ultra-violet  (350  m/x.-400  m/i)  is  actually  quite  high  compared  to 
light  of  550  m/x-600  m/u,  (Jerlov,  1951).  Second,  the  presence  of  suspended  ma- 
terial increases  scattering  to  a  great  degree.  The  consequences  of  this  fact  are 
difficult  to  ascertain :  scattering  increases  exponentially  with  decreasing  wave- 
length, so  that  the  presence  of  suspended  matter  selectively  increases  the  extinction 
of  short-wave-lengths.  However,  there  are  some  secondary  considerations  which 
cannot  be  ignored :  a  plankton-feeding  fish,  for  example,  might  use  ultra-violet  sen- 
sitivity advantageously  to  locate  concentrated  areas  of  suspended  matter  (including 
organisms)  by  short-wave-length  light  scattered  from  them. 

Walls  (1942)  has  advanced  arguments  to  support  the  idea  that  filtering  lenses 
are  a  sort  of  evolutionary  "second  line"  in  the  battle  against  chromatic  aberration. 
Retinal  cone  oil  droplets  are  held  to  be  the  usual  method  of  filtering  short-wave- 
length radiations.  These  are  found  in  turtles  and  birds  (yellow,  red,  orange  and 


384  DONALD  KENNEDY  AND  ROGER  D.  MILKMAN 

colorless),  amphibians  (yellow)  and  some  fishes  (colorless).  Walls  believes  that 
a  group  which  becomes  nocturnal  in  the  course  of  evolution  loses  its  oil  droplets ; 
the  filtering  lens  is  evolved  as  a  substitute  in  secondarily  diurnal  forms.  This,  he 
believes,  explains  the  presence  of  yellow  lenses  in  snakes  and  diurnal  geckoes. 

This  idea  does  not  seem  to  explain  the  situation  adequately.  Wald  and  Zuss- 
man  (1938)  have  shown  that  the  oil  droplets  of  birds  contain  three  carotenoid  pig- 
ments, a  different  one  of  which  is  responsible  for  each  color.  The  yellow  one  is 
xanthophyll,  which  is  also  responsible  for  the  yellow  coloration  of  the  human 
macula  lutea.  Such  droplets  are  unquestionably  filters,  but  they  are  not  filters  de- 
signed for  removing  the  ultra-violet.  Xanthophyll,  for  example,  has  its  absorption 
maximum  near  450  m/*,  and  has  declined  to  a  very  low  absorption  in  the  region  of 
400  m/x  where  chromatic  aberration  begins  to  become  especially  serious.  The  other 
oil-droplet  pigments  have  their  maxima  at  even  longer  wave-lengths.  In  the  human 
eye,  in  which  the  cone-rich  fovea  is  equipped  with  a  xanthophyll  filter,  there  is  also 
a  yellow  lens. 

Other  evidence,  too,  contradicts  the  idea  that  the  filtering  lens  is  functionally 
identical  with  the  oil  droplets  and  replaces  them  when  the  latter  are  lost  in  evolu- 
tion. The  frog,  which  has  been  shown  in  these  experiments  to  possess  a  sharp 
ultra-violet  cut-off  in  its  lens,  also  has  yellow  cone  oil  droplets.  Finally,  we  have 
made  observations  on  the  yellow  cornea  of  the  yellow  perch,  and  find  that  it  owes 
its  coloration  to  the  presence  of  (primarily)  /^-carotene,  a  carotenoid  with  nearly 
the  same  absorption  spectrum  as  xanthophyll.  The  perch,  too,  has  a  lens  filter  for 
ultra-violet.  It  thus  appears  that  the  carotenoid  filters  in  the  eyes  of  vertebrates 
(oil  droplets,  yellow  corneas,  and  macula  lutea}  either  serve  some  special  function 
unrelated  to  the  selective  absorption  of  ultra-violet  by  the  lens,  or  that  they  are 
accessory  filters  which  serve  to  widen  the  band  of  short-wave-length  absorption. 
In  either  case,  they  are  not  adaptively  equivalent  to  an  ultra-violet  filter  in  the  lens. 

Evidence  suggests  that  the  pigment  of  the  primate  lens  is  a  melanin  (Goure- 
vitch,  1949).  Yellow  lenses  of  other  mammals  may  also  owe  their  coloration  to 
a  melanin,  although  little  chemical  characterization  has  been  done.  The  pigment 
can  be  extracted  by  alkali,  but  not  by  water  (Walls,  1940). 

The  lenses  of  the  fishes  and  amphibians  studied  here  owe  their  selective  ab- 
sorption to  an  entirely  different  sort  of  pigment,  which  is  water-soluble.  It  has 
not  been  possible  so  far  to  determine  the  chemical  identity  of  these  lens  pigments ; 
their  behavior  suggests,  at  least,  that  360-pigment  from  a  variety  of  fish  and  320- 
pigment  from  the  butterfish  are  closely  related  chemically. 

Not  many  groups  of  water-soluble  natural  compounds  show  the  type  of  ultra- 
violet spectrum  exhibited  by  these  substances.  The  two  major  groups  which  do 
are  the  pteridines  (Forrest  and  Mitchell,  1954a,  1954b,  1955)  and  some  metabolites 
of  tryptophane  such  as  kynurenine.  There  are  chemical  similarities  between  the 
lens  pigments  and  these  two  classes  of  substances,  but  also  some  marked  differences. 
At  present,  there  is  no  definite  basis  for  deciding  in  which  group  of  compounds  the 
lens  pigments  belong,  although  pteridines  have  been  previously  isolated  from  the 
eyes  and  integument  of  fish  (Pirie  and  Simpson,  1946;  Hiittel  and  Sprengling, 
1943). 

The  data  on  frog  spectral  sensitivity  at  short  wave-lengths  show  clearly  the 
large  effect  which  selective  lens  absorption  has  on  actual  visual  processes.  How- 


VERTEBRATE  LENSES  AS  FILTERS  385 

ever,  there  is  a  quantitative  discrepancy  between  /;;  I'itro  measurements  of  lens  ab- 
sorption and  the  electrophysiological  sensitivity  data.  Spectrophotometric  meas- 
urements on  the  excised  frog  lens  show  that  it  has  a  transmission  at  365  m^  of  less 
than  \°/c  of  the  incident  light.  Comparison  of  the  relative  sensitivity  of  normal 
and  aphakic  frogs  at  365  nip,,  however,  reveals  that  the  normal  animals  are  about 
5%  to  10%  as  sensitive  as  those  lacking  lenses.  The  differences  may  be  explained 
if  it  is  remembered  that  in  the  Spectrophotometric  measurements,  only  a  small  cen- 
tral core  of  lens  tissue  was  measured ;  thus,  this  figure  represents  the  extinction  of 
the  longest  optical  path  through  the  lens.  In  the  intact,  dark-adapted  animal,  with 
its  pupil  dilated,  light  passes  through  the  edges  of  the  lens  as  well,  thus  reducing 
its  effectiveness  as  a  filter  and  accounting  for  the  difference  in  sensitivity. 

The  experiments  show  that  the  scotopic  spectral  sensitivity  function  of  frogs 
clearly  agrees  with  rhodopsin  absorption  down  to  365  mp.,  provided  no  intra-ocular 
filters  intervene,  and  that  an  estimate  of  the  effect  of  such  filters  in  modifying  spec- 
tral sensitivity  may  be  made  by  measuring  their  transmission  in  vitro. 

SUMMARY 

1.  Spectrophotometric  studies  of  fresh  intact  lenses  from  a  variety  of  fish  and 
from  frogs  have  shown  that  they  are  steep  cut-off  fibers  for  ultra-violet  radiations, 
selectively  absorbing  almost  all  light  of  wave-length  shorter  than  400  m/*. 

2.  Certain  species  of  fish  possess  lenses  having  high  transmission  in  the  near 
ultra-violet,  between  320  and  400  m/x ;  these  species  must  be  sensitive  to  this  spec- 
tral region,  since  visual  pigments  absorb  there.     Lenses  of  the  butterfish  show  a 
steep  cut-off  at  about  350  m^. 

3.  There  appears  to  be  a  correlation  between  possession  of  ultra-violet  filtering 
lenses  and  a  requirement  for  acute  vision,  supporting  the  idea  that  they  aid  visual 
acuity  by   eliminating  wave-lengths   which   produce   severe   chromatic   aberration. 
Such  lenses,  however,  cannot  be  regarded  as  functionally  equivalent  to  such  intra- 
ocular carotenoid  filters  as  retinal  oil  droplets  and  macula  lutea  since  they  absorb 
in  quite  different  spectral  regions.     The  theory  that  lens  filters  are  an  evolutionary 
"replacement"  for  oil  droplets  in  secondarily  diurnal  animals  is  thus  not  in  agree- 
ment with  these  findings. 

4.  Substances  responsible  for  the  properties  of  these  lenses  as  filters  have  been 
extracted  with  water  from  the  lens  tissue.     Lenses  which  cut  off  at  400  m/j,  yield 
a  substance  with  an  ultra-violet  absorption  maximum  at  360  m/* ;  those  of  the  butter- 
fish,  which  cut  off  at  350  m/*,  yield  a  substance  with  maximum  absorption  at  320  m/^. 
A  presumed  oxidation  product  of  360-pigment  has  been  obtained  which  is  spectrally 
similar  to  320-pigment  from  the  butterfish.     Both  substances  have  been  character- 
ized as  to  solubility,  ultra-violet  absorption  spectra,  and  chromatographic  behavior, 
but  no  definite  identification  has  been  made. 

5.  Comparison  of  spectral  sensitivity  in  normal  frogs   and  frogs   deprived  of 
their  lenses  has  been  made  by  recording  the  electroretinogram.     The  results  show 
that  the  lens  has  the  anticipated  effect  in  restricting  short-wave-length  sensitivity. 
In  frogs  without  lenses,  scotopic  sensitivity  is  in  good  agreement  with  the  absorption 
of  rhodopsin  down  to  360  m//,,  while  in  normal  animals  sensitivity  declines  sharply 
below  400 


386  DONALD  KENNEDY  AND  ROGER  D.  MILKMAN 

LITERATURE  CITED 

FORREST,  H.  S.,  AND  H.  K.  MITCHELL,  19S4a.     Pteridines  from  Drosophila.    I.  Isolation  of  a 

yellow  pigment.     /.  Amer.  Chem.  Soc.,  76:  5656-5658. 
FORREST,  H.  S.,  AND  H.  K.  MITCHELL,  1954b.     Pteridines  from  Drosophila.     II.  Structure  of 

the  yellow  pigment.     /.  Amer.  Chem.  Soc.,  76 :  5658-5662. 
FORREST,  H.  S.,  AND  H.  K.  MITCHELL,  1955.     Pteridines  from  Drosophila.     III.  Isolation  and 

identification  of  three  more  pteridines.    /.  Amer.  Chem.  Soc.,  77 :  4865-4869. 
GOUREVITCH,  A.,   1949.     Sur  la  nature  chimique  du  pigment  du  crystallin.     C.  R.  Soc.  Biol., 

Paris,  143 :  1426-1427. 
HUTTEL,  R.,  AND  G.  SpRENGLiNG,  1943.     Uber  Ichthyopterin,  eine  blaufluorescierenden  Stoff  aus 

Fischenhaut.     Licb.  Ann.,  554 :  69-82. 
JERLOV,  N.  G.,  1951.     Optical  studies  of  ocean  waters.     Rcpts.  Swedish  Deep-Sea  Expcd.,  Phys. 

Chem.,  3  :  1. 
MILKMAN,  R.  D.,  AND  D.   KENNEDY,   1955.     Modification  of  spectral  sensitivity  by  the   lens. 

Amer.  J.  Physiol.,  183  :  645. 

WALD,  G.,  1939.     The  porphyropsin  visual  system.    /.  Gen.  Physiol.,  22 :  775-794. 
WALD,  G.,  1952.     Alleged  effects  of  the  near  ultra-violet  on  human  vision.     /.  Opt.  Soc.  Amer., 

42:  171-177. 

WALD,  G.,  1955.     The  photoreceptor  process  in  vision.     Amer.  J.  Ophthaluwl.,  40:  18-41. 
WALD,  G.,  P.  K.  BROWN  AND  P.  H.  SMITH,  1955.     lodopsin.     /.  Gen.  Physiol.,  38 :  623-681. 
WALD,  G.,  AND  D.  R.  GRIFFIN,  1947.     The  change  in  refractive  power  of  the  human  eye  in  dim 

and  bright  light.    /.  Opt.  Soc.  Amer.,  37  :  321-336. 
WALD,  G.,  AND  H.  ZUSSMAN,  1938.     Carotenoids  of  the  chicken  retina.     /.  Biol.  Chem.,  122 : 

449-460. 

WALLS,  G.  L.,  1940.     The  pigment  of  the  vertebrate  lens.     Science,  91 :  172-175. 
WALLS,  G.  L.,  1942.     The  visual  cells  and  their  history.     Biol.  Symp.,  7:  203-251. 
WALLS,  G.  L.,  AND  H.  D.  JUDD,  1933.     The  intra-ocular  colour  filters  of  vertebrates.     Brit.  J. 

Ophthaluwl.,  17 :  641-675. 
WEALE,  R.  A.,  1953.    Light  absorption  in  the  crystalline  lens  of  the  cat.    Nature,  173 :  1049-1050. 


RELATIVE  INTENSITY  OF  OYSTER  SETTING  IN  DIFFERENT 
YEARS  IN  THE  SAME  AREAS  OF  LONG  ISLAND  SOUND 

V.  L.  LOOSANOFF  AND  C.  A.  NOMEJKO 

U.  S.  Fish  and  Wildlife  Service,  Milford,  Conn. 

Quantitative  studies  of  marine  bottom  invertebrates  have  been  conducted  since 
the  early  part  of  the  century,  and  the  results  have  substantially  enriched  our  knowl- 
edge and  understanding  of  aquatic  communities.  The  contributions  of  many  work- 
ers to  this  important  branch  of  marine  biology  have  been  reviewed  by  several  au- 
thors, including  Sparck  (1935)  and,  more  recently,  Sanders  (1956). 

Regardless  of  the  progress  made,  there  still  remains  one  aspect  of  this  field 
which  has  been  relatively  neglected  but  which  should  be  of  special  interest  to  many 
students  of  bottom  communities.  In  general,  it  concerns  the  recruitment  of  the 
new  year-classes  of  such  forms  as  mollusks  and  echinoderms  that  have  pelagic 
larvae  which,  after  a  free-swimming  period,  descend  to  the  bottom  and  meta- 
morphose, the  act  commonly  called  setting.  In  particular,  it  deals  with  the  varia- 
tions in  the  intensity  of  setting  of  the  same  species  in  the  same  area  in  different 
years,  and  comparing  these  variations  with  those  of  other,  nearby  areas. 

Our  long-term  studies  of  the  biological  events  of  Long  Island  Sound  give  us 
the  opportunity  to  discuss  certain  aspects  of  this  problem  in  relation  to  the  Ameri- 
can oyster,  Crassostrea  virginlca.  The  conclusions  are  based  on  data  collected  dur- 
ing the  past  12  years,  1944  through  1955,  from  ten  chosen  areas.  The  locations  of 
these  areas,  which  we  shall  call  stations,  are  shown  in  Figure  1.  They  were  con- 
fined to  three  depths—  10,  20  and  30  feet — and  represented  three  major  oyster- 
producing  sections  of  Long  Island  Sound,  namely,  New  Haven,  Milford  and  Bridge- 
port. The  combined  area  of  these  sections  is  approximately  80  square  miles. 

The  intensity  of  setting  at  each  of  the  stations  was  evaluated  by  counting  the 
number  of  recently  set  oysters  on  special  collectors  consisting  of  wire  mesh  bags 
filled  with  old  oyster  shells  (Prytherch,  1930).  This  is  the  standard  method  in  use 
at  our  laboratory  for  over  20  years,  and  with  which  most  oyster  biologists  and 
oystermen  are  now  well  familiar  (Loosanoff  and  Engle,  1940;  Loosanoff,  Engle  and 
Nomejko,  1955).  It  is  important  to  emphasize  that  the  locations  of  the  stations 
remained  the  same  during  the  12  years,  and  that  the  methods  of  determining  the 
intensity  of  setting  were  identical  for  all  stations. 

To  evaluate  the  relative  productivity  of  each  station,  we  employed  a  simple 
ranking  method  by  giving,  each  year,  Rank  1  to  the  most  productive  station,  Rank  2 
to  the  next  most  productive,  and  so  on,  until  the  least  productive  was  given  Rank  10. 
For  example,  for  1944,  Station  1,  the  most  productive,  was  given  Rank  1;  Station 
2,  the  least  productive,  Rank  10;  Station  3,  Rank  9;  etc.  (Table  I). 

To  determine  the  relative  productivity  of  the  stations  during  the  entire  12-year 
period,  we  expressed  the  rank  of  each  station  as  the  sum  of  its  yearly  ranks  (Table 
I).  Naturally,  the  stations  that  were  generally  better  producers  and,  therefore, 

387 


388 


V.  L.  LOOSANOFF  AND  C.  A.  NOMEJKO 


entitled  to  low  ranks,  such  as  Station  9,  showed  lower  sums  than  the  stations  that 
were  less  productive.  On  the  basis  of  this  total  score,  we  gave  a  long-range  rank 
to  each  station. 

The  question  immediately  arose  as  to  whether  these  ranks  would  remain  ap- 
proximately the  same  if  the  stations  were  graded  for  their  performance  only  during 
the  years  of  better  sets,  namely,  1944,  1945,  1946,  1953  and  1955.  However,  our 
analysis  showed  that  the  ranks  of  the  stations  for  these  years  were  not  substantially 
different  from  the  long-range  ranks  based  on  the  12-year  observation  period 
(Table  I). 


FIGURE  1.     Locations  and  depths   (in  feet)   of  ten  stations  established  for  observation  of  oyster 

setting  in  Long  Island  Sound,  1944-1955. 

A  close  study  of  the  data  provided  information  as  to  the  relative  importance  of 
the  stations,  the  depths,  and  the  areas  in  the  different  years.  It  was  established 
that,  excluding  Stations  2,  3  and  4,  each  station  ranked  first  at  least  one  year  out 
of  12.  It  was  also  found  that  a  10-foot  station  ranked  first,  five  times ;  a  20-foot 
station  ranked  first,  three  times ;  and  a  30-foot  station,  four  times.  Thus,  consider- 
ing that  at  the  10-foot  depth  we  have  one  station  more  than  at  20  or  30,  it  appears 
that  Rank  1  was  occupied  by  stations  of  each  of  the  three  depths  the  same  number 
of  times. 

If  we  add  the  sums  of  the  yearly  ranks  of  all  the  stations  at  the  same  depth  and 
then  calculate  the  average  sum  of  the  yearly  ranks  of  these  depth-groups,  we  will 
find  that  the  10-foot  stations  have  a  score  of  65.2 ;  20-foot  stations,  57.3 ;  and  30- 
foot  stations,  75.7.  Thus,  the  20-foot  stations  seem  to  be  generally  somewhat  more 
productive  than  the  others,  while  the  30-foot  stations  appear  to  be  the  least  pro- 


INTENSITY  OF  OYSTER  SETTING 


389 


ductive  of  the  groups  at  all  three  depths.  The  latter  conclusion,  however,  may  not 
be  well  founded  because  the  low  rank  of  the  30-foot  stations  is  chiefly  due  to  the 
history  of  setting  at  Station  3  which,  through  the  12  years,  was  consistently  one  of 
the  poorest,  never  rising  above  sixth  place ;  whereas  another  30-foot  station,  Num- 
ber 10,  was,  in  more  than  half  the  instances,  among  the  five  best  stations,  and  ranked 
first  on  three  occasions  (Table  I). 

TABLE  I 

Rank  crder  of  the  sampling  stations  representing  oyster-setting  areas  of  Long  Island  Sound 

during  the  12-year  period,  1944-1955 


Areas 

Milford 

New  Haven 

Bridgeport 

Stations 

1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

Depth  in  feet 

10 

20 

30 

10 

10 

20 

30 

10 

20 

30 

Years 

1944 

1 

10 

9 

5 

2 

4 

7 

6 

8 

3 

1945 

1 

4 

8 

10 

6 

5 

9 

2 

3 

7 

1946 

6 

9 

8 

10 

5 

1 

3 

7 

2 

4 

1947 

7 

6 

8 

9 

5 

2 

10 

1 

3 

4 

1948 

8 

6.5* 

10 

3 

6.5* 

5 

2 

4 

1 

9 

1949 

8 

4 

9 

3 

5 

7 

1 

2 

6 

10 

1950 

7 

9 

10 

8 

4 

6 

2 

5 

1 

3 

1951 

9 

10 

7 

8 

4 

6 

5 

3 

2 

1 

1952 

1 

5 

10 

7 

4 

6 

8 

3 

2 

9 

1953 

7 

3 

8 

10 

4.5* 

6 

9 

4.5* 

2 

1 

1954 

7 

2.5* 

9.5** 

9.5** 

1 

5 

6 

8 

4 

2.5* 

1955 

4 

5 

6 

10 

7 

9 

8 

3 

2 

1 

Sum  of  12  yearly 

ranks 

66 

74 

102.5 

92.5 

54 

62 

70 

48.5 

36 

54.5 

Long-range  rank, 

12  years 

6 

8 

10 

9 

3 

5 

7 

2 

1 

4 

Rank  for  1944,  1945, 

1946,  1953,  1955 

3 

7 

9 

10 

5 

6 

8 

4 

2 

1 

Indicate  a  tie  between  stations  for  the  same  rank. 


The  data  were  also  used  to  evaluate  the  relative  productivity  of  the  three  areas 
under  observation,  namely,  New  Haven,  Milford  and  Bridgeport  (Fig.  1).  By 
adding  the  ranks  of  all  the  stations,  as  given  in  Table  I,  for  each  area  and  year, 
and  dividing  the  resulting  figure  by  the  number  of  stations,  the  average  station  rank 
for  each  area  was  determined  (Table  II).  Accordingly,  each  area  was  given  a 
yearly  rank,  the  one  with  the  lowest  score  occupying  the  first  or  most  productive 
position.  The  sums  of  the  average  yearly  ranks  for  each  year,  as  shown  at  the 
bottom  of  Table  II,  were  also  determined.  It  was  found  that  the  Bridgeport  area 
occupied  first  rank,  or  the  best  producing  position,  nine  years  out  of  12,  and  never 
held  third  or  last  place.  The  New  Haven  area  was  next,  occupying  first  rank  for 


390 


V.  L.  LOOSANOFF  AND  C.  A.  NOMEJKO 


three  years.  The  Milford  area,  however,  never  reached  the  highest  position,  ranked 
second  only  four  times,  and  was  in  the  third,  or  lowest  position  for  the  remaining 
eight  years. 

We  cannot  offer  a  fully  satisfactory  explanation  for  the  variations  or,  in  some 
instances,  stability  from  year  to  year  in  the  relative  productivity  of  our  stations. 
Such  considerations  as  original  number  of  eggs  discharged ;  mortality  of  larvae  due 
to  diseases,  enemies,  or  lack  of  food ;  and  several  others  are,  of  course,  of  impor- 


TABLE  II 

Average  yearly  station-ranks  of  the  New  Haven,  Milford  and  Bridgeport  areas,  and  general  rank 

of  each  of  these  three  areas  for  each  year  of  1944-1955  period.    Sums  of  average 

yearly  ranks  of  stations  of  each  area,  and  the  ranking  of  the  areas 

during  the  entire  12-year  period  are  also  given 


Areas 

Ranks 

Years 

New 

Milford 

Bridge- 

1 

2 

3 

Haven 

port 

1944 

4.50 

6.67 

5.67 

New  Haven 

Bridgeport 

Milford 

1945 

7.50 

4.33 

4.00 

Bridgeport 

Milford 

New  Haven 

1946 

4.75 

7.67 

4.33 

Bridgeport 

New  Haven 

Milford 

1947 

6.50 

7.00 

2.67 

Bridgeport 

New  Haven 

Milford 

1948 

4.13 

8.17 

4.67 

New  Haven 

Bridgeport 

Milford 

1949 

4.00 

7.00 

6.00 

New  Haven 

Bridgeport 

Milford 

1950 

5.00 

8.67 

3.00 

Bridgeport 

New  Haven 

Milford 

1951 

5.75 

8.67 

2.00 

Bridgeport 

New  Haven 

Milford 

1952 

6.25 

5.33 

4.67 

Bridgeport 

Milford 

New  Haven 

1953 

7.38 

6.00 

2.50 

Bridgeport 

Milford 

New  Haven 

1954 

5.38 

6.33 

4.83 

Bridgeport 

New  Haven 

Milford 

1955 

8.50 

5.00 

2.00 

Bridgeport 

Milford 

New  Haven 

Sum  of 

Bpt.—  9  yrs. 

N.  H.—  5  yrs. 

Mfd.—  8  yrs. 

average 
yearly 

69.64 

80.84 

46.34 

N.  H.—  3  yrs. 
Mfd.—  0  yrs. 

Mfd.  —  4  yrs. 
Bpt.  —  3  yrs. 

N.  H.—  4  yrs. 
Bpt.—  0  yrs. 

ranks 

tance.  Nevertheless,  there  is  little  doubt  that  the  intensity  of  setting  of  oysters  at 
all  stations  depends  to  a  large  extent  upon  the  peculiarities  of  the  inshore  system 
of  water  currents. 

The  complexity  and  characteristics  of  these  currents  in  the  oyster-producing 
section  of  Long  Island  Sound  are  still  relatively  unknown  because  no  detailed 
study  has  ever  been  made.  We  know,  however,  that  planktotrophic  larvae,  with 
comparatively  longer  pelagic  lives,  like  those  of  oysters  and  many  other  pelecypods, 
are  carried  by  water  masses  and  that  their  distribution  is  controlled  by  the  cur- 
rents. Under  certain  conditions  the  direction  of  the  currents  may  be  so  changed 
that  the  larvae  will  be  carried  away  from  the  areas  where  setting  normally  takes 
place,  and  eventually  perish.  In  other  instances,  as  reported  by  Coe  (1953)  for 
Dona.r  gouldi,  an  enormous  increase  in  the  population  of  a  species  may  occur  be- 
cause swarms  of  pelagic  larvae,  about  ready  to  set,  are  unexpectedly  brought  in- 


INTENSITY  OF  OYSTER  SETTING  391 

shore  by  water  currents.  Hence,  it  is  understandable  that  the  productivity  of 
small  areas,  such  as  those  designated  for  our  stations,  should,  in  general,  be  more 
affected  by  minor  changes  in  larvae-carrying  currents  than  that  of  larger  areas, 
such  as  New  Haven,  Milford  or  Bridgeport,  which  cover  many  square  miles  of 
oyster-producing  bottom,  and  should  certainly  display  more  stability  in  maintaining 
their  relative  positions. 

These  observations  emphasize  the  importance  of  studying  the  different  aspects 
of  local  minor  currents,  including  their  direction,  velocity  and  stratifications.  They 
also  indicate  the  importance  of  understanding  the  relationship  between  the  behavior 
of  such  currents  and  the  locations  of  the  spawning  beds,  of  oysters  or  other  mol- 
lusks,  where  the  larvae  originate. 

Our  studies  suggest,  moreover,  that  minor  currents  are  often  extremely  precise 
in  their  behavior.  This  was  well  demonstrated  by  observations  on  the  intensity 
of  setting  of  oysters  at  our  Station  10,  located  several  miles  from  the  shore  and  in 
comparatively  deep  water,  but  where,  nevertheless,  heavy  setting  continued  steadily 
day  after  day  for  as  long  as  three  or  four  weeks  because  the  currents  consistently 
brought  a  supply  of  ready-to-set  larvae  to  that  point.  In  1955,  this  regularity  was 
not  noticeably  affected  even  by  the  strong  winds  of  hurricane  "Connie"  nor  by  the 
winds  and  record-breaking  floods  of  "Diane."  Finally,  they  imply  the  potential 
danger  of  interfering  with  established  combinations  of  the  favorable  ecological  con- 
ditions existing  on  the  bottom  by  modifying  its  contour  so  as  to  change  the  direc- 
tions of  the  local  currents.  Although  these  changes  may  not  affect  commonly 
studied  factors,  such  as  temperature  and  salinity,  they  may,  nevertheless,  so  alter 
the  currents  that  the  larvae  will  be  carried  to  new  areas,  some  of  which  may  not 
be  suitable  for  their  setting. 

We  wish  to  express  our  appreciation  to  Barbara  J.  Myers  for  her  assistance  in 
analyzing  these  data,  and  to  our  colleagues,  Harry  C.  Davis  and  Rita  S.  Riccio,  for 
their  helpful  suggestions  in  preparing  this  paper. 

SUMMARY 

1.  During  the  12  years  of  observations  none  of  the  stations,  representing  rela- 
tively small  bottom  areas,  always  occupied  a  position  among  the  best  oyster  set 
producers. 

2.  If  larger  areas  instead  of  individual  stations  were  compared,  a  definite  tend- 
ency of  the  Bridgeport  area  to  be  more  productive  than  the  others  was  evident. 

3.  There  was  no  evidence  that  the  stations  located  at  a  definite  depth,  such  as 
10,  20  or  30  feet,  consistently  produced  better  sets  of  oysters  than  the  stations  at 
other  depths. 

4.  There  may  be  a  great  variability  in  the  density  of  oyster  set  even  within  a 
given  depth  and  district  in  the  same  year.     For  example,  Stations  4  and  5,  al- 
though located  in  the  same  district  and  at  the  same  depth,  showed  a  rather  different 
standing  with  Long-Range  Ranks  of  9  and  3,  respectively. 

5.  Local  minor  water  currents  are  important  in  the  relative  productivity   of 
bottom  areas. 


392  V.  L.  LOOSANOFF  AND  C.  A.  NOMEJKO 

LITERATURE  CITED 

COE,  W.  R.,  1953.     Resurgent  populations  of  littoral  marine  invertebrates  and  their  dependence 

on  ocean  currents  and  tidal  currents.     Ecology,  34  :  225-229. 
LOOSANOFF,  V.  L.,  AND  J.  B.  ENGLE,   1940.     Spawning  and  setting  of  oysters  in  Long  Island 

Sound  in  1937,  and  discussion  of  the  method  for  predicting  the  intensity  and  time  of 

oyster  setting.     Bull.  U.  S.  Bur.  Fish.,  49:  217-255. 
LOOSANOFF,  V.  L.,  J.  B.  ENGLE  AND  C.  A.  NOMEJKO,  1955.     Differences  in  intensity  of  setting 

of  oysters  and  starfish.     Biol.  Bull.,  109:  75-81. 
PRYTHERCH,  H.  F.,  1930.     Improved  methods  for  the  collection  of  seed  oysters.     App.  IV ,  Kept. 

U.  S.  Coiuin.  of  Fisheries,  for  1930,  47-59. 
SANDERS,  H.  L.,   1956.     Oceanography  of  Long  Island  Sound,   1952-1954.     X.   The  biology  of 

marine  bottom  communities.     Bull.  Bingham  Oceanographic  Coll.,  15:  345-414. 
SPARCK,   T.,    1935.     On   the   importance   of  quantitative   investigation   of   the   bottom   fauna    in 

marine  biology.     /.  Cons.  Int.  E.rplor.  Mer.,  10:  3-19. 


INFLUENCING  THE  CALLING  OF  SEA  ROBINS   (PRIONOTUS 

SPP.)  WITH  SOUND1-   ; 

JAMES  M.  MOULTON 
Boii'doin  College,  Brunszvick,  Maine 

Despite  abundant  evidence  that  fishes  hear  and  produce  sounds  (Fish,  1948, 
1954;  Griffin,  1955;  von  Frisch,  1938),  a  review  of  the  literature  (Moulton  and 
Backus,  1955)  on  attempts  to  influence  fish  movements  with  man-made  sounds  has 
uncovered  reports  only  of  quickened  movements  of  fishes  during  production  of 
such  sounds.  Nor  has  a  biological  significance  of  any  sound  known  to  stem  from 
a  fish,  whether  produced  by  stridulation  of  skeletal  parts  or  by  the  air  bladder, 
been  clearly  demonstrated.  However,  the  apparent  relationship  between  sound- 
production  by  some  species  of  fishes  and  their  respective  breeding  seasons  has  been 
noted  by  a  number  of  authors  (Fish,  1954,  pp.  51,  83;  Goode,  1888.  p.  137;  Mar- 
shall, 1954,  p.  254),  and  the  possible  significance  of  fish  calls  in  bringing  individuals 
of  the  same  species  together  has  been  suggested.  Sounds  are  also  produced  during 
defensive  spine  raising  of  such  fishes  as  groupers,  grunts,  squirrel  fishes  and  sea 
robins. 

During  the  summer  of  1954,  it  was  accidentally  discovered  that  the  production 
of  certain  fish  calls,  later  identified  as  the  calls  of  sea  robins,  could  be  stimulated 
by  transmission  of  certain  sounds  into  the  water,  and  that  the  calling  could  be  sup- 
pressed by  other  sounds  (Moulton,  1955).  The  study  resulting  from  this  finding 
was  pursued  further  during  the  summer  of  1955.  The  observations  yielded  evi- 
dence that  calls  produced  during  the  breeding  season  of  two  species  of  sea  robins 
(Prionotus  carolimts  L.  and  P.  cvolans  L.)  are  produced  as  responses  to  calls  of 
the  same  species,  and  that  by  the  transmission  of  appropriate  sounds,  some  degree 
of  control  over  the  calling  of  sea  robins  may  be  exercised. 

The  sound-generating  equipment  employed  in  the  experiments  here  described 
consisted  of  a  Hewlett-Packard  audio  oscillator  Model  LAJ  or  a  Magnecorder  tape 
recorder  Model  PT6J ;  either  an  Altec  Type  A-323B  or  a  Craftsman  Model  C550 
amplifier,  and  a  QBG  transducer.  The  monitoring  system  was  an  AX- 120  ADP 
or  an  AX-58-C  Rochelle  salt  hydrophone  and  a  Woods  Hole  Suitcase  amplifier. 
Recordings  were  made  on  the  Magnecorder  tape  recorder  at  a  speed  of  15  in./sec. 
and  were  analyzed  on  a  Vibralyzer  vibration  analyzer.  The  experiments  were  per- 
formed from  a  raft  anchored  over  72  feet  of  water  in  Great  Harbor,  Woods  Hole, 
Massachusetts. 

1  Contribution  No.  862  from  the  Woods  Hole  Oceanographic  Institution. 

2  The  work  was  performed  while  the  author  was  a  Research  Fellow  at  the  Woods  Hole 
Oceanographic  Institution  during  the  summers  of  1954  and  1955. 

3  This  study  was  supported  in  part  by  a  grant  from  the  Bowdoin  College  Faculty  Research 
Fund,  established  by  the  Class  of  1928. 

393 


394  JAMES  M.  MOULTON 

THE  SOUNDS  OF  SEA  ROBINS 

The  sound-producing  air  bladder  of  the  sea  robin  has  been  described  by  Fish 
(1954).  It  is  the  apparent  source  of  two  different  calls.  One  of  these  calls  is  a 
vibrant  grunt  produced  when  a  sea  robin  is  handled  in  or  out  of  water,  and  when  a 
sea  robin  is  brought  to  the  surface  by  net  or  by  hook  and  line.  The  grunt  accom- 
panies fin  erection. 

As  determined  by  vibration  analysis,  the  sea  robin  grunt  is  a  single  burst  of 
noise  lasting  about  %0  second.  The  upper  frequency  limit  is  approximately  1.7  kc., 
the  lower  below  44  cps.  The  grunt  is  audible  to  the  unaided  ear  above  the  water 
when  a  sea  robin  is  submerged  four  feet  beneath  the  surface.  Noises  of  frequency 
characteristics  similar  to  those  of  the  grunt  may  be  obtained  by  pressing  the  air 
bladder  through  the  ventral  body  wall  of  the  intact  fish,  and  by  stimulation  of  the 
nerves  to  the  drumming  muscles  located  on  the  lateral  surfaces  of  the  bilobed  air 
bladder. 

The  onset  of  the  breeding  season  of  the  sea  robins  at  Woods  Hole  is  marked 
by  the  production  by  these  fishes  of  a  staccato  call.  Although  this  call  has  been 
monitored  from  sea  robins  contained  within  a  live  car  at  the  surface  of  Great 
Harbor,  no  single  fish  has  been  identified  as  the  source  of  an  individual  call.  This 
call  is  not  produced  under  conditions  that  bring  forth  the  grunt  already  described, 
and  it  is  much  more  frequently  produced  by  fishes  in  the  Harbor  than  by  caged 
specimens. 

The  breeding  season  of  the  sea  robins  at  Woods  Hole  extends  from  June  to 
September,  with  July  and  August  the  height  of  the  season  (Bigelow  and  Schroeder, 
1953).  In  1955,  listening  began  on  29  June.  The  first  staccato  calls  were  heard 
on  5  July,  and  calls  were  heard  on  each  day  of  listening  thereafter  until  work  ter- 
minated for  the  summer  on  30  August.  The  number  of  outbursts  of  calling  and 
the  number  of  calls  comprising  a  single  outburst  increased  rapidly  during  the  first 
half  of  July  (compare  Tables  I  and  II).  During  the  latter  part  of  August  calling 
became  more  infrequent. 

TABLE  I 
Responses  of  sea  robins  to  audio  oscillator  signals  in  Great  Harbor,  15  July,  1955 

Number  of 

signal 
Time  transmissions  Response 

1415  1  2  calls 

1420  1  No  calls 

1430  1  No  calls 

1440  1  No  calls 

1450  1  No  calls 

1500  2  1  call  after  first  signal 

1510  3  1  call  after  first  signal 

1520  2  No  calls 

1530  2  No  calls 

1540  2  1  call  after  first  signal 

1550  3  1  call  after  second  signal 

1600  2  No  calls 

1610  4  1  call  after  signals  one  and  two 

1620  3  No  calls 

1630  3  No  calls 


INFLUENCING  CALLING  OF  SEA  ROBINS  395 

TABLE  II 

Responses  of  sea  robins  to  recordings  of  the  staccato  call  and  to  audio  oscillator  signals, 

Great  Harbor,  20  July,  1955 

Number  and 
type  of  signal 
Time  transmission  Response 

1425  1  recording  2  calls 

1430  1  signal  No  calls 

1435  1  recording  No  calls 

1440  1  signal  No  calls 

1445  1  recording  4  calls 

1450  1  signal  No  calls 

1455  1  recording  No  calls 

1500  1  signal  2  calls 

1505  1  recording  No  calls 

1510  1  signal  No  calls 

1515  1  recording  No  calls 

1520  1  signal  6  calls 

1525  1  recording  No  calls 

1530  1  signal  No  calls 

1535  1  recording  No  calls 

1540  1  signal  No  calls 

1545  1  recording  No  calls 

(5  spontaneous  calls  during  this  interval) 

1550  1  signal  No  calls 

1555  1  recording  No  calls 

1600  1  signal  No  calls 

1605  1  recording  No  calls 

(12  spontaneous  calls  during  this  interval) 

1610  1  signal  No  calls 

1615  1  recording  No  calls 

1620  1  signal  No  calls 

1625  1  recording  No  calls 

1630  4  signals  1  call  after  No.  2 

1635  3  recordings  No  calls 

1640  3  signals  No  calls 

The  staccato  calls  consist  of  pulses  of  noise  usually  produced  in  pairs,  at  an  av- 
erage rate  of  22  pulses/second.  The  paired  arrangement  of  the  pulses  in  a  typical 
call  is  probably  due  to  a  slightly  asynchronous  contraction  of  the  drumming  mus- 
cles on  the  two  lobes  of  the  air  bladder.  The  pairing  of  the  pulses  is  not  dis- 
tinguishable to  the  ear,  but  can  be  seen  on  vibration  analysis.  Absence  of  the 
double  pulses  in  a  portion  of  some  calls  and,  rarely,  throughout  a  call  suggests  that 
one  lobe  of  the  air  bladder  may  be  silent  during  sound  production  by  the  other  lobe. 
The  individual  pulses  of  the  staccato  call  lie  between  500  cps  and  4  kc.  The 
frequencies  of  greatest  intensity  lie  between  700  cps  and  2.5  kc.  The  respective 
intensity  peaks  of  the  paired  pulses  are  at  different  frequencies  on  the  vibragrams, 
separated  by  approximately  1  kc.  This  is  presumably  related  to  a  differential 
resonance  of  the  two  air  bladder  lobes  which  generally  differ  somewhat  in  size.  It 
is  possible  to  obtain  sounds  of  similar  frequency  and  intensity  characteristics  by 
palpitation  of  the  dissected  air  bladder. 


396  JAMES  M.  MOULTON 

INFLUENCING  PRODUCTION  OF  THE  STACCATO  CALL 

With  the  sound-generating  equipment  employed,  it  is  possible  to  transmit  a 
series  of  sound  pulses  crudely  imitative  of  the  staccato  call  of  the  sea  robin  when  the 
audio  oscillator  is  set  at  17  to  40  cps.  (The  QBG  emits  a  considerably  distorted 
wave  train  when  driven  with  a  sine  wave  at  these  frequencies.)  With  transmis- 
sions timed  to  correspond  to  the  duration  of  an  average  call,  2%  to  3  seconds,  pro- 
duction of  the  staccato  call  by  free  sea  robins  was  repeatedly  incited  during  July 
and  August  of  1954  and  1955. 

Tables  I  and  II  present  the  results  of  two  experiments  extending  over  2  hours 
and  15  minutes  on  15  and  20  July,  1955.  On  15  July  (Table  I)  from  one  to  three 
imitations  of  the  staccato  call  were  transmitted  at  ten-minute  intervals,  except  for 
the  second  trial  which  followed  the  first  by  five  minutes.  Of  the  15  trials,  6  were 
followed  immediately  by  calling  of  free  sea  robins.  There  was  no  calling  during 
the  listening  period  other  than  immediately  following  signal  transmissions. 

On  20  July  (Table  II),  playing  of  recordings  of  the  sea  robin  staccato  calling 
into  the  harbor  water  was  alternated  with  transmission  of  imitations  of  the  calling 
at  five-minute  intervals.  During  the  28  tests  of  20  July,  the  calling  of  free  sea 
robins  was  heard  five  times  immediately  following  transmissions,  twice  after  play- 
ing recordings  of  the  calling  and  three  times  following  transmissions  of  the  imita- 
tions. Two  spontaneous  outbursts,  frequent  by  20  July,  were  heard  during  the 
period  of  the  experiment.  As  Table  II  also  indicates,  outbursts  of  several  calls 
were  the  rule  by  20  July,  whereas  earlier  in  the  month  single  or  double  calls  com- 
prised the  characteristic  outburst  in  1955. 

SUPPRESSING  OF  THE  STACCATO  CALL 

Signals  of  200  to  600  cps  transmitted  for  the  approximate  duration  of  a  staccato 
call  interrupt  the  production  of  this  call  by  sea  robins.  (Again  the  QBG  signal 
is  considerably  distorted  as  at  the  lower  frequency.)  Signals  above  2  kc.  have 
never  been  effective  in  suppressing  the  calling.  Signals  from  600  cps  to  2  kc.  are 
variable  in  effectiveness.  Sea  robins  confined  in  a  live  car  and  sea  robins  on  the 
bottom  of  Great  Harbor,  observed  by  an  aqualung  diver,  Mr.  Robert  Weeks  of  the 
Woods  Hole  Oceanographic  Institution,  show  no  obvious  change  in  behavior  dur- 
ing transmission  of  signals  effective  in  suppressing  the  staccato  call. 

DISCUSSION 

Conditions  bringing  forth  the  grunting  of  sea  robins  suggest  that  this  sound  is 
part  of  a  general  alarm  reaction.  It  may  be  of  value  in  nature  as  an  adjunct  to  the 
spiny  armor  of  the  species  in  discouraging  enemies,  but  no  evidence  is  available  on 
this  point. 

That  the  staccato  calling  reaches  its  climax  near  the  peak  of  the  sea  robin 
breeding  season  is  strongly  suggestive  of  a  relation  of  this  calling  to  breeding 
activities,  and  the  possibility  cannot  be  overlooked  that  the  calls  serve  as  a  species 
recognition  device  in  waters  where  visibility  is  rather  poor.  Mr.  Robert  Weeks 
has  informed  me  that  visibility  on  the  bottom  of  Great  Harbor  beneath  the  raft  was 
a  little  over  6  feet  on  10  July,  1955,  and  that  sea  robins  could  be  seen  clearly  within 


INFLUENCING  CALLING  OF  SEA  ROBINS  397 

that  distance  moving  over  the  bottom  of  the  harbor.  The  calling  is  heard  at  night, 
as  well  as  in  the  daytime.  In  a  few  instances  during  the  summer,  production  of 
the  staccato  call  was  heard  to  follow  various  sharp  percussive  sounds — the  discharge 
of  a  high-energy  spark  into  the  water,  the  explosion  of  a  detonating  cap  in  the 
harbor,  and  the  slamming  of  the  live  car  lid  on  the  raft. 

Since  the  first  staccato  calls  of  1955  were  heard  from  fishes  caged  at  the  surface, 
while  sea  robins  characteristically  feed  on  the  bottom,  it  was  thought  that  warming 
of  surface  waters  might  have  initiated  calling  from  surface  specimens  earlier  than 
their  calling  would  ordinarily  have  commenced.  However,  temperatures  taken  with 
a  bathythermograph  during  July  and  August  of  1955  showed  that  there  was  a 
thorough  mixing  of  water  over  the  72-foot  depth  under  the  raft,  and  no  records 
obtained  showed  over  a  two-degree  F.  variation  in  temperature  from  the  surface 
to  the  bottom. 

While  the  significance  of  the  calling  behavior  of  the  sea  robin  to  its  survival 
and  normal  behavior  is  as  yet  undetermined,  the  observations  reported  have  dem- 
onstrated that  sound  is  significant  to  the  behavior  of  sea  robins.  The  work  has 
demonstrated  that  it  is  possible  to  control  sound  production  by  these  marine  fishes 
with  man-made  sounds.  The  findings  stand  as  an  exception  to  the  general  rule 
(Moulton  and  Backus,  1955)  that  production  of  man-made  sounds  causes  only 
quickened  swimming  movements  of  fishes. 

It  should  also  be  of  interest  to  students  of  marine  animal  noises  that  it  is  pos- 
sible to  incite,  without  handling  or  trapping,  the  calling  of  marine  fishes  by  trans- 
mission of  appropriate  signals,  thus  making  it  possible  to  move  experiments  to  the 
natural  environment  from  the  confines  of  laboratory  tanks,  which  under  the  best  of 
conditions  suppress  and  may  otherwise  modify  calling  behavior. 

I  am  grateful  for  highly  valued  criticism  to  Dr.  J.  B.  Hersey,  Mr.  William 
Schevill  and  Dr.  R.  H.  Backus  of  the  Woods  Hole  Oceanographic  Institution,  who 
read  the  manuscript  of  this  paper.  I  am  much  indebted  to  Mr.  Willard  Dow,  as 
to  many  others  of  the  Institution,  for  their  generous  technical  advice. 

SUMMARY 

1.  A  vibrant  grunt  and  a  staccato  call  of  sea  robins  in  the  Woods  Hole  area  are 
described.     Sounds  similar  to  these  can  be  obtained  by  manipulation  of  the  air 
bladder  and  by  stimulation  of  the  nerves  to  the  drumming  muscles. 

2.  It  is  suggested  that  the  sea  robin  grunt  is  part  of  a  general  alarm  reaction, 
and  that  the  staccato  call  is  related  to  the  breeding  behavior  of  the  sea  robin.     It  is 
suggested  that  the  staccato  call  may  serve  as  a  species  recognition  device  in  waters 
where  visibility  is  relatively  poor. 

3.  A  method  of  controlling  production  of  the  staccato  call  is  described.     Pro- 
duction of  the  call  can  be  initiated  by  playing  into  the  water  imitations  of  the  call 
and  recordings  of  the  call  itself.     The  calling  can  be  suppressed  by  playing  of  sig- 
nals of  200  to  600  cps,  and,  less  consistently,  by  playing  of  signals  of  600  cps  to  2  kc. 

4.  The  results  obtained  furnish  an  exception  to  the  general  rule  that  sound 
production  causes  only  quickened  swimming  movements  of  free  fishes,  and  demon- 
strate the  possibility  of  exercising  some  degree  of  control  over  the  behavior  of  fishes 
in  nature  with  man-made  sounds. 


398  JAMES  M.  MOULTON 

LITERATURE  CITED 

BIGELOW,  H.  B.,  AND  W.  C.  ScHROEDER,  1953.     Fishes  of  the  Gulf  of  Maine.     First  revision. 

Fish  and  Wildlife  Service,  Fish.  Bull.,  53. 
FISH,  M.  P.,  1948.     Sonic  fishes  of  the  Pacific.     Project  NR  083-003.     Contr.  N6  ori-195,  t.o. 

i,  between  ONR  and  Woods  Hole  Oceanographic  Institution,  Tech.  Report  No.  2. 
FISH,  M.  P.,   1954.     The  character  and  significance  of  sound  production  among  fishes  of  the 

western  North  Atlantic.     Bull.  Bingham  Oceanographic  Collection,  14,  Art.  3. 
VON  FRISCH,  K.,  1938.     The  sense  of  hearing  in  fish.     Nature,  141 :  8-11. 
GOODE,  G.  B.,  1888.     American  fishes.     Standard  Book  Co.,  New  York. 
GRIFFIN,  D.  R.,  1955.     Hearing  and  acoustic  orientation  in  marine  animals.     Papers  Mar.  Biol. 

and  Oceanogr.,  Deep-Sea  Research,  suppl.  to  Vol.  3,  pp.  406-417. 
MARSHALL,  N.  B.,  1954.     Aspects  of  deep  sea  biology.     Hutchinson's,  London. 
MOULTON,  J.  M.,  1955.     The  eliciting  and  suppressing  of  a  marine  biological  sound.     Bull.  Ecol. 

Soc.  Amer.,  36  :  80. 
MOULTON,  J.  M.,  AND  R.  H.  BACKUS,   1955.     Annotated  references  concerning  the  effects  of 

man-made  sounds  on  the  movements  of  fishes.     Fisheries  Circ.  No.   17,  Dep't  of  Sea 

and  Shore  Fisheries,  Augusta,  Maine. 


CYTOLOGICAL  EVIDENCE  FOR  A  ROLE  OF  THE  CORPUSCLES  OF 
STANNIUS  IN  THE  OSMOREGULATION  OF  TELEOSTS  x 

PRISCILLA  RASQUIN 
The  American  Museum  of  Natural  History,  New  York  24,  Nezv  York 

Until  recently  there  was  no  experimental  evidence  to  show  what  tissue  in  tele- 
osts  was  responsible  for  elaboration  of  the  vital  hormones  of  the  adrenal  cortex. 
For  many  years  adrenal  cortical  function  was  attributed  to  the  corpuscles  of  Stan- 
nius  first  described  by  Stannius  in  1839.  This  was  largely  because  of  their  morpho- 
logical position  on  the  ventral  surface  of  the  kidney,  analogous  to  the  adrenal  posi- 
tion in  other  species  of  the  vertebrate  series,  and  because  they  showed  histological 
characteristics  of  endocrine  function.  Although  Giacomini  (1908),  in  studies 
based  on  histology  and  morphology,  attributed  adrenal  cortical  function  to  secretory 
epithelium  lining  the  cardinal  veins,  he  did  not  relinquish  the  corpuscles  of  Stan- 
nius as  a  part  of  the  adrenal  complex  but  rather  designated  them  as  the  posterior 
interrenal  tissue.  He  called  the  glandular  tissue,  which  is  associated  with  the 
cardinal  veins  in  the  head  kidney,  the  anterior  interrenal  tissue. 

Many  important  factors  have  mediated  against  considering  the  corpuscles  of 
Stannius  as  true  adrenal  tissue.  Pettit  (1896)  demonstrated  compensatory  hyper- 
trophy of  one  corpuscle  after  removal  of  the  other  in  eels.  However,  Vincent 
(1898)  claimed  to  have  extirpated  both  corpuscles  in  eels  without  causing  death  to 
result.  The  inference  is  that  if  the  glands  were  as  vital  in  the  physiology  of  the 
teleost  as  the  adrenals  are  in  the  mammal,  the  eels  would  have  been  unable  to  sur- 
vive without  them.  Garrett  (1942)  confirmed  previous  observations  of  Giacomini 
(1911)  that  the  corpuscles  originate  embryologically  from  evaginations  of  the 
pronephric  ducts  and  not  from  mesothelium  which  provides  the  adrenal  cortical 
anlagen  of  other  vertebrates.  In  certain  forms,  as  Amla,  Garrett  thought  that  the 
glands  might  also  arise  from  mesonephric  tubules.  Rasquin  (1951)  showed  that 
the  corpuscles  were  not  stimulated  by  implantation  of  fresh  carp  pituitary  or  in- 
jection of  mammalian  ACTH  as  was  the  anterior  interrenal  tissue.  Pickford 
(1953)  confirmed  the  fact  that  the  corpuscles  were  not  under  pituitary  control  by 
demonstrating  that  there  was  no  atrophy  of  the  glands  after  hypophysectomy  in  the 
marine  cyprinodont  Fundulus  hcteroclitus,  although  this  investigator  was  also  un- 
able to  find  any  effect  of  hypophysectomy  on  the  anterior  interrenal  tissue.  How- 
ever, Chavin  (1954)  reported  complete  atrophy  of  anterior  interrenal  after  hypo- 
physectomy in  the  goldfish  and  no  reaction  of  the  corpuscles  of  Stannius  to  the 
same  operation. 

Rasquin  (1951)  reported  that  lipids  were  not  demonstrated  in  anterior  inter- 
renal cells  of  Astyana.v  by  the  use  of  osmic  acid  or  Sudan  IV  techniques.  How- 
ever, further  investigation  with  more  modern  techniques  applied  to  paraffin  rather 
than  frozen  sections  has  shown  that  this  is  not  the  case.  The  use  of  Baker's  acid 

1  This  work  was  supported  in  part  by  a  grant  from  the  National  Science  Foundation. 

399 


400  PRISCILLA  RASQUIN 

hematein  stain  with  acridine  red,  as  suggested  by  Rennels  (1953),  has  shown  a 
positive  reaction  for  phospholipids  in  the  anterior  interrenal  tissue  of  the  teleost. 
The  diffuse  nature  of  this  tissue  and  the  fact  that  patches  of  cells  containing  posi- 
tive droplets  alternate  with  those  that  are  negative  in  reaction  make  it  possible  to 
lose  positively  reacting  tissue  in  broken-up  frozen  sections.  The  discovery  that  the 
glandular  cells  of  the  corpuscles  of  Stannius  also  contained  phospholipid  granules 
provided  a  technique  for  studying  the  cellular  reaction  of  the  gland  to  various  ex- 
perimental procedures. 

MATERIALS  AND  METHODS 

A  total  of  135  individuals  of  the  species  Astyanax  mexicanus  (Filippi)  were 
used  in  the  course  of  the  experiment.  All  were  sexually  mature,  between  one  and 
two  years  of  age  and  appeared  in  healthy  and  vigorous  condition.  Experimental 
procedures  involved  the  injections  of  water,  electrolytes,  DCA  and  pitressin.  Table 
I  shows  the  distribution  of  fish  among  the  various  procedures  and  the  times  al- 
lowed to  elapse  between  injection  and  killing.  In  each  group  of  three  or  more 
animals,  the  tissues  from  one  fish  were  fixed  in  Bouin's  fluid  and  stained  in  Harris' 
hematoxylin  and  eosin ;  the  tissues  from  the  remaining  fish  in  each  group  were  fixed 
in  calcium-formol  and  stained  with  acid  hematein  (Baker,  1946).  The  fish  were 
killed  by  decapitation  and  the  musculature  from  one  side  of  the  body  and  the  air 
bladder  were  removed  before  placing  the  body  in  the  fixing  fluid.  About  an  hour 
later  the  kidneys,  containing  the  corpuscles  of  Stannius,  were  dissected  out  and  re- 
turned to  fresh  fluid.  This  procedure  insured  rapid  fixation  of  the  rather  labile 
granules  of  the  glandular  tissue.  All  tissues  were  imbedded  in  paraffin  and  sec- 
tioned at  five  microns  and  some  were  counterstained  with  acridine  red. 

The  volume  of  all  fluid  injections  was  0.05  cc.  except  for  those  of  pitressin-and- 
water,  where  0.15  cc.  was  used  and  the  injections  were  made  into  the  abdominal 
cavity.  Glass-distilled  water  was  used,  alone  and  for  dissolving  sodium  and  po- 
tassium chloride.  The  implantation  of  dry  DCA  pellets  was  also  made  intraperi- 
toneally.  These  contained  75  mg.  each,  and,  inasmuch  as  this  amount  was  far  too 
great  for  the  small  fish,  the  pellets  were  broken  up  and  small  pieces  were  used. 
With  this  method  there  is  no  way  of  measuring  the  amount  of  hormone  absorbed 
by  any  one  fish.  However,  pieces  of  pellet  were  observed  in  all  implanted  fish 
at  the  time  of  death,  indicating  a  continuous  supply  of  hormone  throughout  the 
experimental  period. 

Two  series  of  injections  were  made  with  pitressin  for  a  study  of  the  reaction 
to  antidiuretic  hormone.  The  first  of  these  consisted  of  one  pressor  unit  in  0.05  cc. 
aqueous  solution  in  each  fish.  The  second  series  consisted  of  the  same  amount  of 
hormone  diluted  to  0.15  cc.  with  glass-distilled  water,  for  the  purpose  of  giving 
an  additional  stimulus  of  water  load  in  the  fish. 

Weights  of  Astyana.r  of  this  age  group  range  between  one  and  two  and  one-half 
grams.  Weighing  the  fish  either  before  or  after  killing  was  avoided,  first  because 
prompt  fixation  was  necessary,  and  secondly,  because  the  fright  caused  by  extra 
handling  might  have  had  some  effect  on  granulation  in  the  cells  to  be  studied. 

In  addition,  one  Astyanax  was  used  for  each  of  the  following  methods :  the 
pyridine  extraction  test  (Baker,  1946)  to  verify  the  phospholipid  content  of  the 
tissues  reacting  positively  to  acid  hematein,  Cowdry's  modification  of  Bensley's 


OSMOREGULATION  IX  TELEOSTS 


401 


TABLE  I 

Numbers  of  Astyanax  used  and  duration  of  experimental  procedures 


Experimental  procedure 
Implantation  of  DCA  pellets 


Daily  injections  0.05  cc.  distilled  water 


Daily  injections  2.0  mg.  sodium  chloride 


Daily  injections  0.5  mg.  potassium  chloride 


Single  injection  1.0  mg.  potassium  chloride 


Single  injection  aqueous  pitressin  1  unit 


Single  injection  1  unit  pitressin  plus  0.1  cc. 
water 


Nos.  of 
fish 

No.  of  days 
before  sacrifice 

3 

1 

3 

3 

3 

5 

3 

8 

3 

10 

3 

18 

3 

25 

6 

75 

3 

1 

3 

3 

3 

5 

4 

7 

3 

1 

3 

3 

3 

5 

3 

7 

3 

1 

3 

3 

3 

5 

3 

7 

3 

30  minutes 

3 

1  hour 

1 

2  hours 

4 

24  hours 

3 

30  minutes 

3 

1  hour 

3 

2  hours 

3 

4  hours 

3 

6  hours 

3 

24  hours 

3 

30  minutes 

3 

1  hour 

3 

2  hours 

3 

4  houfs 

3 

6  hours 

3 

24  hours 

Tests  for  pyridin  extraction,  mitochondria,  4 

and  ascorbic  acid 

method  for  mitochondria  as  given  by  Jones  ( 1950 )  to  ascertain  the  nature  of  the 
grannies  in  the  cells  of  the  corpuscles  of  Stannius,  and  Bourne's  (1936)  method 
to  discover  the  presence  or  absence  of  ascorbic  acid  in  the  same  glands. 

Lastly,  ten  fish  were  injected  with  0.1  cc.  distilled  water  five  days  a  week  for 
four  weeks  and  ten  others  were  allowed  to  live  in  1  °/c  sodium  chloride  for  ten  days. 
The  corpuscles  of  Stannius  of  all  these  were  studied  after  staining  with  Baker's 
acid  hematein. 


402 


PRISCILLA  RASQUIN 


* 

^i^^^^^^Mfli 

*    J* 


• 


Cells  of  the  corpuscles  of  Stannius  of  Astyanax  mcxicanns  stained  with  Baker's  acid  hema- 
tein  after  various  experimental  procedures.     Magnification  1200  X. 

FIGURE  1.     Normal  untreated  fish  showing  blackened  granules  in  the  cytoplasm. 
FIGURE  2.     Cells  unstained  after  pyridin  extraction  test,  indicating  the  blackened  granules 
to  be  composed  of  phospholipid. 


OSMOREGULATION  IN  TELEOSTS  403 

EXPERIMENTAL  RESULTS 

Many  teleost  tissues  reacted  positively  to  Baker's  acid  hematein  stain :  red  blood 
cells,  myelin  sheaths  of  nerves,  zymogen  granules  in  exocrine  pancreas,  granules  in 
cells  of  both  anterior  interrenal  and  corpuscles  of  Stannius,  and  granules  of  the 
coarse  granular  eosinophiles  found  in  the  connective  tissues  and  sometimes  in  the 
blood  of  teleosts.  The  only  tissues  that  remained  positive  after  pyridin  extraction 
were  the  erythrocytes  and  some  of  the  large  granules  in  the  anterior  interrenal  cells. 

Figure  1  is  a  photomicrograph  showing  the  positive  reaction  to  Baker's  acid 
hematein  stain  in  the  cells  of  the  corpuscle  of  Stannius  of  a  normal,  untreated 
Astyana.v.  The  black  material  is  made  up  of  phospholipid  granules  and  possibly 
also  mitochondria.  Figure  2  shows  the  corpuscle  cells  devoid  of  any  stained  granu- 
lation after  application  of  the  pyridin  extraction  test;  the  dark  stained  objects  are 
erythrocytes.  The  corpuscle  is  normally  made  up  of  small  granular  cells  that  are 
greater  both  in  amount  of  cytoplasm  and  size  of  nucleus  at  the  periphery  than  at 
the  center  of  the  gland.  Sometimes  the  gland  has  a  cord-like  appearance  caused 
by  two  lines  of  cells  on  either  side  of  a  capillary.  The  nuclei  are  distal  to  the 
blood  vessel,  and  the  cytoplasmic  granules  crowded  into  the  part  of  the  cell  ad- 
jacent to  the  capillary  wall.  At  other  times,  probably  associated  with  less  activity, 
no  cord-like  or  acinar  arrangement  can  be  detected  and  the  cells  appear  to  be 
crowded  within  the  confines  of  the  connective  tissue  capsule  without  any  obvious 
architecture.  Bobin  (1949),  using  Sudan  Black  B  and  osmic  acid,  has  also  dem- 
onstrated the  lipid  nature  of  the  cellular  granules  of  the  corpuscles  in  the  European 
eel.  In  this  species,  she  was  able  to  distinguish  both  mitochondria,  which  were 
rod-like  or  slightly  filamentous,  and  secretory  granules,  which  were  spherical.  A 
similar  distinction  was  not  apparent  in  Astyana.r.  When  stained  for  mitochondria, 
the  cells  wrere  found  to  be  crowded  with  these  organelles  which  were  spherical  and 
smaller  than  the  granules  stained  with  acid  hematein.  The  size  difference,  however, 
may  be  an  artifact  related  to  the  different  fixing  and  staining  process.  The  proba- 
bility is  that  acid  hematein  stains  both  types  of  inclusions  at  the  same  time.  After 
the  corpuscle  cells  are  degranulated  by  experimental  procedures  there  is  a  simul- 
taneous loss  of  so  much  cytoplasm  that  mere  non-reactivity  of  mitochondria  cannot 
be  responsible  for  the  loss  of  staining  reaction.  The  application  of  acid  silver  ni- 
trate for  demonstration  of  ascorbic  acid  resulted  in  only  very  rare  stained  granules 
in  occasional  corpuscle  cells.  However,  Fontaine  and  Hatey  (1955)  have  found 
a  high  content  of  ascorbic  acid  in  these  glands  in  the  salmon. 

Effects  of  desoxycorticosterone  acetate  (DCA) 

Implantation  of  DCA  pellets  brought  about  an  enlargement  of  the  cells  of  the 
corpuscles  with  a  simultaneous  increase  in  number  and  size  of  cytoplasmic  granules. 

FIGURE  3.  Increase  in  granulation  in  corpuscle  cells  of  a  fish  that  had  received  injections 
of  water  five  days  a  week  for  four  weeks. 

FIGURE  4.  Decrease  in  granulation  in  corpuscle  cells  of  a  fish  that  had  lived  in  1%  saline 
for  ten  days. 

FIGURE  5.  Decrease  in  granulation  6  hours  after  injection  of  one  unit  undiluted  aqueous 
pitressin. 

FIGURE  6.  Increase  in  granulation  6  hours  after  injection  of  one  unit  pitressin  plus  an 
added  water  load. 


404  PRISCILLA  RASQUIN 

The  hypertrophy  of  the  cells  with  their  heavy  granulation,  particularly  at  the  pe- 
riphery of  the  glands,  was  observed  as  early  as  three  days  after  implantation.  After 
18  days,  heavy  granulation  was  seen  in  all  the  cells  throughout  the  gland.  At  the 
same  time,  the  cord-like  arrangement  of  the  cellular  elements  along  the  capillaries 
was  pronounced,  particularly  noticeable  under  the  low  power  of  the  microscope. 
This  reaction  was  maintained  throughout  the  75-day  period.  The  hypophyses  of 
the  three  animals  killed  18  days  after  implantation  were  sectioned  and  stained  with 
Masson's  trichrome  stain.  Histological  study  revealed  that  these  glands  were  ap- 
parently normal  in  every  detail.  Prolonged  administration  of  DCA  had  no  such 
effect  on  the  transitional  lobes  as  was  observed  by  Rasquin  and  Atz  ( 1952 )  after 
injection  of  cortisone  in  the  same  species.  Administration  of  cortisone  brought 
about  an  inversion  of  the  ratio  of  acidophils  to  basophils  with  subsequent  marked 
acidophilia  of  the  lobe. 

Effects  of  water 

The  same  results  in  the  cells  of  the  corpuscles  of  Stannius,  enlargement  and 
heavy  granulation,  were  obtained  by  injections  of  distilled  water.  However,  study 
of  the  glands  on  the  first  and  third  days  after  injections  were  started  showed  an 
initial  shrinkage  of  the  cells,  causing  spaces  to  occur  between  them,  and  there  was 
some  evidence  of  degranulation  on  the  first  day.  From  the  fifth  day  onward  the 
cells  were  hypertrophied  and  heavily  granulated.  The  granulations  were  evident 
even  in  the  hematoxylin  and  eosin-stained  sections  where  they  were  markedly  acido- 
philic.  Figure  3  is  a  photomicrograph  of  the  corpuscle  of  a  fish  injected  five  days 
a  week  for  four  weeks  with  distilled  water.  Heavy  granulation  is  very  evident 
here.  Furthermore,  hypertrophy  of  the  entire  gland  was  seen  in  most  of  the  ten 
fish  subjected  to  this  procedure ;  sometimes  the  hypertrophy  occurred  in  only  one 
corpuscle  so  that  the  hypertrophied  organ  would  be  twice  the  size  of  the  other  one 
in  the  same  animal. 

Effects  of  sod  in  111  chloride 

Continued  sodium  chloride  injection  at  a  dosage  of  2  mg.  per  day  brought  about 
only  slight  hypertrophy  of  the  cells  of  the  corpuscles  of  Stannius  and  granulation 
appeared  about  the  same  as  that  seen  in  normal  glands.  However,  the  glands  in 
the  fish  that  lived  10  days  in  \%  saline  showed  degranulation  of  the  cells.  This 
reaction  is  seen  in  Figure  4. 

Effects  of  potassium  chloride 

Because  of  the  toxicity  of  potassium  chloride  the  daily  dose  had  to  be  reduced 
to  0.5  mg.  in  order  to  ensure  survival.  Doses  of  one  mg.  each  were  fatal,  the  fish 
dying  between  two  and  24  hours  after  injection.  Some  of  these  were  preserved 
for  study  (Table  I).  After  one  injection  of  0.5  mg.,  the  cells  of  the  Stannius  cor- 
puscles appeared  large  and  heavily  granulated.  Subsequently  degranulation  oc- 
curred and  the  cells  were  much  smaller  in  size.  In  addition,  the  cord-like  arrange- 
ment of  the  cells  was  disrupted  and  red  blood  cells  were  scarce  as  a  result  of  de- 
creased blood  supply.  Degranulation  was  obvious  in  all  fish  dead  24  hours  after 
the  one-mg.  dose.  In  sections  stained  with  hematoxylin  and  eosin,  it  could  be 


OSMOREGULATION  IN  TELEOSTS  405 

plainly  seen  that  the  degranulation  resulted  in  considerable  loss  of  cytoplasm  from 
the  cells.  Nuclei  were  crowded  together,  especially  in  the  center  of  the  gland 
where  they  were  virtually  denuded  of  cytoplasm.  In  the  corpuscles  of  the  fish  re- 
ceiving the  smaller  daily  doses  complete  degranulation  was  not  seen ;  some  glands 
contained  more  stained  granules  than  others  but  in  general,  all  the  cells  were  smaller 
than  normal  and  the  granulation  was  fine  and  usually  confined  to  a  small  area  about 
the  nucleus. 

Effects  of  pitrcssin 

The  cells  of  the  corpuscles  of  Stannius  reacted  differently  to  the  two  procedures 
employed  for  pitressin  administration.  With  pitressin  alone,  the  cells  were  de- 
granulated  and  decreased  in  size  although  this  was  not  so  extreme  as  when  po- 
tassium chloride  was  used.  One-half  hour  after  injection,  the  cells  showed  a  very 
fine  granulation  distributed  mostly  in  a  narrow  ring  around  the  nucleus.  The 
same  picture  was  obtained  after  one  hour  except  that  the  hematoxylin  and  eosin- 
stained  sections  showed  the  nuclei  to  be  somewhat  shrunken  and  hyperchromatic. 
After  two  hours  the  granules  seemed  larger  and  more  numerous  and  this  slightly 
heavier  granulation  persisted  up  to  six  hours  after  injection.  By  24  hours,  however, 
the  gland  had  returned  to  its  normal  appearance.  Figure  5  represents  the  cor- 
puscle cells  six  hours  after  injection  of  pitressin. 

In  great  contrast  to  Figure  5  is  Figure  6  which  represents  the  corpuscle  cells 
six  hours  after  the  injection  of  diluted  pitressin.  The  hypertrophied  cells  with 
heavy  black  granulation  were  typical  of  all  the  corpuscles  from  one  to  six  hours 
after  injection.  After  only  one-half  hour  the  cells  appeared  small  and  granulation 
was  fine  and  confined  mainly  to  a  ring  around  the  nucleus,  as  described  for  the  in- 
jections of  pitressin  alone.  After  24  hours,  the  corpuscle,  although  still  heavily 
granulated,  had  begun  to  take  on  a  more  normal,  lighter  stained  appearance. 

All  the  experimental  procedures,  with  the  exception  of  pitressin  injections, 
served  to  decrease  the  staining  response  of  mitochondria  in  kidney  tubules.  In  the 
case  of  DCA  administration,  the  staining  reactivity  returned  to  the  mitochondria  of 
the  tubules  after  75  days,  indicating  that  the  fish  had  made  some  physiological  ad- 
justment to  long  continued  administration  of  this  hormone.  The  kidney  tubules  of 
all  fish  included  in  the  pitressin-injected  group  showed  deeply  stained  mitochondria, 
especially  noticeable  in  the  more  distal  parts  of  the  tubules,  the  intermediate  seg- 
ments and  the  ureters. 

DISCUSSION 

Much  of  the  literature  pertaining  to  the  corpuscles  of  Stannius  is  now  of  his- 
torical interest  only.  A  full  bibliography  up  to  1946  was  published  by  Aboim. 
The  most  recent  contribution  is  by  Bauchot  (1953)  who  studied  the  comparative 
anatomy  of  the  glands  in  47  different  species  including  both  marine  and  fresh  water 
forms,  attempting  to  relate  their  anatomical  location  to  phylogeny.  He  concluded 
that  the  most  primitive  position  of  the  corpuscles  is  an  anterior  one  about  midway 
of  the  length  of  the  kidney,  and  the  most  evolved,  a  posterior  one,  much  nearer 
the  vent,  although  there  were  exceptions,  as  in  the  salmonids  and  Solea  where 
the  location  of  the  corpuscles  was  not  compatible  with  the  systematic  position  of 


406  PRISCILLA  RASQUIN 

the  fishes.  This  author  also  considered  the  number  of  corpuscles  to  have  a  phylo- 
genetic  significance.  Thus  the  holostean,  Ainia,  possesses  between  40  and  50  cor- 
puscles and  the  salmonids  anywhere  from  six  to  14,  while  the  usual  number  for 
most  teleosts  is  two.  In  Astyana.v  the  number  was  found  to  vary  between  two  and 
four,  although  two  was  by  far  the  most  common.  Garrett  (1942)  also  thought  the 
large  number  of  corpuscles  was  a  more  primitive  condition,  the  advanced  condition 
of  two  major  corpuscles  being  produced  by  the  fusion  of  many  smaller  ones.  Gar- 
rett, after  demonstrating  the  origin  of  the  corpuscles  from  the  pronephric  duct,  sug- 
gested a  homology  of  the  glands  with  a  part  of  the  Mullerian  duct  and  Bauchot  is 
in  agreement  with  this  suggestion.  Some  of  the  reasoning  behind  this  idea  is  con- 
cerned with  the  fact  that  the  chondrosteans,  in  which  the  corpuscles  of  Stannius  are 
absent,  have  reduced  and  non-functional  Mullerian  ducts,  while  the  holosteans  and 
teleosteans,  in  which  there  are  remnants  of  the  Mullerian  ducts,  possess  the  cor- 
puscles of  Stannius. 

In  general,  two  kinds  of  changes  were  brought  about  in  the  cells  of  the  corpuscles 
by  the  experimental  procedures.  Degranulation,  loss  of  cytoplasm  and  consequent 
decrease  in  size  of  cells  accompanied  the  administration  of  potassium  chloride,  un- 
diluted pitressin,  and  long-continued  immersion  in  saline.  Hypertrophy  of  cells 
with  increase  in  numbers  and  size  of  blackened  granules  accompanied  the  adminis- 
tration of  water,  diluted  pitressin  and  DCA.  The  non-reactivity  of  the  cells  after 
sodium  chloride  injection  may  be  owing  to  the  fact  that  the  dosage  was  too  small  to 
have  an  effect.  Unfortunately,  little  is  known  about  the  action  of  DCA  in  fish. 

Final  interpretation  of  these  results  must  await  further  study,  particularly  by 
investigators  who  have  physiological  techniques  at  their  disposal.  It  is  possible 
that  the  corpuscles  were  responding  merely  to  the  increased  water  load,  that  de- 
granulation  after  administration  of  potassium  chloride  was  owing  to  the  toxic  ef- 
fects of  the  potassium  ion  and  that  the  degranulation  after  undiluted  pitressin  was 
an  initial  release  of  secretion  unaccompanied  by  further  immediate  stimulation.  It 
seems  fairly  obvious  from  these  results  that  the  corpuscles  of  Stannius  have  some 
function  in  osmoregulation,  inasmuch  as  changes  in  the  granulation  are  accompanied 
by  changes  in  the  metabolic  activity  of  the  kidney  tubules. 

If  the  function  of  the  corpuscles  has  to  do  with  water  excretion  it  might  help 
to  explain  why  various  investigators  have  been  unable  to  demonstrate  water  reten- 
tion in  teleosts  after  administration  of  posterior  lobe  hormones.  Burgess,  Harvey 
and  Marshall  (1933)  were  unable  to  demonstrate  any  effect  on  urine  flow  in  the 
catfish,  Anieiurus  nebulosus,  with  0.2  to  2.0  units  of  pitressin  per  kilogram.  Their 
graph  shows  a  slight  increase  in  water  diuresis  for  the  catfish  after  pitressin  injec- 
tion, probably  without  statistical  significance.  Boyd  and  Dingwall  (1939),  using 
pituitrin,  were  unable  to  cause  an  increase  in  weight  in  young  carp,  although  com- 
parable doses  of  the  hormone  acted  positively  on  frogs  to  increase  the  weight  as  a 
consequence  of  water  retention.  Fontaine  and  Raffy  (1950)  thought  that  the 
failure  might  have  been  due  to  the  use  of  mammalian  hormone  and  therefore  they 
repeated  the  experiments  with  preparations  made  from  the  pituitaries  of  fish,  carp, 
eels,  etc.  Their  fish  posterior  pituitary  preparations  proved  to  be  potent  in  causing 
water  retention  in  frogs,  but  negative  results  were  still  obtained  in  the  fish. 

Callamand  et  al.  (1951)  reported  that  the  hypophysis  was  not  concerned  with 
osmoregulation  in  eels  inasmuch  as  they  were  able  to  place  hypophysectomized 


OSMOREGULATION  IN  TELEOSTS  407 

Angitilla  back  and  forth  from  fresh  water  to  sea  water  and  even  into  water  with 
twice  the  salinity  of  sea  water  without  any  deleterious  effects.  On  the  other  hand, 
Pickford  (1953)  found  that  hypophysectomized  Fundulns  hctcroclitns  were  unable 
to  survive  in  fresh  water  or  diluted  sea  water,  although  this  species  is  normally 
euryhaline.  Burden  (1956)  was  able  to  keep  these  fish  alive  in  fresh  water  by 
replacement  therapy  of  Fundulns  pituitary  material.  He  postulated  the  secretion  of 
an  unknown  factor  by  the  Fundulns  pituitary  which  regulates  the  salt  balance  of  the 
fish  in  fresh  water.  Other  investigators  (Matthews,  1933;  Abramowitz,  1937) 
have  reported  no  difficulty  maintaining  hypophysectomized  Fundulns  in  fresh  water. 

Neurosecretory  material  in  the  hypophysis  and  hypothalamus  of  a  teleost  was 
first  described  by  Scharrer  (1932).  Since  then  Arvy,  Fontaine  and  Gabe  (1954) 
have  shown  that  neurosecretory  material  in  the  hypothalamo-hypophyseal  systems 
of  Phoxinus  and  Anguilla  can  be  depleted  by  subjecting  the  fish  to  hypertonic  solu- 
tions, indicating  a  sensitivity  of  the  neurosecretory  apparatus  to  the  need  for  re- 
taining water  in  the  internal  environment.  Rasquin  and  Stoll  (1955)  have  shown 
that  neurosecretion  may  be  withheld  in  the  brain  nuclei  after  injection  of  pitressin, 
indicating  a  reaction  to  the  antidiuretic  principle,  even  though  it  has  not  yet  been 
demonstrated  physiologically. 

Interpretations  of  cellular  activity  in  the  corpuscles  of  Stannius  for  this  report 
depend  mainly  on  the  reaction  of  the  cells  to  Baker's  acid  hematein  stain  for  phos- 
pholipids.  Unfortunately  the  significance  of  phospholipin  in  cellular  metabolism 
is  not  yet  thoroughly  understood.  Among  several  theories  reviewed  by  Sinclair 
(1934),  one  considers  that  phospholipids  are  increased  during  cellular  activity,  par- 
ticularly in  actively  secreting  glands  such  as  the  salivary  glands  and  the  corpus  lu- 
teum.  Rennels  (1953)  also  believes  that  phospholipids  play  an  important  role  in 
the  secretory  activity,  citing  the  staining  reaction  of  hypophyseal  acidophils,  adrenal 
cortical  cells  and  mitochondria.  He  points  out  that  different  phases  of  activity  of 
both  secretory  granules  and  mitochondria  are  accompanied  by  positive  or  negative 
reactions  to  the  stain.  After  gonadectomy,  mitochondria  of  the  delta  cells  of  the 
rat  hypophysis  showed  an  increased  activity  presumably  associated  with  increased 
secretory  function  of  the  cells,  even  though  the  secretory  granules  of  these  cells  have 
no  affinity  for  the  stain. 

Cain  and  Harrison  (1950)  have  also  suggested  that  histochemically  demon- 
strable phospholipid  is  connected  with  some  special  metabolic  activity.  In  a  cyto- 
logical  study  of  the  adrenal  cortical  cells  in  the  rat,  they  have  shown  that  mitochon- 
dria have  an  affinity  for  acid  hematein  during  the  phase  of  active  secretion  in  the 
cell,  and  that  after  discharge  of  secretory  products,  the  mitochondria  become  nega- 
tive to  the  stain.  The  mitochondria  become  positive  to  the  stain  before  the  lipid 
droplets,  but  the  droplets  are  not  formed  from  the  mitochondria ;  rather  they  are 
separate  and  distinct  within  the  cytoplasm.  Therefore,  for  the  present  report,  the 
increase  in  positive  staining  response  of  the  corpuscles  of  Stannius  has  been  inter- 
preted as  an  indication  of  increased  metabolic  activity.  This  interpretation  is 
strengthened  by  simultaneous  hypertrophy  of  the  cells  and  of  the  whole  organ  with 
increased  stainable  granulation. 

All  these  results  strongly  indicate  the  presence  of  a  special  mechanism  antago- 
nistic to  the  antidiuretic  hormone  in  teleosts,  and  this  may  possibly  be  produced 
by  the  corpuscles  of  Stannius  which  are  not  found  in  other  vertebrates. 


408  PRISCILLA  RASQUIN 

SUMMARY 

1.  The  effects  of  DCA,  pitressin,  water,  and  sodium  and  potassium  chloride  on 
the  cytology  of  the  corpuscles  of  Stannius  were  studied  by  means  of  Baker's  acid 
hematein  stain  for  phospholipids.     The  fresh  water  characin  Astyana.v  nic.ricanus 
was  used. 

2.  Two  kinds  of  changes  in  the  cells  of  the  corpuscles  were  brought  about  by 
the  experimental  procedures :  degranulation,  loss  of  cytoplasm  and  consequent  de- 
crease in  size  of  cells  accompanied  the  administration  of  potassium  chloride,  un- 
diluted pitressin  and  long  continued  immersion  in  \%  sodium  chloride,  and  hyper- 
trophy of  cells  with  increase  in  numbers  and  size  of  blackened  granules  accompanied 
the  administration  of  water,  diluted  pitressin  and  DCA. 

3.  Loss  of  staining  reaction  in  mitochondria  of  kidney  tubules  was  associated 
with  increased  secretory  activity  in  the  corpuscles  of  Stannius  except  in  the  case  of 
long  continued  DCA  administration  and  administration  of  pitressin. 

4.  The  results  are  interpreted  as  indicating  a  function  of  the  corpuscles  of  Stan- 
nius in  the  osmoregulation  of  these  fish  possibly  connected  with  excretion  of  excess 
water. 

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OSMOREGULATION  IN  TELEOSTS  409 

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ON  THE  ECOLOGY  OF  THE  LOWER  MARINE  FUNGI  l>  - 

HELEN  S.  VISHNIAC 
Department  of  .Microbiology,  Yale  University,  Neiv  Haven  11,  Connecticut 

The  lower  marine  fungi  (i.e.,  Myxomycetes  and  aquatic  Phycomycetes)  have 
generally  been  described  as  occurring  on  plant  and  animal  hosts.  While  several 
forms  have  been  described  as  saprophytes,  the  only  genus  known  to  occur  on  debris 
is  Labyrinthula  (Sparrow,  1936).  All  other  described  species  are  endobiotic  or 
epibiotic  with  rhizoids  penetrating  living  or  dead  host  cells.  They  are  also  sporadic 
in  occurrence.  The  application  of  a  semi-quantitative  plating  technique  to  sea 
water  has  now  established  that  lower  fungi  are  far  more  common  in  littoral  waters 
than  previous  studies  indicate,  and  has  suggested  a  new  ecological  niche  for  these 
fungi. 

The  plating  technique  which  we  used  consisted  of  spreading  samples  of  sea  water 
with  a  bent  glass  rod  on  the  surface  of  a  solid  isolation  medium  (Table  I). 

TABLE  I 

Isolation  Medium 

Sea  water  80  ml./  100  ml. 

Gelatin  hydrolysate  0.1% 

Glucose  (added  aseptically)  0.1% 

Liver  1:20  0.001% 
B-vitamins 

Agar  1.5% 

adjusted  to  pH  7.5 

Marine  mineral  base  (Vishniac,  1955)  was  sometimes  substituted  for  sea  water. 
Gelatin  hydrolysate  and  the  B-vitamin  mixture  were  prepared  as  by  Vishniac  and 
Watson  (1953).  Liver  extract  concentrate  1:20  was  obtained  from  the  Nutri- 
tional Biochemicals  Co. 

The  moisture  content  of  the  medium  is  critical :  the  agar  plates  should  be  dried 
overnight,  but  not  allowed  to  stand  for  more  than  two  days.  Just  before  use  the 
plates  are  spread  with  2000  units  of  Penicillin  G  (Squibb,  buffered)  and  0.5  mg. 
of  dihydrostreptomycin  sulfate  (Wyeth)  in  concentrated  aqueous  solution.  These 
plates  will  then  absorb  a  0.2-ml.  sea  water  sample  in  an  hour  or  two.  After  inocu- 
lation, the  plates  were  incubated  at  20  degrees  or  less.  Such  plates  support  the 
growth  of  lower  fungi,  yeasts,  molds,  and  some  diatoms,  but  few  or  no  bacteria. 
We  have  found  unfortunately  incomplete  suppression  of  bacterial  growth  when 
water  samples  taken  from  City  Point,  New  Haven,  near  the  sewage  disposal  plant, 

1  Contribution  No.  869  from  The  Woods  Hole  Oceanographic  Institution. 

2  It  is  a  pleasure  to  acknowledge  the  hospitality  of  Dr.  J.  Ryther  and  other  members  of 
The  Woods  Hole  Oceanographic  Institution,  the  kindness  of  Dr.  W.  R.  Taylor  in  identifying 
Polysiphonia  urccolata,  and  the  excellent  technical  assistance  of  Miss  E.  A.  Adair.     This  study 
was  aided  by  a  contract  between  Yale  University  and  the  Office  of  Naval  Research,  Department 
of  the  Navy,  NR  135-241.     Reproduction  in  whole  or  part  is  permitted  for  any  purpose  of  the 
United  States  Government. 

410 


ECOLOGY  OF  LOWER  MARINE  FUNGI  411 

were  plated.  Colonies  of  lower  fungi  visible  to  the  naked  eye  appear  in  a  week 
or  ten  days.  Colonies  were  counted  at  30  X  and  further  examined  at  100  X. 

The  results  of  spreading  triplicate  0.2-ml.  samples  of  sea  water  taken  in  June, 
1956  from  waters  in  and  around  Woods  Hole,  Mass,  are  given  in  Table  II.  The 
number  of  species  represented  is  probably  a  minimum  figure,  since  only  colonies 
which  were  markedly  distinct  in  color,  texture,  or  size  of  thallus  were  picked  for 
further  study.  Yeasts,  molds  and  diatoms  were  not  counted  regularly.  Yeasts 
were  rare.  From  0-5  colonies  of  molds,  mainly  Penicillium  spp.  of  uncertain 
provenance,  were  found  on  those  plates  for  which  molds  were  counted.  The  pres- 
ence of  molds  was  not  correlated  with  the  presence  of  lower  fungi. 

It  is  evident  from  the  poor  agreement  between  triplicate  platings  in  Table  II 
that  this  technique  is  quantitative  only  within  an  order  of  magnitude.  The  pro- 
cedure suffers  from  the  following  defects  : 

TABLE  II 

Occurrence  of  lower  fungi  in  sea  water 
Origin  of  sample  Colonies/plate  No.  species 

1.  Sea  water  tap,  W.H.O.I.  0,0,0  0 

2.  Great  Harbor  0,  0,  0  0 

3.  Eel  Pond  94,  34,  20  7 

Tap  water,  from  bowls  with  algae 

4.  Algae  washed  ca.  5  hrs.  23,  8,  20  5 

5.  Algae  washed  ca.  24  hrs.  15,  10,  1  6 

(another  collection) 

Water  expressed  from  algae 

6.  Pilings,  U.  S.  Fish  and  Wildlife  Station  62,  5,  56  2 

7.  Rocks,  Red  Spindle  (Grassy  Island)  66,  214,  35  7 

( 1 )  Spreading  the  sample,  necessitated  by  the  aerobic  nature  and  poor  tempera- 
ture tolerance  of  the  desired  forms,  entails  the  loss  of  3  to  8%  of  a  0.2-ml.  sample. 
The  amount  of  sample  remaining  on  the  glass  rod  was  determined  by  weighing  the 
salt  remaining  on  the  glass  rod  after  spreading  a  20%  NaCl  solution. 

(2)  Either  a  thallus  or  a  spore  may  give  rise  to  a  colony.     The  ecologic  impli- 
cations of  the  presence  of  a  thallus  or  a  spore  in  the  sample  are  quite  different. 

(3)  A  thallus  may  produce  zoospores  in  the  interval  between  spreading  and 
drying  of  the  sample,  giving  rise  to  several  colonies.     The  occasional  appearance 
of  groups  of  colonies  of  the  same  form  on  a  plate  was  presumed  to  have  this  cause. 

(4)  Not  every  viable  spore  or  thallus  present  in  the  sample  may  give  rise  to  a 
colony.     The  conditions  provided  for  growth  were  chosen  after  study  of  a  limited 
number  of  marine  forms — members  of  the  genera  Labyrinthula,  Sirolpidiuni,  Thrau- 
stochytriinn,  and  three  unidentified  isolates.     Obviously,  forms  with  other  require- 
ments may  exist.     We  have  been  particularly  interested  in  assessing  the  probable 
extent  of  the  selective  action  of  the  medium. 

It  is  probable  that  forms  with  additional  nutritional  requirements  would  grow 
sufficiently  on  the  medium  used  to  form  a  countable  colony.  The  ability  of  an  in- 
dividual organism,  previously  well  nourished,  to  form  a  colony  in  the  absence  of 
required  nutrients  is  inversely  proportional  to  the  quantity  of  nutrient  required. 
Generally,  absence  of  a  suitable  carbon  source  is  felt  first,  amino  acids  required  as 


412  HELEN  S.  VISHNIAC 

growth  factors  next,  and  lastly  the  absence  of  added  vitamins — some  of  which  may 
be  stored  in  quantities  sufficient  for  many  generations.  It  is  highly  improbable 
that  suitable  carbon  sources  for  any  of  the  fungi  with  which  we  were  concerned 
were  lacking.  As  a  source  of  amino  acids,  gelatin  hydrolysate  is  inferior,  since  it 
is  poor  in  methionine  and  in  the  aromatic  amino  acids.  Nevertheless,  we  have 
maintained  methionine-requiring  Phycomycetes  on  gelatin  hydrolysate  media  for 
over  a  year  of  semi-monthly  transfers.  The  feasibility  of  isolating  forms  requiring 
growth  factors  not  present  in  this  medium  was  demonstrated  by  the  isolation  of 
sterol-requiring  Labyrinthidas  from  two  of  the  sea  water  samples  used  in  this  study 
(4  and  7,  Table  II).  Syntrophism  may  occur  (though  no  obvious  examples  were 
seen)  on  these  plates. 

An  experimental  approach  to  this  problem  was  made  by  plating  a  sample  of  sea 
water  from  Ulva  colonies  on  pilings  at  City  Point,  New  Haven,  in  triplicate  on  the 
isolation  medium,  on  the  isolation  medium  without  liver  extract,  on  the  isolation 
medium  without  liver  extract,  vitamins,  or  glucose,  and  on  a  medium  containing 
glucose,  glutamate,  and  thiamine  as  its  only  organic  constituents.  At  the  same  time 
isolates  known  to  require  liver  extract  and  known  to  require  glucose,  amino  acids, 
and  vitamins  were  plated  on  these  media.  Of  the  media  used,  only  the  glucose- 
glutamate-thiamine  agar  gave  significantly  lower  sea  water  counts.  This  medium 
also  failed  to  support  typical  colony  formation  by  isolates  known  to  require  growth 
factors.  The  isolates  requiring  liver  extract  failed  to  form  typical  colonies  on  the 
medium  from  which  liver,  glucose,  and  vitamins  were  omitted  also,  though  the 
omission  of  liver  extract  alone  did  not  affect  their  growth  under  the  conditions  used. 

It  is,  on  the  other  hand,  quite  possible  that  fungi  exist  which  did  not  form  rec- 
ognizable colonies  because  they  were  inhibited  by  the  ingredients  of  the  medium. 
Representative  colonies  were  picked  from  the  isolation  plates  made  at  Woods  Hole 
into  tubes  of  semi-solid  (0.1%  agar)  isolation  medium  for  further  study.  It  then 
developed  that,  in  semi-solid  media,  each  of  the  organic  constituents  of  the  medium 
was  somewhat  inhibitory  to  some  of  the  fungi  isolated.  The  results  of  the  com- 
parative plating  of  City  Point  sea  water  outlined  above  and  of  similarly  plating  iso- 
lates known  to  be  inhibited  by  ingredients  of  the  isolation  medium  indicated  that, 
for  these  fungi,  the  inhibitions  are  relieved  by  growth  on  a  solid  agar  surface. 
The  validity  of  this  conclusion  must  be  restricted  to  the  fungi  examined. 

In  principle,  these  defects  are  not  unique  to  our  procedure ;  they  require  re- 
statement here  because  this  is  the  first  application  of  plating  techniques  to  the  lower 
marine  fungi,  indeed  to  ecologic  studies  of  any  aquatic  Phycomycetes,  and  because 
they  bear  on  the  conclusions  to  be  drawn  from  our  results.  The  results  of  Table  II 
suggest,  first,  that  the  presence  of  these  fungi  is  correlated  with  the  organic  content 
of  the  water  examined,  since  the  highly  polluted  Eel  Pond  is  richer  than  Great 
Harbor  (on  the  incoming  tide).  Second,  in  Great  Harbor  (and  in  the  Hole), 
fungi  are  associated  with  algae :  they  may  be  isolated  from  water  taken  from  finger 
bowls  in  which  algae  were  being  kept  under  a  constant  drip  of  previously  fungus- 
free  tap  water.  They  may  be  isolated  from  water  squeezed  by  hand  from  masses 
of  attached  algae  growing  on  rocks  and  pilings. 

The  organic  content  of  polluted  waters,  such  as  those  of  the  Eel  Pond,  could 
reasonably  be  expected  to  support  a  population  of  free-living  non-filamentous  fungal 
saprophytes,  just  as  of  bacterial  saprophytes.  We  have  calculated,  from  cell  counts 
of  representative  cultures,  that  the  amount  of  soluble  organic  material  required  to 
produce  a  single  thallus  of  the  common  holocarpic  or  monocentric  marine  Phycomy- 


ECOLOGY  OF  LOWER  MARINE  FUNGI  413 

cetes  is  of  the  order  of  1  m/x  g.  But  the  development  of  techniques  for  establishing 
directly  the  presence  of  a  free-living  fungus  population  would  be  very  desirable. 
Two  instances  of  association  with  organic  debris  were  noted.  One  colony  of  a 
monocentric  Phycomycete  was  found  on  a  plate  on  a  stray  grain  of  pine  pollen. 
One  species  (an  undescribed  myxoid  form  here  referred  to  as  isolate  "S"),  of  which 
1-22  colonies  were  found  on  every  plate  containing  lower  fungi,  formed  colonies 
which  were  as  often  as  not  centered  on  a  microscopic  bit  of  nondescript  organic 
debris. 

In  view  of  the  known  endo-  and  epibiotic  habit  of  marine  fungi,  the  apparent 
association  with  algae  required  further  investigation.  The  algae  with  which  the 
sea  water  samples  of  Table  II  were  associated  were  examined  microscopically  for 
the  presence  of  fungal  thalli.  As  might  have  been  expected  from  the  experience 

TABLE  III 

Fungi  associated  with  algal  surfaces 

No.  species 
No.  fragments  fungi/fragment 

1.  Algae  from  rocks  at  Red  Spindle  (Grassy  Island) 

Ectocarpus  1 

Antithamnion  8  1—3 

Polysiphonia  4  1-3 

Ceramium  3  1-3 

2.  Algae  from  rocks  of  Pine  Island 

Ectocarpus  4  1-2 

Elachistea  5  1 

Punctaria 

Chorda  filum  1  3 

Callithamnion  18  1-3 

Antithamnion  4  2 

1  0 

Polysiphonia  3  0 

Bryopsis  10  1-2 

4  0 

of  previous  investigators  (see  Petersen,  1905;  Sparrow,  1934,  1936;  Kobayashi  and 
Ookubo,  1953),  recognizable  thalli  were  rare.  The  one  species  which  Sparrow 
(1936)  found  epidemic  later  in  the  summer — Ectrogella  perforans  in  Licmophora — 
was  conspicuously  absent.  The  only  form  found  during  these  examinations  was 
Petersenia  lobata  (?)  on  Polysiphonia  urceolata  (fide  W.  R.  Taylor)  collected  at 
the  Red  Spindle  and  allowed  to  rest  for  12  days  in  a  ringer  bowl  under  dripping  sea 
water  (as  suggested  by  Sparrow).  Five  days  later  the  infection  had  disappeared. 
The  results  of  plating  small  pieces  of  algae  showed,  in  marked  contrast,  that  the 
algal  surface  not  contaminated  by  lower  fungi  was  rare.  Bits  of  algae,  usually 
strands  approximately  one  cm.  long,  were  cut  from  the  collected  plants,  drawn 
gently  over  the  edge  of  a  petri  dish  to  remove  water  and  placed  on  the  surface  of 
the  usual  antibiotic-isolation  medium  agar.  No  fungal  thalli  (possibly  because  of 
their  small  size)  could  be  recognized  at  100  X  on  the  algal  fragments  at  the  time 
of  plating.  Neither  fungal  thalli  nor  rhizoids  were  seen  within  the  cells  of  these 
algae  at  any  time,  though  the  specimens  were  examined  at  430  X  at  the  end  of  the 
incubation  period.  The  association  of  lower  fungi  with  fragments  of  algae  col- 
lected at  the  Red  Spindle  (Grassy  Island)  in  Great  Harbor  and  at  Pine  Island 


414  HELEN  S.  VISHNIAC 

(off  Nonamesset  Island,  in  the  Hole)  is  shown  in  Table  III.  Molds  and  yeasts 
were  observed  fairly  frequently;  specimens  in  this  series  which  were  obscured  by 
such  forms  have  been  omitted  from  the  tabulation. 

Are  these  fungi  on  the  algal  surface  or  in  the  surface  film  of  water  surrounding 
the  algal  fragment?  If  the  surface  film  of  water  is,  at  a  guess,  about  0.01  ml.,  one 
would  expect  to  find  of  the  order  of  1-10  viable  units  of  lower  fungi  in  it.  The 
results  of  Table  III  are  not  inconsistent  with  this  estimate,  but  are  unfortunately 
not  quantitative,  because  discrete  colonies  are  rarely  formed  around  algal  fragments. 
But  we  are  inclined  to  consider,  for  two  reasons,  that  the  fungi  found  in  association 
with  algal  fragments  resemble  many  of  the  marine  bacteria  in  being  present,  as 
thalli,  on  the  surface  of  the  alga.  First,  we  have  attempted  (in  a  limited  number 
of  trials)  to  wash  the  fungi  away  with  sterile  sea  water.  The  results,  even  with 
such  algae  as  Bryopsis  phiuwsa  and  PolysipJwnia  urccolata  which  were  free  of  the 
forest  of  hairs  and  epiphytes  found  on  most  marine  algae,  were  poor.  No  more 
fragments  were  free  of  fungi  after  washing  than  before,  and  essentially  the  same 
type  of  fungi  was  present.  Secondly,  although  both  holocarpic  and  eucarpic  Phy- 
comycetes  were  among  the  forms  associated  with  algal  surfaces,  the  predominant 
form  was,  as  in  sea  water  samples,  the  myxoid  surface-loving  "S."  Every  algal 
fragment  listed  in  Table  III  as  having  associated  fungi  had  "S"  associated  with  it. 
The  association  of  "S"  with  bits  of  organic  debris  has  already  been  noted.  "S"  was 
also  found  to  be  associated  with  two  of  six  copepods  plated  as  part  of  a  rather  un- 
productive plankton  haul  from  Great  Harbor.  Another  surface-loving  form,  La- 
bwinthula  sp.,  occurred  less  frequently  on  Polysiplwnia,  Centillium,  and  Ectocarpits. 

CONCLUSIONS  AND  SUMMARY 

Our  data  therefore  suggest  that  the  lower  marine  fungi  occupy  essentially  the 
same  ecologic  niche  as  the  marine  saprophytic  bacteria.  These  fungi  can  be  found 
in  suitably  polluted  sea  water  in  numbers  of  the  order  of  1-500,000  viable  units/liter 
but  less  than  5000/liter  in  more  open  waters  of  Woods  Hole.  The  fungus  count 
increases,  as  has  long  been  noted  for  bacteria  (Gazert,  1906),  in  the  presence  of 
macroscopic  algae.  They  also  resemble  marine  bacteria  in  their  association  with 
surfaces.  As  a  group,  these  fungi  differ  from  marine  bacteria  in  being  strongly 
aerobic.  One  may  justifiably  wonder  as  to  the  basis  of  their  success  in  competition 
with  bacteria  in  this  niche.  Studies,  now  in  progress,  of  the  individual  species  of 
fungi  concerned  may  clarify  this  question. 

LITERATURE  CITED 

GAZERT,   H.,   1906.     Untersuchungen   iiber   Meeresbakterien   und   ihren   Einfluss   auf  den    Stoff- 

wechsel  im  Meere.     Deutsche  Sitdpolar-E.rpcdition,  1901-03,  Berlin,  7:  235-296. 
KOBAYASIII,  Y.,  AND  M.  OoKUBO,  1953.     Studies  on  the  marine  Phycomycetes.     Bull.  Nat.  Scl. 

Mns.,  Tokyo,  33  :  53-65. 
PETERSEN,  H.  E.,  1905.     Contributions  a  la  connaissance  des  Phycomycetes  marins  (Chytridinae 

Fischer).     Ovcrsigt  k.  dansk.  vidcnsk.  Sclskabs  Forliandl.,  1905:  439-488. 
SPARROW,    F.    K.,    JR.,    1934.     Observations    on    marine    phycomycetes    collected    in    Denmark. 

Dansk.  hot.  Ark.,  8  (6)  :  1-24. 
SPARROW,  F.  K.,  Jr.,  1936.     Biological  observations  on  the  marine  fungi  of  Woods  Hole  waters. 

Biol.  Bull.,  70 :  236-263. 

VISHNIAC,  H.  S.,  1955.     Marine  mycology.     Trans.  N.  Y.  Acad.  Sci.,  17:  352-360. 
VISHNIAC,  H.  S.,  AND  S.  W.  WATSON,  1953.     The  steroid  requirements  of  Labyrinthula  vitcl- 

lina  var.  pacifica.     J.  Gen.  Microbiol.,  8 :  248-255. 


THE  EFFECT  AND  AFTER-EFFECT  OF  VARIED  EXPOSURE 
TO  LIGHT  ON  CHICKEN  DEVELOPMENT 

WILBOR  O.  WILSON,  ALLEN  E.  WOODARD  AND  HANS  ABPLANALP 
Poultry  Husbandry  Department,  University  of  California,  Dai'is,  California 

The  basic  factor  necessary  for  initiating  development  of  the  gonads  of  birds, 
according  to  Rowan  (1938),  appears  to  be  length  of  day.  Intensity  above  a  cer- 
tain low  threshold,  he  says,  appears  to  be  of  no  significance.  Frequency  of  light 
stimulation  plays  an  important  role  according  to  Benoit  (1936)  and,  more  recently, 
Staffe  (1951),  Kirkpatrick  and  Leopold  (1952),  and  Farner  ct  al.  (1953a).  On 
the  other  hand,  with  uninterrupted  lighting  early  growth  was  delayed  and  repro- 
ductive performance  adversely  affected,  as  shown  by  studies  of  Lamoreux  (1943)^ 
Callenbach  ct  al.  (1944),  and  Ringrose  and  Potter  (1953). 

Since  most  of  the  studies  cited  above  involved  wild  birds,  it  remains  to  be  seen 
whether  their  results  are  directly  applicable  to  the  domestic  fowl ;  this  is  particu- 
larly true  for  strains  of  chickens  that  have  been  bred  for  egg  production.  Previous 
work  on  the  effect  of  intermittent  lighting  on  laying  hens  by  Wilson  and  Abplanalp 
(1956)  has  indicated  that  egg  production  can  be  maintained  with  very  small 
amounts  of  light  energy,  provided  it  is  given  intermittently  ( less  than  six  one-minute 
intervals  in  24  hours).  These  results  tend  to  support  earlier  findings  by  Staffe 
(1951),  who  demonstrated  that  short  light  flashes  from  1500- watt  lamps  were 
effective  in  stimulating  laying  hens  to  increase  winter  egg  production. 

The  present  study  was  conducted  in  order  to  gain  further  information  about  the 
effects  of  intermittent  lighting  upon  the  development  of  chickens.  It  was,  however, 
restricted  to  an  investigation  of  early  growth  and  the  onset  of  sexual  maturity  in 
pullets. 

MATERIAL  AND  METHODS 

Two  experiments  were  conducted  with  Single  Comb  White  Leghorn  stock  of 
the  University  of  California  at  Davis.  In  the  first,  pedigreed  chicks  were  hatched 
on  December  4.  1953.  The  purpose  of  the  test  was  to  study  the  effect  of  supple- 
mentary light  on  the  development  of  chickens.  Each  hen's  chicks  were  distributed 
as  equally  as  possible  among  six  experimental  groups.  Each  lot  of  chicks  was 
then  brooded  and  reared  in  15  X  15-foot  pens  of  a  house  with  open  fronts.  Natural 
light  was  given  to  all  six  pens,  but  three  received  supplementary  artificial  light  from 
a  continuously  burning  100- watt  incandescent  bulb.  Three  brooding  methods  were 
used  with  the  two  light  treatments  as  follows : 

Group  Brooder  type  Light  regime 

1  4  infrared  lamps  Natural  light  only 

2  Electric  Natural  light  only 

3  Gas  Natural  light  only 

4  4  infrared  lamps  Natural  light  +  100  W  continuous 

5  Electric  Natural  light  +  100  W  continuous 

6  Gas  Natural  light  +  100  W  continuous 

415 


415       WILBOR  O.  WILSON,  ALLEN  E.  WOODARD  AND  HANS  ABPLANALP 


The  gas  brooders  provided  a  somewhat  higher  room  temperature  than  either  of  the 
other  types. 

Brooding  was  discontinued  when  the  chicks  were  6  weeks  old.  A  week  later, 
at  7  weeks  of  age,  the  chicks  were  scored  for  feathering.  Four  grades  were  used 
in  assessing  completeness  of  feathering,  ranging  from  1  for  poorest  feathering  to 
4  for  best  performance.  Both  sexes  were  scored. 

At  the  same  time  a  few  males  from  large  families  were  killed  in  order  to  deter- 
mine comb  and  testis  weights.  Body  weight  was  measured  first  at  7  weeks  of  age 
and  from  then  on  at  4-week  intervals.  Age  at  first  egg  and  the  average  weight 
of  the  first  three  eggs  were  determined  whenever  possible. 

The  second  experiment  was  to  determine  effects  of  intensity  and  frequency  of 
lighting  upon  growth  and  sexual  development  of  pullets.  Pullet  chicks  were 
hatched  on  July  1,  1955,  and  all  brooded  alike  up  to  5  weeks  of  age.  The  following 
brooding  and  lighting  regime  was  used : 

0-1  weeks  of  age:  10- watt  bulb;  continuous  light 
1-3  weeks  of  age  :  40- watt  bulb ;  continuous  light 
3-5  weeks  of  age:  10- watt  bulb;  continuous  light 

Shielded  incandescent  light  bulbs  were  the  only  source  of  heat. 

At  5  weeks  of  age,  and  in  some  cases  again  at  90  days  of  age,  the  experimental 
lots  of  birds  were  subjected  to  changes  in  lighting  regime,  according  to  the  follow- 
ing plan : 


Group 

Light  intensity  (foot-candles) 

Total  hrs.  light  in 
24  lours 

Number  of  light  periods 
in  24  hours  after 
90  days  of  age 

35-90  days 

After  90  days 

A 

0.5-30.0 

0.5-30.0 

14.0 

1 

B 

0.0-0.4 

0.5-30.0 

14.0 

1 

C 

0.0-0.4 

0.0-0.4 

1.5 

12;  6;  3;  2. 

D 

0.5-30.0 

0.0-0.4 

1.5 

12;6;3;2. 

E 

0.5-30.0 

0.4-6.6 

1.5 

12;6;3;2. 

As  indicated  in  the  table  above,  groups  C,  D,  and  E  were  each  divided  into  four 
subgroups  at  90  days  of  age,  and  the  latter  were  subjected  to  light  periods  of  vary- 
ing frequency,  while  the  total  duration  of  lighting  was  held  constant.  The  12  light 
periods  per  24  hours  consisted  of  7.5  minutes  each,  followed  by  52.5  minutes  of 
darkness.  Similar  regularly  spaced  periods  of  light  were  used  where  six  and  three 
stimuli  were  given.  In  the  case  of  two  light  periods,  however,  45  minutes  of  light 
was  alternately  followed  by  7  hours,  15  minutes  and  by  15  hours,  15  minutes  of 
darkness.  Group  A  served  as  control  and  was  held  under  "cool  white"  fluorescent 
lighting  with  14  hours  of  light  per  day.  Light  intensities  varied  between  0.5  and 
30.0  foot-candles  according  to  the  location  of  individual  laying  cages. 

Four  windowless  climatic  chambers  were  used  for  this  experiment ;  they  have 
been  described  in  detail  by  Wilson  and  Abplanalp  (1956).  Each  chamber  was  sub- 
divided into  two  sections  with  a  partition  of  black  sisalkraft,  in  order  to  allow  repli- 
cation of  treatments.  Thus,  each  lighting  regime  was  given  in  two  different  cham- 
bers. A  diagrammatic  outline  of  this  arrangement  is  given  in  Figure  1. 

Temperatures  were  held  constantly  at  approximately  80°  F.     The  pullets  were 


AFTER-EFFECT  OF  LIGHT  ON  CHICKENS 


417 


L 
,  J. 


/.  5  firs 
5/c/cry 
O-6.6  f.C. 


/.  5  firs. 
2/J0& 

o-6.ef.c 


/.$  firs 

6/cfay 
O-6.6  /CC. 


/.  5  firs 
J/Vby 
O-6.6  f.C. 


.  .  r 


/  6  firs 
/2/c/ay 
O-6.6  fC. 


O-6.6  / 


o-66/:c 


/2/cfoj, 

O-6.6>CC 


firs 

*/**</ 

.5-3O.O  fC 

Con  fro/ 


FIGURE  1.  Floor  plan  of  experimental  rooms  showing  location  of  replications.  Total  light 
per  day,  number  of  light  periods  per  24  hours,  and  intensity  in  foot-candles  are  given  in  order 
for  each  subgroup. 

placed  in  individual  10-inch  cages  when  they  were  5  weeks  old.  Two  double  rows 
of  cages  were  arranged  in  step  fashion,  which  meant  that  the  birds  in  the  upper  rows 
were  somewhat  closer  to  the  light  source  than  those  in  the  lower  cages.  The  top 
rows  were  18  inches  from  the  light  source,  while  the  bottom  ones  were  3  feet  dis- 
tant. Light  intensities  varied  between  0.0  and  0.4  foot-candles  for  the  lower  tiers 
and  between  0.4  and  6.6  foot-candles  for  the  upper  ones. 

Experimental  lighting  was  held  at  suboptimal  intensities  in  order  to  bring  out 
more  clearly  the  possible  effects  of  frequency  of  light  periods. 

The  following  traits  were  observed  and  analyzed:  1)  Body  weights  at  12,  20, 
and  28  weeks  of  age;  2)  age  at  first  egg  for  individual  pullets;  and  3)  average 

TABLE  I 

Effect  of  supplementary  light  on  growth,  feathering  and  sexual  development  of  December-hatched  chicks. 

Number  of  individuals  in  parenthesis 


C-22 

Body  weight,  females  gms. 

Testes  wt. 
at  7  weeks 
(gms.) 

Feather 
score 

Aver,  age 
at  1st  egg 
(days) 

Aver.  wt. 
1st  3  eggs 
(gms.) 

7  wk. 

12  wk. 

16  wk. 

20  wk. 

No  artificial  lights 

(189) 
436 

(189) 
1023 

(189) 
1322 

(189) 
1605 

(36) 
.240 

(553) 
3.47 

(189) 

157.7 

41.0 

100-watt  lights  cont. 

(190) 
452 

(190) 
1006 

(190) 
1303 

(190) 
1519 

(32) 
.284 

(545) 
3.21 

(190) 
163.8 

43.6 

Difference 

-16* 

17 

19 

86** 

-.044** 

.26** 

-6.1* 

-2.6** 

*  =  P  <  .05. 
**  =  P  <  .01. 


418      WILBOR  O.  WILSON,  ALLEN  E.  WOODARD  AND  HANS  ABPLANALP 


TABLE  II 

Relation  of  light  intensity  at  different  stages  of  growth  and  age  at  first  egg.     Group  A  and  B  received 
14  hours  of  light  after  90  days  of  age,  other  groups  1\  hours 


No 

Light  treatment 
foot-candles 

Body  weight  (gms.) 

Median 

Aver.  wt. 

At  248 
davs  % 

Between 
35-90  days 

After 
90  days 

12  wk. 

20  wk. 

28  wk. 

egg  (days) 

(gms.) 

maturity 

A 

10 

0.5-30. 

0.5-30. 

1031. 

1514. 

1744. 

153. 

39.7 

100 

B 

10 

0-0.4 

0.5-30. 

990. 

1505. 

1779. 

161. 

42.2 

100 

C 

40 

0-0.4 

0-0.4 

1017. 

1391. 

1707. 

191. 

48.9 

85 

D 

40 

0.5-30. 

0-0.4 

1013. 

1466. 

1838. 

179. 

47.5 

72 

E 

40 

0.5-30. 

0.4-6.6 

958. 

1426. 

1705. 

169. 

45.1 

95 

weight  of  first  three  eggs  laid  (whenever  possible).     Median  age  at  first  egg  was 
used  as  a  measure  of  sexual  maturity  of  entire  groups  of  pullets. 

RESULTS 

The  results  of  the  first  test  are  shown  in  Table  I.  Continuous  light  added  to 
natural  illumination  apparently  favors  early  growth  of  chicks  up  to  at  least  7  weeks 
of  age.  Later  on,  and  most  conspicuously  at  20  weeks  of  age,  the  effects  of  con- 
tinuous light  supplement  are  just  the  opposite.  Significantly  higher  body  weights 
were  found  at  20  weeks  for  the  birds  brooded  under  natural  light  only. 

Comb  weight  of  7-week-old  males  shows  little  or  no  effect  as  the  result  of  differ- 
ent light  treatments,  but  testis  weights  responded  in  the  same  way  as  body  weight. 
Added  continuous  lighting  resulted  in  significantly  heavier  testes  at  7  weeks  of  age. 

Continuous  light  tends  to  retard  feathering  and  sexual  maturity  of  pullets  (but 
not  of  males).  The  6-day  differences  in  maturity  between  supplemented  and  con- 
trol groups  was  highly  significant.  This  delay  in  age  at  first  egg  was  associated 
with  a  somewhat  higher  weight  of  first  eggs. 

The  results  of  the  second  experiment  are  given  in  Tables  II  and  III.  In  Table 
II  the  data  are  arranged  according  to  total  amount  of  daily  lighting  and  light  in- 
tensities. Table  III,  on  the  other  hand,  shows  the  effects  of  increasing  frequencies 
of  light  periods  with  a  given  amount  of  light  applied  after  90  days  of  age. 

TABLE  III 
Frequency  of  light  intervals/ 24  hrs.  in  relation  to  growth  and  age  at  first  egg 


Light  foot- 
candles 

Light  periods/24  hrs. 

Total 
light/24 
hrs. 

No. 

Aver,  body  weight  (gms.) 

Median 
age  at  1st 
egg  (days) 

Aver.  wt. 
1st  3  eggs 
(gms.) 

Number 

Length 

12  wk. 

20  wk. 

28  wk. 

0-6.6 

12 

1\  min. 

90  mill. 

30 

991. 

1437. 

1751. 

168.0 

46.5 

0-6.6 

6 

15  min. 

90  min. 

30 

980. 

1410. 

1736. 

174.0 

46.1 

0-6.6 

3 

30  min. 

90  min. 

30 

1011. 

1481. 

1826. 

190.5 

48.0 

0-6.6 

2 

45  min. 

90  min. 

30 

1002. 

1381. 

1750. 

189.0 

47.6 

0.5-30. 

1 

14  hrs. 

14  hrs. 

20 

1010. 

1510. 

1759. 

160.0 

41.2 

AFTER-EFFECT  OF  LIGHT  ON  CHICKENS  419 

The  observed  body  weights  of  pullets  do  not  show  any  clear-cut  effects  of  either 
light  intensity,  amount  of  light,  or  lighting  frequency. 

Sexual  maturity  as  measured  by  median  age  at  first  egg,  however,  was  strongly 
affected  by  differences  in  lighting,  both  before  and  after  90  days  of  age.  The  con- 
trol treatment  (group  A)  with  highest  light  intensity  during  early  and  late  periods 
of  development  matured  earliest.  Group  B,  with  low  light  intensity  to  90  days  of 
age  but  high  intensity  thereafter,  matured  8  days  later.  Similarly,  groups  E,  D,  and 
C  show  consistently  adverse  effects  of  reduced  light  intensities  upon  age  at  first  egg. 

The  influence  of  light  intensity  on  maturity  is  apparently  operative  over  a  con- 
siderable period  of  early  development  and  is  not  merely  restricted  to  a  period  very 
close  to  the  onset  of  egg  production.  This  can  best  be  shown  by  regrouping  the 
results  of  median  age  at  sexual  maturity. 

Light  before  90  Li8ht  after  90  days  of  age 

days  of  age  Dim  Bright  Difference 

Dim  191  161  30 

Bright  179  153  26 

Difference  12 

Here  it  may  be  noted  that  bright  light  administered  before  pullets  were  90  days  old 
advanced  sexual  maturity  by  only  12  and  8  days,  while  intensive  lighting  after  90 
days  of  age  produced  effects  of  30  and  26  days.  This  clearly  indicates  that  the  in- 
fluence of  light  intensity  upon  sexual  maturity  becomes  more  pronounced  the  closer 
toward  onset  of  lay  it  can  operate. 

Table  III  shows  the  effects  of  more  frequent  light  periods  on  age  at  first  egg. 
In  each  case  the  birds  received  a  total  of  90  minutes  of  dim  light  in  24  hours.  The 
results  are  clear-cut  and  show  that  light  is  more  effective  in  stimulating  sexual  de- 
velopment the  more  frequently  it  is  applied.  Thus,  12  short  periods  of  dim  light 
permitted  pullets  to  mature  almost  as  early  as  one  14-hour  period  of  intensive  light. 
The  same  amount  of  dim  light  given  in  only  two  doses,  on  the  other  hand,  resulted 
in  extremely  late  maturity.  These  findings  are  shown  more  clearly  in  Figure  2. 
The  data  represent  medians  for  the  replicate  lots.  Each  lot  contained  subgroups 
which  differed  in  light  intensity.  See  regime  for  groups  C,  D  and  E. 

DISCUSSION 

The  first  experiment  confirmed  that  growth  of  the  domestic  fowl  can  be  influ- 
enced by  light.  All-night  lights  may  have  aided  early  growth  in  this  experiment 
by  providing  the  chicks  more  opportunity  to  feed.  Frequent  feedings  may  be  par- 
ticularly helpful  when  the  crop  capacity  of  chicks  is  still  poorly  developed. 

The  adverse  effects  of  continuous  lighting  after  7  weeks  of  age,  as  found  in  this 
study,  are  in  agreement  with  findings  by  Tomhave  (1954). 

The  results  of  the  second  test  with  respect  to  growth  after  12  weeks  of  age  are 
not  conclusive.  They  fail  to  substantiate  reports  by  Clegg  and  Sanford  (1951) 
and  by  Barott  and  Pringle  (1951),  who  found  that  intermittent  lighting  has  a  bene- 
ficial effect  on  early  growth  of  chickens  prior  to  12  weeks. 

In  this  study  it  has  been  found  that  continuous  light  has  adverse  effects  on 
feathering,  becoming  more  serious  as  the  birds  become  older.  Similar  effects  of 
continuous  light  on  turkeys  have  been  reported  by  Mueller  ct  al.,  1951. 


420      WILBOR  O.  WILSON,  ALLEN  E.  WOODARD  AND  HANS  ABPLANALP 

The  role  of  continuous  light  in  the  sexual  development  of  chicks  appears  to  be 
a  complex  one.  While  the  7-week-old  cockerels  showed  increased  testes  weight 
under  continuous  lighting,  the  pullets  appeared  to  be  delayed  in  their  development. 
Unfortunately,  there  were  no  testes  measurements  available  for  cockerels  near  ma- 
turity; hence,  it  is  difficult  to  assess  the  possible  importance  of  refractory  behavior 
of  pullets.  Evidence  of  retarded  growth  at  20  weeks  of  age  indicates,  however, 
that  both  cockerels  and  pullets  may  have  been  delayed  sexually  near  the  point  of 
maturity. 


196  - 


Medions    for    replicote   iots 


4  6  8  10  12 

FREQUENCE  OF  LIGHT  PERIODS /24   HOURS-    Total  Daily  Light  =l/2     Hours 
FIGURE  2.     The  relation  of  frequency  of  light  periods  and  age  at  first  egg. 

From  the  results  of  the  second  experiment,  as  well  as  from  an  earlier  report 
by  Wilson  and  Abplanalp  (1956),  it  is  clear  that  the  total  amount  of  light  is  not 
the  sole  determinant  in  stimulating  sexual  development  of  pullets.  Aside  from  the 
adverse  effects  of  continuous  lighting  (which  may  possibly  be  due  to  a  nervous 
fatigue  of  the  animal),  it  has  also  been  demonstrated  here  that  light  intensity  and 
the  distribution  and  frequency  of  light  stimuli  are  important  variables  that  must  be 
considered  in  the  problem  of  light  stimulation. 

The  present  results  show  that  under  limiting  conditions  of  low  light  intensities 
and  short  periods  of  light  exposure,  the  rate  of  sexual  development  increases  with 
longer  total  daily  exposure  to  light,  greater  light  intensity,  and  more  frequent  stimu- 
lation. These  relationships  may  not  hold  when  either  light  intensity  or  total  dura- 
tion of  light  exposure  is  increased  beyond  certain  thresholds.  Nevertheless,  they 


AFTER-EFFECT  OF  LIGHT  ON  CHICKENS  421 

permit  certain  conclusions  of  practical  value.  Thus,  where  natural  light  is  to  be 
supplemented,  it  would  seem  reasonable  to  use  frequent  but  short  intervals  of  ar- 
tificial lighting  in  place  of  continuous  lighting  or  of  light  periods  adjoining  the 
natural  day. 

Several  attempts  have  been  made  recently  to  rationalize  the  response  of  birds  to 
light.  Wolfson  (1953)  interprets  his  data  as  supporting  the  hypothesis  that  the 
total  daily  dose  of  light  determines  the  response.  He  postulates,  however,  that  the 
proportion  of  light  exposure  to  darkness  in  a  given  cycle  is  the  critical  factor  in 
determining  the  response  rather  than  the  daily  dose  of  light. 

Kirkpatrick  and  Leopold  (1952),  in  agreement  with  Jenner  and  Engels  (1952), 
maintain  that  the  dark  period  per  se  is  a  major  controlling  factor  in  the  response 
of  birds  to  light.  Such  an  interpretation  appears  primarily  different  in  terminology 
and  emphasis  but  seems  to  add  little  to  an  understanding  of  the  problem.  It  has 
been  criticized  by  Hammond  (1953)  and  by  Farner  et  al.  (1953b). 

In  the  light  of  this  study,  the  theory  advanced  by  Farner  et  al.  (1953b)  seems 
the  simplest  and  most  suitable  for  explaining  the  action  of  light  in  reproduction  of 
birds.  In  brief,  it  postulates  that  there  exists  a  light-sensitive  gonadotropic  mecha- 
nism capable  of  activation  almost  immediately  upon  onset  of  lighting;  it  remains 
active  throughout  the  light  period  and  even  for  some  time  following  termination  of 
the  latter.  This  theory  has  helped  to  explain  the  effectiveness  of  extremely  short 
photoperiods  (a  total  of  6  minutes  in  24  hours)  in  maintaining  egg  production  of 
chickens  (Wilson  and  Abplanalp,  1956).  We  believe  this  theory  suitable  to  ex- 
plain the  current  findings  which  show  the  increasing  effectiveness  of  a  given  amount 
of  light  when  given  in  numerous  small  doses. 

The  formulation  of  general  theories  on  the  basis  of  published  evidence  is  seri- 
ously hampered  by  non-uniformity  in  experimental  material,  procedures,  and  ter- 
minology. On  the  basis  of  Farner's  theory  and  present  findings,  one  may  attempt 
to  interpret  light  response  by  means  of  three  independent  main  effects  and  their 
interactions,  namely : 

1.  Total  daily  amount  of  light  exposure 

2.  Light  intensity 

3.  Frequency  of  light  intervals. 

Additional  assumptions  are  needed  in  order  to  explain  refractoriness  and  perhaps 
seasonal  changes  in  reproduction  of  birds,  but  the  present  experiments  do  not  per- 
mit any  new  interpretation  of  their  role. 

SUMMARY 
The  present  experiments  were  designed  to  determine  the  effects  of : 

a.  Total  daily  amount  of  light 

b.  Intensity  of  light 

c.  Frequency  of  light  intervals 

on  the  growth  and  development  of  Leghorn  chickens. 

a.  Total  daily  amount  of  light  exposures:  The  first  test  dealt  with  the  effect  of 
supplementing  natural  light  with  continuous  light.  Continuous  light  improved  body 
weight  of  all  birds  and  testes  size  of  males  at  7  weeks,  but  impaired  feather  devel- 
opment. Continuous  lighting  delayed  sexual  maturity  of  pullets,  and  growth  in 
both  sexes  was  retarded  until  they  reached  20  weeks  of  age. 


422      WILBOR  O.  WILSON,  ALLEN  E.  WOODARD  AND  HANS  ABPLANALP 

In  the  second  experiment  under  suboptimal  light  intensities,  light  exposure  has 
no  effect  on  body  size  after  12  weeks  of  age.  Rate  of  sexual  development  was  in- 
creased by  larger  daily  exposures  to  light.  These  effects  were  more  pronounced 
when  treatments  were  applied  to  pullets  over  90  days  old  than  during  earlier  stages 
of  development. 

b.  Intensity  of  light:  Three  light  intensities  were  applied  to  growing  birds: 
0.0-0.4  foot-candles,  0.4-6.6  foot-candles,  and  0.5-30.0  foot-candles.     Growth  was 
not  affected  by  lower  light  intensities,  but  sexual  maturity  was  delayed. 

c.  Frequency  of  light  intervals:  Body  weight  was  unaffected  by  lighting  fre- 
quency.    Sexual  maturity,  however,  was  significantly  advanced  when  suboptimal 
light  exposure  and  light  intensities  were  applied  in  frequent  but  small  doses.     Thus, 
it  was  found  that  12  periods  of  7.5  minutes  of  dim  light  per  day  produced  a  rate  of 
sexual  development  almost  equal  to  the  rate  with  14  hours  per  day  of  normal  light- 
ing.    These  results  are  taken  as  further  proof  that  the  after-effects  of  light  on  the 
reproductive  mechanisms  of  chickens  are  considerable 

LITERATURE  CITED 

BAROTT,  H.  G.,  AND  E.  M.  PRINGLE,  1951.     Effect  of  environment  on  growth  and  feed  and  water 
'  consumption  of  chickens.     IV.  The  effect  of  light  on  early  growth.     /.  Nutrition,  45  : 
265-274. 

BENOIT,  J.,   1936.     Facteurs  externes  et  internes  de  1'activite   sexuelle.     I.   Stimulation   par   la 
'  lumiere  de  1'activite  sexuelle  chez  le  canard  et  la  cane  domestique.     Bull.  Biol.  France 
Bdgique,  70 :  487-533. 

CALLENBACH,  E.  W.,  J.  E.  NICHOLAS  AND  R.  R.  MURPHY,  1944.  Influence  of  light  on  age  at 
sexual  maturity  and  ovulation  rate  of  pullets.  Penn.  Agr.  Expt.  Sta.  Bull.  461. 

CLEGG,  R.  E.,  AND  P.  E.  SANFORD,  1951.  The  influence  of  intermittent  periods  of  light  and  dark 
on  the  rate  of  growth  of  chicks.  Poultry  Sci.,  30 :  760-762. 

EARNER,  D.  S.,  L.  R.  MEWALDT  AND  S.  D.  IRVING,  1953a.  The  role  of  darkness  and  light  in  the 
activation  of  avian  gonads.  Science,  118:  351-352. 

EARNER,  D.  S.,  L.  R.  MEWALDT  AND  S.  D.  IRVING,  1953b.  The  role  of  darkness  and  light  in  the 
photoperiodic  response  of  the  testes  of  white-crowned  sparrows.  Biol.  Bull.,  105: 
434-441. 

HAMMOND,  J.,  JR.,  1953.  Photoperiodicity  in  animals:  The  role  of  darkness.  Science.  117: 
389-390. 

JENNER,  C.  E.,  AND  W.  L.  ENGELS,  1952.  The  significance  of  the  dark  period  in  the  photo- 
periodic  response  of  male  juncos  and  white-throated  sparrows.  Biol.  Bull.,  103: 
345-355. 

KIRKPATRICK,  C.  M.,  AND  A.  C.  LEOPOLD,  1952.  The  role  of  darkness  in  sexual  activity  of  the 
quail.  Science,  116:  280-281. 

LAMOREUX,  W.  F.,  1943.  The  influence  of  different  amounts  of  illumination  upon  the  body- 
weight  of  birds.  Ecology,  24:  79-84. 

MUELLER,  C.  D.,  F.  MOULTRIE,  L.  F.  PAYNE,  H.  D.  SMITH  AND  R.  E.  CLEGG,  1951.  The  effect 
of  light  and  temperature  on  molting  in  turkeys.  Poultry  Sci.,  30 :  829-838. 

RINGROSE,  R.  C.,  AND  L.  M.  POTTER,  1953.  Artificial  light  delays  pullet  maturity.  New  Hamp. 
Agr.  Exp.  Sta.  Bull.  402  (p.  42). 

ROWAN,  W.,  1938.     Light  and  seasonal  reproduction  in  animals.     Biol.  Rev.,  13 :  374-402. 

STAFFE,  A.,  1951.     Belichtung  und  Legeleistung  beim  Huhn.     Experientla,  7:  399-400. 

TOMHAVE,  A.  E.,  1954.  Influence  of  artificial  lights  during  rearing  on  the  egg  production  of 
October  hatched  New  Hampshires.  Poultry  Sci.,  33 :  725-729. 

WILSON,  W.  O.,  AND  H.  ABPLANALP,  1956.  Intermittent  light  stimuli  and  egg  production  in 
chickens.  Poultry  Sci.,  35:  532-538. 

WOLFSON,  A.,  1953.  Gonadal  and  fat  response  to  a  5  :  1  ratio  of  light  to  darkness  in  the  white- 
throated  sparrow.  Condor,  55  :  187-192. 


INDEX 


A  BDOMINAL  nerve  cord,  cockroach,  prop- 
erties of  connective  tissue  sheath  of,  278. 

ABPLANALP,  H.     See  W.  O.  WILSON,  415. 

Absorption  of  light  by  lower  vertebrate  lenses, 
375. 

Acclimation  of  oxygen  consumption  by  cock- 
roach, 53. 

Acclimation,  thermal,  of  mollusc,  129. 

Accumulation  of  radionuclides  by  fishes,  336, 
352. 

Acetylcholine  and  frog  brain  oxygen  consump- 
tion, 314. 

Acmaea,  distribution  and  acclimation  of,  129. 

Actin  participation  in  actomyosin  contraction, 
290. 

Action  potential  of  cockroach  nerve  cord,  278. 

Action  potential  of  vertebrate  lens,  375. 

Activation  of  Urechis,  Nereis  and  Asterias  eggs, 
313. 

Activity  of  neurosecretory  cells  in  crayfish,  62. 

Aerobiosis  of  marine  fungi,  410. 

After-effect  of  light  on  chicken  development, 
415. 

AIRTH,  R.  L.,  AND  L.  R.  BLINKS.  A  new  phy- 
coerythrin  from  Porphyra,  321. 

Albinism,  inheritance  of  in  snails,  45. 

Algae,  association  of  with  marine  fungi,  410. 

Algae,  brine,  culture  of,  223,  230. 

Algae,  pigments  of,  321. 

ALLEN,  M.  D.     See  N.  E.  KEMP,  293,  305. 

Alloxan,  radioactive,  uptake  and  distribution 
of  in  toadfish  tissues,  300. 

American  cockroach,  oxygen  consumption  of, 
53. 

Amphibian  lens  sensitivity,  375. 

Amylase,  chromatographic  study  of,  298. 

Anatomy  of  digenetic  trematode,  248. 

ANDERSON,  J.  C.  Relations  between  metabo- 
lism and  morphogenesis  during  regenera- 
tion in  Tubifex.  II.,  179. 

ANDERSON,  J.  M.  The  innervation  of  muscle 
fibers  in  the  extrinsic  stomach-retractor 
strands  of  the  starfish,  Asterias,  297. 

ANDERSON,  J.  M.  Observations  on  autotomy 
in  the  starfish,  Asterias,  297. 

Annelid,  regeneration  of,  179. 

Annual  Report  of  the  Marine  Biological  Labo- 
ratory, 1. 

Antarctic  bryozoa,  123. 

Antigens  of  Paramecium,  358. 

Areal  differences  in  oyster  setting,  387. 


Arginine  biosynthesis  in  Escherichia,  319. 

Artemia,  growth  of,  230. 

Asterias,  membrane  potential  and  resistance  of 

eggs  of,  153. 

Asterias,  method  of  for  opening  bivalves,  114. 
Astynax,  osmoregulation  of,  399. 
Autotomy  in  starfish,  297. 
Avian  development,  effect  of  light  on,  415. 
Axon  of  squid,  conduction  velocity  in,  295. 
Azygia,  morphology  and  life-history  of,  248. 

T3ANKSIOLA,  contact  chemoreceptors  in,  92. 

BARR,  L.     See  J.  W.  GREEN,  290. 

Behavioral  change  in  population  of  Nassarius, 

291. 
BILEAU,  SR.  M.  CLAIRE  OF  THE  SAVIOR.     The 

uptake  of  1-131   by  the  thyroid  gland  of 

turtles  after  treatment  with  thiourea,  190. 
Bisexuality  in  echinoids,  328. 
Bivalves,  opening  of  by  sea  stars,  114. 
Bladder,  air,  role  of  in  sound  production  by  fish, 

393. 

Blastema  formation  in  Tubifex,  179. 
Blastoderms,  chick,  respiratory  metabolism  of, 

77. 

BLINKS,  L.  R.     See  R.  L.  AIRTH,  321. 
Blocking  time  of  cockroach  nerve  cord,  278. 
Blowfly,  ingestion  of  carbohydrates  by,  204. 
BOOLOOTIAN,  R.  A.,  AND  A.  R.  MOORE.     Her- 

maphroditism  in  echinoids,  328. 
BOROUGHS,  H.,  S.  J.  TOWNSLEY  AND  R.  \V. 

HIATT.     The  metabolism  of  radionuclides 

by  marine  organisms.     I,  II.,  336,  352. 
Brine  algae,  culture  of,  223. 
BROCKWAY,  A.  P.     The  effects  of  x-irradiation 

on   the   pupae   of   the   yellow   mealworm, 

Tenebrio,  297. 

Bryozoa,  marine,  studies  on,  123. 
BUCK,  J.     See  J.  W.  HASTINGS,  101. 
Budding  of  Hydra,  effect  of  x-irradiation  on, 

240. 

CADDIS  flies,  chemoreceptors  in,  92. 

CAGLE,  J.     See  A.  K.  PARPART,  294. 

CAIN,  G.  L.     Studies  on  cross-fertilization  and 

self-fertilization  in  Lymnaea,  45. 
Calling  of  sea  robins,  393. 
Cancer   therapy,    interpretation    of   action   of 

certain  chemical  agents  in,  291. 


423 


424 


INDEX 


Carangoides,  metabolism  of  radionuclides  by, 
336. 

Carbohydrate  ingestion  by  blowfly,  204. 

CARLSON,  F.  D.  See  P.  G.  LENHERT,  293; 
R.  E.  THIES,  295. 

Cell  types,  neurosecretory,  in  crayfish,  62. 

CHAET,  A.  B.  Chromatographic  study  of 
crystalline  style  amylase,  298. 

CHAET,  A.  B.  Mechanism  of  toxic  factor  re- 
lease, 298. 

CHASE,  A.  M.  A  combined  effect  of  urea  and 
borate  buffer  on  uricase  activity,  299. 

Chemoreception  in  blowfly,  204. 

Chemoreceptors  in  Trichoptera,  92. 

CHENEY,  R.  H.  Dimethylated  dioxypurines 
and/or  x-ray  inhibition  of  Arbacia  egg 
development,  299. 

Chick  blastoderms,  respiratory  metabolism  of, 
77. 

Chicken  development,  effect  of  light  on,  415. 

Chorion  of  Fundulus  egg,  development  of,  293. 

Chromatophorotropic  principles  of  Uca,  312. 

Chromosome  behavior  during  oogenesis  of 
rotifer,  364. 

Chrysemys,  uptake  of  radio-iodine  by  thyroid 
of,  190. 

CLAFF,  C.  L.,  F.  N.  SUDAK  AND  N.  R.  STONE. 
Experimental  hypothermia  and  carbon 
dioxide  production  in  the  white  rat,  288. 

Clams,  opening  of  by  starfish,  114. 

Cleavage  furrows  in  Arbacia  eggs,  induction  of, 
317. 

Cleavage  of  Ilyanassa  egg,  distribution  of  mito- 
chondria and  lipid  droplets  during,  300. 

Cleavage  time  of  Arbacia  egg,  effect  of  argon  on 
at  high  pressures,  303. 

CLEMENT,  A.  C.,  AND  F.  E.  LEHMANN.  The 
distribution  of  mitochondria  and  lipid 
droplets  during  early  cleavage  in  Ilyanassa, 
300. 

CLOWES,  G.  H.  A.     See  M.  E.  KRAHL,  307. 

Cockroach,  oxygen  consumption  of,  53. 

Cockroach  abdominal  nerve  cord,  properties  of 
connective  tissue  sheath  of,  278. 

COHEN,  M.  J.  Sensory  and  motor  relation- 
ships of  a  crustacean  central  ganglion,  318. 

Cold,  acclimation  of  Acmaea  to,  129. 

Cold,  effect  of  on  oxygen  consumption  of  cock- 
roach, 53. 

COLWIN,  A.  L.,  L.  H.  COLWIN  AND  D.  E. 
PHILPOTT.  Sperm  entry  in  Hydroides  and 
Saccoglossus  studied  by  electron  micro- 
scopy, 289. 

COLWIN,  L.  H.,  A.  L.  COLWIN  AND  D.  E. 
PHILPOTT.  Electron  microscope  studies 
of  the  egg  surfaces  and  membranes  of 
Hydroides  and  Saccoglossus,  289. 

Comparative  physiology  of  nervous  system, 
278. 


Connective  tissue  sheath  of  cockroach  abdomi- 
nal nerve  cord,  278. 

Contact  chemoreceptors  in  Trichoptera,  92. 

Contractility  of  glycerinated  Vorticellae,  319. 

COOPERSTEIN,  S.  J.,  A.  LAZAROW  AND  W. 
LAUFER.  The  uptake  and  distribution  of 
radioactive  alloxan  in  islet  and  other  tis- 
sues of  the  toadfish,  300. 

Copulation  in  snails,  45. 

Corpuscles  of  Stannius,  role  of  in  teleost  osmo- 
regulation,  399. 

Cortex  of  Fundulus  egg,  relation  of  to  formation 
of  perivitelline  space,  304. 

Cortical  cytoplasmic  changes  after  fertilization 
of  Fundulus  eggs,  305. 

Coryphaena,  metabolism  of  radionuclides  by, 
336. 

COWGILL,  R.  W.  Phosphorylase  system  in  the 
lobster,  300. 

Crassostrea,  setting  in,  387. 

CRAVEN,  G.  See  G.  T.  SCOTT,  294 ;  R.  DE  VOE, 
296. 

Crayfish  neurosecretory  cell  types,  62. 

Cross-fertilization  in  Lymnaea,  45. 

Crustacean,  neurosecretory  cell  types  in,  62. 

Culture  of  brine  algae,  223. 

Currents,  water,  role  of  in  oyster  setting,  387. 

Cyanide  effects  on  Tubifex  metabolism,  179. 

Cyclic  activity  of  turtle  thyroid,  190. 

Cytochrome  oxidase  activity  in  chick  embryos, 
77. 

Cytology  of  corpuscles  of  Stannius,  399. 

Cytology  of  oogenesis  in  rotifer,  364. 

CZERLINSKI,  G.  H.     See  1.  M.  KLOTZ,  306. 

~P)CA,  effect  of  on  cytology  of  corpuscles  of 
Stannius,  399. 

DEHNEL,  P.  A.,  AND  E.  SEGAL.  Acclimation  of 
oxygen  consumption  to  temperature  in 
Periplaneta,  53. 

Dehydrogenase  activity  during  Asterias  de- 
velopment, 305. 

Density  of  oyster  setting,  387. 

Depth  in  relation  to  oyster  setting,  387. 

DETHIER,  V.  G.,  D.  R.  EVANS  AND  M.  V. 
RHOADES.  Some  factors  controlling  the  in- 
gestion of  carbohydrates  by  the  blowfly, 
204. 

Developing  ova,  effect  of  accelerating  and  re- 
tarding factors  on,  309. 

Development,  chicken,  effect  of  light  on,  415. 

Development  of  Arbacia  egg,  inhibition  of  by 
dimethylated  dioxypurines  and/or  x-rays, 
299. 

DIBBELL,  D.,  AND  H.  HoLTZER.  The  action  of 
Nessler's  reagent  and  ATP  on  extracted 
and  denatured  muscle,  301. 

Differentiation  of  chick  embryos,  77. 


[NDEX 


425 


Digenetic    trematode,    morphology    and    lite- 

history  of,  248. 

Dimorphism  in  echinoids,  328. 
Directions,  flight,  of  homing  terns,  235. 
Distribution  of  Bryozoa,  123. 
Dolphin,  metabolism  of  radionuclides  by,  330. 
Dunaliella,  culture  of,  223,  230. 
DURAND,  J.  B.     Neurosecretory  cell  types  and 

their  secretory  activity  in  the  crayfish,  62. 

gCHINODERM,  hermaphroditism  in,  328. 

Echinoderm  eggs,  membrane  potential  and  re- 
sistance of,  153. 

Echinoids,  hermaphroditism  in,  328. 

Ecological  implications  of  radioactivity  in  the 
sea,  336,  352. 

Ecological  relationships  of  phyto-  and  zoo- 
plankton,  230. 

Ecology  of  Acmaea,  129. 

Ecology  of  bryozoa,  123. 

Ecology  of  lower  marine  fungi,  410. 

Ecology  of  oyster  setting,  387. 

Ecology  of  salt  ponds,  223,  230. 

Effect  of  light  on  chicken  development,  415. 

Egg,  starfish,  membrane  potential  and  resist- 
ance of,  153. 

Egg  formation  in  Habrotrocha,  364. 

Egg  surfaces  and  membranes  of  Hydroides  and 
Saccoglossus,  289. 

Electric  current,  effects  of  on  contraction  of 
Spirogyra  chloroplasts,  310. 

Electrical  properties  of  starfish  eggs,  153. 

Electrolytes,  effect  of  on  cytology  of  corpuscles 
of  Stannius,  399. 

Electrophysiology  of  vertebrate  lens,  375. 

ELLIOTT,  A.  M.     See  C.  RAY,  JR.,  310. 

ELLIOTT,  A.  M.,  AND  D.  E.  OUTKA.  Fermen- 
tation studies  in  9  varieties  of  Tetra- 
hymena,  301. 

ELLIOTT,  A.  M.,  AND  J.  W.  TREMOR.  Electron 
microscope  studies  of  conjugating  Tetra- 
hymena,  302. 

Embryos,  chick,  respiratory  metabolism  of,  77. 

Endocrine  gland  (thyroid)  of  turtle,  190. 

Enzyme  activity  of  chick  blastoderm,  77. 

Enzymes,  role  of  in  firefly  light  production,  101. 

Estradiol,  effect  of  on  invertebrate  metabolism 
in  vitro,  318. 

Euthynnus,  metabolism  of  radionuclides  by, 
336. 

EVANS,  D.  R.     See  V.  G.  DETHIER,  204. 

Exochella,  morphology  and  distribution  of,  123. 

Explantation  of  chick  embryos,  77. 

pEEDING  of  Trichoptera,  92. 

Feeding  reaction  of  blowfly,  204. 
Fertilization,  cross-  and  self-,  in  Lymnaea,  45. 


Fertilization  of  starfish  ei  mlirane  po- 

tential and  resistance  before  and  after,  153. 

Fertilized  Arbacia  eggs,  change  in  rate  of  release 
ul  K-42  in,  296.' 

Fertilized  egg  of  Arbacia,  hyaline  polymer  nf, 
294. 

FIGGE,  F.  H.  J.,  AND  R.  WICHTERMAX.  In- 
fluence of  hematoporphyrin  and  phenol  on 
x-radiation  sensitivity  of  Paramecium,  302. 

Filtering  action  of  vertebrate  lenses,  375. 

FINGER,  I.  Immobilizing  and  precipitating 
antigens  of  Paramecium,  358. 

Firefly  pseudoflash,  101. 

Fish  lens  spectral  sensitivity,  375. 

Fish,  marine,  influencing  calling  of  by  sound, 
393. 

Fish,  osmoregulation  of,  399. 

Fishes,  metabolism  of  radionuclides  by,  336, 
352. 

Fixation  and  staining  of  Ilyanassa  eggs,  307. 

Flight  directions  of  homing  terns,  235. 

FRASER,  R.  C.  The  presence  and  significance 
of  respiratory  metabolism  in  streak- 
forming  chick  blastoderms,  77. 

FRIEDL,  F.     See  G.  C.  STEPHENS,  312,  313. 

FRINGS,  H.,  AND  M.  FRINGS.  The  location  of 
contact  chemoreceptors  sensitive  to  su- 
crose solutions  in  adult  Trichoptera,  92. 

Frog  lens  spectral  sensitivity,  375. 

Fungi,  marine,  ecology  of,  410. 

r^ENETIC  nature  of  cyclic  variations  in 
turtle  thyroid  activity,  190. 

Genetics  of  snails,  45. 

GIBOR,  A.     The  culture  of  brine  algae,  223. 

GIBOR,  A.  Some  ecological  relationships  be- 
tween phyto-  and  zooplankton,  230. 

Glucose-utilization  pathways  in  eggs,  307. 

Glutathione,  post-irradiation  treatment  <  '1 
Hydra  with,  240. 

GOLDSMITH,  T.  H.,  AND  D.  R.  GRIFFIN.  Fur- 
ther observations  of  homing  terns,  235. 

Gonad  size  in  Acmaea,  129. 

GREEN,  J.  W.,  M.  HARSCH,  L.  BARR  AND  C.  L. 
PROSSER.  Ionic  regulation  in  the  fiddler 
crabs,  Uca  sp.,  290. 

GRIFFIN,  D.  R.     See  T.  H.  GOLDSMITH,  235. 

GROSS,  P.  R.  Amphibian  yolk:  chemistry  and 
infrastructure,  287. 

GROSS,  P.  R.,  D.  E.  PHILPOTT  AND  S.  NASS. 
Electron  microscopy  of  the  mitotic  appara- 
tus in  dividing  Arbacia  eggs,  290. 

GRUNDFEST,  H.     Sec  A.  TYLER,  153. 

Gustatory  responses  of  blowflies,  204. 

GUTTMAN,  B.     See  G.  C.  STEPHENS,  312. 

t_J  AGERMAN,  D.  D.     Invertebrate  metabo- 
lism in  vitro  not  affected  by  estradiol,  318. 
HARSCH,  M.     See  J.  W.  GREEN,  290. 


426 


INDEX 


HASTINGS,  J.  W.,  AND  J.  BUCK.  The  firefly 
pseudoflash  in  relation  to  photogenic 
control,  101. 

HAYASHI,  T.,  R.  ROSENBLUTH,  P.  SATIR  AND  M. 
VOZICK.  Participation  of  actin  in  acto- 
myosin  contraction,  290. 

HAYWOOD,  C.  The  effect  of  argon  at  high 
pressures  on  the  cleavage  time  of  the  sea 
urchin,  Arbacia,  303. 

Heart  physiology  of  marine  fish,  316. 

Heart  rate  of  Acmaea,  129. 

Heat,  acclimation  of  Acmaea  to,  129. 

Heat,  effect  of  on  oxygen  consumption  of  cock- 
roaches, 53. 

HEILBRUNN,  L.  V.,  AND  W.  L.  WILSON.  An 
interpretation  of  the  action  of  certain 
chemical  agents  used  in  cancer  therapy, 
291. 

Hemerythrin,  nature  of  metal-to-protein  bond 
in,  293. 

Hemerythrin  and  hemocyanin,  chemical  nature 
of  bound  oxygen  in,  306. 

Hemocyanin,  Busycon,  crystallization  of,  306. 

Hemoglobin  of  Petromyzon,  molecular  weight 
of,  293. 

Hermaphroditism  in  echinoids,  328. 

HIATT,  R.  W.     See  H.  BOROUGHS,  336,  352. 

Histology  of  neurosecretory  cell  types  in  cray- 
fish, 62. 

HOLTZER,  H.     See  D.  DIBBELL,  301. 

HOLTZER,  H.,  J.  LASH  AND  S.  HOLTZER.  The 
enhancement  of  somitic  muscle  maturation 
by  the  embryonic  spinal  cord,  303. 

Homing  terns,  flight  directions  of,  235. 

Hormone  activity  in  crayfish,  62. 

Hsu,  W.  S.     Oogenesis  in  Habrotrocha,  364. 

HUVER,  C.  W.  The  relation  of  the  cortex  to 
the  formation  of  a  perivitelline  space  in 
the  eggs  of  Fundulus,  304. 

Hydra,  twinning  in,  269 

Hydra,  x-irradiation  of,  240. 

Hypothermia  and  carbon  dioxide  production 
in  white  rat,  288. 

Hypoxic  glow  in  fireflies,  101. 

| -131,  uptake  of  by  turtle  thyroid,  190. 

Immobilizing  antigens  of  Paramecium,  358. 

Immunology  of  Paramecium,  358. 

Indophenol  oxidase  in  chick  embryo,  77. 

Influencing  calling  of  sea  robins,  393. 

Ingestion  of  carbohydrates  by  blowfly,  204. 

Inheritance  of  albinism  in  snails,  45. 

Inhibiting  agents  in  Tubularia,  315. 

Initial  flight  directions  of  homing  terns,  235. 

Innervation  of  Asterias  stomach-retractor 
muscle  fibers,  297. 

Insect,  metabolism  of  in  relation  to  tempera- 
ture, 53. 


Insect  abdominal  nerve  cord,  properties  of  con- 
nective tissue  sheath  of,  278. 

Insects,  chemoreception  in,  92. 

Insemination  in  Lymnaea,  45. 

Insemination  of  starfish  eggs,  effect  of  on  mem- 
brane potential  and  resistance,  153. 

Intensity  of  oyster  setting,  387. 

Intertidal  mollusc,  microgeographic  variation 
in,  129. 

lodoacetate,  effect  of  on  Tubifex  regeneration, 
179. 

Ion  exchange  in  Ulva,  294. 

Ion  replacement  in  Ulva,  296. 

Ionic  regulation  in  fiddler  crab,  290. 

Ionizing  radiations,  effects  of  on  Hydra,  240. 

Isotopes,  metabolism  of  by  marine  organisms, 
336,  352. 

1  ENNER,  C.  E.  The  occurrence  of  a  crystal- 
line style  in  the  marine  snail,  Nassarius, 
304. 

JENNER,  C.  E.  Seasonal  resorption  of  the 
copulatory  organ  in  Nassarius  and  Lit- 
torina,  305. 

JENNER,  C.  E.  A  striking  behavioral  change 
leading  to  the  formation  of  extensive 
aggregations  in  a  population  of  Nassarius, 
291. 

JENNER,  C.  E.  The  timing  of  reproductive 
cessation  in  geographically  separated  popu- 
lations of  Nassarius,  292. 

\T  AO,  C.  Y.  Absence  of  membrane  potential 
in  presence  of  asymmetrical  ion  distri- 
bution in  the  Fundulus  egg,  292. 

KAO,  C.  Y.     See  A.  TYLER,  153. 

KELTCH,  A.  K.     See  M.  E.  KRAHL,  307. 

KEMP,  N.  E.,  AND  M.  D.  ALLEN.  Electron 
microscopic  observations  on  changes  in  the 
cortical  cytoplasm  after  fertilization  of 
Fundulus  eggs,  305. 

KEMP,  N.  E.,  AND  M.  D.  ALLEN.  Electron 
microscopic  observations  on  the  develop- 
ment of  the  chorion  of  Fundulus,  293. 

KENNEDY,  D.,  AND  R.  D.  MILKMAN.  Selective 
light  absorption  by  the  lenses  of  lower 
vertebrates,  and  its  influence  on  spectral 
sensitivity,  375. 

KlVY-ROSENBERG,    E.,    AND    B.    W.    ZWEIFACH. 

Dehydrogenase  activity  in  developmental 
stages  of  Asterias  as  measured  with  tetra- 
zolium  salts,  305. 

KLOTZ,  I.  M.,  AND  T.  A.  KLOTZ.  The  chemical 
nature  of  bound  oxygen  in  hemerythrin 
and  in  hemocyanin,  306. 

KLOTZ,  I.  M.,  AND  T.  A.  KLOTZ.  The  nature 
of  the  metal-to-protein  bond  in  hemeryth- 
rin, 293. 


INDEX 


427 


KLOTZ,  I.  M.,  T.  A.  KI.OTX  AND  G.  H.  CZKRLIN- 
SKI.  Crystallization  of  Busycon  heino- 
cyanin,  306. 

KRAHL,  M.  E.,  A.  K.  KELTCH,  C.  P.  WALTERS 
AND  G.  H.  A.  CLOWES.  Pathways  of 
glucose-C14  utilization  in  eggs  of  Arbacia, 
Mactra  and  Chaetopterus,  307. 

Kuhlia,  metabolism  of  radionuclides  by,  336. 

T    AMPYRID  fireflies,  photogenic  control  in, 

^     101. 

LASH,  J.     Sec  H.  HOLTZER,  303. 

LAUFER,  W.     See  S.  J.  COOPERSTEIN,  300. 

LAVOIE,  M.  E.  How  sea  stars  open  bivalves, 
114. 

LAZAROW,  A.     See  S.  J.  COOPERSTEIN,  300. 

LEES,  A.  D.  Methods  for  investigating  the 
locations  of  the  photoperiodic  receptors  in 
insects,  319. 

LEHMANN,  F.  E.  Improved  fixing  and  staining 
methods  for  cellular  structures  in  Ilyanassa 
eggs,  307. 

LEHMANN,  F.  E.     See  A.  C.  CLEMENT,  300. 

LENHERT,  P.  G.,  W.  E.  LOVE  AND  F.  D. 
CARLSON.  The  molecular  weight  of  hemo- 
globin from  Petromyzon,  293. 

Lenses  of  lower  vertebrates,  selective  light 
absorption  by,  375. 

LEVINE,  L.  Contractility  of  glycerinated 
Yorticellae,  319. 

Life-history  of  Azygia,  248. 

Light,  effect  of  on  chicken  development,  415. 

Light  absorption  by  lenses  of  lower  vertebrates, 
375. 

Light  production  of  firefly,  101. 

Location  of  chemoreceptors  in  Trichoptera,  92. 

Long  Island  Sound,  relative  intensity  of  oyster 
setting  in,  387. 

LOOSANOFF,  V.  L.,  AND  C.  A.  XoMEjKO.  Rela- 
tive intensity  of  oyster  setting  in  different 
years  in  the  same  areas  of  Long  Island 
Sound,  387. 

Loss  of  radionuclides  by  fishes,  336,  352. 

LOVE,  W.  E.     See  P.  G.  LENHERT,  293. 

LOWER,  G.  G.     See  D.  E.  PHILPOTT,  294. 

Luminescence  of  fireflies,  101. 

Lymnaea,  cross-  and  self-fertilization  in,  45. 

Lynch,  W.  F.  Factors  inhibiting  metamor- 
phosis in  tadpoles  of  Amaroecium,  308. 

\  \  AAS,  W.  K.  Regulation  of  arginine  bio- 
synthesis in  Escherichia,  319. 

Magnesium  sulfate,  post-irradiation  treatment 
of  Hydra  with,  240. 

Magnifying  the  invisible,  294. 

Marine  bryozoa,  studies  on,  123. 

Marine  fish,  influencing  calling  of  by  sound, 
393. 

Marine  fungi,  ecology  of,  410. 


Marine  organisms,  metabolism  <>t  radionuclides 
by,  336,  352. 

Marine  red  alga,  new  pigment  from,  321. 

MARSLAND,  D.     See  A.  M.  ZIMMERMAN,  317. 

Maturation  of  rotifer  egg,  364. 

Maturity,  chicken,  effect  of  light  on  develop- 
ment of,  415. 

MEINKOTH,  N.  A.  A  North  American  record 
of  Rhopalura,  a  parasite  of  the  nemertean 
Amphiporus,  308. 

Melanophore-dispersing  hormone  of  Uca,  rate 
of  disappearance  of  from  blood,  313. 

Membrane  potential  and  resistance  of  starfish 
egg,  153. 

Mendelian  inheritance  in  snails,  45. 

MENKIN,  V.,  G.  MENKIN  AND  L.  MENKIN. 
Studies  on  accelerator  and  retarding 
factors  of  one  species  on  the  developing 
ova  of  an  unrelated  form,  309. 

MENKIN,  V.,  G.  MENKIN  AND  W.  ROGERS. 
Further  studies  on  some  factors  concerned 
in  the  regulation  of  cell  division,  309. 

Metabolism  during  regeneration  of  Tubifex, 
179. 

Metabolism  of  chick  embryos,  77. 

Metabolism  of  cockroach  in  relation  to  tem- 
perature, 53. 

Metabolism  of  radionuclides,  336,  352. 

Metamorphosis  of  Amaroecium  tadpoles,  308. 

Metamorphosis  of  oysters,  387. 

Microgeographic  variation  in  mollusc,  129. 

Microinjection  of  Asterias  eggs,  153. 

Micromanipulation  of  Arbacia  eggs,  316. 

MILKMAN,  R.  D.     See  D.  KENNEDY,  375. 

Mitosis,  factors  concerned  in  regulation  of ,  309. 

Mitosis  in  Arbacia,  electron  microscopy  of,  290. 

Modification  of  x-ray  injury  to  Hydra,  240. 

Molecular  weight  of  new  phycoerythrin,  321. 

Mollusc,  cross-  and  self-fertilization  in,  45. 

Mollusc,  microgeographic  variation  in,  129. 

Mollusc,  setting  in,  387. 

Molluscs,  opening  of  by  starfish,  114. 

Molting  cycle  of  crayfish  in  relation  to  neuro- 
secretion,  62. 

MONROY,  A.     See  A.  TYLER,  296,  153. 

MOORE,  A.  R.     See  R.  A.  BOOLOOTIAN,  328. 

Morphogenesis  of  Tubifex,  179. 

Morphology  of  Azygia,  248. 

Morphology  of  bryozoa,  123. 

MOULTON,  J.  M.  Influencing  the  calling  of  sea 
robins  with  sound,  393. 

Mouthparts  of  Trichoptera,  92. 

Muscle,  action  of  Nessler's  reagent  and  ATP 
on  extracted  and  denatured,  301. 

Muscle,  concentration  of  strontium-89  in,  336. 

Muscle  maturation,  somitic,  enhancement  of  by 
embryonic  spinal  cord,  303. 

Myosin,  frog,  ATPase  activity  of,  320. 


428 


INDEX 


Mytilus,  opening  of  by  starfish,  114. 
Myxomycetes,  ma  ••  ''logy  ot,  410. 

JSJASS,    S.     Amphibian    yolk:    the    phospho- 

protein  phosphatase  system,  287. 
NASS,  S.     See  P.  R.  GROSS,  290. 
Navigation  of  terns,  235. 
Neothunnns,  metabolism  of  radionuclides  by, 

336. 
Nerve  cord,  cockroach,  properties  ot  con 

tissue  sheath  of,  278. 
Neurophysiology  of  cockroach  abdominal  n< 

d,  278. 

Neurosccretory  cell  types  in  cravli--h,  <>-. 
Nitrogen  supply,  importance  ot  for  growth  ot 

brine  algae,  22.-!. 

Noise  production  by  sea  robins,  393. 
XOMKJKO,  C.  A.     SeeV.  T,.  LOOSANOFF,  3X7. 

QOGENESIS  in  Habrotrocha, 

(Opening  of  bivalves  by  s,  114. 

<  >n  onectes,  neurosecretory  cell  types  in 

Orientation  of  homing  terns,  235. 

Osmbregulation  of  teleosi. -.  3"'). 

OSIERHOUT,  W.  J.  \".  Effects  of  electric  cur- 
rent on  the  contraction  of  the  chloroplasts 
of  Spiro^yra,  310. 

OUTKA,  LX     See  A.  M.  ELI  .  01. 

Ova,  starfish,  n  potential  and  resist- 

ance ol,  153. 

Oxygen,  role  of  in  mvtly  light  production.  101. 
nnption  '  h,  53. 

Oxygen  consumption  of  Tubilex,  17'). 

( )yster  settiir     ,         isity  of,  387. 

( )y>ters.  openin     •  -  arfish,  114. 

IJADAYVER,  J.     Sec  A.  M.  ZIMMERMAN,  317. 

I  'araincciiim,  ai;  "lS;. 

site,  moi  ,  'I  life-history  ot,  24S. 

Parasite  of  n  >^. 

Parasites  <>i  <  ")5. 

Parasiti  fish,  carbohydrates  metaboli/ed 

by,  311. 

Parasites  of  Lit  hod  for  recognition 

of,  316. 

PARK,  H.  D.  Modification  of  x-ray  injury  to 
Hydra  littoralis  by  post-irradiation  treat- 
ment: with  magnesium  sulfate  and  gluta- 
thione,  240. 

PARPART,  A.  K.,  AND  J.  CAGLE.  Hyaline 
polymer  of  the  fertili/ed  egg  of  Arbacia, 
2<>4. 

Pelmatohydra,  twinning  in,  26'). 

Periplaneta,  oxv  '  •  umption  ot,  53. 

Permeability  of  cockroach  abdominal  nerve 
cord,  278. 

pH,  effect  of  on  growth  of  brine  algae,  223. 


PFIILPOTT,  D.  E.     See.  A.  L.  COLVVIN,  289;  L.  H. 
COLWIN,  289;  P.  R.  GROSS,  290. 

PHILPOTT,  D.  E.,  AND  G.  G.  LOWER.  Magni- 
fying the  invisible,  294. 

Phormia  regina,  ingestion  of  carbohydrates  by, 
204. 

Phosphate,  importance  ot  for  brine  algal 
growth,  223. 

Phosphorylase  system,  in  lobster,  300. 

Photinus,  photogenic  control  in,  101. 

Photogenic  control  in  lirefly,  101. 

Photoperiodic  receptors  of  insects,  319. 

Photuris,  photogenic  control  in,  101. 

Phryganea,  contact  chemoreceptors  in,  92. 

Phycoerythrin  from  Porphyra,  321. 

Phycomycetes,  ecology  of,  410. 

Phyto-  and  zooplankton,  ecological  relation- 
ships of,  230. 

Pigmentation  of  lower  vertebrate  eyes,  375. 

Pigments  of  algae,  321. 

1'itressin,  effect  of  on  cytology  of  corpuscles  of 
Stannius,  399. 

Planktonic  existence  of  Nassarius,  effect  of 
substrate  on  duration  of,  312. 

I'M  i  \  centropus,  contact  chemoreceptors  in,  92. 

I'fi i  vmonas,  culture  of,  223,  230. 

Polluted  sea  water,  presence  of  fungi  in,  410. 

Porphyra,  new  phycoerythrin  from,  321. 

Post-irradiation  treatment  of  hydra,  240. 

Potassium,  role  of  in  cockroach  nerve  cord 
ri  induct  ion,  278. 

Potassium  content  of  Asterias  eggs,  153. 

Potential,  ai  tion,  in  scallop  eye  retina,  310. 

Potential,  membrane,  of  Fundulus  egg,  292. 

Potential,  membrane,  of  starfish  egg,  153. 

Potential,  slow  action,  of  vertebrate  lens,  375. 

Precipitating  antigens  of  Paramecium,  358. 

Preference-aversion  tests  in  blowfly,  204. 

Pressure-centrifuge  studies  on  mast  cells,  317. 

Prionotus,  influencing  calling  of,  393. 

••Productivity  of  bottom  areas  of  Long  Island 
Sound,  387. 

Properties  of  connective  tissue  sheath  of  cock- 
roach nerve  cord,  278. 

PROSSER,  C.  L.     See  J.  W.  GREEN,  290. 

Protective  effect  of  magnesium  sulfate  and 
glutathione  against  radiation  damage  to 
hydra,  240. 

Protozoan,  antigens  of,  358. 

Pseudo-flash,  firefly,  101. 

Ptilostomis,  contact  chemoreceptors  in,  92. 

Pyrophorus,  photogenic  control  in,  101. 

D  ADIO-IODINE,  uptake  of  by  turtle  thy- 
roid, 190. 

Radionuclides,  metabolism  of,  336,  352. 
R  \SQUIN,  P.     Cytological  evidence  for  a  role 

of  the  corpuscles  of  Stannius  in  the  osino- 
regulation  ot  teleosts,  399. 


INDEX 


429 


RATLIFF,  F.  Retinal  action  potentials  in  the 
eye  of  the  scallop,  310. 

RAY,  C,  JR.,  AND  A.  M.  ELLIOTT.  Strain  dif- 
ferences in  viability  following  conjugation 
within  variety  9  of  Tetrahymena,  310. 

READ,  C.  P.  Carbohydrate  metabolized  by 
cestode  parasites  of  dogfish,  311. 

Recovery  time  of  cockroach  nerve  cord,  278. 

Red  algae,  new  pigment  from,  321. 

Regeneration  of  Tubifex,  179. 

Reproduction  of  Pelmatohydra  twins,  269. 

Reproductive  cessation  in  Nassarius,  290. 

Resistance,  membrane,  of  starfish  egg,  153. 

Respiration  of  cockroaches,  45. 

Respiratory  metabolism  of  chick  embryos,  77. 

RHOADES,  M.  V.     See  V.  DETHIER,  204. 

ROEDER,  K.  D.     See  B.  M.  TWAROG,  278. 

Roentgen  irradiation  of  hydra,  240. 

ROGERS,  \V.     See  V.  MENKIN,  309. 

ROGICK,  M.  Studies  on  marine  bryozoa. 
VIII.,  123. 

Role  of  corpuscles  of  Stannius  in  teleost  osmo- 
regulation,  399. 

ROSENBLUTH,  R.     See  T.  HAYASHI,  290. 

Rotifer,  oogenesis  in,  364. 

RUGH,  R.     See].  WOLF,  288. 

RUGH,  R.,  AND  J.  WOLF.  Recovery  from  x- 
irradiation  effects  at  the  cellular  level,  311. 

C  ALINITY  tolerance  of  brine  algae,  223. 

SATIR,  P.     See  T.  HAYASHI,  290. 

SCHELTEMA,  R.  S.     The  effect  of  substrate  on 

the    length     of    planktonic    existence    in 

Nassarius,  312. 

SCOTT,  G.  T.     See  R.  DE  YOE,  296. 
SCOTT,  G.  T.,  R.   DE  VOE  AND  G.   CRAVEN. 

Sodium  ion  exchange  in  Ulva,  294. 
Sea  robins,  influencing  calling  of,  393. 
Sea  stars,  method  of  for  opening  bivalves,  114. 
Sea  urchin,  hermaphroditism  in,  328. 
Seasonal  activity  of  turtle  thyroid,  190. 
Seasonal    resorption    of    copulatory    organ    in 

Nassarius  and  Littorina,  305. 
Secretory  activity  of  crayfish  cells,  62. 
SEGAL,  E.  Microgeographic  variation  as  ther- 
mal acclimation  in  an  intertidal  mollusc, 

129. 

SEGAL,  E.     See  P.  A.  DEHNEL,  53. 
Selective  light  absorption  by  lenses  of  lower 

vertebrates,  375. 
Sensitivity  of  Trichoptera  chemoreceptors  to 

sucrose,  92. 
Sensitivity,  spectral,  of  lower  vertebrate  lenses, 

375. 
Sensory  control   of   blowfly   carbohydrate   in- 

gestion,  204. 
Sensory  and  motor  relationships  of  crustacean 

central  ganglion,  318. 


Serotypes  of  Paramecium,  364. 

Setting,  oyster,  relative  intensity  of,  387. 

Sexual  characteristics,  chicken,  effect  of  light 
on  development  of,  415. 

Size  in  relation  to  cockroach  oxygen  consump- 
tion, 53. 

Skipjack,  metabolism  of  radionnclides  by,  336. 

Snail,  cross-  and  self-fertilization  in,  45. 

Sodium,  role  of  in  cockroach  nerve  cord  con- 
duction, 278. 

Sodium  content  of  Asterias  eggs,  153. 

Sound,  influencing  calling  of  sea  robins  with, 
393. 

Spawning  behavior  of  Acmaea,  129. 

Spectral  sensitivity  of  lower  vertebrate  lenses, 
375. 

Sperm  entry  in  Hydroidesand  Saccoglossus,  289. 

Sperm  viability  in  snails,  45. 

Spermatozoa  in  dogfish  oviducal  gland,  314. 

Stannius,  corpuscles  of,  role  of  in  teleost  osmo- 
regulation,  399. 

Starfish,  method  of  for  opening  bivalves,  114. 

Starfish  egg,  membrane  potential  and  resistance 
of,  153. 

Stephanoptera,  culture  of,  223,  230. 

STEPHENS,  G.  C.,  F.  FRIEDL  AND  B.  GUTTMAN. 
Electrophoretic  separation  of  chromato- 
phorotropic  principles  of  the  fiddler  crab, 
Uca,  312. 

STEPHENS,  G.  C.,  A.  STRICKHOLM  AND  F. 
FRIEDL.  The  rate  of  disappearance  of  the 
melanophore-dispersing  hormones  from  the 
blood  of  the  fiddler  crab,  313. 

Stichococcus,  culture  of,  223,  230. 

STONE,  N.  R.     See  C.  L.  CLAFF,  288. 

Streak-forming  chick  blastoderm,  metabolism 
of,  77. 

STRICKHOLM,  A.     See  G.  C.  STEPHENS,  313. 

Strongylocentrotus,  hermaphroditism  in,  328. 

STUNKARD,  H.  XV.  The  morphology  and  life- 
history  of  the  digenetic  trematode,  Azygia, 
248. 

STUNKARD,  H.  XV.  Studies  on  parasites  of  the 
green  crab,  Carcinides,  295. 

Style,  crystalline,  occurrence  of  in  Nassarius, 
304." 

Sucrose,  sensitivity  of  Trichoptera  chemore- 
ceptors to,  92. 

SUDAK,  F.  N.     See  C.  L.  CLAFF,  288. 

Sugars,  ingestion  of  by  blowfly,  204. 

"pAXONOMY  of  bryozoa,  123. 

Taxonomy  of  digenetic  trematode,  248. 

Teleosts,  osmoregulation  of,  399. 

TEMIN,     H.     M.     Studies    on     activation     of 

Urechis,  Nereis  and  Asterias  eggs,  313. 
Temperature,  role  of  in  growth  of  brine  algae, 

223. 


430 


INDEX 


Temperature  acclimation  of  Acmaea,  129. 

Temperature  effect  on  oxygen  consumption  of 
cockroach,  53. 

Temporal  differences  in  oyster  setting,  387. 

Terns,  homing,  initial  flight  directions  of,  235. 

TE  WINKEL,  L.  E.  Spermatozoa  in  the  ovi- 
ducal  gland  of  the  smooth  dogfish,  Mus- 
telus,  314. 

Tetrahymena,  fermentation  studies  in,  301. 

Tetrahymena,  strain  differences  in  viability 
following  conjugation  of,  310. 

Thermal  acclimation  of  mollusc,  129. 

TRIES,  R.  E.,  AND  F.  D.  CARLSOX.  Conduc- 
tion velocity  in  the  giant  axon  of  the  squid, 
Loligo,  295. 

Thiourea  treatment  of  turtles,  190. 

Thyroid,  turtle,  uptake  of  1-131  by,  190. 

Tilapia,  metabolism  of  radionuclides  by,  336, 
352. 

Tissue  culture  of  chick  embryos,  77. 

TOKAY,  E.  Acetylcholine  and  frog  brain 
oxygen  consumption,  314. 

Topography  as  factor  in  homing  response  of 
terns,  235. 

TOWNSLEY,  S.  J.     See  H.  BOROUGHS,  336,  352. 

Toxic  factor  release,  mechanism  of,  298. 

Tracheal  end  cells  in  fireflies,  role  of  in  light 
production,  101. 

Trematode,  morphology  and  life-history  of, 
248. 

TREMOR,  J.  W.     See  A.  M.  ELLIOTT,  302. 

Trichoid  sensilla  in  Trichoptera,  92. 

Trichoptera,  chemoreceptors  of,  92. 

Tubifex,  metabolism  and  morphogenesis  during 
regeneration  of,  179. 

Tuna,  metabolism  of  radionuclides  by,  336. 

TURNER,  C.  L.  Twinning  and  reproduction  of 
twins  in  Pelmatohydra,  269. 

Turtle  thyroid  gland,  uptake  of  1-131  by,  190. 

TWAROG,  B.  M.,  AND  K.  D.  ROEDER.  Proper- 
ties of  the  connective  tissue  sheath  of  the 
cockroach  abdominal  nerve  cord,  278. 

TWEEDELL,  K.  S.  A  comparison  of  two  in- 
hibiting agents  in  Tubularia,  315. 

Twinning  in  Pelmatohydra,  269. 

TYLER,  A.,  AND  A.  MONROY.  Change  in  rate 
of  release  of  K-42  upon  fertilization  in  eggs 
of  Arbacia,  296. 

TYLER,  A.,  A.  MONROY,  C.  Y.  KAO  AND  H. 
GRUNDFEST.  Membrane  potential  and 
resistance  of  the  starfish  egg  before  and 
after  fertilization,  153. 

JJ  LTRAVIOLET  absorption  by  frog  and  fish 

lenses,  375. 
Uptake  of  1-131  by  turtle  thyroid,  190. 


Uptake  of  radionuclides  by  fishes,  336,  352. 
Uricase  activity,  combined  effect  of  urea  and 
borate  buffer  on,  299. 

VARIABILITY  of  reaction  of  turtle  thyroid 

to  thiourea  treatment,  190. 
Vertebrates,   lower,   selective  light  absorption 

by  lenses  of,  375. 
DE  VILLAFRANCA,  G.  W.     The  ATPase  activity 

of  frog  myosin,  320. 
YISHNIAC,  H.  S.     On  the  ecology  of  the  lower 

marine  fungi,  410. 

Visual  pigments  of  lower  vertebrate  lenses,  375. 
DE  VOE,  R.     See  G.  T.  SCOTT,  294. 
DE  VOE,   R.,  G.  T.  SCOTT  and  G.   CRAVEN. 

The  reversible  replacement  of  potassium 

by  rubidium  ion  in  Ulva,  296. 
VOZICK,  M.     See  T.  HAYASHI,  290. 

^y  ALTERS,  C.  P.     See  M.  E.  KRAHL,  307. 

WICHTERMAN,  R.  Attempts  to  breed  an  x-ray 
resistant  clone  of  Paramecium,  315. 

WICHTERMAN,  R.     See  F.  H.  J.  FIGGE,  302. 

WIERCINSKI,  F.  J.  The  micromanipulation  of 
Arbacia  eggs,  316. 

WILBER,  C.  G.  The  physiology  of  the  heart 
in  marine  fish,  316. 

WILLEY,  C.  H.  A  rapid  method  for  recog- 
nition of  specimens  of  Littorina  infected 
with  trematode  larvae,  316. 

WILSOX,  W.  L.     See  L.  V.  HEILBRUNN,  291. 

WILSON,  W.  ().,  A.  E.  WOODARD  AND  H. 
ABPLANALP.  The  effect  and  after-effect 
of  varied  exposure  to  light  on  chicken 
development,  415. 

WOLF,  J.     See  R.  RUGH,  311. 

WOLF,  J.,  AND  R.  RUGH.  The  relation  of 
gonad  hormones  to  x-irradiation  sensitivity 
in  mice,  288. 

WOODARD,  A.  E.     See  W.  O.  WILSON,  415. 

Worm,  regeneration  of,  179. 

^-IRRADIATION,  recovery  from  effects  of, 

at  cellular  level,  311. 
X-irradiation  sensitivity  of  mice,  288. 
X-irradiation  of  Tenebrio,  297. 
X-organ  of  crayfish,  62. 
X-ray  injury  to  hydra,  modification  of,  240. 
X-ray  resistant  clone  of  Paramecium,  attempts 

to  breed,  315. 
X-ray    sensitivity    of    Paramecium,    effect    of 

hematoporphyrin  and  phenol  on,  302. 


INDEX  431 

"VOLK,    amphibian,    chemistry    and     ultra-  ZIMMERMAN,    A.    M.,    J.    I'ADAWKR    AXD    D. 

structure  of,  287.  MARSLAND.     Pressure-centrifuge     studies 

Yolk,   amphibian,    phosphoprutcin    and    phus-  on  mast  cells,  317. 

phatase  system  of,  287.  Zoo-  and   phytoplankton,   ecological   relation- 
ships of,  230. 

^IMMERMAN,  A.  M.,  AND  D.  MARSLANU.  ZWEIFACH,  B.  \V.     .SVr  E.  KIVY-ROSENBERG, 

Induction  of  premature  cleavage  furrows  305. 

in  the  eggs  of  Arbacia,  317. 


Volume  111 


Number  1 


THE 


BIOLOGICAL  BULLETIN 


*v-\ 


PUBLISHED  BY 

THE   MARINE   BIOLOGICAL  LABORATORY 


Editorial  Board 


T.  H.  BULLOCK,  University  of  California, 

Los  Angeles 

E.  G.  BUTLER,  Princeton  University 

K.  W.  COOPER,  University  of  Rochester 

L.  V.  HEILBRUNN,  University  of  Pennsylvania 

M.  E.  KRAHL,  University  of  Chicago 

J.  H.  LOCHHEAD,  University  of  Vermont 


E.  T.  MOUL,  Rutgers  University 
ARTHUR  W.  POLLISTER,  Columbia  University 
MARY  E.  RAWLES,  Johns  Hopkins  University 
BERTA  SCHARRER,  Albert  Einstein  College 

of  Medicine 

J.  H.  WELSH,  Harvard  University 
A.  R.  WHITING,  University  of  Pennsylvania 


DONALD  P.  COSTELLO,  University  of  North  Carolina 

Managing  Editor 


AUGUST,    1956 


Marine  Biological  Laboratory 


3LIBR 


AUG27 


WOODS  HOLE,  MASS. 


Printed  and  Issued  by 

LANCASTER  PRESS,  Inc. 

PRINCE  8C  LEMON  STS. 

LANCASTER,  PA. 


aiimimiiiiimimiimimimiiiiimimmimiiiiiimmiimiiiiimmm 


CLEANING  COMPOUND 


for  Glass,   Porcelain  and   Metal  Ware  . 


Penetrates  and  loosens  "fro- 
zen" glass  joints,  stopcocks  and 
stoppers 

Not  harmful  to  hands 

Particularly  efficient  in  remov- 
ing dried  blood,  organic  resi- 
dues and  wax  pencil  marks 


THOMAS  CLEANING  COMPOUND,  General 
Laboratory  Purpose.  A  fine  white  powder 
especially  formulated  for  general  laboratory 
use  from  effective  synthetic  detergents,  care- 
fully buffered  and  blended  without  use  of 
harsh  alkalies  or  ingredients  which  affect 
sensitive  skin.  Useful  for  cleaning  glassware, 
porcelain  or  metal  ware.  Particularly  efficient 
in  removing  dried  blood,  organic  residues, 
and  wax  pencil  marks,  and  for  making  soak 
solution  to  facilitate  freeing  frozen  ground 
glass  joints,  stopcock  plugs  and  glass  stoppers. 

Totally  soluble  in  hot,  cold,  hard  or  soft 
water.  Produces  surfaces  free  from  film, 
streaks  and  hard  water  deposits.  When  used 
in  usual  concentration  of  Bounce  to  1  gallon, 
or  in  somewhat  higher  concentration  for 
stubborn  deposits,  in  either  hot  or  cold  water, 
the  pH  of  the  wash  water  is  constant  at 
approximately  9.0  pH. 

Contains  a  surface  active  (wetting)  agent, 
to  facilitate  rinsing,  which  is  stable  in  acids 
or  alkalies,  and  a  sequestering  agent  which 
disperses  the  metal  ions  in  tap  water  such  as 
calcium,  magnesium,  etc.,  without  precipita- 
tion; both  additives  combine  effectively  to 
emulsify  organic  residues,  oils  and  greases, 


lUlllllllllllllllllllllllllK 


3298. 

etc.,  and  disperse  finely  divided  deposits  such 
as  activated  charcoal,  organic  or  inorganic 
pigments,  and  various  catalysts  used  in 
organic  synthesis. 

Packed  in  convenient  rectangular  canisters 
containing  3  Ibs.,  and  in  25  Ib.  and  100  Ib. 
fiber  drums.  The  canisters  have  metal  top  and 
bottom,  and  friction-fit,  oval  metal  cap, 
4  x  2^2  inches  wide,  which  contains  a  hinged 
pouring  spout  for  convenient  dispensing. 
Overall  dimensions  4>£  x  3  x  8  inches  high. 


3298.    Thomas    Cleaning    Compound,    General 
Laboratory  Purpose,  as  above  described. 

Per  3  Ib.  canister,  with  pourout 1.86 

Per  original  ease  containing  12  x  3  Ib.  canisters 

(36  Ibs.) 17.86 

Per  case  in  lots  of  3  original  cases  (108  Ibs.) . .  16.74 
Per  case  in  lots  of  30  original  cases 

(1080  Ibs.) 15.62 

Per  drum  containing  25  Ibs 9.45 

Per  100  Ibs.  in  lots  of  4  x  25  Ib.  drums 34.00 

Per  100  Ibs.  in  lots  of  1  x  100  Ib.  drum .  .  .  .30.62 
Per  100  Ibs.  in  lots  of  20  x  25  Ib.  drums 

(500  Ibs.) 32.30 

Per  100  Ibs.  in  lots  of  40  x  25  Ib.  drums 

(1000  Ibs.) 30.40 


Sample  of  Thomas  Cleaning  Compound  senf  upon  request. 


A.H.T.CO. 


ARTHUR  H.  THOMAS  COMPANY 

More  and  more  laboratories  rely  on  Thomas  /    Laboratory   Apparatus   and  Reagents 

BOX  779  •  PHILADELPHIA  5.  PA. 


y  /    Lab 
I  P.O. 


iimiimimmiiiiimimiimimimiiimimmiimimmimiimmiimmmmmm 


BIOLOGY  MATERIALS 

The  Supply  Department  of  the  Marine  Biological  Labora- 
tory has  a  complete  stock  of  excellent  plain  preserved  and 
injected  materials,  and  would  be  pleased  to  quote  prices  on 
school  needs. 


PRESERVED  SPECIMENS 

for 

Zoology,  Botany,  Embryology, 
and  Comparative  Anatomy 

LIVING  SPECIMENS 

for 
Zoology  and  Botany 

including  Protozoan  and 
Drosophila  Cultures,  and 
Animals  for  Experimental  and 
Laboratory  Use. 

MICROSCOPE  SLIDES 

for 

Zoology,  Botany,  Embryology, 
Histology,  Bacteriology,  and 
Parasitology. 

CATALOGUES  SENT  ON  REQUEST 


Supply  Department 

MARINE 
BIOLOGICAL  LABORATORY 

Woods  Hole,  Massachusetts 


CONTENTS 


Page 
Annual  Report  of  the  Marine  Biological  Laboratory 1 

CAIN,  GERTRUDE  L. 

Studies  on  cross-fertilization  and  self-fertilization  in  Lymnaea 
stagnalis  appressa  Say 45 

DEHNEL,  PAUL  A.,  AND  EARL  SEGAL 

Acclimation  of  oxygen  consumption  to  temperature  in  the 
American  cockroach  (Periplaneta  americana) 53 

DURAND,  JAMES  B. 

Neurosecretory  cell  types  and  their  secretory  activity  in  the 
crayfish 62 

FRASER,  RONALD  C. 

The  presence  and  significance  of  respiratory  metabolism  in 
streak-forming  chick  blastoderms 77 

FRINGS,  HUBERT,  AND  MABLE  FRINGS 

The  location  of  contact  chemoreceptors  sensitive  to  sucrose 
solutions  in  adult  Trichoptera 92 

HASTINGS,  J.  WOODLAND,  AND  JOHN  BUCK 

The  firefly  pseudoflash  in  relation  to  photogenic  control.  ...   101 

LAVOIE,  MARCEL  E. 

'  How  sea  stars  open  bivalves 114 

ROGICK,  MARY 

Studies   on   marine   bryozoa.     VIII.  Exochella   longirostris 
Jullien  1888 123 

SEGAL,  EARL 

Microgeographic  variation  as  thermal  acclimation  in  an  inter- 
tidal  mollusc 129 

TYLER,  ALBERT,  ALBERTO  MONROY,  C.  Y.  KAO  AND   HARRY 

GRUNDFEST 

Membrane  potential  and  resistance  of  the  starfish  egg  before 
and  after  fertilization .  153 


IdH    1AZG    P